Compositions and methods for effective in vivo delivery

Abstract

Disclosed herein are compositions, methods, kits, and systems for efficient delivery of cargo (for example, therapeutic cargo) into cells, illustratively for in vivo delivery. The present disclosure provides lipid-containing particles (for example, virus-like particles) for delivery of therapeutic cargo. The present disclosure also provides polynucleotides encoding the lipid-containing particles provided herein, which may be useful for producing the lipid-containing particles. Also provided are methods for editing nucleic acid molecules in cells using the lipid-containing particles provided herein, as well as cells and kits that include lipid-containing particles.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 285,995, filed December 3, 2021, U.S. Provisional Patent Application No. 63 / 298,621, filed January 11, 2022, U.S. Provisional Patent Application No. 63 / 298,611, filed January 11, 2022, U.S. Provisional Patent Application No. 63 / 298,626, filed January 11, 2022, and U.S. Provisional Patent Application No. 63 / 423,372, filed November 7, 2022, each of which is incorporated herein by reference.

[0002] Federally funded research This invention was made with government support under Grant Nos. UG3AI150551, U01AI142756, R35GM118062, RM1HG009490, R01EY009339, and T32GM095450 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Retroviruses can serve as attractive scaffolds for virus-like particles (VLPs). Retroviral capsids generally lack the rigid symmetry requirements of non-enveloped icosahedral viruses (Zhang et al., 2015), suggesting increased structural flexibility for incorporating non-native protein cargo. In addition, retroviral tropism can be modulated by pseudotyping virions with different envelope glycoproteins, which can enable targeting of VLPs to specific cell types (Cronin et al., 2005). Previous studies have demonstrated that fusing a desired protein cargo to the C-terminus of the retroviral gag polyprotein is sufficient to directly package the cargo protein into retroviral particles (Kaczmarczyk et al., 2011; Voelkel et al., 2010). More recently, a similar strategy has been applied to package Cas9 RNPs within retroviral particles (Mangeot et al., 2019). However, there remains a need for VLPs with therapeutic levels of in vivo delivery efficiency. Summary of the Invention

[0004] overview In one aspect, the present disclosure provides a lipid-containing particle comprising: (a) a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule; (b) a fusion protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and (c) a therapeutic cargo.

[0005] In another aspect, the present disclosure provides a composition comprising: (a) a first nucleic acid molecule encoding a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule; and (b) a second nucleic acid molecule encoding a fusion protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES) and a cargo, wherein the cargo comprises a therapeutic cargo or a binding partner for a therapeutic cargo. In another aspect, the present disclosure provides a nucleic acid molecule encoding a fusion protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES) and a cargo, wherein the cargo comprises a therapeutic cargo or a binding partner for a therapeutic cargo.

[0006] In yet another aspect, the present specification describes a lipid-containing particle comprising: (a) a human endogenous retrovirus (HERV) envelope protein, (b) a humanized viral envelope protein, or a non-immunogenic cell fusion molecule; (c) a fusion protein comprising a plasma membrane-localized protein coupled to a cleavable linker; and (d) a therapeutic cargo. In some embodiments, the fusion protein comprises an NES.

[0007] In another aspect, the present disclosure provides a method for producing a lipid-containing particle described herein, comprising: (a) providing a system expressing (i) a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule; (ii) a fusion protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and iii. a cargo, wherein the system produces a lipid-containing particle; and optionally, (b) harvesting and purifying the lipid-containing particle.

[0008] In a further aspect, the present disclosure describes a method for producing a lipid-containing particle as described herein, comprising: (a) providing a system for expressing (i) a fusion protein comprising a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule; (ii) a plasma membrane-localized protein coupled to a cleavable linker; and iii. a cargo, wherein the system produces lipid-containing particles; and optionally (b) harvesting and purifying the lipid-containing particles. In some embodiments, the fusion protein comprises an NES.

[0009] In another aspect, the present disclosure provides a method of producing a lipid-containing particle described herein, comprising: (a) providing a system expressing a fusion protein comprising (i) a humanized retroviral structural protein; a human endogenous retroviral (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, and (ii) a nuclear export sequence (NES), wherein the system produces lipid-containing particles; and, optionally, (b) harvesting and purifying the lipid-containing particles.

[0010] In yet another aspect, the present disclosure provides a method for producing lipid-containing particles as described herein, comprising: (a) providing a system for expressing a fusion protein comprising (i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally a HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein; (ii) a cleavable linker; and (iii) a cargo, wherein the system produces lipid-containing particles; and optionally (b) harvesting and purifying the lipid-containing particles. In some embodiments, the fusion protein further comprises an NES.

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

[0012] In various embodiments, the therapeutic cargo can comprise a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, a small molecule compound, or any combination thereof. In some embodiments, the therapeutic cargo does not comprise a nuclease, a reverse transcriptase, a base editor, or a prime editor. In some embodiments, the therapeutic cargo is a protein (by way of example only, a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, or a reverse transcriptase). In some embodiments, the therapeutic cargo is a nucleic acid molecule (by way of example only, DNA, RNA, retrotransposon, aptazyme, aptamer, or ribozyme). In some embodiments, the therapeutic cargo is an epigenetic editor, a restriction endonuclease (optionally a Type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, retrotransposon, an oligonucleotide, an aptazyme, aptamer, ribozyme, a small molecule compound, or any combination thereof.

[0013] In various embodiments, the fusion protein can comprise a plasma membrane-localized protein, an NES, and a therapeutic cargo, arranged in order from N-terminus to C-terminus. The fusion protein can further comprise a cleavable linker, optionally wherein the cleavable linker is positioned between the plasma membrane-localized protein and the therapeutic cargo, optionally wherein the cleavable linker is positioned between the NES and the therapeutic cargo, and optionally wherein the fusion protein further comprises a nuclear localization sequence (NLS) C-terminal to the cleavable linker. In some embodiments, the NLS is positioned within the fusion protein so that it remains attached to the therapeutic cargo after cleavage of the cleavable linker, enabling delivery of the therapeutic cargo to the nucleus of the cell.

[0014] In various embodiments, the plasma membrane-localized protein can include a human endogenous retroviral (HERV) structural protein, optionally HERV gag; a humanized viral structural protein; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein.

[0015] In various embodiments, the therapeutic cargo is a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a Type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof.

[0016] In another aspect, the present disclosure provides lipid-containing particles comprising a lipid membrane encapsulating a protein core, wherein the protein core comprises a group-specific antigen (gag) protease (pro) polyprotein and cleavage products, the lipid-containing particles further comprising a therapeutic cargo, the therapeutic cargo being present inside the protein core, and the cleavage products comprising (i) a gag nucleocapsid protein sequence and (ii) a nuclear export sequence (NES) and lacking the therapeutic cargo. In some embodiments, the therapeutic cargo is fused to a nuclear localization sequence (NLS).

[0017] In another aspect, the present disclosure provides a lipid-containing particle comprising a lipid membrane encapsulating a protein core, wherein the protein core comprises a group-specific antigen (gag) protease (pro) polyprotein and a fusion protein, wherein the fusion protein comprises the following: a gag nucleocapsid protein sequence, a therapeutic cargo, a cleavable linker, and a nuclear export sequence (NES), wherein the cleavable linker is positioned between the therapeutic cargo and the NES.

[0018] In another aspect, the disclosure provides a population of lipid-containing particles, wherein the population comprises lipid-containing particles comprising a lipid membrane encapsulating a protein core, the protein core comprising a group-specific antigen (gag) protease (pro) polyprotein and a fusion protein, the population comprising lipid-containing particles comprising a therapeutic cargo, the therapeutic cargo being present inside the protein core, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein being at least 1.5 among the population of lipid-containing particles, the fusion protein comprising a first polypeptide and a second polypeptide, and the first polypeptide comprising a sequence of a gag nucleocapsid protein and the second polypeptide comprising a sequence of the therapeutic cargo.

[0019] In another aspect, the disclosure provides a composition comprising: (i) a first polynucleotide comprising a nucleic acid sequence encoding a group-specific antigen (gag) protease (pro) polyprotein; and (ii) a second polynucleotide comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises the following: a sequence of a gag nucleocapsid protein, a therapeutic cargo, a cleavable linker, and a nuclear export sequence (NES), wherein the cleavable linker is positioned between the therapeutic cargo and the NES. In some embodiments, the composition is a pharmaceutical composition.

[0020] In another aspect, the present disclosure provides a fusion protein comprising a gag nucleocapsid protein sequence, a therapeutic cargo, a cleavable linker, and a nuclear export sequence (NES); wherein the cleavable linker is positioned between the therapeutic cargo and the NES.

[0021] In another aspect, the present disclosure provides methods of using the lipid-containing particles provided herein, for example, in methods of nucleic acid editing.

[0022] In another aspect, the present disclosure provides cells for producing the lipid-containing particles provided herein. In some embodiments, the cells contain any of the nucleic acids encoding components of the lipid-containing particles provided herein.

[0023] In another aspect, the present disclosure provides kits comprising any of the lipid-containing nanoparticles, nucleic acid sequences, fusion proteins, and compositions provided herein.

[0024] It should be understood that the foregoing concepts, and additional concepts described below, can be arranged in any suitable combination, as the disclosure is not limited in this respect. Furthermore, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.

[0025] Incorporation by Reference 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.

[0026] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative modes in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0027] Brief description of the drawings [Figure 1] Figure 1A shows a schematic diagram of a base-edited virus-like particle (BE-VLP). As shown in the diagram, a base editor protein is fused to the C-terminus of the murine leukemia virus (MLV) gag polyprotein via a linker that is cleaved by MLV protease during particle maturation.

[0028] Figure 1B shows two graphs summarizing the base editing efficiency of version 1 (v1) BE-VLPs. Adenine base editing efficiency of v1 BE-VLPs at two genomic loci in HEK293T cells (referred to as "HEK2" and "HEK3," respectively). The protospacer position of the targeted adenine is indicated by a subscript (i.e., A5 = adenine at position 5), where the PAM is at positions 21-23. Data are shown as individual data points and mean ± standard error (sem) for n = 3 independent biological replicates.

[0029] [Figure 2A-B] Figure 2A shows a schematic diagram of v1 engineered VLPs (eeVLPs) and v2 eVLPs. More efficient linker cleavage in v2 BE-VLPs can result in improved cargo release after VLP maturation.

[0030] Figure 2B shows a graph summarizing the adenine base editing efficiency of v1 and v2 BE-eVLPs at position A7 of the BCL11A enhancer site in HEK293T cells.

[0031] [Figure 2C-D] FIG. 2C shows a schematic demonstrating that improved localization of cargo, as enabled in v3 eVLPs in production cells, leads to more efficient incorporation into eVLPs.

[0032] Figure 2D shows a schematic demonstrating that introducing a 3xNES motif upstream of a cleavable linker can promote cytoplasmic localization of gag-3xNES-cargo in production cells, but can promote nuclear localization of a free adenine base editor (ABE) cargo in transduced cells.

[0033] [Figure 2E-F] Figure 2E shows a graph summarizing the adenine base editing efficiency of v2.4 and v3 BE-eVLPs at position A7 of the BCL11A enhancer site in HEK293T cells.

[0034] Figure 2F shows a schematic demonstrating that optimal gag-cargo:gag-pro-pol stoichiometry can balance the amount of cargo protein per particle with the amount of MMLV protease required for efficient particle maturation.

[0035] [Figure 2G]Figure 2G shows a graph summarizing the adenine base editing efficiency of v3.4 eVLPs with different gag-ABE:gag-pro-pol stoichiometries at position A7 of the BCL11A enhancer site in HEK293T cells. The legend displays the % gag-ABE plasmid of the total amount of gag-ABE and gag-pro-pol plasmids. As shown in Figure 2B, Figure 2E, and Figure 2G, values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0036] [Figure 3A-B] Figure 3A is a graph quantifying the amount of BE molecules per eVLP by anti-Cas9 and anti-MLV(p30) ELISA. Values ​​and error bars reflect the mean ± standard error of n=3 independent replicates.

[0037] Figure 3B is a graph quantifying relative sgRNA abundance by RT-qPCR using sgRNA-specific primers, normalized to v1 sgRNA abundance. Values ​​and error bars reflect the mean ± standard error of n=3 technical replicates.

[0038] [Figure 3C-D] Figures 3C-3D show graphs comparing the editing efficiency of v1, v2.4, v3.4, and v4 BE-eVLPs at the BCL11A enhancer site in HEK293T cells (Figure 3C) and the Dnmt1 site in NIH 3T3 cells (Figure 3D). Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0039] [Figure 3E-F]Figure 3E is a graph summarizing the adenine base editing efficiency in HEK293T cells of either a single v4 BE-eVLP targeting the HEK2 or BCL11A enhancer locus separately, or multiple v4 BE-eVLPs targeting both loci simultaneously.

[0040] Figure 3F is a graph summarizing the adenine base editing efficiency of FuG-B2-pseudotyped v4 BE-eVLPs in Neuro-2a cells or 3T3 fibroblasts.

[0041] [Figure 3G] Figure 3G shows a graph summarizing adenine base editing efficiencies at three on-target genomic loci and their corresponding Cas-dependent off-target sites in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmid. OT1 = off-target site 1, OT2 = off-target site 2, OT3 = off-target site 3.

[0042] [Figure 3H] Figure 3H is a graph summarizing the frequency of Cas-independent off-target editing at six off-target R-loops in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmid. OTRL = off-target R-loop.

[0043] [Figure 3I-J] Figure 3I is a graph quantifying the amount of molecules of BE-encoding DNA per v4 BE-eVLP detected by qPCR of lysed eVLPs or lysis buffer only.

[0044] Figure 3J is a graph quantifying the amount of BE-encoding DNA detected by qPCR of lysates from HEK293T cells either treated with v4 BE-eVLPs or transfected with a BE-encoding plasmid. As shown in Figures 3E-J, data are presented as individual data points and means ± standard errors for n=3 independent biological replicates.

[0045] [Figure 4A-B] FIG. 4A is a graph summarizing the correction efficiency of the COL7A1 (R185X) mutation in patient-derived primary human fibroblasts.

[0046] Figure 4B is a graph summarizing the correction efficiency of the Idua(W392X) mutation in primary mouse fibroblasts. As shown in Figure 4A-B, values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0047] [Figure 4C] Figure 4C is a graph summarizing adenine base editing efficiency at the B2M and CIITA loci in primary human T cells. Data are shown as individual data points and means ± standard errors for n=3 independent biological replicates.

[0048] [Figure 5A] Figure 5A is a schematic diagram of P0 ICV injection of v4 BE-eVLPs. Dnmt1-targeting v4 BE-eVLPs were co-injected with lentivirus encoding EGFP-KASH. Tissues were harvested 3 weeks post-injection, and the cortex and midbrain were isolated. Nuclei were dissociated for each tissue and analyzed by high-throughput sequencing as bulk unsorted (all nuclei) or GFP+ nuclei.

[0049] [Figure 5B]Figure 5B is a graph summarizing adenine base editing efficiency at the Dnmt1 locus in bulk unsorted (all nuclei) and GFP+ populations. Data are shown as individual data points and mean ± standard error for n=4 mice.

[0050] [Figure 6A] Figure 6A shows a schematic diagram of systemic injection of BE-eVLPs. Pcsk9-targeting BE-eVLPs were injected retroorbitally into 6-7 week-old C57BL / 6J mice. Organs were harvested 1 week after injection, and genomic DNA from unsorted cells was sequenced.

[0051] [Figure 6B-C] Figure 6B is a graph summarizing adenine base editing efficiency at the Pcsk9 exon 1 splice donor in mouse liver after systemic injection of v1 BE-VLPs or v4 BE-eVLPs. Data are shown as individual data points and mean ± standard error for n=3 mice (v1 BE-VLPs and v4 BE-eVLPs at 4x10 VLPs) or n=4 mice (v4 BE-eVLPs at 7x10 eVLPs).

[0052] Figure 6C shows the 7 x 10 11

[0023] Figure 11 is a graph summarizing adenine base editing efficiency at the Pcsk9 exon 1 splice donor in the heart, kidney, liver, lung, muscle, and spleen of mice after systemic injection of v4 BE-eVLPs. Data are shown as mean ± standard error for individual data points and for n = 4 mice (treated) or n = 3 mice (untreated).

[0053] [Figure 6D]Figure 6D is a graph summarizing quantification of DNA sequencing reads containing A·T→G·C mutations within protospacer positions 4-10 for 14 CIRCLE-seq-designated off-target loci from the livers of v4 BE-eVLP-treated, AAV-treated, and untreated mice. Data are shown as individual data points and means ± standard errors for n = 4 mice (BE-eVLP), n = 5 mice (AAV), or n = 3 mice (untreated). vg = viral genome.

[0054] [Figure 6E] Figure 6E is a graph summarizing the quantification of serum Pcsk9 levels as measured by ELISA. Data are presented as mean ± standard error for individual data points and n = 4 mice (treated) or n = 3 mice (untreated).

[0055] [Figure 7A] Figure 7A is a schematic of Rpe65 exon 3 encompassing the R44X mutation (labeled and shown in gray in the schematic), which can be corrected by an A·T→G·C transversion at position A6 of the protospacer (labeled and underlined in the schematic; PAM sequence (AGT) in italics). The sequences shown (top to bottom) correspond to SEQ ID NOs: 5-6.

[0056] [Figure 7B-C] Figure 7B is a schematic diagram of subretinal injection. Five weeks after injection, phenotypic rescue was assessed via ERG, and tissue was subsequently harvested for sequencing.

[0057] Figure 7C is a graph summarizing adenine base editing efficiency at protospacer positions A3, A6, and A8 in genomic DNA from rd12 mice. Data are shown as individual data points and means ± standard errors for n = 6 mice (both treatment groups) or n = 4 mice (untreated).

[0058] [Figure 7D-E] Figure 7D is a graph summarizing the allele frequency distribution of genomic DNA collected from treated rd12 mice. Data are shown as mean ± standard error for n = 6 mice. 8e-LV = ABE8e-NG-LV, 8e-eVLP = v4 ABE8e-NG-eVLP.

[0059] Figure 7E is a graph summarizing scotopic a- and b-wave amplitudes measured by ERG after overnight dark adaptation. Data are shown as individual data points and means ± standard errors for n = 8 mice (wild-type), n = 6 mice (ABE8e-NG-LV and v4 ABE8e-NG-eVLP), or n = 4 mice (naive).

[0060] [Figure 7F-G] Figure 7F is a graph summarizing adenine base editing efficiency at protospacer positions A3, A6, and A8 in genomic DNA collected from rd12 mice. Data are shown as individual data points and means ± standard errors for n = 6 mice (v4 ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated). P values ​​were calculated using a two-tailed t-test.

[0061] Figure 7G is a graph summarizing the allele frequency distribution of genomic DNA collected from treated rd12 mice. Data are shown as mean ± standard error for n = 6 mice (v4 ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated). 7.10-LV = ABE7.10-NG-LV, 7.10-eVLP = v4 ABE7.10-NG-eVLP.

[0062] [Figure 7H-I]Figure 7H is a graph summarizing scotopic a- and b-wave amplitudes measured by ERG after overnight dark adaptation. Data are shown as individual data points and means ± standard errors for n = 8 mice (wild-type), n = 7 mice (v4 ABE7.10-NG-eVLP), n = 5 mice (ABE7.10-NG-LV), or n = 4 mice (naive). P values ​​were calculated using a two-tailed t-test.

[0063] FIG. 7I shows images of Western blots of protein extracts from RPE tissues of wild-type, untreated, v4 ABE7.10-NG-eVLP-treated, and ABE7.10-NG-LV-treated mice.

[0064] [Figure 7J] Figure 7J shows representative ERG waveforms from wild-type, untreated, ABE7.10-NG-LV-treated, and v4 ABE7.10-NG-eVLP-treated mice.

[0065] [Figure 8A-B] Figure 8A shows an image of immunoblot analysis of proteins from purified BE-VLPs using anti-Cas9, anti-p30, and anti-VSV-G antibodies to verify VLP production.

[0066] Figure 8B is a graph summarizing the adenine base editing efficiency of v1 BE-VLPs at position A7 of the BCL11A enhancer site in HEK293T cells. Values ​​and error bars reflect the mean ± standard error of n = 3 independent biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0067] [Figure 8C]Figure 8C is a schematic diagram of an immature BE-VLP with ABE8e fused to the gag structural protein. Various MMLV protease cleavage sites were inserted between gag and ABE8e to determine the optimal cleavable sequence that facilitates release of ABE8e from gag during proteolytic virion maturation. Arrows indicate the cleavage sites. The sequences shown are PRSSLY (SEQ ID NO: 7), PALTP (SEQ ID NO: 8), VQAL (SEQ ID NO: 9), VLTQ (SEQ ID NO: 10), PLQVL (SEQ ID NO: 11), TLNIERR (SEQ ID NO: 12), TSTLL (SEQ ID NO: 13), and MENSS (SEQ ID NO: 14).

[0068] [Figure 8D-E] FIG. 8D shows a representative image of a Western blot assessing cleaved ABE8e versus full-length gag-ABE8e in purified v2 BE-eVLP variants.

[0069] Figure 8E is a graph summarizing densitometry-based quantification of cleaved ABE8e fractions from Western blots. Data are shown as individual data points and mean ± standard error for n = 3 technical replicates.

[0070] [Figure 9A] Figure 9A shows a schematic of the v2.4 and v3 BE-eVLP constructs. Three HIV NESs were fused to either the C- or N-terminus of the gag-ABE fusion. A protease-cleavable linker was incorporated between the ABE and NES sequences such that the final BE cargo lacked the NES after proteolytic virion maturation. The sequences shown are TSTLL (SEQ ID NO: 13), MENSS (SEQ ID NO: 14), MSKLL (SEQ ID NO: 15), ATVVS (SEQ ID NO: 16), PLQVL (SEQ ID NO: 11), TLNIERR (SEQ ID NO: 12), IRKIL (SEQ ID NO: 17), and FLDG (SEQ ID NO: 18).

[0071] [Figure 9B-C]Figure 9B shows representative immunofluorescence images of producer cells transfected with the v2.4 gag-ABE construct or the v3.4 gag-3 xNES-ABE construct. Forty-eight hours after transfection, cells were fixed with paraformaldehyde and stained with anti-tubulin antibody to stain the cytoskeleton, DAPI to stain the nucleus, and anti-Cas9 antibody to visualize the gag-ABE fusion, as indicated in the legend. The scale bar represents 50 μm.

[0072] Figure 9C shows a graph summarizing automated image analysis-based quantification of cytoplasmic localization of the v2.4 gag-ABE or v3.4 gag-3 xNES-ABE constructs. Data are presented as individual data points and mean ± standard error for n = 3 technical replicates. P values ​​were calculated using a two-tailed t-test.

[0073] [Figure 10A-C] Figure 10A shows a representative negative stain transmission electron micrograph (TEM) of v4 BE-eVLPs. The scale bar indicates 200 nm.

[0074] Figures 10B-10C show quantification of protein content of v1, v2.4, v3.4, and v4 BE-eVLPs as measured by anti-Cas9 or anti-MLV(p30) ELISA. Data are shown as individual data points and mean ± standard error for n=3 technical replicates.

[0075] [Figure 10D-G] Figure 10D shows a graph comparing particle number-normalized editing efficiency of v1, v2.4, v3.4, and v4 BE-VLPs at the BCL11A enhancer site in HEK293T cells. Data are shown as mean ± standard error for n=3 biological replicates.

[0076] Figure 10E shows a graph summarizing cell viability after v4 BE-eVLP treatment of HEK293T cells and NIH 3T3 fibroblasts. Data are presented as mean ± standard error for n=3 biological replicates.

[0077] Figure 10F is a graph summarizing the indel frequencies produced by v1 Cas9-VLPs and v4 Cas9-eVLPs at the EMX1 locus in HEK293T cells. Data are shown as mean ± standard error for n = 3 biological replicates. Data were fitted to a four-parameter logistic curve using nonlinear regression.

[0078] Figure 10G shows a graph summarizing the adenine base editing efficiency of VSV-G pseudotyped v4 BE-eVLPs in Neuro-2a cells or 3T3 fibroblasts. Data are shown as individual data points and mean ± standard error for n = 3 biological replicates.

[0079] [Figure 11A] FIG. 11A shows the experimental timeline for the orthogonal R-loop assay.

[0080] [Figure 11B-C] Figure 11B is a graph summarizing on-target editing control for orthogonal R-loop experiments. Data are shown as individual data points and mean ± standard error for n = 3 biological replicates.

[0081] Figure 11C is a graph summarizing cell viability following v4 BE-eVLP treatment of RDEB fibroblasts. Data are presented as mean ± standard error for n=3 biological replicates.

[0082] [Figure 11D]Figure 11D is a graph quantifying DNA sequencing reads containing A·T → G·C mutations within positions 4-10 of the protospacer for 10 previously identified off-target loci from genomic DNA of fibroblasts derived from a v4-BE-eVLP-treated RDEB patient. The gray dotted line represents the highest observed background mutation rate of 0.1%. Data are shown as individual data points and mean ± standard error for n = 3 biological replicates.

[0083] [Figure 12] Figures 12A-12B show flow cytometry analysis for nuclei sorting from mouse brain after P0 ICV injection, related to Figures 5A-5B. Figure 12A shows a representative flow cytometry graph. Singlet nuclei were gated based on FSC / BSC ratio and DyeCycle Ruby signal. Row 1 demonstrates the gating strategy for GFP-negative samples. Bulk nuclei correspond to events that passed gate D for singlet nuclei. Figure 12B is a graph summarizing the percentage of GFP-positive nuclei measured by flow cytometry after P0 ICV injection. Data are shown as mean + standard error for n=3 biological replicates.

[0084] [Figure 13] FIG. 13A shows a graph summarizing plasma aspartate transaminase (AST) and alanine transaminase (ALT) levels one week after v4 BE-eVLP injection.

[0085] Figures 13B-13C show representative images of hematoxylin and eosin staining of the livers of (Figure 13B) untreated mice and (Figure 13C) v4 BE-eVLP-treated mice at 1 week post-injection. Representative examples of each are shown. Scale bars represent 50 μm.

[0086] [Figure 14A]Figures 14A-C show the results of sequencing analysis of RPE cDNA after v4 BE-eVLP or lentivirus treatment. Figure 14A shows that v4 BE-eVLP and lentivirus treatment resulted in a 50-60% A·T→G·C transversion at the target adenine (A6) of the Rpe65 transcript. Data are shown as individual data points and mean ± standard error for n = 6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n = 4 (ABE7.10-NG-LV and untreated) replicates. [Figure 14B] Figures 14B-C show off-target A-to-G RNA editing by v4 BE-eVLPs and lentiviruses as measured by high-throughput sequencing of Mcm3ap (Figure 14B) and Perp (Figure 14C) transcripts. Data are shown as individual data points and mean ± standard error for n=6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n=4 (ABE7.10-NG-LV and naive) replicates. [Figure 14C] Figures 14B-C show off-target A-to-G RNA editing by v4 BE-eVLPs and lentiviruses as measured by high-throughput sequencing of Mcm3ap (Figure 14B) and Perp (Figure 14C) transcripts. Data are shown as individual data points and mean ± standard error for n=6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n=4 (ABE7.10-NG-LV and naive) replicates. DETAILED DESCRIPTION OF THE INVENTION

[0087] Detailed Description The implementation of some methods disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of those skilled in the art, unless otherwise specified. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMA Usubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR 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 (RI Freshney ed. (2010)).

[0088] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor polypeptide" includes a plurality of chimeric transmembrane receptor polypeptides.

[0089] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with 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 in the context of 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. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0090] As used herein, "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 non-limiting examples are prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (including, by way of example, plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, and mosses), algal cells (including, by way of example, Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.). Examples of cells that can be used include cells from organisms such as: C. Agardh, and the like), seaweed (for example, kelp), fungal cells (for example, yeast cells, cells from mushrooms), animal cells, cells from invertebrates (for example, Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (for example, fish, amphibians, reptiles, birds, mammals), mammalian cells (for example, pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not be derived from a naturally occurring organism (for example, cells may be synthetically produced, sometimes referred to as artificial cells).

[0091] The term "antigen," as used herein, refers to a molecule or fragment thereof that can be bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent, such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent, such as an immunological protein (for example, an antibody). An antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce antibodies that are capable of binding to that antigen.

[0092] The term "antibody" as used herein refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody encompasses antibodies (by way of example only, monoclonal and polyclonal antibodies) as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Derivatives, variants, or fragments thereof can refer to functional derivatives or fragments that retain the binding specificity (by way of example only, fully and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), minibodies, diabodies, and single-domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody encompasses antigen-binding fragments of antibodies and optimized, engineered, or chemically conjugated antibodies. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized in fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies).

[0093] The term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates such as 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, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides 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, and Texas A&M.Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; and Boehringer Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2'-dATP available from 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, and Cascade Blue available from Molecular Probes, Eugene, Oreg. Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, RhodamineNucleotides can also be labeled or marked by chemical modification. The chemically modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (for example, bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (for example, biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (for example, biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0094] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably to refer to polymeric, single-stranded, double-stranded, or multi-stranded forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can be present in a cell-free environment. A polynucleotide can be a gene or a 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 contain one or more analogs (by way of example, altered backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (such as rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, a locus (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0095] The term "gene," as used herein, refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) and their corresponding nucleotide sequences involved in encoding an RNA transcript. When used herein with reference to genomic DNA, the term includes intervening non-coding and regulatory regions and can encompass the 5' end and 3' end. In some uses, the term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequence (e.g., an "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, e.g., a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" encompasses not only the transcribed sequence but also 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 within the genome of an organism. A gene can refer to a "foreign gene" or a non-native gene. A non-native gene can refer to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location within the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions, and / or deletions (as an example, a non-native sequence).

[0096] The terms "target polynucleotide" and "target nucleic acid," as used herein, refer to a nucleic acid or polynucleotide targeted by a cargo of the present disclosure. A target polynucleotide can be DNA (for example, exogenous or endogenous). DNA can refer to a template that produces an mRNA transcript and / or various regulatory regions that regulate transcription of mRNA from a DNA template. A target polynucleotide can be a portion of a larger polynucleotide, such as a chromosome or a region of a chromosome. A target polynucleotide can refer to an extrachromosomal sequence (for example, 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 a template that encodes a protein. A target polynucleotide comprising RNA can encompass various regulatory regions that regulate protein translation from an mRNA template. A target polynucleotide can encode a gene product (for example, DNA encoding an RNA transcript or RNA encoding a protein product) or can include regulatory sequences that regulate expression of a gene product. Generally, the term "target sequence" refers to a nucleic acid sequence on a single strand of a target nucleic acid. The target sequence can be a part of a gene, a regulatory sequence, genomic DNA, cell-free nucleic acid including cfDNA and / or cfRNA, cDNA, fusion gene, and RNA including mRNA, miRNA, rRNA, etc., as well as others. When targeted by a cargo, the target polynucleotide can result in altered gene expression and / or activity. When targeted by a cargo, the target polynucleotide can result in an edited nucleic acid sequence. The target nucleic acid can include a nucleic acid sequence that cannot be related to any other sequence in a nucleic acid sample by a single nucleotide substitution. The target nucleic acid can include a nucleic acid sequence that cannot be related to any other sequence in a nucleic acid sample by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions.In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides of the 5' end of the target nucleic acid. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides of the 3' end of the target nucleic acid.

[0097] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Up-regulated," with respect to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence compared to its expression level in the 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 compared to its expression in the wild-type state.

[0098] The terms "complement," "complements," "complementary," and "complementarity," as used herein, generally refer to a sequence that is perfectly complementary to and capable of hybridizing to a given sequence. In some cases, a sequence hybridized to a given nucleic acid is said to be the "complement" or "reverse-complement" of a given molecule when its base sequence over a given region is capable of binding complementary to that of its binding partner, e.g., when AT, AU, GC, and GU base pairs are formed. Generally, 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 (as an example, more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art) over hybridization to non-target sequences during a hybridization reaction. Typically, hybridizable sequences share a degree of sequence complementarity over all or part of their respective lengths, such as 25% to 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 purposes of assessing percent complementarity, can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (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 (e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (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 for the chosen algorithm, including default parameters.

[0099] Complementarity can be complete or substantial / sufficient. Complete complementarity between two nucleic acids can mean that the two nucleic acids are capable of forming a duplex in which all bases in the duplex are bound to complementary bases by Watson-Crick pairing. Substantial or sufficient complementarity can mean that the sequence of one strand is not completely and / or completely complementary to the sequence of the opposing strand, but sufficient binding occurs between the bases of the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm of the hybridized strands, or by empirical determination of the Tm using routine methods.

[0100] The term "regulating," as used herein in reference to expression or activity, 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.

[0101] 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). The term does not imply a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical synthesis, enzymatic synthesis, or whether it is naturally occurring. The term applies to naturally occurring amino acid polymers and amino acid polymers containing at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The term encompasses amino acid chains of any length, including full-length proteins and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. The terms "amino acid" and "amino acids," as used herein, generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural and unnatural amino acids, which have been chemically modified to include a group or chemical moiety that does not naturally occur on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0102] The term "variant," as used herein with respect to a polypeptide, refers to a polypeptide that is related to, but not identical to, a wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Variants include polypeptides that contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide. Variants also include derivatives of wild-type polypeptides and fragments of wild-type polypeptides.

[0103] The term "percent (%) identity" as used herein refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent identity (i.e., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways within the skill of those skilled in the art, using publicly available computer software such as, for example, BLAST, ALIGN, or Megalign (DNASTAR) software. The 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 at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0104] The Cas protein referred to herein can be a type of protein or polypeptide. The Cas protein can refer to a nuclease. The Cas protein can refer to an endoribonuclease. The Cas protein can refer to any modified (e.g., truncated, mutated, extended) polypeptide sequence or homolog of a Cas protein. The Cas protein can be codon-optimized. The Cas protein can be a codon-optimized homolog of a Cas protein. The Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., more active than a wild-type homolog of the protein or polypeptide). The Cas protein can be a type II Cas protein. The Cas protein can be Cas9. The Cas protein can be a type V Cas protein. The Cas protein can be Cpf1 or Cas12a. The Cas protein can be C2c1. The Cas protein can be C2c3. The Cas protein can be a type VI Cas protein. The Cas protein can be C2c2 or Cas13a. The Cas protein can be Cas13b. The Cas protein can be Cas13c. The Cas protein can be Cas13d. The Cas protein can be Cas14. The Cas protein (including, by way of example, a variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-directed polypeptide) can bind to a target nucleic acid. The Cas protein (including, by way of example, a variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonuclease) can bind to a target RNA or DNA.

[0105] The term "crRNA," as used herein, can generally refer to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., crRNA from S. pyogenes). A crRNA can generally refer to a nucleic acid having up to 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., crRNA from S. pyogenes, S. aureus, etc.). A crRNA can also refer to modified forms of crRNA that can include nucleotide changes, such as deletions, insertions, or substitutions, variants, mutations, or chimeras. The crRNA can be a nucleic acid having at least about 60% sequence identity to a wild-type exemplary crRNA (e.g., crRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six contiguous nucleotides. For example, the 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 (e.g., crRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six contiguous nucleotides.

[0106] The term "tracrRNA," as used herein, generally refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from S. pyogenes). tracrRNA can refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from S. pyogenes, S. aureus, etc.). tracrRNA sequences can refer to modified forms of tracrRNA that can include nucleotide changes, such as deletions, insertions, or substitutions, variants, mutations, or chimeras. A tracrRNA can refer to a nucleic acid that can be at least about 60% identical to a wild-type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six 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 six contiguous nucleotides.

[0107] As used herein, "guide nucleic acid" can refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to site-specifically bind to a nucleic acid sequence. A targeted or target nucleic acid can comprise nucleotides. A guide nucleic acid can comprise nucleotides. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid can be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore may not be complementary to the guide nucleic acid can be referred to as a non-complementary strand. A guide nucleic acid can comprise a polynucleotide strand and can be referred to as 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 strands and can be referred to as a "dual guide nucleic acid." A dual guide nucleic acid can comprise a crRNA and a tracrRNA. Unless otherwise specified, the term "guide nucleic acid" can be inclusive, referring to both single and dual guide nucleic acids.

[0108] A guide nucleic acid can include a segment that can be referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence." The nucleic acid targeting segment can include a subsegment that can be referred to as a "protein binding segment" or a "protein binding sequence" or a "Cas protein binding segment."

[0109] The term "targeting sequence" as used herein refers to the nucleotide sequence and corresponding amino acid sequence that encodes the targeting polypeptide that mediates the localization (or retention) of protein to a subcellular location, for example, the plasma membrane or membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome, or other organelle.For example, targeting sequence can direct protein (for example, receptor polypeptide or adaptor polypeptide) to the nucleus by using nuclear localization signal (NLS); outside the nucleus of cell, for example, cytoplasm, by using nuclear export signal (NES); mitochondria by using mitochondrial targeting signal; endoplasmic reticulum (ER) by using ER retention signal; peroxisome by using peroxisome targeting signal; plasma membrane by using membrane localization signal; or combinations thereof.

[0110] As used herein, a "nuclear localization domain" can refer to a nuclear localization signal or other sequence or domain that is capable of crossing the nuclear membrane and 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."

[0111] As used herein, a "nuclear export domain" or "nuclear export sequence" (NES) or "nuclear export signal" (NES) can refer to a nuclear export signal or other sequence or domain that is present in a protein and is capable of targeting the protein for translocation from the cell nucleus to the cytoplasm via the nuclear pore complex using nuclear transport. The 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."

[0112] In eukaryotic cells, protein transport between the nucleus and cytoplasm can be mediated by the karyopherin-β family of transport factors, also known as importins and exportins. The direction of nucleocytoplasmic transport can be directed by a nuclear targeting signal within the cargo protein. A nuclear localization sequence (NLS) can direct the protein into the nucleus, and a nuclear export sequence (NES) can directly translocate the protein from the nucleus to the cytoplasm. The NES can directly bind to the transport karyopherin CRM1 (also known as exportin 1), which can escort the cargo protein through the nuclear pore complex.

[0113] In some embodiments, an NES that can be used in the subject compositions, methods, kits, or systems is 8-15 residues in length and has a Φ 2,3 -Φ2-X 2,3 -Φ3-X-Φ4, where Φn represents Leu, Val, Ile, Phe, or Met, and X can be any amino acid. In some cases, the NES that can be used in the compositions, methods, kits, or systems of the presently disclosed subject matter is described in Xu D, et al. Mol Biol Cell. 2012 Sep;23(18):3673-6, such as those described in the extended Kosugi consensus sequences (Class 1a, Φ1-X3-Φ2-X2-Φ3-X-Φ4; Class 1b, Φ1-X2-Φ2-X2-Φ3-X-Φ4; Class 1c, Φ1-X3-Φ2-X3-Φ3-X-Φ4; Class 1d, Φ1-X2-Φ2-X3-Φ3-X-Φ4; Class 2, Φ1-X-Φ2-X2-Φ3-X-Φ4; Class 3, Φ1-X2-Φ2-X3-Φ3-X2-Φ4; where Φn=L, V, I, F, or M, and A, C, T, and W can be one of Φn). In some cases, NESs that can be used in the compositions, methods, kits, or systems of the presently disclosed subject matter are aligned with three sequence patterns: Φ1-X 1,2,3 -Φ2-[^W]2-Φ3-[^W]-Φ4 (Type 1), Φ1-X 2,3It conforms to the NES consensus with -Φ2-[^W]3-Φ3-[^W]-Φ4 (type 2), and Φ1-X2-Φ2-X[^W]2-Φ3-[^W]2-Φ4 (type 3), where [^W] is any of the 20 amino acids excluding Trp, and Ala and Thr residues can be used only once at either position Φ1 or Φ2.

[0114] As used herein, "fusion" can refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., a portion). Fusions can include one or more of the same non-native sequences. Fusions can include one or more different non-native sequences. Fusions can be chimeric. Fusions can include a nucleic acid affinity tag. Fusions can include a barcode. Fusions can include a peptide affinity tag. Fusions can provide subcellular localization of site-specific polypeptides (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an endoplasmic reticulum (ER) retention signal, and the like). Fusions can provide non-native sequences (e.g., an affinity tag) that can be used for tracking or purification. Fusions can be small molecules such as biotin, or dyes such as Alexa fluor dyes, Cyanine 3 dyes, and Cyanine 5 dyes.

[0115] The fusion can refer to any protein that has a functional effect. For example, the fusion protein can comprise a methyltransferase activity, a demethylase activity, a dismutase activity, an alkylating activity, a depurinating activity, an oxidizing activity, a pyrimidine dimer forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity or a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, a remodeling activity, a protease activity, an oxidoreductase activity, a transferase activity, a hydrolase activity, a lyase activity, an isomerase activity, a synthase activity, a synthetase activity, or a demyristoylating activity. The effector protein can modify a genomic locus. In some embodiments, the fusion protein does not comprise nuclease activity. In some embodiments, the fusion protein does not comprise deaminase activity. In some embodiments, the fusion protein does not comprise polymerase activity (e.g., reverse transcriptase activity).

[0116] As used herein, "non-native" can refer to a nucleic acid or polypeptide sequence not found in a native nucleic acid or protein. Non-native can refer to an affinity tag. Non-native can refer to a fusion. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. The non-native sequence may exhibit and / or encode an activity (by way of example, an enzymatic activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, a ubiquitination 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 may be linked to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to produce a chimeric nucleic acid and / or polypeptide sequence encoding the chimeric nucleic acid and / or polypeptide.

[0117] The terms "subject," "individual," and "patient," used interchangeably herein, refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also encompassed.

[0118] The terms "treatment" and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. For example, treatment can include administering a system or cell population disclosed herein. Therapeutic benefit refers to any therapeutically relevant improvement or effect in 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 who reports one or more physiological symptoms of the disease, although the disease, condition, or symptom may not yet be manifested.

[0119] The term "effective amount" or "therapeutically effective amount" refers to the amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells) comprising a system of the present disclosure, that is sufficient to produce a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to the amount of a composition that is sufficient to delay the onset of symptoms, halt progression, or reduce or alleviate at least one symptom of a disorder treated by a method of the present disclosure.

[0120] Delivery Vehicle In some aspects, the present disclosure relates to a delivery vehicle for delivering therapeutic cargoes and / or other molecules into cells in vitro, ex vivo, or in vivo. In some cases, the delivery vehicle of the present disclosure has high efficiency for in vivo delivery of therapeutic cargoes and / or other molecules into cells of a subject. In some cases, the delivery vehicle of the present disclosure includes lipid-containing particles such as virus-like particles, exosomes, lipid nanoparticles, proteolipid vehicles, extracellular vesicle mimics, and membrane vesicles. The delivery vehicles disclosed herein (for example, lipid-containing particles) can be highly efficient for in vivo delivery of cargoes upon administration to a subject, for example, most of the cargo loaded into the delivery vehicle is delivered to the target cells and to the desired subcellular location of the target cells (for example, the cell nucleus or cytoplasm). In some cases, the delivery vehicle can be used to deliver a genome editing system to the target cells and have high efficiency for in vivo gene editing performed by the genome editing system. In some cases, the delivery vehicle is used to deliver an expression construct encoding a therapeutic protein (by way of example, an antibody, transcription factor, or chimeric antigen receptor (CAR)) to cells of a subject, and can have high expression efficiency of the therapeutic protein in the subject.

[0121] In some cases, the lipid-containing particles provided herein comprise a lipid-based outer layer surrounding the lumen. Cargo can be loaded into the lipid-containing particles inside the lumen. In some cases, cargo is loaded into the lipid-containing particles by attaching to the outer lipid-based layer. The outer lipid-based layer can be a single lipid layer or a lipid bilayer consisting of two layers of lipid molecules. In some cases, the lipid-containing particles have one or more fusion proteins inserted or attached to the outer lipid layer. The fusion protein helps the lipid-containing particles fuse with the membrane of the target cell, and thus can deliver the cargo loaded in the lipid-containing vesicles to the target cell.

[0122] Typical sizes of lipid-containing particles are about 10 nm to about 1000 nm, for example, 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 m, 50nm to 300nm, 50nm to 400nm, 50nm to 500nm, 50nm to 600nm, 50nm to 800nm, 100nm to 200nm, 100nm to 300nm, 100nm to 400nm, 100nm to 500nm, 100nm to 600nm, 100nm to 800nm, 200nm to 300nm, 200nm to 400nm, 200nm to 500nm, 200nm to 600nm, 200nm to 800nm, 400nm to 600nm, 400nm to 800nm, or 600nm to 800nm. In some cases, the lipid-containing particle comprises a virus-like particle, lipid nanoparticle, or proteolipid vehicle and has a size of about 10 nm to about 100 nm, for example, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 80 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 80 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, or about 40 nm to about 80 nm.In some cases, the lipid-containing particles include exosomes and have a size of about 50 nm to 200 nm, for example, about 50 nm to 80 nm, about 50 nm to 100 nm, about 50 nm to 120 nm, about 50 nm to 150 nm, about 50 nm to 160 nm, about 50 to 180 nm, about 60 nm to 80 nm, about 60 nm to 100 nm, about 60 nm to 120 nm, about 60 nm to 160 nm, about 60 nm to 160 nm, or about 60 nm to 180 nm. The size may be about 0 nm, about 60 nm to 180 nm, about 80 nm to 100 nm, about 80 nm to 120 nm, about 80 nm to 160 nm, about 80 nm to 180 nm, about 80 nm to 180 nm, about 100 nm to 120 nm, about 100 nm to 150 nm, about 100 nm to 180 nm, about 120 nm to 150 nm, about 120 nm to 180 nm, about 150 nm to 180 nm, or about 150 nm to 200 nm.

[0123] In some aspects, provided herein are a delivery vehicle (e.g., a lipid-containing particle) comprising a cell fusion molecule (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule); a fusion protein comprising a plasma membrane-localized protein (e.g., coupled to a nuclear export sequence (NES)); and a cargo (e.g., a therapeutic cargo or a binding partner for a therapeutic cargo).

[0124] In some aspects, provided herein are a delivery vehicle (e.g., a lipid-containing particle) comprising a cell fusion molecule (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule); a fusion protein comprising a plasma membrane-localized protein (e.g., coupled to a cleavable linker); and a cargo (e.g., a therapeutic cargo or a binding partner for a therapeutic cargo).

[0125] In some aspects, provided herein is a delivery vehicle (e.g., a lipid-containing particle) that includes a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein or a human endogenous retroviral (HERV) structural protein, e.g., a HERV gag, a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane recruitment protein), and a nuclear export sequence (NES).

[0126] In some aspects, provided herein is a delivery vehicle (e.g., a lipid-containing particle) that includes a fusion protein comprising i) a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein; a human endogenous retroviral (HERV) structural protein, e.g., a HERV gag; a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane recruitment protein), a cleavable linker, and a cargo (e.g., a therapeutic cargo or a binding partner for a therapeutic cargo).

[0127] fusion proteins In several aspects, fusion proteins suitable for assembling cargo into a delivery vehicle, e.g., a lipid-containing particle, and delivering the cargo into a cell are disclosed herein. The fusion protein can include a plasma membrane-localized protein (e.g., a retroviral gag protein, a human endogenous retroviral gag protein, or a pleckstrin homology domain) fused to a cargo protein (e.g., a therapeutic cargo). In some cases, the fusion protein includes a cargo that is a binding partner for the therapeutic cargo (e.g., the binding partner can directly bind to the therapeutic cargo, or the binding partner can bind to another molecule that couples to or interacts with the therapeutic cargo). For example, the fusion protein can include a plasma membrane-localized protein (e.g., a retroviral gag protein, a human endogenous retroviral gag protein, or a pleckstrin homology domain) coupled to a nucleic acid binding protein that can bind to, e.g., a nucleic acid molecule, e.g., RNA (e.g., mRNA), or DNA. In some cases, the fusion protein is suitable for delivery by a delivery vehicle disclosed herein.

[0128] The plasma membrane-localized proteins disclosed herein can be derived from a virus, a human, or any other suitable source. In some cases, the plasma membrane-localized protein is an endogenous human protein.

[0129] In some cases, the fusion proteins disclosed herein include a nuclear localization sequence (NLS), which facilitates delivery of the fusion protein, or cargo released from the fusion protein (illustratively, released from the fusion protein after cleavage of a cleavable linker), to the nucleus of a target cell.

[0130] In some cases, the fusion polypeptides disclosed herein include at least one NLS sequence, for example, two or more, three or more, four or more, or five or more NLS sequences. In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus and / or C-terminus of the fusion protein (for example, within the following 50 amino acids). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus of the fusion protein (for example, within the following 50 amino acids). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the C-terminus of the fusion protein (for example, within the following 50 amino acids). In some cases, one or more NLS sequences (three or more, four or more, or five or more NLS sequences) are located at or near both the N-terminus and C-terminus of the fusion protein (for example, within the next 50 amino acids). In some cases, an NLS sequence is located at the N-terminus of the fusion protein and an NLS sequence is located at the C-terminus of the fusion protein.

[0131] In some cases, the cargo protein is delivered as part of a fusion protein disclosed herein, operably linked to, for example, a structural protein (for example, a human endogenous retrovirus (HERV) structural protein or a plasma membrane recruitment domain). In some embodiments, one or more NLS sequences are located at or near one or both ends of the cargo protein sequence of the fusion protein. For example, in some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus and / or C-terminus of the cargo protein sequence (for example, within the following 50 amino acids). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus of the cargo protein sequence (for example, within the following 50 amino acids). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the C-terminus of the cargo protein sequence (for example, within the next 50 amino acids). In some cases, one or more NLS sequences (three or more, four or more, or five or more NLS sequences) are located at both the N-terminus and C-terminus of the cargo protein sequence or near both the N-terminus and C-terminus of the cargo protein sequence (for example, within the next 50 amino acids). In some cases, an NLS sequence is located at the N-terminus of the cargo protein sequence and an NLS sequence is located at the C-terminus of the cargo protein sequence.

[0132] In some cases, the fusion proteins disclosed herein include 1 to 10 NLS sequences (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 NLS sequences). In some cases, the fusion proteins include (are fused to) 2 to 5 NLS sequences (e.g., 2 to 4, or 2 to 3 NLSs). Non-limiting examples of NLS sequences include NLS sequences derived from the SV40 virus large T antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 19); an NLS from nucleoplasmin (such as the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 20)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 21) or RQRRNELKRSP (SEQ ID NO: 22); an hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 23); an IBB domain from importin-alpha, having the sequence RMRIZFKNKGKDT AELRRRRVE VS VELRK AKKDEQILKRRN V (SEQ ID NO: 24); a fibroid T protein, having the sequences VSRKRPRP (SEQ ID NO: 25) and PPKKARED (SEQ ID NO: 26); a human p53 sequence PQPKKKPL (SEQ ID NO: 27); and a mouse c-abl The sequence SALIKKKKKMAP (SEQ ID NO: 28) of IV; the sequences DRLRR (SEQ ID NO: 29) and PKQKKRK (SEQ ID NO: 30) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 31) of hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 32) of mouse MxI protein; the sequence KRKGDE VDGVDEV AKKKS KK (SEQ ID NO: 33) of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 34) of steroid hormone receptor (human) glucocorticoid, as well as sequences having at least 80% identity with the aforementioned sequences. In some cases, the NLS comprises the amino acid sequence MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 35).

[0133] Other non-limiting examples of NLS sequences include KRTADGSEFESPKKKRKV (SEQ ID NO: 36), KKTELQTTNAENKTKKL (SEQ ID NO: 37), KRGINDRNFWRGENGRKTR (SEQ ID NO: 38), RKSGKIAAIVVKRPRK (SEQ ID NO: 39), and MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 35), SPKKKRKVEAS (SEQ ID NO: 40), AGCCCCAAGAAgAAGAGaAAGGTGGAGGCCAGC (SEQ ID NO: 41), and GPKKKRKVAAA (SEQ ID NO: 42), as well as Cokol et al., EMBO Rep., 2000, 1(5):411-415 and Freitas et al., Current Genomics, 2009, 10(8):550-7; Lu, J., et al., Cell Commun Signal 19, 60(2021); international publication No. WO / 2001 / 038547 (each of which is incorporated herein by reference in its entirety); WO 2001 / 038547, as well as any of the sequences having at least 80% identity to the foregoing sequences.

[0134] In some cases, the fusion proteins disclosed herein include a nuclear export sequence (NES). In some cases, the NES promotes localization of the fusion protein in the cytosol of the target cell relative to the nucleus.

[0135] In some cases, the fusion polypeptides disclosed herein include at least one NES sequence, for example, two or more, three or more, four or more, or five or more NES sequences. In some cases, the one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the N-terminus and / or C-terminus of the fusion protein (for example, within the next 50 amino acids). In some cases, the fusion proteins disclosed herein contain only one NES sequence. In some cases, the fusion proteins disclosed herein contain three NES sequences. In some cases, the one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the N-terminus of the fusion protein (for example, within the next 50 amino acids). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the C-terminus of the fusion protein (for example, within the next 50 amino acids). In some cases, one or more NES sequences (three or more, four or more, or five or more NES sequences) are located at both the N-terminus and C-terminus of the fusion protein (for example, within the next 50 amino acids). In some cases, an NES sequence is located at the N-terminus of the fusion protein and an NES sequence is located at the C-terminus of the fusion protein.

[0136] In some cases, the cargo protein is delivered as part of a fusion protein disclosed herein, for example, operably linked to a structural protein (for example, a human endogenous retroviral structural protein or a plasma membrane recruitment domain). In some embodiments, one or more NES sequences are located at or near one or both ends of the cargo protein sequence inside the fusion protein. For example, in some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at the N-terminus and / or C-terminus of the cargo protein sequence or near the N-terminus and / or C-terminus of the cargo protein sequence (for example, within the following 50 amino acids). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at the N-terminus of the cargo protein sequence or near the N-terminus of the cargo protein sequence (for example, within the following 50 amino acids). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at the C-terminus of the cargo protein sequence or near the C-terminus of the cargo protein sequence (for example, within 50 amino acids of the following). In some cases, one or more NES sequences (three or more, four or more, or five or more NES sequences) are located at both the N-terminus and C-terminus of the cargo protein sequence or near both the N-terminus and C-terminus of the cargo protein sequence (for example, within 50 amino acids of the following). In some cases, an NES sequence is located at the N-terminus of the cargo protein sequence and an NES sequence is located at the C-terminus of the cargo protein sequence. In some cases, the fusion proteins disclosed herein contain only one NES sequence. In some cases, the fusion protein contains only one NES sequence, and the NES sequence is located at or near the N-terminus of the cargo protein (for example, within 50 amino acids of the N-terminus).

[0137] In some embodiments, the fusion protein comprises one NES sequence and two NLS sequences. In some of these embodiments, the NES sequence, the NLS sequence, and the cargo protein sequence are arranged in N- to C-terminal order as follows: NES-NLS-cargo protein-NLS. In some embodiments, the fusion protein comprises two or more NES sequences and two NLS sequences. In some of these embodiments, the NES sequence, the NLS sequence, and the cargo protein sequence are arranged in N- to C-terminal order as follows: n X NES (n≧2)-NLS-cargo protein-NLS.

[0138] In some cases, the fusion proteins disclosed herein include 1 to 10 NES sequences (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 NES sequences). In some cases, the fusion proteins include (are fused to) 2 to 5 NES sequences (e.g., 2 to 4, or 2 to 3 NES sequences).

[0139] In some cases, an NES sequence that can be used in a fusion protein includes LQLPPLERLTL (SEQ ID NO: 43) derived from the HIV-1 Rev protein and a sequence having at least 80% identity thereto. In some cases, an NES sequence includes LALKLAGLDL (SEQ ID NO: 44) derived from PKIα and a sequence having at least 80% identity thereto. In some cases, an NES sequence disclosed herein includes, for example, a sequence such as those described in la Cour T, 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 / LRN) can be used in the fusion proteins disclosed herein, for example, for the purpose of packaging cargo proteins into lipid-containing particles, such as virus-like particles.

[0140] In some cases, the fusion protein comprises a cleavable linker between two or more components. Illustratively, the fusion protein may comprise a cleavable linker between a cargo protein sequence and a plasma membrane-localized protein sequence (for example, a retroviral gag protein sequence). In some cases, the cleavable linker separates the plasma membrane-localized protein sequence from the NLS sequence and / or NES sequence at its N-terminus or C-terminus. The cleavable linker may separate the cargo protein sequence from the plasma membrane-localized protein sequence, the NLS sequence, and / or the NES sequence at its N-terminus or C-terminus. The cleavable linker sequences provided herein may be cleavable sequences that are recognized and cleaved by viral proteases, bacterial proteases, or eukaryotic proteases (for example, proteases derived from plants, animals, or fungi). In some cases, the cleavable sequence is recognized by a retroviral protease (pro, for example, a pro from Moloney Murine Leukemia Virus (MMLV) or Friend Murine Leukemia Virus (FMLV)). Non-limiting examples of cleavable sequences that can be used in the fusion protein include TSTLL MENSS (SEQ ID NO: 1), PRSSLYPALTP (SEQ ID NO: 2), VQALVLTQ (SEQ ID NO: 3), and PLQVLTLNIERR (SEQ ID NO: 4), as well as sequences having at least 80% identity to the foregoing.

[0141] In some cases, the fusion proteins disclosed herein also include one or more non-cleavable linkers operably linking the components together. The non-cleavable linker can be any suitable linker sequence used in fusion protein constructs, such as, for example, a peptide linker consisting of glycine (Gly) and serine (Ser) residues. In some embodiments, the non-cleavable linker comprises an amino acid sequence selected from the group consisting of (GS)x (SEQ ID NO: 45), (GGS)x (SEQ ID NO: 46), (GGGGS)x (SEQ ID NO: 47), (GGSG)x (SEQ ID NO: 48), and (SGGG)x (SEQ ID NO: 49), where x is an integer between 1 and 50.

[0142] In some embodiments, the fusion protein (before cleavage) comprises the following structure: [gag nucleocapsid polyprotein]-[3X NES]-[cleavable linker]-[NLS]-[therapeutic cargo]-[NLS].

[0143] In some cases, the fusion protein has one of the following configurations of components located in order from N-terminus to C-terminus: [plasma membrane-localized protein]-[n*NES]-[cleavable linker]-[m1*NLS]-[cargo protein]-[m2*NLS]; [plasma membrane-localized protein]-[cleavable linker]-[m1*NLS]-[cargo protein]-[m2*NLS]-[n*NES]; [plasma membrane-localized protein]-[cleavable linker 1]-[m1*NLS]-[cargo protein]-]-[m2*NLS]-[cleavable linker 2]-[n*NES]; and [plasma membrane-localized protein]-[cleavable linker 1]-[m1*NLS]-[cargo protein]-[m2*NLS]; [m1*NLS]-[cargo protein]-[m2*NLS]-[cleavable linker]-[n*NES]-[plasma membrane-localized protein]; [n*NES]-[m1*NLS]-[cargo protein]-[m2*NLS]-[cleavable linker]-[plasma membrane-localized protein]; [n*NES]-[cleavable linker 1]-[m1*NLS]-[cargo protein]-[m2*NLS]-[cleavable linker 2]-[plasma membrane-localized protein]; and [m1*NLS]-[cargo protein]-[m2*NLS]-[cleavable linker]-[plasma membrane-localized protein]; where n, m1, and m2 are each integers ranging from 0 to 10 and represent the number of repeats of the respective sequences to which they refer. The non-cleavable linker sequence may or may not be present in any of the configurations described above between any two adjacent components.

[0144] Cleavage of fusion proteins (e.g., gag therapeutic cargo) In some embodiments, the fusion protein provided herein (e.g., a gag therapeutic cargo) comprises: (a) a gag nucleocapsid protein sequence, (b) a therapeutic cargo sequence, (c) a cleavable linker, and (d) a nuclear export sequence (NES). In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease, a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, a small molecule compound, or any combination thereof. Illustratively, the therapeutic cargo can be a base editor comprising a nuclease domain. In certain embodiments, the therapeutic cargo does not comprise a nuclease, a polymerase (e.g., a reverse transcriptase), a base editor, or a prime editor. In some embodiments, the therapeutic cargo is a protein (by way of example, a nuclease, base editor, prime editor, epigenetic editor, restriction endonuclease (optionally a Type IIS restriction enzyme), recombinase, transcription factor, antibody, chimeric antigen receptor, T cell receptor, or reverse transcriptase). In some embodiments, the therapeutic cargo is a nucleic acid molecule (by way of example, DNA, RNA, retrotransposon, aptazyme, aptamer, or ribozyme). In some embodiments, the therapeutic cargo is an epigenetic editor, restriction endonuclease (optionally a Type IIS restriction enzyme), recombinase, transcription factor, antibody, chimeric antigen receptor, T cell receptor, organelle, nucleic acid molecule, DNA, RNA, retrotransposon, oligonucleotide, aptazyme, aptamer, ribozyme, small molecule compound, or any combination thereof.

[0145] In some embodiments, the cleavable linker is positioned between the sequence of the therapeutic cargo and the NES. When the fusion protein is packaged into the lipid-containing particle (for example, a virus-like particle) provided herein, it can be cut off at the cleavable linker, and for example, the cleavable linker can be recognized by the protease (pro) protein of the lipid-containing particle (for example, a virus-like particle). When the fusion protein is cleaved at the cleavable linker, the therapeutic cargo can be released into the protein core as a separate protein. As a result, in some cases, the lipid-containing particle (for example, a virus-like particle) provided herein includes a cleavage product that includes the gag nucleocapsid protein and the NES, but lacks the therapeutic cargo. In some of these embodiments, cleavage at the cleavable linker results in a relatively small amount of fusion protein in the lipid-containing particle that has not been cleaved at the cleavable linker compared to the amount of fusion protein initially packaged into the lipid-containing particle, illustratively at most about 50%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 35 ... The fusion protein is present in an amount of about 18%, at most about 16%, at most about 14%, at most about 12%, at most about 10%, at most about 8%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.8%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, at most about 0.05%, or at most about 0.01%. In some embodiments, the fusion protein that is not cleaved by the cleavable linker does not remain in the lipid-containing particle.

[0146] In some embodiments, after cleavage, the fusion protein comprises the structure [gag nucleocapsid protein]-[3X NES]. In some embodiments, the cleavage product comprises the structure [NLS]-[therapeutic cargo]-[NLS].

[0147] In some embodiments, as a result of the cleavage of the fusion protein at the cleavable linker, there is a significant amount of therapeutic cargo present inside the protein core of the lipid-containing particle, apart from the fusion protein or cleavage product.In some embodiments, there is a relatively large amount of therapeutic cargo present inside the protein core of the lipid-containing particle compared to the fusion protein in the lipid-containing particle.In some embodiments, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is at least 1.5.In some cases, the ratio is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20.In some cases, the ratio is at least 100.In some cases, the ratio is at least 1000.In some cases, the ratio is at least 10,000. In some cases, the ratio is from about 1.5 to about 100, for example, from about 2 to about 100, from about 5 to about 100, from about 10 to about 100, from about 2 to about 80, from about 5 to about 80, from about 10 to about 80, from about 2 to about 60, from about 5 to about 60, from about 10 to about 60, from about 2 to about 50, from about 5 to about 50, from about 10 to about 50, from about 2 to about 40, from about 5 to about 40, from about 10 to about 40, from about 2 to about 30, from about 5 to about 30, from about 10 to about 30, from about 2 to about 20, from about 5 to about 20, from about 10 to about 20, from about 20 to about 100, from about 30 to about 100, from about 40 to about 100, from about 50 to about 100, or from about 80 to about 100, etc. In some cases, the ratio is from about 100 to about 1000, for example, from about 100 to about 800, from about 100 to about 600, from about 100 to about 500, from about 100 to about 400, from about 100 to about 300, from about 100 to about 200, from about 200 to about 1000, from about 200 to about 800, from about 200 to about 600, from about 200 to about 500, from about 200 to about 400, from about 200 to about 300, from about 400 to about 1000, from about 400 to about 800, from about 400 to about 600, or from about 400 to about 500.

[0148] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.2%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, at least about 99.95%, or at least about 99.99% of the sequence of the therapeutic cargo in the lipid-containing particle is present in the form of the therapeutic cargo located inside the protein core, separate from the fusion protein or cleavage product. In some embodiments, there is about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.95%, about 99.99%, or about 100% of the sequence of the therapeutic cargo in the lipid-containing particle is present in the form of the therapeutic cargo positioned inside the protein core. The percentage of the therapeutic cargo sequence present in the form of a separate therapeutic cargo located inside the protein core can be calculated as the percentage of the amount of therapeutic cargo located inside the protein core to the sum of the amount of therapeutic cargo located inside the protein core and the amount of fusion protein in the lipid-containing particle that contains the therapeutic cargo sequence.

[0149] The amount of therapeutic cargo or fusion protein, respectively, within the lipid-containing particle can be measured by any technique known to those skilled in the art for quantitative measurement of proteins or protein sequences, for example, Western blots or Jess blots using antibodies against sequences or epitopes of the therapeutic cargo can be performed to distinguish and measure the amount of therapeutic cargo and fusion protein.

[0150] In some cases, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is measured at the population level of the same or similar lipid-containing particles (e.g., virus-like particles), illustratively one or more preparations of such lipid-containing particles. In some aspects, a population of lipid-containing particles (e.g., virus-like particles) is provided herein. In some embodiments of the population of lipid-containing particles, the lipid-containing particles comprise a lipid membrane encapsulating a protein core, and the protein core comprises a group-specific antigen (gag) protease (pro) polyprotein and a fusion protein comprising a therapeutic cargo sequence and a gag protein sequence. In some of these embodiments, the lipid-containing particles further comprise a therapeutic cargo located inside the protein core, separate from the fusion protein. In some embodiments, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is at least 1.5 among the population of lipid-containing particles. In some embodiments, the ratio among the population of lipid-containing particles is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20. In some cases, the ratio between the populations of lipid-containing particles is at least 100. In some cases, the ratio between the populations of lipid-containing particles is at least 1000. In some cases, the ratio between the populations of lipid-containing particles is at least 10,000. In some cases, the ratio between the populations of lipid-containing particles is between 1.5 and 100, for example, between 2 and 100, between 5 and 100, between 10 and 100, between 2 and 80, between 5 and 80, between 10 and 80, between 2 and 60, between 5 and 60, between 10 and 60, between 2 and 50, between 5 and 50, between 10 and 50, between 2 and 40, between 5 and 40, between 10 and 40, between 2 and 30, between 5 and 30, between 10 and 30, between 2 and 20, between 5 and 20, between 10 and 20, between 20 and 100, between 30 and 100, between 40 and 100, between 50 and 100, or between 80 and 100, etc.In some cases, the ratio between populations of lipid-containing particles is between 100 and 1000, for example, between 100 and 800, between 100 and 600, between 100 and 500, between 100 and 400, between 100 and 300, between 100 and 200, between 200 and 1000, between 200 and 800, between 200 and 600, between 200 and 500, between 200 and 400, between 200 and 300, between 400 and 1000, between 400 and 800, between 400 and 600, or between 400 and 500.

[0151] In some embodiments, among a population of lipid-containing particles, there is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.2%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, at least about 99.95%, or at least about 99.99% of the sequences of the therapeutic cargo in the lipid-containing particles are present in the form of the therapeutic cargo located inside the protein core. In some embodiments, among a population of lipid-containing particles, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.95%, about 99.99%, or about 100% of the sequences of the therapeutic cargo in the lipid-containing particles are present in the form of the therapeutic cargo positioned inside the protein core. The percentage of a therapeutic cargo sequence in a population of lipid-containing particles can be calculated as the percentage of the amount of therapeutic cargo located inside the protein core among the population of lipid-containing particles relative to the sum of the amount of therapeutic cargo located inside the protein core and the amount of fusion protein in the lipid-containing particle that contains the therapeutic cargo sequence.

[0152] In some of these embodiments, at a population level, there is a relatively small amount of fusion protein in the lipid-containing particles that has not been cleaved at the cleavable linker compared to the amount of fusion protein that was initially packaged into the population of lipid-containing particles as a result of cleavage at the cleavable linker, illustratively up to about 40%, up to about 35%, up to about 30%, up to about 25%, or up to about 50% of the fusion protein that was initially packaged into the population of lipid-containing particles prior to cleavage. The fusion protein is present in an amount of at most about 20%, at most about 18%, at most about 16%, at most about 14%, at most about 12%, at most about 10%, at most about 8%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.8%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, at most about 0.05%, or at most about 0.01%.

[0153] In other embodiments, at the population level, only trace amounts of fusion protein remain in a population of lipid-containing particles that are not cleaved by the cleavable linker, illustratively at most about 100, 80, 60, 50, 40, 30, 20, 10, 8, 6, 4, 2, or 1 such uncleaved fusion protein (e.g., virus-like particle) per each lipid-containing particle, or at most about 1 such uncleaved fusion protein (e.g., virus-like particle) per 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000, 5000, 10^4, 10^5, or 10^6 lipid-containing particles in the population.

[0154] In some embodiments, as a result of the cleavage of the fusion protein at the cleavable linker, there is a significant amount of therapeutic cargo present inside the protein core of the lipid-containing particle, apart from the fusion protein or cleavage product.In some embodiments, there is a relatively large amount of therapeutic cargo present inside the protein core of the lipid-containing particle compared to the fusion protein in the lipid-containing particle.In some embodiments, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is at least 1.5.In some cases, the ratio is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20.In some cases, the ratio is at least 100.In some cases, the ratio is at least 1000.In some cases, the ratio is at least 10,000. In some cases, the ratio is between 1.5 and 100, for example, between 2 and 100, between 5 and 100, between 10 and 100, between 2 and 80, between 5 and 80, between 10 and 80, between 2 and 60, between 5 and 60, between 10 and 60, between 2 and 50, between 5 and 50, between 10 and 50, between 2 and 40, between 5 and 40, between 10 and 40, between 2 and 30, between 5 and 30, between 10 and 30, between 2 and 20, between 5 and 20, between 10 and 20, between 20 and 100, between 30 and 100, between 40 and 100, between 50 and 100, or between 80 and 100, for example. In some cases, the ratio is between 100 and 1000, for example, between 100 and 800, between 100 and 600, between 100 and 500, between 100 and 400, between 100 and 300, between 100 and 200, between 200 and 1000, between 200 and 800, between 200 and 600, between 200 and 500, between 200 and 400, between 200 and 300, between 400 and 1000, between 400 and 800, between 400 and 600, or between 400 and 500.

[0155] Plasma membrane-localized proteins In some cases, the plasma membrane localization proteins described herein form the basic structure of the delivery vehicle, for example, at least a portion of the delivery vehicle. In some cases, the plasma membrane localization proteins described herein also promote the self-assembly of the delivery vehicle (for example, VLP). For example, the plasma membrane localization proteins can form a membrane enclosure to promote the localization to the plasma membrane and packaging of the delivery vehicle (for example, virus-like particle).

[0156] In some cases, the plasma membrane-localized protein is, for example, a viral protein derived from a virus. In some cases, the plasma membrane-localized protein is a mammalian protein, for example, a mammal, for example, a human protein. In some cases, the plasma membrane-localized protein is a human endogenous protein.

[0157] In some cases, the plasma membrane-localized protein is a polyprotein derived from a virus, a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof. Illustratively, the plasma membrane-localized protein comprises a retroviral gag protein, e.g., a retroviral polyprotein comprising one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. In some cases, the gag protein is derived from Friend Murine Leukemia Virus (FMLV). In some cases, the retroviral gag polyprotein is a gag polyprotein of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of a human immunodeficiency virus.

[0158] Non-limiting examples of plasma membrane-localized proteins include human papillomavirus (HPV) L1 protein, HPV L2 protein, hepatitis B virus (HBV) core protein, chikungunya virus (CHIKV) C-E3-E2-6k-E1, human immunodeficiency virus (HIV) gag-pol, HIV gag, respiratory syncytial virus (RSV) M, RSV NP, human metapneumovirus (HMPV) M, influenza M1, Zika virus (ZIKV) C, ZIKV prM / M, dengue virus (DENV) C-prM, West Nile virus (WNV) prME protein, WNV CprME protein, filovirus VP40 or Z protein, baculovirus P1 protein, rotavirus VP7, rotavirus VP2 protein, rotavirus VP6 protein, SARS M protein, SARS E protein, SARS The present invention also includes an N protein, a porcine circovirus type 2 (PCV2) capsid, a baculovirus VP2 protein, a baculovirus VP5 protein, a baculovirus VP3 protein, or a baculovirus VP7 protein, a hepatitis C virus (HCV) core protein, an Ebola nucleocapsid, a parovirus VP1 protein, a parovirus VP2 protein, a Newcastle disease virus (NDV) M protein, a hepatitis E virus (HeV) M protein, a Nipah virus (NIV) M protein, a human polyomavirus 2 (JCPyV) VP1 protein, a human parainfluenza virus type 3 (HPIV3) M protein, an HPIV3N protein, or a mumps virus (MuV) M protein, a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof.

[0159] In some cases, the plasma membrane-localized protein sequence comprises a human endogenous retrovirus (HERV) gag protein. In some cases, the plasma membrane-localized protein sequence comprises a pleckstrin homology (PH) domain. Non-limiting examples of plasma membrane-localized protein sequences can include those listed in Table 2.

[0160] Cell fusion / envelope proteins The cell fusion protein disclosed herein can refer to a protein present on the outer membrane of a delivery vehicle (for example, inserted into, attached to, or anchored to the lipid layer) and promoting fusion of the vehicle with the delivery membrane, for example, the target cell membrane. In some cases, the cell fusion protein mediates the tropism of the delivery vehicle, for example, the preferential fusion of the delivery vehicle with one or more specific cell types. In some cases, the cell fusion protein causes mixing between lipids in the delivery vehicle and lipids in the target cell. In some cases, the lipid-containing particle includes a human endogenous retrovirus (HERV) envelope protein, a humanized viral envelope protein, or a non-immunogenic cell fusion molecule. Non-limiting examples of HERV envelope proteins can include those listed in Table 1.

[0161] In some cases, the cell fusion protein comprises a mammalian protein. In some cases, the cell fusion protein comprises a viral protein. In some embodiments, the cell fusion protein comprises a mammalian protein or a homolog of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a non-mammalian protein, for example, a viral protein or a homolog of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a native protein or a derivative of a native protein, a synthetic protein, a fragment thereof, a variant thereof, a protein fusion comprising one or more cell fusion proteins or fragments, and any combination thereof.

[0162] The non-immunogenic cell fusion proteins provided herein can be reduced in immunogenicity to human subjects compared to proteins xenogenic to human subjects. For example, the non-immunogenic cell fusion proteins can be humanized to reduce immunogenicity to human subjects. In some embodiments, the cell fusion proteins can be modified to reduce immune reactivity. For example, the cell fusion proteins can be decorated with molecules that reduce immune interactions, such as PEG, such as those described in Croyle MA, et al., J Virol. 2004 Jan;78(2):912-21, which is incorporated herein by reference in its entirety. Thus, in some embodiments, the envelope protein comprises PEG, for example, a PEGylated polypeptide. Amino acid residues in cell fusion proteins targeted by the immune system may be modified so that they are not recognized by the immune system, such as those described in Lech PJ, et al., Virology. 2014 Apr; 454-455: 237-46; and Kneissl S, et al., PLoS One. 2012; 7(10): e46667, each of which is incorporated herein by reference in its entirety. In some embodiments, the protein sequence of the cell fusion protein is modified to resemble an amino acid sequence found in humans (humanization). In some embodiments, the protein sequence of the cell fusion protein is modified to a protein sequence that binds less strongly to the MHC complex. In some embodiments, the cell fusion protein is derived from a virus or organism that does not infect humans (and against which humans have not been vaccinated), increasing the likelihood that the patient's immune system is naive to the cell fusion protein (e.g., there is a negligible humoral or cell-mediated adaptive immune response to the cell fusion protein) (doi:10.1006 / mthe.2002.0550, doi:10.1371 / journal.ppat.1005641, doi:10.1038 / gt.2011.209, DOI 10.1182 / blood-2014-02-558163).In some embodiments, the glycosylation of the envelope protein is altered to modify immune interactions or reduce immune reactivity.

[0163] In some cases, the cell fusion protein comprises a sequence selected from Nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or a derivative thereof.

[0164] In some cases, the cell fusion protein comprises a mammalian protein. Examples of mammalian cell fusion proteins may include, but are not limited to, SNARE family proteins such as vSNARE and tSNARE, syncytin proteins such as Syncytin-1 and Syncytin-2, myomaker, myomixer, myomerger, FGFRL1 (fibroblast growth factor receptor-like 1), Minion, isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for example, as disclosed in U.S. Patent No. 6,099,857), gap junction proteins such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (for example, as disclosed in U.S. Patent Application Publication No. 2007 / 0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells, its homolog, its fragment, or its variant, and protein fusions comprising one or more proteins or fragments thereof. In some embodiments, the cell fusion protein comprises a curvature-generating protein, such as, for example, Epsin1, dynamin, or a protein containing a BAR domain, such as those described in Kozlov et al., CurrOp StrucBio 2015 2015 Aug;33:61-67; Zimmerberg et al., Nat Rev Mol Cell Biol. 2006 Jan;7(1):9-19; Richard et al., Biochem J. 2011 Dec 1;440(Pt 2):185-193, each of which is incorporated herein by reference in its entirety.

[0165] In some cases, the cell fusion protein includes a non-mammalian protein, for example, a viral cell fusion protein. In some embodiments, the viral cell fusion protein is a class I viral membrane cell fusion protein, a class II viral cell fusion protein, a class III viral membrane cell fusion protein, a viral cell fusion protein, or other viral cell fusion protein, or a homolog thereof, a fragment thereof, a variant thereof, or a protein fusion comprising one or more proteins or fragments thereof. Examples of class I viral cell fusion proteins that can be used in the VLPs disclosed herein include, but are not limited to, baculovirus F proteins, such as F proteins from the genus nuclear polyhedrosis virus (NPV), including Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinin from orthomyxoviruses, F proteins from paramyxoviruses (including measles (Katoh et al. BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and filovirus and coronavirus cell fusion proteins. In some embodiments, class II viral cell fusion proteins, such as dengue E glycoprotein, have a structural signature of a beta sheet that forms an elongated ectodomain that reassembles into a trimerized hairpin. In some embodiments, class II viral cell fusion proteins lack a central coiled-coil. Examples of class II viral cell fusion proteins that can be used in the VLPs disclosed herein include, but are not limited to, tick-borne encephalitis E (TBEV E), Semliki Forest virus E1 / E2, and cell fusion proteins from Sindbis, rubella, and dengue viruses. In some embodiments, class III viral cell fusion proteins, such as vesicular stomatitis virus G glycoprotein, combine structural signatures found in class I and class II.In some embodiments, the Class III viral cell fusion protein comprises a helix (e.g., forming a six-helix bundle for protein folding similar to Class I viral cell fusion proteins) and three sheets with amphipathic fusion peptides at their termini, reminiscent of Class II viral cell fusion proteins. Examples of Class III viral cell fusion proteins that can be used in the VLPs disclosed herein include, but are not limited to, rhabdovirus G (e.g., vesicular stomatitis virus protein G (VSV-G)), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB), Epstein-Barr virus glycoprotein B (EBV gB), thogotovirus G, baculovirus gp64 (e.g., Autographa California multiplex NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G). In some embodiments, the class IV viral cell fusion protein is a fusion-associated small transmembrane (FAST) protein (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012) Electronic Thesis and Dissertation Repository. Paper 388), which is encoded by non-enveloped reoviruses. In some embodiments, the class IV viral cell fusion protein is small enough not to form a hairpin (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0166] Non-limiting examples of other viral cell fusion proteins that can be used in the VLPs disclosed herein include, but are not limited to, the following: viral syncytia proteins, such as influenza hemagglutinin (HA) or mutants thereof, or fusion proteins thereof; human immunodeficiency virus type 1 cell fusion protein (HIV-1 ENV), gp120 from HIV binding LFA-1 to form lymphocyte syncytia, HIV gp41, HIV gp160, or HIV transactivator of transcription (TAT); viral glycoprotein VSV-G, a viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family; glycoproteins gB and gH-gL of varicella-zoster virus (VZV); murine leukemia virus (MLV)-10A1; gibbon ape leukemia virus glycoprotein (GaLV); G-type glycoproteins in rabies, mokola, vesicular stomatitis virus, and togavirus; murine hepatitis virus JHM surface spike protein; porcine respiratory coronavirus spike and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; measles virus F and H, HN, or G genes; canine distemper virus, Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus, and respiratory syncytial virus; human herpesvirus 1 and simian varicella virus gH, along with the chaperone protein gL; human, bovine, and simian herpesvirus gB; envelope glycoproteins of Friend murine leukemia virus and Mason Pfizer monkey virus; mumps virus hemagglutinin neuraminidase and glycoproteins F1 and F2; membrane glycoproteins from Venezuelan equine encephalitis; paramyxovirus F protein; SIV gp160 protein; Ebola virus G protein; or Sendai virus fusion protein, or its homolog morbillivirus, fragments thereof, variants thereof, or any combination thereof.In some cases, the viral cell fusion protein comprises a measles virus hemagglutinin (HA) protein and / or measles virus fusion glycoprotein, an influenza virus neuraminidase (NA) protein, a measles virus F protein, an influenza virus HA protein, a Moloney virus MLV-A protein, a Moloney virus MLV-E protein, a baboon endogenous retrovirus (BAEV) cell fusion protein, an Ebola virus glycoprotein, a foamy virus cell fusion protein, or a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof.

[0167] Non-limiting examples of other viral cell fusion proteins that can be used in the VLPs disclosed herein include, but are not limited to, the hemagglutinin (HA) or neuraminidase (NA) proteins derived from Orthomyxoviridae influenza A, E protein E1 and E2 subunits (together and separately in complexes) of Togaviridae CHIV; S, E, or MN proteins from Coronaviridae - SARS and COVID19; F or G proteins from Paramyxoviridae Nipah virus; GP protein from Filoviridae Ebola; E protein from Flaviviridae Dengue virus; Gn and Gc proteins (together and separately in complexes) from Fenuiviridae - Sand fly fever virus; Arenaviridae Lassau virus a GP protein from a virus; a Gn and Gc protein (together and separately incorporated in a complex) from a hantavirus (Hantaviridae); a G protein from a Borna disease virus (Bornaviridae); a Gn and Gc protein (together and separately incorporated in a complex) from a Crimean-Congo hemorrhagic fever virus (Bunyaviridae); an S, M, or L protein from a hepatitis B virus (Hepadnaviridae); a cell fusion protein from herpes simplex virus 1 (Herpesviridae); an EV protein from variola virus (Poxviridae); an S, L, or M protein from hepatitis D; or a glycoprotein from a hepatitis E virus (Hepeviridae), or a homolog thereof, a fragment thereof, a variant thereof, and a protein fusion comprising one or more proteins or fragments thereof.

[0168] In some embodiments, the cell fusion protein is derived from a paramyxovirus, ie, Nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbilivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein.

[0169] In some embodiments, the cell fusion protein is derived from Poxvirus.Additional exemplary cell fusion protein is disclosed in U.S. Patent No. 9,695,446, U.S. Patent No. 2004 / 0028687, U.S. Patent No. 6,416,997, U.S. Patent No. 7,329,807, U.S. Patent No. 2017 / 0112773, U.S. Patent No. 2009 / 0202622 and U.S. Patent No. 2004 / 0009604, and International Publication No. 2006 / 027202 and International Publication No. 2020102709, each of which is incorporated herein by reference in its entirety.

[0170] In some embodiments, cell fusion proteins include EFF-1, AFF-1, gap junction proteins, such as connexins (Cn43, GAP43, CX43, etc.) (DOI: 10.1021 / jacs.6b05191), other tumor-associated proteins, homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

[0171] The cell fusion proteins disclosed herein can be retargeted by mutating amino acid residues in the fusion protein (e.g., hemagglutinin protein). In some embodiments, the envelope protein is randomly mutated. In some embodiments, the envelope protein is rationally mutated. In some embodiments, the envelope protein is subjected to directed evolution.

[0172] The cell fusion proteins disclosed herein can be retargeted by covalently conjugating a targeting moiety. For example, a cell fusion protein can be covalently conjugated to a targeting moiety by expressing a chimeric protein comprising an envelope protein linked to a targeting moiety. The target of the targeting moiety includes any peptide (e.g., a receptor) that is presented on the target cell. In some cases, the target is expressed at a higher level on the target cell than on non-target cells.

[0173] Targeting moieties can be selected to target specific tissue types, such as muscle, brain, liver, pancreas, and lung, or to target diseased tissues, such as tumors. In particularly preferred embodiments of the present invention, exosomes are targeted to brain tissue.

[0174] Specific, non-limiting examples of targeting moieties include muscle-specific peptides discovered by phage display to target skeletal muscle, a 29-amino acid fragment of the rabies virus glycoprotein that binds to the acetylcholine receptor, or a fragment of nerve growth factor that targets its receptor to target neurons; and secretin peptides that bind to the secretin receptor can be used to target biliary epithelium and pancreatic epithelium. Alternatively, immunoglobulins and their derivatives, including scFv antibody fragments, can also be expressed as fusion proteins that target specific antigens, such as VEGFR, for cancer gene therapy. Alternatively, natural ligands for receptors can be expressed as fusion proteins to confer specificity, such as NGF, which binds to NGFR and confers neuron-specific targeting.

[0175] Targeting moieties can include, for example, antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies, such as sdAbs (either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds, such as fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T cell receptors (TCRs). Cell fusion proteins may be retargeted by noncovalently conjugating a targeting moiety to the fusion protein or targeting protein (e.g., hemagglutinin protein). For example, fusion proteins can be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell and redirect fusion activity to cells displaying the antibody's target.

[0176] Targeting moieties can be, by way of example, humanized antibody molecules, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (such as, by way of example, Zybodies®); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (such as, by way of example, shark single domain antibodies, such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (such as, by way of example, Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem dyes. These may include abodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®.

[0177] In some embodiments, the targeting moiety linked to the cell fusion protein binds to a cell surface marker on the target cell, such as, for example, a protein, a glycoprotein, a receptor, a cell surface ligand, an agonist, a lipid, a sugar, a class I transmembrane protein, a class II transmembrane protein, or a class III transmembrane protein.

[0178] In some cases, the delivery vehicles disclosed herein (by way of example only, VLPs, exosomes, or lipid nanoparticles) also display a targeting moiety that is not conjugated to a cell fusion protein or other protein, to redirect the fusion activity of the delivery vehicle toward the cell bound by the targeting moiety or to affect the homing of the delivery vehicle toward the target cell.

[0179] Virus-like particles (e.g., "lipid-containing particles") Disclosed herein, in some aspects, are compositions, methods, and systems relating to virus-like particles that can be utilized to deliver cargo into cells.

[0180] The virus-like particles (VLPs) disclosed herein can comprise one or more virally derived proteins, such as structural and envelope proteins of the VLP. In some cases, the virally derived proteins are present as part of a fusion protein that forms the VLP.

[0181] In some cases, the loading capacity of the VLPs disclosed herein is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 80-fold, or 100-fold higher than conventional VLPs.

[0182] Structural proteins of VLPs In some cases, the structural proteins described herein form at least a portion of the basic structure of the virus-like particle, for example, the capsid that encapsulates the lumen of the VLP. The structural proteins of the virus-like particle can include a plasma membrane-localized protein. In some cases, the plasma membrane-localized proteins described herein also promote self-assembly of the VLP, for example, by forming a membrane enclosure, thereby promoting plasma membrane localization and packaging of the virus-like particle. In some cases, the structural proteins described herein promote the release of the VLP from the production cell in which the VLP is produced.

[0183] In some cases, the structural protein of the VLP (e.g., a plasma membrane-localized protein) is, for example, a viral protein derived from a virus. In some cases, the structural protein of the VLP is, for example, a mammalian protein derived from a mammal, for example, a human. In some cases, the structural protein of the VLP is a human endogenous protein.

[0184] In some cases, the structural protein (e.g., a plasma membrane-localized protein) of the VLP is a polyprotein derived from a virus, a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof. Illustratively, the structural protein (e.g., a plasma membrane-localized protein) of the VLP comprises a retroviral gag protein, e.g., a retroviral polyprotein comprising one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. In some cases, the gag protein is derived from Friend murine leukemia virus (FMLV). In some cases, the retroviral gag polyprotein is a gag polyprotein of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of a human immunodeficiency virus.

[0185] Non-limiting examples of structural proteins of VLPs (including, by way of example, plasma membrane-localized proteins) include human papillomavirus (HPV) L1 protein, HPV L2 protein, hepatitis B virus (HBV) core protein, chikungunya virus (CHIKV) C-E3-E2-6k-E1, human immunodeficiency virus (HIV) gag-pol, HIV gag, respiratory syncytial virus (RSV) M, RSV NP, human metapneumovirus (HMPV) M, influenza M1, Zika virus (ZIKV) C, ZIKV prM / M, dengue virus (DENV) C-prM, West Nile virus (WNV) prME protein, WNV CprME protein, filovirus VP40 or Z protein, baculovirus P1 protein, rotavirus VP7, rotavirus VP2 protein, rotavirus VP6 protein, SARS M protein, SARS E protein, SARS The present invention also includes an N protein, a porcine circovirus type 2 (PCV2) capsid, a baculovirus VP2 protein, a baculovirus VP5 protein, a baculovirus VP3 protein, or a baculovirus VP7 protein, a hepatitis C virus (HCV) core protein, an Ebola nucleocapsid, a parovirus VP1 protein, a parovirus VP2 protein, a Newcastle disease virus (NDV) M protein, a hepatitis E virus (HeV) M protein, a Nipah virus (NIV) M protein, a human polyomavirus 2 (JCPyV) VP1 protein, a human parainfluenza virus type 3 (HPIV3) M protein, an HPIV3N protein, or a mumps virus (MuV) M protein, a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof.

[0186] envelope proteins In some cases, the VLPs disclosed herein comprise an outer lipid-based membrane ("envelope"). In some cases, the envelope comprises a single layer of lipids. In some cases, the envelope comprises a lipid bilayer. In some cases, the envelope further comprises a cell fusion protein (also referred to as the "envelope protein" of the VLP) that is inserted into, attached to, or anchored to the lipid layer.

[0187] The envelope protein can promote fusion of the VLP to a membrane, for example, a cell membrane. In some cases, the envelope protein mediates the tropism of the VLP, for example, the preferential fusion of the VLP to one or more specific types of cells. In some cases, the envelope protein causes mixing between lipids in the VLP and lipids in the target cell. The envelope protein can be any of the cell fusion proteins disclosed above. In some cases, the envelope protein can be a fusion protein containing a targeting moiety disclosed above.

[0188] In some cases, the envelope protein of the VLP is engineered to pseudotype the VLP for a particular property, for example, a particular tropism for a selected group of cells. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from hepatitis B virus (HBV) glycoprotein, hepatitis C virus (HCV) glycoprotein, Marburg virus glycoprotein, Ebola virus glycoprotein, and VSV-G glycoprotein; and the target cell is a liver cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus glycoprotein, human parainfluenza virus glycoprotein, and VSV-G; and the target cell is a lung cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is measles virus hemagglutinin and / or measles virus fusion glycoprotein; and the target cell is CD34 + In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from measles virus hemagglutinin and / or measles virus fusion glycoprotein, HTLV-1 glycoprotein, and VSV-G glycoprotein; and the target cell is a CD8 +The target cell is a T cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from HIV-1 envelope, HTLV-1 glycoprotein, measles virus hemagglutinin, and VSV-G glycoprotein; and the target cell is a CD4+ T cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as, but not limited to, Ross River virus glycoprotein or VSV-G; and the target cell is a skeletal muscle cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G; and the target cell is a visual cell (e.g., in a retinal cell, a photoreceptor cell, etc.). In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G; and the target cell is an auditory cell (by way of example, a hair cell, a cochlear cell, etc.). In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, for example, but not limited to, the viral glycoprotein is selected from rabies glycoprotein, Mokola virus glycoprotein, Semliki Forest virus glycoprotein, Sindbis virus glycoprotein, Venezuelan equine encephalitis virus glycoprotein, influenza hemagglutinin glycoprotein, and VSV-G; and the target cell is a central nervous system cell (by way of example, a neuron (by way of example, an excitatory and inhibitory neuron) and a glial cell (by way of example, an oligodendrocyte, an astrocyte, and a microglia)).

[0189] Nucleic acid editing efficiency In some embodiments, the lipid-containing particles (by way of example only) provided herein have improved nucleic acid editing efficiency when contacting a cell and editing a nucleic acid molecule inside the cell.

[0190] By way of example, in some cases, lipid-containing particles provided herein that include a fusion protein that includes an NES or a cleavage product that includes an NES can have higher nucleic acid editing efficiency, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 800%, or 1000% higher, compared to a corresponding lipid-containing particle that is otherwise the same but does not have an NES in the fusion protein or cleavage product.

[0191] In some cases, the lipid-containing particles provided herein comprise a fusion protein comprising a cleavable linker positioned between the therapeutic cargo sequence and the NES, and such lipid-containing particles have a higher nucleic acid editing efficiency, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 800%, or 1000% higher, compared to a corresponding lipid-containing particle that is otherwise identical but in which the therapeutic cargo sequence and the NES are positioned on the same side of the cleavable linker in the fusion protein.

[0192] In some cases, the lipid-containing particles provided herein contain cleavage products that contain the sequence of the gag nucleocapsid protein and a nuclear export sequence (NES) and lack a therapeutic cargo, and such lipid-containing particles have a higher nucleic acid editing efficiency, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 800%, or 1000% higher, compared to a corresponding lipid-containing particle that is otherwise identical except that the cleavage product contains the gag nucleocapsid protein but lacks both the NES and the therapeutic cargo.

[0193] In some cases, in the lipid-containing particles provided herein, the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is at least 1.5, and such lipid-containing particles have a higher nucleic acid editing efficiency, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 800%, or 1000% higher, compared to a corresponding lipid-containing particle that is otherwise the same except that the ratio of the amount of therapeutic cargo present inside the protein core to the amount of fusion protein is lower than the ratio in the lipid-containing particle provided herein, for example, less than 1.5.

[0194] The nucleic acid editing efficiency of lipid-containing particles provided herein can be measured using any suitable technique known to those skilled in the art.In some cases, this is measured by calculating the percentage of cells in which target nucleic acid molecules are edited by a given population of lipid-containing particles contacted with a population of cells relative to the total number of cells in the population.In some cases, nucleic acid editing efficiency is normalized by the amount of lipid-containing particles in the population, for example, the number of lipid-containing particles, the concentration of lipid-containing particles, or the volume of the liquid composition containing lipid-containing particles that contacts cells.In some cases, the nucleic acid editing efficiency of lipid-containing particles provided herein is compared with that of corresponding lipid-containing particles by assays in which the nucleic acid editing performance of both lipid-containing particles is tested at a series of different amounts, for example, number, concentration, or volume.In this way, a dose-response curve can be plotted for the lipid-containing particles tested, and the relative nucleic acid editing efficiency can be derived from this dose-response curve.Exemplary methods for measuring nucleic acid editing efficiency include the methods described in the examples of the present disclosure.

[0195] Human endogenous VLPs and humanized VLPs In some aspects, provided herein are virus-like particles, such as non-viral human endogenous virus-like particles (heVLPs), or humanized VLPs comprising humanized viral components (such as humanized viral structural proteins or humanized viral envelope proteins), that have reduced or no immunogenicity to human subjects.

[0196] Unlike virus-like particles according to some embodiments of the present disclosure, the heVLPs or humanized VLPs described herein can package protein cargo by integrating all production DNA into the genomic DNA of the production cell line. Once the cell line is created, protein-delivering heVLPs can be produced in a constitutive or inducible manner. Protein cargo is packaged into heVLPs by fusing a selected human endogenous GAG protein or other plasma membrane-localized protein (also referred to herein as a "plasma membrane recruitment domain") to the protein-based cargo.

[0197] The heVLP or humanized VLP system described herein has the potential to be simpler, more efficient, and safer than conventional artificially derived lipid / gold nanoparticle and viral particle-based delivery systems because the heVLP or humanized VLP is composed of human-derived or humanized components. The cargo inside the particle may or may not be human-derived, but the heVLP or humanized VLP contains human-derived or endogenous human or synthetic non-immunogenic components.

[0198] The "synthetic" components include surface scFv / nanobody / darpin peptides that have been demonstrated to be non-immunostimulatory and can be used to enhance targeting and cellular uptake of heVLPs. This means that the outer surface of the particles lacks components that can be significantly immunostimulatory, which can minimize immunogenicity and antibody neutralization of these particles.

[0199] In some cases, except for the cargo, the heVLPs provided herein do not contain exogenous viral components unique to other VLPs, which represents an important and novel advancement in the technology. Additionally, heVLPs can (but do not need to) utilize chemical-based dimerization agents, and heVLPs can be integrated with biomolecules and chemicals, including, by way of example, specialized single- and / or double-stranded DNA molecules (e.g., plasmids, minicircles, closed-end linear DNA, AAV The AAV particle may be capable of packaging and delivering cargo molecules including therapeutic or diagnostic agents, including DNA, episomes, bacteriophage DNA, homologous recombination repair templates, etc.), single-stranded and / or double-stranded RNA molecules (including, by way of example, single guide RNAs, prime editing guide RNAs, messenger RNAs, transfer RNAs, long non-coding RNAs, circular RNAs, RNA replicons, circular or linear splicing RNAs, microRNAs, small interfering RNAs, short hairpin RNAs, piwi-interacting RNAs, toehold switch RNAs, RNAs to which RNA-binding proteins can bind, bacteriophage RNAs, internal ribosome entry site-containing RNAs, etc.), proteins, chemical compounds and / or molecules (including, by way of example, small molecules), and combinations of the cargoes listed above (including, by way of example, AAV particles).

[0200] The heVLPs described herein differ from conventional retroviral particles, virus-like particles (VLPs), exosomes, and other previously described extracellular vesicles that can be loaded with cargo because, at least, heVLPs can be produced by strategic overexpression of human-derived components in human cells, heVLPs have a vast variety of possible cargoes and loading strategies, heVLPs lack restrictive DNA / RNA length constraints, heVLPs lack pol and exogenous gag-derived proteins, and heVLPs have a unique cell entry mechanism.

[0201] Described herein are compositions and methods for cargo delivery that can be used with a diverse array of protein and nucleic acid molecules, including genome editing, epigenome modulation, transcriptome editing, and proteome modulation reagents, that are applicable to many disease therapies.

[0202] 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 in heVLP-producing cells from an exogenous source such as a plasmid or a stably integrated transgene) (e.g., as shown in Table 1) or other human endogenous envelope proteins on the outside; and human endogenous GAG proteins, other plasma membrane-localized proteins (e.g., as shown in Table 2), and / or biomolecular / chemical cargo placed in the core of the heVLP inside the membrane (e.g., within the lumen surrounded by the phospholipid bilayer).

[0203] In some aspects, provided herein are humanized VLPs comprising a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV / glycoprotein(s) (e.g., overexpressed in heVLP-producing cells from an exogenous source such as a plasmid or a stably integrated transgene) (e.g., as shown in Table 1), or other human endogenous envelope proteins on the outside; and viral structural proteins (e.g., retroviral gag protein) on the inside of the membrane (e.g., within the lumen surrounded by the phospholipid bilayer).

[0204] In some aspects, provided herein are humanized VLPs comprising a membrane comprising a phospholipid bilayer having one or more viral envelope proteins disclosed herein, and a human endogenous GAG protein, other plasma membrane-localized protein, and / or biomolecule / chemical cargo located in the core of the heVLP inside the membrane (e.g., within the lumen surrounded by the phospholipid bilayer).

[0205] The cargo may or may not be fused to a human endogenous GAG or other plasma membrane-localized protein. In some cases, the heVLP or humanized VLP does not contain non-human gag and / or pol proteins. In some cases, the heVLP or humanized VLP does not express gag and / or pol proteins except for gag proteins encoded in the human genome or gag proteins encoded by consensus sequences derived from gag proteins found in the human genome. Human-derived GAG or other plasma membrane-localized proteins fused to the cargo can be overexpressed in heVLP-producing cells from an exogenous source, such as a plasmid or a stably integrated transgene.

[0206] Human endogenous GAG proteins and human pleckstrin homology (PH) domains can localize to biological membranes. The PH domain can interact with phosphatidylinositol lipids and proteins within biological membranes, such as PIP2, PIP3, the bg subunit of GPCRs, and PKC. However, in addition to localization to phospholipid bilayers, human endogenous GAG proteins can also drive budding and particle formation. This dual functionality of human endogenous GAGs can enable cargo packaging and particle budding / formation. One such human endogenous GAG protein used for this purpose is the human Arc protein, which can be fused to protein-based cargo and recruit the cargo to the cytosolic side of the phospholipid bilayer. These human endogenous GAG phospholipid bilayer recruitment domains can be fused to the N- or C-terminus of protein-based cargo via a polypeptide linker of variable length, regardless of the location of one or more nuclear localization sequences (NLSs) within the cargo. In some cases, the linker between the protein-based cargo and the human endogenous GAG phospholipid bilayer recruitment domain is a polypeptide linker composed primarily of glycine and serine, ranging from 5 to 20 amino acids in length, for example 8 to 12 amino acids in length, and for example 10 amino acids in length. [Table 1] [Table 2]

[0207] Human endogenous GAG or other phospholipid bilayer recruitment domains can localize cargo to the phospholipid bilayer, and this protein cargo is packaged into heVLPs or humanized VLPs that bud from the producing cells into the extracellular space. In this application, 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 promote the localization of cargo to the cytosolic face of the plasma membrane in heVLP or humanized VLP-producing cells, and also enable the cargo to localize to the nucleus of transduced cells without the use of exogenous retroviral GAG or chemical and / or light-based dimerization systems. HeVLP delivery of Cas9 is significantly more efficient when fused to a human endogenous GAG protein than when fused to a PH plasma membrane-localized protein or when not fused at all.

[0208] When heVLPs are produced via transient transfection of a production cell line, they can also package and deliver combinations of DNA and RNA. DNA transfected into cells possesses size-dependent mobility, such that a fraction of the transfected DNA remains in the cytosol, while another fraction of the transfected DNA localizes to the nucleus. 44 46 One fraction of the transfected DNA in the nucleus expresses the components required to make heVLPs, and the other fraction in the cytosol / near the plasma membrane is encapsulated into and delivered in heVLPs.

[0209] Combinations of foreign DNA, foreign RNA, and proteins (foreign and / or endogenous proteins) are referred to as type 1 cargo (T1 heVLPs), foreign RNA and proteins (foreign and / or endogenous proteins) are referred to as type 2 cargo (T2 heVLPs), foreign DNA and proteins (foreign and / or endogenous proteins) are referred to as type 3 cargo (T3 heVLPs), and proteins (foreign and / or endogenous proteins) are referred to as type 4 cargo (T4 heVLPs). Thus, T1 contains DNA, RNA, + / - foreign proteins, T2 contains RNA + / - foreign proteins, T3 contains DNA + / - foreign proteins, and T4 is a particle with or without foreign protein cargo. Therefore, since there is no "foreign cargo," T4 without foreign proteins is considered an "empty particle." "Exogenous cargo" refers to cargo that is not endogenous to the producing cell and can be packaged and / or incorporated into heVLPs. In addition, T1-T4 heVLPs can package exogenous chemical molecules in addition to the types of cargo present in the T1-T4 heVLPs. RNA in this context can be, for example, a single guide RNA (sgRNA), a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), and / or an mRNA encoding the cargo. As used herein, "small molecule" refers to a small organic or inorganic molecule with a molecular weight of less than about 3,000 daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 daltons (Da).The small molecule can be, for example, at least about 100 Da to about 3,000 Da (for example, about 100 to about 3,000 Da, about 100 to about 2,500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1,500, about 500 to about 1,000, about 300 to about 1,000 Da, or about 100 to about 250 Da).

[0210] The cargo is limited by the diameter of the particle, which in some embodiments ranges from 150 nm to 500 nm, by way of example only.

[0211] Other non-limiting examples of heVLPs, human endogenous viral structural proteins, and plasma membrane-localized proteins include those described in WO 2020 / 252455, the entire contents of which are incorporated herein by reference in their entirety.

[0212] In some embodiments, for efficient recruitment of cargo to heVLPs or humanized VLPs, the cargo preferably comprises a covalent or non-covalent connection to a human endogenous GAG or other plasma membrane recruitment domain, as shown in Table 2. Covalent connections can include, for example, direct protein-protein fusions produced from a single reading frame, inteins capable of forming peptide bonds, other proteins capable of forming covalent connections at R groups, and / or RNA splicing. Non-covalent connections can include, for example, DNA / DNA hybrids, DNA / RNA hybrids, and / or RNA / RNA hybrids (nucleic acids that base pair with other nucleic acids via hydrogen bonding interactions), protein domains that dimerize or multimerize with or without the need for chemical compounds / molecules to induce protein-protein binding (such as DmrA / DmrB / DmrC (Takara Bio), FKBP / FRB, dDZF, and leucine zippers), single-chain variable fragments, nanobodies, affibodies, proteins that bind to DNA and / or RNA, proteins with quaternary structure interactions, optogenetic protein domains that can dimerize or multimerize in the presence of certain wavelengths of light, and / or split proteins that naturally reconstitute.

[0213] In some embodiments, the cargo comprises a fusion with a dimerization domain or a protein-protein binding domain, which may or may not require a molecule to trigger dimerization or protein-protein binding.

[0214] In some embodiments, the producer cells are FDA-approved cell lines, allogeneic cells, and / or donor-derived autologous cells. In some embodiments, the complete or active peptide domain of human CD47 may be incorporated into the surface of the heVLP to reduce immunogenicity. Examples of AAV proteins encompassed herein are AAV REP 52, REP 78, and VP 1-3. The capsid site into which a protein can be inserted is T138, starting from the VP1 amino acid count. A dimerization domain can illustratively be inserted at this point within the capsid. Examples of dimerization domains encompassed herein, which may or may not require a small molecule inducer, are dDZF1, dDZF2, DmrA (Takara Bio), DmrB (Takara Bio), DmrC (Takara Bio), FKBP, FRB, GCN4 scFv, 10x / 24x GCN4, GFP nanobody, and GFP. Examples of split inteins encompassed herein are Npu DnaE, ​​Cfa, Vma, and Ssp DnaE. Other examples of split proteins encompassed herein that create covalent bonds together are Spy Tag and Spy Catcher. Examples of RNA-binding proteins encompassed herein are MS2, Com, and PP7. Examples of synthetic DNA-binding zinc fingers encompassed herein are ZF6 / 10, ZF8 / 7, ZF9, MK10, zinc finger 268, and zinc finger 268 / NRE. Examples of proteins that multimerize as a result of quaternary structure encompassed herein are E. coli ferritin and other chimeric forms of ferritin. Examples of optogenetic "light-inducible proteins" encompassed herein are Cry2, CIBN, and Lov2-Ja. Examples of transduction-enhancing peptides encompassed herein are L17E, Vectofusin-1 (Miltenyi Biotec), KALA, and various forms of nisin.

[0215] In another embodiment, the produced and isolated T1-T4 heVLPs can be loaded with biological or chemical molecular cargos using nucleofection, lipid, polymer, or CaCh transfection, sonication, freeze-thawing, incubation at various temperatures, and / or heat shock of purified particles mixed with cargo. These techniques are adapted from techniques employed to load cargo into exosomes for therapeutic or research applications. For example, 100 μg of heVLPs or humanized VLPs can be resuspended in 200-450 μL of 50 mM trehalose in PBS, mixed with cargo at the desired concentration, and electroporated (GenePulser II Electroporation System with volume expander, Bio-Rad, Hercules, CA, USA) in a 0.4 cm cuvette at 0.200 kV and 125 μF.

[0216] Preferably, heVLPs or humanized VLPs are harvested from the cell culture medium supernatant 36-48 hours after transfection, or when the heVLPs or humanized VLPs are at their maximum concentration in the producer cell medium (the producer cells excrete particles into the medium, and at some point the particle concentration in the medium will be optimal for particle harvesting). The supernatant can be purified by any method known in the art, for example, centrifugation, ultracentrifugation, sedimentation, ultrafiltration, and / or chromatography. In some embodiments, the supernatant is first filtered to remove particles larger than 1 pm, for example, through a 0.45 pm pore size polyvinylidene fluoride hydrophilic membrane (Millipore Millex-HV) or a 0.8 pm pore size mixed cellulose ester hydrophilic membrane (Millipore Millex-AA). After filtration, the supernatant can be further purified and concentrated, for example, by ultracentrifugation, for example, at a speed of 80,000 to 100,000 x g at a temperature between 1°C and 5°C for 1 to 2 hours, or at a speed of 8,000 to 15,000 x g at a temperature between 1°C and 5°C for 10 to 16 hours. After this centrifugation step, the heVLPs or humanized VLPs are concentrated in the form of a centrifugal mass (pellet), which can be resuspended to a desired concentration, mixed with a transduction enhancement reagent, subjected to buffer exchange, or used as is. In some embodiments, the heVLP-containing supernatant or humanized VLP-containing supernatant can be filtered, precipitated, centrifuged, and resuspended into a concentrated solution. For example, polyethylene glycol (PEG), for example, PEG8000, or antibody-bead conjugates that bind to heVLP or humanized VLP surface proteins or membrane components can be used to precipitate the particles.

[0217] The purified particles are stable and can be stored at 4°C for up to 1 week or at -80°C for several years without any apparent loss of activity.

[0218] Preferably, the heVLPs or humanized VLPs are resuspended or buffer exchanged so that the particles are suspended in a suitable carrier. In some embodiments, buffer exchange can be performed by ultrafiltration (Sartorius Vivaspin 500 MWCO 100,000).

[0219] Exosomes In some aspects, the delivery vehicle disclosed herein is an exosome. In several aspects, compositions, methods, and systems related to exosomes that can be utilized to deliver cargo into cells are disclosed herein. The term "exosome," as used herein, can refer to small membrane-bound vesicles (30-100 nm) of endosomal origin. In some cases, exosomes are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. In some cases, the exosomes described herein are derived from B lymphocytes, dendritic cells (DCs), mesenchymal stromal cells (MSCs), amniotic epithelial (AE) cells, and / or placenta-derived cells.

[0220] Source cells according to the present invention can be selected from a wide range of cells, illustratively mesenchymal stem cells or stromal cells or fibroblasts (which can be obtained, by way of example, from bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, tooth buds, umbilical cord blood, skin tissue, etc.), amniotic cells, and more specifically amniotic epithelial cells, myeloid suppressor cells. In general, both primary cells and cell lines are suitable sources of exosomes. Non-limiting examples include, by way of illustration, human embryonic kidney (HEK) cells, pericytes, lymphocytes, endothelial and epithelial cells from different organs, such as the trachea, lungs, gastrointestinal tract, urinary tract, etc., dendritic cells (DCs), or other cells from the hematopoietic system, such as macrophages, monocytes, B or T cells, NK cells, neutrophils, eosinophils, mast or basophils, erythrocytes or erythrocyte precursor cells, platelets, and megakaryocytes, to name a few, cells from different origins, such as placenta-derived cells (e.g., decidual placental cells), syncytiotrophoblasts, and amniotic epithelial cells, and cells from the CNS and PNS, such as microglia, astrocytes, oligodendrocytes and Schwann cells, ependymal cells, and neurons, to name a few, adipocytes from brown or white fat, muscle cells of both smooth and skeletal origin, and cardiomyocytes. Generally, exosomes can be derived from essentially any cell source, whether primary cell source or cell line. Exosome source cells can be any embryonic, fetal, or adult stem cell type, including induced pluripotent stem cells (iPSCs) and other stem or progenitor cells derived by any method. When treating neurological diseases, primary neurons, astrocytes, oligodendrocytes, microglia, and neural progenitor cells, for example, may be used as source cells. Source cells can be allogeneic, autologous, or even xenogeneic in nature to the patient being treated; that is, the cells can be derived from the patient themselves, or from unrelated, matched, or unmatched donors.In certain circumstances, allogeneic cells may be preferable from a medical standpoint, as they can provide immunomodulatory effects that may not be available from a patient's autologous cells for a particular indication.

[0221] In some cases, exosomes are produced by many different types of cells, including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs), and most cells. In some cases, exosomes are also produced by, for example, glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells. In some cases, exosomes for use in accordance with the present application can be derived from any suitable cell, including the cells identified above. Exosomes have also been isolated from physiological fluids such as plasma, urine, amniotic fluid, and malignant effusions.

[0222] In some cases, exosomes are derived from immature DCs. In some cases, exosomes produced from immature DCs do not express MHC-II, MHC-I, or CD86. Thus, such exosomes do not stimulate naive T cells to a significant extent and cannot induce a response in a mixed lymphocyte reaction. Therefore, exosomes produced from immature dendritic cells can be ideal candidates for use in delivering cargo, for example, therapeutic cargo.

[0223] In some cases, the exosomes are obtained from any autologous patient-derived, heterologous haploidentical, or xenogeneic stem cells to reduce or avoid the production of an immune response in the patient to whom the exosomes are delivered. Any exosome-producing cell can be utilized for this particular purpose.

[0224] In some cases, exosomes are produced by many different types of cells and have also been isolated from physiological fluids. Thus, according to the present disclosure, exosomes can be obtained from any suitable cell type discussed above or by isolation from physiological fluids. Typically, the methods of the present invention involve isolating exosomes from cell culture medium or tissue supernatant.

[0225] The exosome produced by cells can be collected from culture medium by any suitable method.Typically, exosome preparation can be prepared from cell culture or tissue supernatant by centrifugation, filtration or a combination of these methods.For example, exosome can be prepared by differential centrifugation, i.e., low-speed (less than 20,000 g) centrifugation to pellet larger particles, followed by high-speed (more than 100,000 g) centrifugation to pellet exosomes, size filtration with suitable filter (for example, 0.22 μm filter), gradient ultracentrifugation (for example, using sucrose gradient), or a combination of these methods.

[0226] In some cases, exosomes are loaded with cargo, for example, therapeutic cargo, for example, proteins, nucleic acid molecules, or small molecules. In some cases, exosomes are prepared and then loaded with the desired therapeutic cargo for delivery.

[0227] In some aspects, the exosomes disclosed herein are engineered to target a desired cell type or tissue. This targeting can be achieved by expressing a targeting moiety on the surface of the exosome that binds to a cell surface moiety expressed on the surface of the targeted cell. In some cases, the targeting moiety is a peptide that is typically expressed as a fusion protein with a transmembrane protein expressed on the surface of the exosome.

[0228] In some cases, exosomes are targeted to specific cell types or tissues by expressing targeting moieties such as peptides on their surface. Suitable peptides are peptides that bind to cell surface moieties, such as receptors or their ligands, found on the cell surface of the targeted cells. Non-limiting examples of suitable targeting moieties include short peptides, scFvs, and complete proteins, as long as the targeting moiety can be expressed on the surface of the exosome and does not interfere with the insertion of membrane proteins into the exosome. Typically, the targeting peptide is heterologous to the transmembrane exosomal protein. The peptide targeting moiety may typically be less than 100 amino acids in length, for example, less than 50 amino acids in length, less than 30 amino acids in length, or a minimum length of 10, 5, or 3 amino acids.

[0229] Targeting moieties can be selected to target specific tissue types, such as muscle, brain, liver, pancreas, and lung, or to target diseased tissues, such as tumors. In particularly preferred embodiments of the present invention, exosomes are targeted to brain tissue.

[0230] Specific, non-limiting examples of targeting moieties include muscle-specific peptides discovered by phage display to target skeletal muscle, a 29-amino acid fragment of the rabies virus glycoprotein that binds to the acetylcholine receptor, or a fragment of nerve growth factor that targets its receptor to target neurons; and secretin peptides that bind to the secretin receptor can be used to target biliary epithelium and pancreatic epithelium. Alternatively, immunoglobulins and their derivatives, including scFv antibody fragments, can also be expressed as fusion proteins that target specific antigens, such as VEGFR, for cancer gene therapy. Alternatively, natural ligands for receptors can be expressed as fusion proteins to confer specificity, such as NGF, which binds to NGFR and confers neuron-specific targeting.

[0231] Peptide targeting moieties can be expressed on the surface of exosomes by expressing them as fusion proteins with exosome transmembrane proteins. Numerous proteins are known to be associated with exosomes; that is, they are incorporated into exosomes as they are formed. In some cases, the targeting moiety includes or is derived from a transmembrane protein. Examples include, but are not limited to, Lamp-1, Lamp-2, CD13, CD86, flotillin, syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, integrin alpha 4, LiCAM, LFA-1, Mac-1 alpha and beta, Vti-1A and B, CD3 epsilon and zeta, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II), immunoglobulins, MHC-I or MHC-II components, TCR beta, and tetraspanins. In particularly preferred embodiments of the present invention, the transmembrane protein is selected from Lamp-1, Lamp-2, CD13, CD86, flotillin, and syntaxin-3. In some cases, the targeting moiety encompasses or is derived from an amino acid sequence variation, alteration, modification, or derivatization of the proteins discussed above. It will be understood that such variation, alteration, modification, or derivatization of the polypeptides as described herein is subject to the requirement that the polypeptide retain any additional required activity or characteristic, as may be specified in subsequent sections of this disclosure.

[0232] Targeting moieties can include, for example, antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies, such as sdAbs (either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds, such as fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T cell receptors (TCRs). Cell fusion proteins may be retargeted by noncovalently conjugating a targeting moiety to the fusion protein or targeting protein (e.g., hemagglutinin protein). For example, fusion proteins can be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell and redirect fusion activity to cells displaying the antibody's target.

[0233] Targeting moieties can be, by way of example, humanized antibody molecules, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (such as, by way of example, Zybodies®); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (such as, by way of example, shark single domain antibodies, such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (such as, by way of example, Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem dyes. These may include abodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®.

[0234] In some embodiments, the targeting moiety linked to the membrane protein binds to a cell surface marker on the target cell, such as, for example, a protein, a glycoprotein, a receptor, a cell surface ligand, an agonist, a lipid, a sugar, a class I transmembrane protein, a class II transmembrane protein, or a class III transmembrane protein.

[0235] In some cases, a targeting moiety is introduced into an exosome by expressing a fusion protein comprising the targeting moiety and an exosomal transmembrane protein in the cells used to produce the exosomes. Expression of this fusion protein in the cells allows the fusion protein to be incorporated into exosomes as they are produced from the cells.

[0236] In some cases, targeting moieties disclosed herein that are applicable to exosomes can also be used with other lipid-containing particles disclosed herein, such as, by way of example, virus-like particles, lipid nanoparticles, and proteolipid vehicles.

[0237] For example, a polynucleotide construct, such as a DNA plasmid expressing a fusion protein, is transfected into cells. Any suitable method can be used to introduce the polynucleotide construct into cells. The polynucleotide construct includes a suitable promoter sequence so that the encoded fusion protein is expressed in the cell. A signal peptide sequence is also included so that the protein is incorporated into the membrane of the endoplasmic reticulum when produced. The membrane protein is then subsequently translocated to the exosome / lysosome compartment before being incorporated into exosomes. The signal sequence is typically the signal peptide sequence for an exosomal transmembrane protein.

[0238] In some cases, exosomes produced by cells can be collected from culture medium by any suitable method.Typically, exosome preparations can be prepared from cell culture or tissue supernatant by centrifugation, filtration, or a combination of these methods.For example, exosomes can be prepared by differential centrifugation, i.e., low-speed (less than 20,000 g) centrifugation to pellet larger particles, followed by high-speed (more than 100,000 g) centrifugation to pellet exosomes, size filtration with suitable filter (e.g., 0.22 μm filter), gradient ultracentrifugation (e.g., using sucrose gradient), or a combination of these methods.

[0239] In some cases, a specific targeting moiety does not need to be incorporated into the exosome. For example, the exosome may be administered directly to the site where therapy is needed. Alternatively, for example, if the exosome contains genetic material encoding an immunogen, direct targeting to a specific site may not be necessary, and delivery, e.g., intradermal or intramuscular, may be sufficient to generate a desired immune response without targeting the exosome to any particular cell type. In some cases, the targeting moiety is not incorporated into the surface of the exosome. However, the exosome is selected to be more likely to target a specific tissue type. For example, exosomes derived from different cells may have a natural affinity for specific cell subtypes required by physiological function, such as the well-established affinity of mature dendritic cell-derived exosomes for T cells. This affinity may be utilized to specifically deliver the cargo mentioned above to tissues.

[0240] In some cases, exosomes are produced from cells modified to express a chimeric polypeptide receptor, for example, a chimeric antigen receptor (CAR). In some cases, exosomes are produced from cells genetically modified to produce a chimeric polypeptide receptor comprising (i) an extracellular recognition domain, (ii) at least one protease cleavage site, and (iii) an intracellular transcription factor, wherein binding of the extracellular recognition domain to its target induces proteolytic cleavage of the at least one protease cleavage site and endogenous transcription by the intracellular transcription factor of at least one polynucleotide encoding a gene product comprising at least one exosomal polypeptide. In some cases, the gene product further comprises a protein of interest, for example, an antibody, single-chain antibody, or any other antibody derivative, a bispecific T cell engager (BiTE), a receptor, a cytokine such as an interleukin, an enzyme such as a caspase or a granzyme, Cas, Cas9, a checkpoint inhibitor, a costimulatory inhibitor, an RNA-binding protein, a membrane transporter such as NPC-1, a splicing factor, a protein associated with a cellular organelle, a lysosomal enzyme, a transcription factor, a mitochondrial protein, an intracellular protein, an antiviral protein, or an antibacterial protein. In some cases, when the protein of interest is an RNA-binding protein, the cells in which the exosomes are produced are further genetically modified to include an RNA cargo molecule selected from the group consisting of mRNA, sgRNA, shRNA, miRNA, shRNA, siRNA, lncRNA, ncRNA, piRNA, piwiRNA, circRNA, tRNA, rRNA, crRNA, and any combination thereof. In some cases, the genetic modification is an in vitro or ex vivo genetic modification.In some cases, the cells from which the exosomes are produced are effector immune cells such as T cells, cytotoxic CD8+ T cells, CD4+ T cells, regulatory T cells, natural killer (NK) cells, B cells, plasma cells, dendritic cells (DCs), macrophages, monocytes, neutrophils, epithelial cells, endothelial cells, microglia, astrocytes, neurons, stem cells, bone marrow-derived mesenchymal stromal cells, Wharton's jelly-derived MSCs, or any other cell type. In some cases, the extracellular recognition domain of the chimeric polypeptide receptor is an antibody, an antibody derivative, a single-chain fragment, a single-chain antibody, a nanobody, a peptide, a ligand for a receptor, an adhesion molecule, a receptor, an interleukin receptor, an extracellular matrix component, or any combination thereof. In some cases, the at least one protease cleavage site is at least one of an S1, S2, and / or S3 cleavage site. In some cases, the fusion polypeptide is a chimeric Notch polypeptide comprising, from the N-terminus to the C-terminus and via a covalent linkage, the following: (i) an extracellular recognition domain not naturally occurring in Notch receptor polypeptides; (ii) a Notch regulatory region comprising a transmembrane domain comprising Lin12-Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; and (iii) an intracellular transcription factor heterologous to the Notch regulatory region, wherein binding of the extracellular recognition domain to its target induces cleavage at the S2 and S3 protease cleavage sites, thereby releasing the intracellular transcription factor that activates transcription of the polynucleotide. In some cases, the Notch regulatory region further comprises a heterodimerization domain comprising the S2 proteolytic cleavage site. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the fusion polypeptide comprises at least one linker. In some cases, the polynucleotide further comprises a transcriptional control element responsive to a transcription factor operably linked to the coding sequence.In some cases, the cells are genetically modified to produce at least two fusion polypeptides that differ between the fusion polypeptides in at least one of (i) an extracellular recognition domain, (ii) a protease cleavage site, and (iii) an intracellular transcription factor. In some cases, the extracellular recognition domains of the fusion polypeptides differ from each other.

[0241] In some cases, the loading of protein cargo into exosomes disclosed herein is achieved by expressing a tri-domain polypeptide construct in the source cells from which the exosomes are produced. In some cases, such a polypeptide construct comprises (i) at least one protein of interest (POI), (ii) at least one multimerization domain, and (iii) at least one exosome sorting domain. The design of the tri-domain polypeptide construct can enable highly efficient loading of the POI into exosomes and also drive increased production of exosomes from the source cells.

[0242] The multimerization polypeptide domain is a key component for achieving this increased loading of the resulting exosomes; interestingly, such multimerization domains may be selected from a wide variety of different species and may also exhibit relatively different mechanisms of action (as an example, they may be heterodimerization domains, or homotrimerization domains, homopentamerization domains, etc.). In some cases, the multimerization domain is a homomultimerization domain, as these allow for simple design of fusion proteins and, importantly, support the controlled loading of a single type of fusion polypeptide construct into exosomes (as opposed to multiple fusion constructs). The multimerization domain can be a dimerization domain, trimerization domain, tetramerization domain, or essentially any higher-order multimerization domain, as long as the domain is capable of promoting the interaction of at least two domains (and the polypeptides of which they form a part).By way of illustration, a non-limiting list of multimerization domains includes the following domains: the leucine zipper homodimerization domain of GCN4 from S. cerevisiae, the retro-leucine zipper homodimerization domain of GCN4 from S. cerevisiae, the fold-on homodimerization domain of fibritin (from T4 bacteriophage), the fragment X heterodimerization domain of phosphoprotein (from human respiratory syncytial virus A), the human alpha helix coiled-coil oligomerization domain of the collagen superfamily, the leucine zipper heterodimerization domain of Fos and Jun (human), the cardiac phospholamban (Cardiac transmembrane homopentameric domain of phospholamban (human), homodimerization domain of parathyroid hormone (human), transmembrane homodimerization domain of glycophorin A (human), trimerization domain of Gp41 (from HIV), C-terminal homodimerization domain of oncoprotein E7 (from HPV 45), and EVH2 homotetramerization domain of vasodilator-stimulated phosphoprotein (human), mitochondrial antiviral signaling protein CARD filament, and / or any combination thereof.

[0243] The multimerization domain can be placed in several different locations in the polypeptide construct. For example, the multimerization domain can be placed between the POI sequence and the exosome sorting domain sequence, within or adjacent to the exosome sorting domain sequence, and / or within or adjacent to the POI sequence. Overall, the design of the tri-domain polypeptide construct (both in terms of the selection of the multimerization domain and its location in the construct, and in terms of the selection of the exosome sorting domain and its position in the construct) is important for determining where the polypeptide will ultimately end up in exosomes after production in exosome-source cells. For example, it is possible to enrich the POI on the surface of exosomes by selecting tetraspanin exosome sorting proteins (for example, CD9, CD63, or CD81) or any other exosome membrane protein (such as Lamp2b). Conversely, by selecting exosome-sorting proteins that are typically present in the exosome lumen, such as ALIX or syntenin, it is essentially possible to enrich the polypeptide construct (and thereby the POI) inside the exosome. Of course, the polypeptide construct may be present simultaneously on the outside and inside of the exosome and in the exosome membrane. Furthermore, in preferred embodiments, the fusion polypeptide construct may contain various types of linkers between different domains, i.e., between at least one POI, at least one multimerization domain, and at least one exosome-sorting domain. The linker may, for example, be a GS (i.e., glycine-serine) linker, i.e., a linker containing the amino acids glycine and serine, or any other type of suitable linker domain that ensures that the activity of different domains is not limited when present in the fusion polypeptide construct.

[0244] A typical tri-domain fusion polypeptide construct according to the invention can be generally described as follows (the following designations should not be construed as illustrating any C-terminal and / or N-terminal orientation, but are merely for illustrative purposes): POI multimerization domain-exosome sorting domain

[0245] The exosome sorting domain of the present disclosure can be selected from any one of the following proteins: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82 , CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin (IL-1) receptor receptors, immunoglobulins, MHC-I or MHC-II components, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD27 3, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, and any combination thereof.

[0246] In some cases, the exosomes are loaded with cell-penetrating peptides, such as those described in U.S. Patent Application Publication No. 20190388347, which is incorporated herein by reference in its entirety.

[0247] Non-limiting examples of exosomes, source cells from which exosomes are produced, cargo that can be delivered in exosomes, methods of loading cargo into exosomes, and methods of producing exosomes are described in U.S. Patent Application Publication Nos. US20070298118, US20180177727, US20200062813, US20200206360, US20200023012, and US20160137716. , US20170173113, US20130053426, US20190167810, US20190388347, US20190224331, US20160137716, US20210188903, US20210069254, and US20200407418, each of which is incorporated by reference in its entirety.

[0248] Lipid Nanoparticles or Proteolipid Vehicles In some aspects, disclosed herein are compositions, methods, and systems related to lipid nanoparticles that can be utilized to deliver cargo into cells. In some aspects, disclosed herein are compositions, methods, and systems related to proteolipid vehicles that can be utilized to deliver cargo into cells.

[0249] Lipid nanoparticles can provide a biocompatible and biodegradable delivery system for the therapeutic cargoes disclosed herein. In some cases, the lipid nanoparticles disclosed herein include nanostructured lipid carriers (NLCs), polymer nanoparticles (PNPs), or lipid-polymer nanoparticles (PLNs). NLCs are modified solid lipid nanoparticles (SLNs) that retain the characteristics of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of therapeutic delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. In this way, the two components enhance drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs.

[0250] Non-limiting examples of lipid nanoparticles disclosed herein include those described in J.A. Zuris et al., Nat Biotechnol. 2014 Oct 30;33(1):73-80; Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021); International Publication Nos. WO2019 / 067992, WO2017 / 173054, WO2015 / 095340, WO2014 / 136086, and WO2019 / 217941, each of which is incorporated by reference in its entirety.

[0251] cargo As used herein, "cargo" can refer to, for example, one or more of a chemical substance, such as a small molecule compound, a combination of DNA, RNA, and protein, a combination of RNA and protein, a combination of DNA and protein, or a protein, for use in therapy or diagnosis, or for genome editing, epigenome modulation, and / or transcriptome modulation. Additionally, endogenous RNA and proteins from a production cell can be packaged and / or incorporated into a delivery vehicle (for example, a VLP, for example, a heVLP or a humanized VLP).

[0252] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein is a polypeptide, including, by way of example, a nuclear transport polypeptide, a nucleic acid binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integration polypeptide, a targeting endonuclease (including, by way of example, a zinc finger nuclease (ZFN), a transcription-activator-like nuclease (TALEN), Cas9 or a homolog thereof), a recombinase, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, a cytochrome P450 ... The cargo may comprise a polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein-binding polypeptide, a lipid-binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, a cell death polypeptide, or any combination thereof. In some embodiments, the cargo contained in the delivery vehicle disclosed herein comprises a protein that targets a protein in a cell for degradation. In some cases, the cargo contained in the delivery vehicle disclosed herein comprises a chimeric antigen receptor (CAR), an antibody, a T-cell receptor or a functional fragment thereof, or any combination thereof.

[0253] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein comprises a polynucleotide, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule. In some cases, the polynucleotide encodes a polypeptide, such as those described in the paragraph above. In some cases, the polynucleotide comprises a napR / DNAbp programming nucleic acid molecule, as described below.

[0254] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein comprises a ribonucleoprotein (RNP) complex formed between one or more proteins and one or more polynucleotides. Illustratively, the cargo can comprise an RNP complex formed by a nucleic acid programmable R / DNA binding protein (napR / DNAbp), described below, and a napR / DNAbp programming nucleic acid molecule, such as a Cas protein and a guide RNA.

[0255] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein includes ribozymes, aptamers, aptazymes, peptides, oligonucleotides, antibody mimetics, peptidomimetics, antibody-drug conjugates, antibiotics, carbohydrates, ribosomes, mitochondria, and other therapeutic molecules such as small molecule compounds.

[0256] In some embodiments, the cargo contained in and delivered by the delivery vehicle disclosed herein includes polypeptides, such as enzymes, structural polypeptides, signal transduction 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, targeting endonucleases (such as zinc finger nucleases, transcription activator-like nucleases (TALENs), Cas9 and their homologs), recombinases, and any combination thereof. In some embodiments, the protein targets proteins in cells for degradation. In some embodiments, the protein targets proteins in cells 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 protein or a chimeric protein.

[0257] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein may be a decoy protein for binding to a pathogenic target protein; a peptide or protein for inducing endosomal escape, such as HA2; a peptide or protein for targeting exosomes to a tissue or organ or cell type of interest; an antibody, intrabody, single-chain variable fragment (scFv), affibody, bispecific or multispecific antibody or binder, receptor, etc.; an enzyme such as alpha-glucosidase and / or glucocerebrosidase for enzyme replacement therapy; a transport protein such as NPC1 or cystinosin; an exosome targeting protein such as an endosomal target protein; Peptides or proteins for optimizing in vivo behavior (for example, their circulation time or immune system recognition), for example, CD47 and / or CD55, or portions of these proteins; cytokines or chemokines; targeting peptides or proteins such as RVG peptides, VSV-G peptides, p-selectin binding peptides, or e-selectin binding peptides; cell penetrating peptides (CPPs) (for example, Tat, penetratin, TP10, CADY); or tumor suppressors.

[0258] In some cases, the cargo contained in and delivered by the delivery vehicle disclosed herein comprises an immunogenic molecule such as a vaccine. The vaccine can be a peptide antigen, RNA (for example, mRNA or circRNA), or DNA (for example, a DNA molecule encoding an antigen). The cargo can also include an adjuvant that enhances the immunogenicity of the vaccine composition.

[0259] In some cases, the cargo protein loaded onto the delivery vehicle functions to bind to another cargo molecule (by way of example, a nucleic acid molecule, a protein, an RNP, etc.) that is delivered by the delivery vehicle.

[0260] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein is a small molecule, for example, an ion (for example, Ca 2+ , Cl - , Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron-withdrawing compounds, electron-donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is a pharmaceutical agent that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in a cell for degradation. In some embodiments, the small molecule targets a protein in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolysis-targeting chimeric molecule (PROTAC).

[0261] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein includes proteins, nucleic acids, or metabolites, such as mixtures of multiple polypeptides, multiple nucleic acids, and multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (such as the Cas9-gRNA complex); multiple transcription factors, multiple epigenetic factors, reprogramming factors (such as Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.

[0262] In some embodiments, the cargo contained in and delivered by the delivery vehicles disclosed herein comprises one or more organelles, such as, by way of example, a chondrisome, a mitochondrion, a lysosome, a nucleus, a cell membrane, a cytoplasm, an endoplasmic reticulum, a ribosome, a vacuole, an endosome, a spliceosome, a polymerase, a capsid, an acrosome, an autophagosome, a centriole, a glycosome, a glyoxysome, a hydrogenosome, a melanosome, a mitosome, a myofibril, a cnidocyst, a peroxisome, a proteasome, a vesicle, a stress granule, a network of organelles, and any combination thereof.

[0263] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein is RNA (viral or heterologous), DNA (single-stranded, double-stranded), green fluorescent protein, nuclease, iron oxide NPs (IONPs), taxol, Alexa Fluor® 488, porphyrin, doxorubicin, fluorescein, DOTA chelator, RNA (messenger, micro, small interfering molecule), ricin toxin A chain, HIV-1 Tat peptide, alkaline phosphatase, green fluorescent protein, quantum dot 585, methacrylic acid (monomer, polymer), CpG DNA, fluorescent protein, luciferase, nickel, biotin, fluorescent polymethacrylic acid, gadopentetate, polymethacrylic acid, CRISPR (Cas9 and guide RNA), green fluorescent protein or mCherry, CellB protein, [NiFe]hydrogenase, ziconotide peptide, three enzyme cascade, cascade) (genetically linked), alcohol dehydrogenase, polystyrene sulfonic acid, RNA, green or teal fluorescent protein, Pseudozyma antarctica lipase B, horseradish peroxidase, DOTAC10 micelles with Gd(III) or Zn(II), Gd(DOTA), fluorescent probes, doxorubicin, DAPI, acridine orange, propidium iodide, proflavine, iron oxide NPs, Gd(III), or Tb(III). In some cases, cargoes contained in the delivery vehicles disclosed herein include those described in Rohovie, MJ, et al., Bioengineering & Translational Medicine, 2:43-57, which is incorporated herein by reference in its entirety.

[0264] In some cases, the cargo contained in and delivered by a delivery vehicle of the present disclosure is at least 10 amino acids (aa) long, at least 20 aa long, at least 30 aa long, at least 50 aa long, at least 80 aa long, at least 100 aa long, at least 150 aa long, at least 200 aa long, at least 250 aa long, at least 300 aa long, at least 350 aa long, at least 400 aa long, at least Includes polypeptides having a length of 500 aa, at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa, or at least 5000 aa. In some cases, the cargo contained in a delivery vehicle of the present disclosure comprises a polypeptide having a length of about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa, or about 5000 aa.

[0265] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein is at least 20 aa in length, at least 30 aa in length, at least 50 aa in length, at least 80 aa in length, at least 100 aa in length, at least 150 aa in length, at least 200 aa in length, at least 250 aa in length, at least 300 aa in length, at least 350 aa in length, at least 400 aa in length, at least 500 aa in length, at least 600 aa in length, at least 700 aa in length, at least 800 aa in length, at least 900 aa in length, at least 100 aa in length, at least 150 aa in length, at least 200 aa in length, at least 250 aa in length, at least 300 aa in length, at least 350 aa in length, at least 400 aa in length, at least 500 aa in length, at least 1000 aa in length, at least 1500 aa in length, at least 1000 aa in length, at least 1500 aa in length, at least 200 aa in length, at least 250 aa in length, at least 300 aa in length, at least 350 aa in length, at least 400 aa in length, at least 500 aa in length, at least 10 ... These include polynucleotides that encode polypeptides having a length of at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa, or at least 5000 aa. In some cases, the cargo contained in a delivery vehicle of the present disclosure comprises a polynucleotide encoding a polypeptide having a length of about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa, or about 5000 aa.

[0266] In some cases, the polypeptide contained in and delivered by the delivery vehicles disclosed herein 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 100 kDa, at least 150 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 10 ... kDa, at least 120 kDa, at least 150 kDa, at least 180 kDa, at least 200 kDa, at least 220 kDa, at least 250 kDa, at least 280 kDa, at least 300 kDa, at least 320 kDa, at least 350 kDa, at least 400 kDa, at least 500 kDa, at least 600 kDa, at least 700 kDa, at least 800 kDa, at least 900 kDa, or at least 1000 kDa. In some cases, the polypeptide contained in and delivered by the delivery vehicles disclosed herein 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, The protein forms is 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.

[0267] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein is at least 50 nucleotides in length, at least 80 nucleotides in length, at least 100 nucleotides in length, at least 150 nucleotides in length, at least 200 nucleotides in length, at least 250 nucleotides in length, at least 300 nucleotides in length, at least 350 nucleotides in length, at least 400 nucleotides in length, at least 500 nucleotides in length, at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, at least 90 ...00 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 100 nucleotides in length, at least 1 The present invention includes single-stranded polynucleotides having a length of at least 10,000 nucleotides, at least 1200 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 8000 nucleotides, at least 10000 nucleotides, at least 12000 nucleotides, at least 14000 nucleotides, or at least 15000 nucleotides.In some cases, the cargo contained in the delivery vehicle of the present disclosure is about 20 nucleotides in length, about 30 nucleotides in length, about 50 nucleotides in length, about 70 nucleotides in length, about 80 nucleotides in length, about 100 nucleotides in length, about 120 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 500 nucleotides in length, about 600 nucleotides in length, about 700 nucleotides in length, about 800 nucleotides in length, about 90 ... The present invention also includes single-stranded polynucleotides encoding polypeptides having a length of about 10,000 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.

[0268] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein is at least 50 nucleotides in length, at least 80 nucleotides in length, at least 100 base pairs in length, 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 length. In some cases, the cargo contained in the delivery vehicle of the present disclosure is 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 and double-stranded polynucleotides encoding polypeptides having a length of 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.

[0269] In some cases, the cargo contained in and delivered by the delivery vehicles disclosed herein does not include a nuclease, reverse transcriptase, base editor, or prime editor.

[0270] nuclease Any suitable nuclease can be delivered by the delivery vehicle disclosed herein, which contains either a nuclease or a polynucleotide encoding the nuclease. In some embodiments, the nuclease delivered by the delivery vehicle disclosed herein (for example, a lipid-containing particle) is not a nucleic acid programmable DNA binding protein (napDNAbp) (for example, a guide RNA-programmable Cas protein, such as Cas9 protein). Suitable nucleases include CRISPR-associated (Cas) proteins, or type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides (e.g., Cas9 or Cas14), type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides (e.g., Cpf1 / Cas12a, C2c1, or c2c3), and type VI CRISPR-associated (Cas) polypeptides (e.g., C2c2 / Cas13a, Cas13b, Cas13c, C2c2 / Cas13b, Cas13c, C2c2 / Cas13c, C2c2 / Cas13a, Cas ... as13d); zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (go) proteins (by way of example only, prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivatives thereof; any variants thereof; and any fragments thereof.

[0271] In some embodiments, the cargo in the delivery vehicle disclosed herein is a non-naturally occurring CRISPR (clustered regularly interspaced short palindromic repeats) / Cas( C RISPR- asThey contain or encode CRISPR-associated (Cas) proteins or Cas nucleases that function in the CRISPR-associated (CRISPR) system. In bacteria, this system can provide adaptive immunity against foreign DNA (Barrangou, R., et al., "CRISPR provides acquired resistance against viruses in prokaryotes," Science (2007) 315:1709-1712; Makarova, K.S., et al., "Evolution and classification of the CRISPR-Cas systems," Nat Rev Microbiol (2011) 9:467-477; Garneau, J.E., et al., "The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA," Nature (2010) 468:67-71; Sapranauskas, R., et al., "The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli," Nucleic Acids Res (2011) 39:9275-9282).

[0272] One or more components of the CRISPR / Cas system (e.g., modified and / or unmodified) delivered by the delivery vehicle disclosed herein can be utilized as a genome manipulation tool in a wide range of organisms, including various mammals, animals, plants, and yeast. The CRISPR / Cas system can include a guide nucleic acid, such as a guide RNA (gRNA), complexed with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid editing. The RNA-guided Cas protein (e.g., a Cas nuclease, such as Cas9 nuclease) can specifically bind to a target polynucleotide (e.g., DNA) in a sequence-dependent manner.Cas proteins can cleave DNA if they possess nuclease activity (Gasiunas, G., et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc Natl Acad Sci USA (2012) 109:E2579-E286; Jinek, M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-821; Sternberg, SH, 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 have been widely used for programmable genome editing in various organisms and model systems (Cong, L., et al., “Multiplex genome engineering using CRISPR-Cas systems,” Science (2013) 339:819-823; Jiang, W., et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31:233-239; Sander, JD & Joung, JK, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).

[0273] In some cases, the Cas protein delivered by the delivery vehicle of the present disclosure is mutated and / or modified relative to the wild-type Cas protein to produce a nuclease-deficient protein or a protein with reduced nuclease activity. A nuclease-deficient protein can retain the ability to bind to DNA but may lack or have reduced nucleic acid cleavage activity. A cargo protein or protein encoded by a cargo nucleic acid molecule containing 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., decreasing, increasing, or eliminating). The Cas protein binds to a target polynucleotide and prevents transcription by physical interference or edits the nucleic acid sequence to produce a non-functional gene product.

[0274] In some embodiments, the cargo in a delivery vehicle disclosed herein comprises or encodes a Cas protein that forms a complex with a guide nucleic acid, such as a guide RNA (gRNA). In some embodiments, the cargo in a delivery vehicle disclosed herein comprises or encodes a Cas protein that forms a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the cargo in a delivery vehicle disclosed herein comprises or encodes an RNA-binding protein (RBP), optionally complexed with a guide nucleic acid, such as a guide RNA (e.g., an sgRNA), that is capable of forming a complex with a Cas protein.

[0275] One or more components of any suitable CRISPR / Cas system can be delivered by the delivery vehicle of the present disclosure. CRISPR / Cas systems can be referred to using various nomenclature systems. Exemplary nomenclature systems are provided in Makarova, K. Set al., "An updated evolutionary classification of CRISPR-Cas systems," Nat Rev Microbiol (2015) 13:722-736 and Shmakov, S. et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Mol Cell (2015) 60:1-13. The CRISPR / Cas system can be a Type I system, a Type II system, a Type III system, a Type IV system, a Type V system, a Type VI system, or any other suitable CRISPR / Cas system. The CRISPR / Cas system used herein can be a Class 1, Class 2, or any other suitable classified CRISPR / Cas system. The determination of Class 1 or Class 2 can be based on the gene encoding the effector module. Class 1 systems generally have a multi-subunit crRNA-effector complex, while Class 2 systems generally have a single protein such as Cas9, Cpf1, C2c1, C2c2, C2c3, or a crRNA-effector complex. Class 1 CRISPR / Cas systems can use a complex of multiple Cas proteins to achieve regulation. Class 1 CRISPR / Cas systems can include, for example, Type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), Type III (e.g., III, IIIA, IIIB, IIIC, IIID), and Type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can use a single, large Cas protein to achieve regulation.Class 2 CRISPR / Cas systems can include, for example, Type II (including, by way of example, II, IIA, and IIB) and Type V CRISPR / Cas types. CRISPR systems can be complementary to each other and / or can provide functional units in trans to facilitate CRISPR gene localization.

[0276] The cargo delivered by the delivery vehicle of the present disclosure can include or encode a class 1 or class 2 Cas protein. The Cas protein can be a type I, type II, type III, type IV, type V, or type VI Cas protein. The Cas protein can include one or more domains. Non-limiting examples of domains include a guide nucleic acid recognition and / or binding domain, a nuclease domain (e.g., DNase or RNase domain, RuvC, HNH), a DNA-binding domain, an RNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. The guide nucleic acid recognition and / or binding domain can interact with the guide nucleic acid. The nuclease domain can include catalytic activity for nucleic acid cleavage. The nuclease domain can lack catalytic activity to prevent nucleic acid cleavage. The Cas protein can be a chimeric Cas protein fused to another protein or polypeptide. For example, the Cas protein can be a chimera of various Cas proteins, including domains from different Cas proteins.

[0277] Non-limiting examples of Cas proteins that can be delivered by the delivery vehicles of the present disclosure include c2c1, Cas13a (formerly C2c2), Cas13b, Cas13c, Cas13d, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas10, Cas10d, Cas14, Cas10, Cas10d, CasF, CasG, CasH, Cas12 a (formerly Cpf1), Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, as well as homologs or modified versions thereof. Non-limiting examples of mutant Cas9 proteins or Cas9 variants include SpG, SpRY, eSpCas9(1.1), SpCas9-HF1, nSpCas9, SpCas9(H840A), dSpCas9, SpCas9(N863A), SpCas9(D839A), SpCas9(H983A), and those described in Chuang CK et al., Int J Mol Sci. 2021 Sep 13;22(18):9872, which are incorporated herein by reference in their entireties.

[0278] Another example of a Cas protein that can be delivered by the delivery vehicle of the present disclosure includes Cas14. Cas14 proteins or polypeptides (also referred to as "CasZ" proteins or polypeptides) can bind to and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) target nucleic acids and / or polypeptides associated with target nucleic acids (e.g., methylation or acetylation of histone tails) (e.g., in some cases, CasZ proteins include active fusion partners, and in some cases, CasZ proteins provide nuclease activity). In some cases, the Cas14 protein or polypeptide is a naturally occurring protein (e.g., naturally occurring in prokaryotic cells) (e.g., CasZ protein). In other cases, the Cas14 protein or polypeptide is not a naturally occurring polypeptide (e.g., Cas14 proteins are variant Cas14 proteins, chimeric proteins, and the like). The Cas14 protein contains three 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 Cas14 protein, but which form the RuvC domain when the protein is produced and folded. Naturally occurring Cas14 proteins function as endonucleases that catalyze cleavage at specific sequences in target nucleic acids (e.g., double-stranded DNA (dsDNA)). Sequence specificity is provided by an associated guide RNA that hybridizes to a target sequence within the target DNA. A naturally occurring Cas14 guide RNA is a crRNA, where the crRNA contains (i) a guide sequence that hybridizes to a target sequence in the target DNA and (ii) a protein-binding segment that binds to the Cas14 protein.Non-limiting examples of Cas14 proteins include those described in U.S. Patent Application 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 cargo disclosed herein comprises a Cas14 polypeptide or a nucleic acid molecule encoding a Cas14 polypeptide. In some cases, the cargo disclosed herein comprises a Cas14a polypeptide or a nucleic acid molecule encoding a Cas14a polypeptide. In some cases, the cargo disclosed herein comprises a Cas14b polypeptide or a nucleic acid molecule encoding a Cas14b polypeptide. In some cases, the cargo disclosed herein comprises a Cas14c polypeptide or a nucleic acid molecule encoding a Cas14c polypeptide.

[0279] Cas proteins can be derived from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelluliruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Leptotrichia wadeii, Leptotrichia wadeii F0279, Rhodobacter capsulatus SB1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442, Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium MA2020, Clostridium aminophilum DSM 10710, Paludibacter propionicigenes WB4, Carnobacterium gallinarum DMS4847, Carnobacterium gallinarum DSM4847, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).

[0280] Cas proteins are found in 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.

[0281] The Cas proteins disclosed herein can be wild-type or modified forms of Cas proteins. The Cas proteins can be active variants, inactive variants, or fragments of wild-type or modified Cas proteins. The Cas proteins can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof, relative to the wild-type version of the Cas protein. The Cas protein can be a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity to a wild-type exemplary Cas protein. A Cas protein can be a polypeptide having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas protein. A variant or fragment 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 portion thereof. While lacking nucleic acid cleavage activity, the variant or fragment can form a complex with a guide nucleic acid and be targeted to a nucleic acid locus.

[0282] Cas proteins can contain one or more nuclease domains, such as a DNase domain. For example, Cas9 proteins can contain a RuvC-like nuclease domain and / or an HNH-like nuclease domain. The RuvC and HNH domains can each cut different domains of double-stranded DNA to create double-strand breaks in DNA. Cas proteins can contain only one nuclease domain (for example, Cpf1 contains a RuvC domain but lacks an HNH domain).

[0283] The 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.

[0284] Cas proteins can be modified to optimize regulation of gene expression. Cas proteins 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 a Cas protein can be modified, deleted, or inactivated to remove domains that are not essential for protein function, or to optimize (e.g., enhance or reduce) the activity of the Cas protein to regulate gene expression, or the Cas protein can be truncated.

[0285] In some embodiments, the cargo delivered by the delivery vehicle of the present disclosure comprises a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcription activation or repression of target DNA sequence.In some embodiments, the cargo comprises or encodes a nuclease-null RNA binding protein derived from an RNA nuclease that can induce transcription activation or repression of target RNA sequence.For example, the cargo can comprise or encode a Cas protein that lacks cleavage activity.

[0286] The Cas protein can be a fusion protein. For example, the Cas protein can be fused to a heterologous functional domain. The heterologous functional domain can include a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a repressor domain. The Cas protein can also be fused to a heterologous polypeptide that provides increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally within the Cas protein.

[0287] The gene regulation can be any gene of interest.It is intended that the gene homologues of the genes described herein are covered.For example, the gene can exhibit a certain identity and / or homology with the genes disclosed herein.Therefore, it is intended that the gene that 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. Thus, it is also contemplated that genes exhibiting or exhibiting 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.

[0288] The Cas protein can be provided in any form. For example, the Cas protein can be provided in the form of a protein, such as a Cas protein alone or complexed with a guide nucleic acid. The Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA.

[0289] Nucleic acids encoding Cas proteins delivered by the delivery vehicles of the present disclosure can be codon-optimized for efficient translation into proteins in a particular cell or organism.

[0290] In some embodiments, the Cas protein is a dead Cas protein. A dead Cas protein can be a protein that lacks nucleic acid cleavage activity.

[0291] The Cas protein can include a modified form of a wild-type Cas protein. The modified form of a wild-type Cas protein can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the Cas protein. For example, the modified form of the Cas protein can have less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of a wild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified form of the Cas protein cannot have substantial nucleic acid cleavage activity. When the Cas protein is in a modified form that does not have substantial nucleic acid cleavage activity, the Cas protein can be referred to as enzymatically inactive and / or "dead" (abbreviated as "d"). Dead Cas proteins (e.g., dCas, dCas9) can bind to target polynucleotides but may not cleave the target polynucleotide. In some aspects, the dead Cas protein is a dead Cas9 protein.

[0292] The dCas9 polypeptide can associate with a single guide RNA (sgRNA) to activate or suppress transcription of target DNA. The sgRNA can be introduced into cells expressing an engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs targeting the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell. The nucleic acid targeted by the guide RNA can be any expressed in cells, such as immune cells. The targeted nucleic acid can be a gene involved in immune cell regulation. In some embodiments, the nucleic acid is associated with cancer. The cancer-associated nucleic acid can be a cell cycle gene, a cell response gene, an apoptosis gene, or a phagocytosis gene. The recombinant guide RNA can be recognized by a CRISPR protein, a nuclease-null CRISPR protein, a variant thereof, a derivative thereof, or a fragment thereof.

[0293] 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 the target polynucleotide. An enzymatically inactive site-specific polypeptide can include an enzymatically inactive domain (for example, a nuclease domain). Enzymatically inactive can refer to the absence of activity. Enzymatically inactive can refer to the substantial absence of activity. Enzymatically inactive can refer to the essentially absence of activity. Enzymatically inactive can refer to less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% of the activity of the wild-type (for example, nucleic acid cleavage activity, wild-type Cas9 activity).

[0294] One or more nuclease domains of a Cas protein (e.g., RuvC, HNH) can be deleted or mutated so that they are no longer functional or contain reduced nuclease activity (e.g., inactivated or dead Cas, or "dCas"). For example, in a Cas protein containing 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 double-stranded DNA, but not a double-strand break. Such a nickase can cleave either the complementary strand or the non-complementary strand, but not both. When all of the nuclease domains of a Cas protein (for example, both the RuvC and HNH nuclease domains in the Cas9 protein; the RuvC nuclease domain in the Cpfl protein) are deleted or mutated, the resulting Cas protein may have reduced or no ability to cleave both strands of double-stranded DNA. An example of a mutation that can convert a Cas9 protein into a nickase is the D10A (aspartic acid 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 Cas9 into a nickase. Examples of mutations that can convert a Cas9 protein into a dead Cas9 are the D10A (aspartic acid to alanine at position 10 of Cas9) mutation in the RuvC domain and the H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) mutation in the HNH domain of Cas9 from S. pyogenes.

[0295] The dead Cas protein can contain one or more mutations compared to the wild-type version of the protein. The mutations can result in less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of one or more of the multiple nucleic acid cleavage domains of the wild-type Cas protein. The mutations can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave a complementary strand of a target nucleic acid but reducing their ability to cleave a non-complementary strand of the target nucleic acid. The mutations can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave a non-complementary strand of the target nucleic acid but reducing their ability to cleave a complementary strand of the target nucleic acid. The mutations can result in one or more of the multiple nucleic acid cleavage domains lacking the ability to cleave both complementary and non-complementary strands of the target nucleic acid. The residues mutated in the nuclease domain can correspond to one or more catalytic residues of a nuclease. For example, residues in a wild-type exemplary S. pyogenes Cas9 polypeptide, such as AsplO, His840, Asn854, and Asn856, can be mutated to inactivate one or more of the nucleic acid cleavage domains (e.g., the nuclease domains). The residues mutated in the nuclease domain of the Cas protein can correspond to residues AsplO, His840, Asn854, and Asn856 in the wild-type S. pyogenes Cas9 polypeptide, as determined, for example, by sequence and / or structural alignment.

[0296] By way of non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or the corresponding mutations in any of the Cas proteins) can be mutated, such as, for example, D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. Mutations other than alanine substitutions may be suitable.

[0297] The D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce a Cas9 protein that substantially lacks DNA cleavage activity (e.g., a dead Cas9 protein). The H840A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity. The N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity. The N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity.

[0298] In some embodiments, the Cas protein is a Class 2 Cas protein. In some embodiments, the Cas protein is a Type II Cas protein. In some embodiments, the Cas protein is 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 derived from S. pyogenes (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is a Cas9 protein derived from S. aureus (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified version of a Cas9 protein derived from S. pyogenes or S. Aureus. In some embodiments, the Cas9 protein is derived from a Cas9 protein derived from S. pyogenes or S. Aureus. For example, a Cas9 protein of S. pyogenes or S. Aureus lacking cleavage activity.

[0299] Cas9 can generally refer to a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to wild-type or modified forms of the Cas9 protein, which can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0300] In some embodiments, the cargo comprises or encodes a "zinc finger nuclease" or "ZFN." ZFN refers to a fusion between a cleavage domain, such as the cleavage domain of FokI, and at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs) capable of binding to polynucleotides such as DNA and RNA. Heterodimerization of two individual ZFNs at a specific position in a polynucleotide with a specific orientation and spacing can result in cleavage of the polynucleotide. For example, a ZFN that binds to DNA can induce a double-strand break in the DNA. To enable the two cleavage domains to dimerize and cleave the DNA, the two individual ZFNs can bind to opposite strands of DNA with their C-termini separated by a specific distance. In some cases, a linker sequence between the zinc finger domain and the cleavage domain can require that the 5' ends of each binding site are separated by approximately 5 to 7 base pairs. In some cases, the cleavage domain is fused to the C-terminus of each zinc finger domain. Exemplary ZFNs are Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8): 805-7; U.S. Patent No. 6,534,261; U.S. Patent No. 6,607,882; U.S. Patent No. 6,746,838; No. 6,824,978; No. 6,866,997; No. 6,933,113; No. 6,979,539; No. 7,013,219; No. 7,030,215; No. 7 ,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and those described in U.S. Patent Application Publication Nos. 2003 / 0232410 and 2009 / 0203140.

[0301] In some embodiments, the protein encoded by the cargo protein or cargo nucleic acid molecule, including ZFN, can generate double-strand breaks in target polynucleotides such as DNA. The double-strand breaks in DNA can result in DNA break repair, which allows for the introduction of gene modification(s) (for example, nucleic acid editing). DNA break repair can occur through non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template containing homologous arms can be provided at the flanking site of target DNA. In some embodiments, ZFN is a zinc finger nickase, which induces site-specific single-strand DNA breaks or nicks, thus resulting in HDR. For example, a description of zinc finger nickase can be found in Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33. In some embodiments, the ZFN binds to a polynucleotide (by way of example, DNA and / or RNA), but the polynucleotide is incapable of being cleaved.

[0302] In some embodiments, the cleavage domain of a protein encoded by a cargo protein or cargo nucleic acid molecule, including ZFNs, comprises a modified form of a wild-type cleavage domain. The modified form of the cleavage domain can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the cleavage domain. For example, the modified form of the cleavage domain can have less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of the wild-type cleavage domain. The modified form of the cleavage domain cannot have substantial nucleic acid cleavage activity. In some embodiments, the cleavage domain is enzymatically inactive.

[0303] In some embodiments, the cargo protein or the protein encoded by the cargo nucleic acid molecule comprises a "TALEN" or "TAL effector nuclease." TALEN refers to an engineered transcription activator-like effector nuclease, which generally contains a central domain and a cleavage domain of DNA-binding tandem repeats. TALENs can be produced by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain. In some cases, the DNA-binding tandem repeats are 33-35 amino acids long and contain two hypervariable amino acid residues at positions 12 and 13 that can recognize at least one specific DNA base pair. A transcription activator-like effector (TALE) protein can be fused to a nuclease, such as wild-type or mutant FokI endonuclease or the catalytic domain of FokI. For example, several mutations to FokI have been made for use in TALENs to improve cleavage specificity or activity. Such TALENs can be engineered to bind to any desired DNA sequence. TALEN can be used to generate double-strand breaks in target DNA sequences, and then undergo NHEJ or HDR to produce genetic modifications (for example, nucleic acid sequence editing). In some cases, a single-strand donor DNA repair template is provided to promote HDR. Detailed descriptions of TALENs and their use for gene editing can be found, by way of example, in U.S. Patent Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55.

[0304] In some embodiments, TALENs are engineered to reduce nuclease activity. In some embodiments, the nuclease domain of TALENs comprises a modified form of a wild-type nuclease domain. The modified form of the nuclease domain can comprise amino acid changes (for example, deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain cannot have substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive.

[0305] In some embodiments, a Transcription Activator-Like Effector (TALE) protein is capable of modulating transcription and is fused to a nuclease-free domain. In some embodiments, a Transcription Activator-Like Effector (TALE) protein is designed to function as a transcription activator. In some embodiments, a Transcription Activator-Like Effector (TALE) protein is designed to function as a transcription repressor. For example, the DNA-binding domain of a Transcription Activator-Like Effector (TALE) protein can be fused (e.g., linked) to one or more transcription activation domains or one or more transcription repression domains. Non-limiting examples of transcription activation domains include the herpes simplex VP16 activation domain and tetrameric repeats of the VP16 activation domain, e.g., the VP64 activation domain. Other examples include VP16, VP32, VP64, VPR, p65, RTA, KRAB, or P65HSF1. Non-limiting examples of transcription repression domains include Kruppel-associated box domains.

[0306] In some embodiments, the cargo protein or the protein encoded by the cargo nucleic acid molecule comprises a meganuclease. Meganuclease generally refers to a rare-cutting endonuclease or homing endonuclease that can be highly specific. Meganucleases can recognize DNA target sites at least 12 base pairs in length, for example, 12 to 40 base pairs in length, 12 to 50 base pairs in length, or 12 to 60 base pairs in length. Meganucleases can be modular DNA-binding nucleases, such as any fusion protein, that include at least one catalytic domain of an endonuclease and at least one DNA-binding domain or protein that specifies a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single-stranded or double-stranded DNA. Meganucleases can be monomeric or dimeric. In some embodiments, meganucleases are naturally occurring (found in nature) or wild-type, while in other instances, meganucleases are non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, meganucleases of the present disclosure include I-CreI meganuclease, I-CeuI meganuclease, I-MsoI meganuclease, I-SceI meganuclease, variants thereof, derivatives thereof, and fragments thereof.Detailed descriptions of useful meganucleases and their applications in gene editing can be found in, for example, Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066, and U.S. Patent Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0307] In some embodiments, the nuclease domain of the meganuclease comprises a modified form of a wild-type nuclease domain. The modified form of the nuclease domain can include amino acid changes (for example, deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain can have less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain cannot have substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive. In some embodiments, the meganuclease can bind to DNA but cannot cleave DNA.

[0308] Targetable 3' overhang nucleases In some cases, the cargo delivered by the delivery vehicle of the present disclosure comprises a nuclease that generates a 3' overhang double-strand break in DNA, for example, a type IIS restriction enzyme or a functional domain of a type IIS restriction enzyme. As used herein, the term "type IIS restriction enzyme" refers to a restriction enzyme that recognizes an asymmetric DNA sequence and cuts outside of the recognition sequence. In one embodiment, the restriction enzyme is Acul.

[0309] In some cases, the cargo comprises a targetable nuclease fusion protein comprising a dimerization-dependent nuclease domain, for example, a type IIS restriction enzyme domain. Illustratively, the targetable nuclease fusion protein comprises a dimerization-dependent nuclease domain, wherein the domain generates a 3' overhang double-strand break in DNA; and a DNA binding domain (DBD). In some cases, the dimerization-dependent nuclease domain is a type IIS restriction enzyme nuclease domain, for example, an Acul nuclease domain.

[0310] In some cases, the DBD is a protein or protein domain that binds to its target nucleic acid in a sequence-dependent manner. In some cases, the DBD disclosed herein is either a zinc finger array or dCas9.

[0311] In some cases, the nuclease fusion protein is a zinc finger nuclease fusion protein. The zinc finger nuclease fusion protein described herein comprises a nuclease domain that generates a 3' overhang double-strand break in DNA upon dimerization (i.e., nuclease activity is "dimerization-dependent"); an optional amino acid linker; and a zinc finger domain that comprises one or more carboxy- or amino-terminal zinc finger(s). Monomeric forms of zinc finger nuclease fusion proteins that comprise one or more carboxy- or amino-terminal zinc finger(s) can join together to form a dimer either upon or before binding to a target site, thereby activating nuclease cleavage. The zinc finger nuclease fusion proteins described herein can be used to frequently generate insertion / deletion mutations (indels) through repair of nuclease-induced DNA breaks by non-homologous end joining.

[0312] Zinc finger nuclease fusion proteins can also be used to copy, integrate, or insert an exogenous nucleic acid sequence of interest into a target site in a genomic locus of a cell. In some embodiments, the methods provided herein include providing an exogenous nucleic acid "donor template" sequence and another nucleic acid sequence encoding a zinc finger nuclease fusion protein or the zinc finger nuclease fusion protein itself to the nucleus of a cell. In some cases, both the exogenous nucleic acid "donor template" sequence and the zinc finger nuclease fusion protein are delivered by a delivery vehicle provided herein. The exogenous nucleic acid donor template sequence includes terminal sequences homologous to sequences within the target site in the genomic locus. Zinc fingers can be designed to specifically recognize and bind to genomic target sites. Upon binding to the target site, the dimerized nuclease domain of the fusion protein(s) can generate a 3' overhanging double-stranded break within the target site and induce homologous recombination repair between the sequence surrounding the break and the exogenous nucleic acid sequence, thereby copying, incorporating, and / or inserting the exogenous nucleic acid sequence into the target site in the genomic locus of the cell.

[0313] A zinc finger nuclease fusion protein can include any nuclease domain that is capable of producing a 3' overhang double-strand break in DNA upon dimerization.

[0314] The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain, including, but not limited to, an Acul, Alol, Bpml, Bael, or Mmel nuclease domain. In some instances, the Acul nuclease domain can have the amino acid sequence:

[0315] Exemplary nucleotide and amino acid sequences encoding Acul are known in the art and can be located, for example, in GenBank Accession No. HQ327692.1.

[0316] In some embodiments, the type IIS restriction enzyme nuclease domain includes an isoschizomer of Acul, such as Eco57I. The nucleotide and amino acid sequences encoding Eco57I can be located, for example, in the UniProt database reference number P25239.

[0317] Exemplary nucleotide and amino acid sequences encoding Alol are known in the art and can be located, for example, in GenBank Accession No. AJ312389.1.

[0318] Exemplary nucleotide and amino acid sequences encoding Bpml are known in the art and can be located, for example, in GenBank Accession No. ADK30556.1. Exemplary nucleotide and amino acid sequences encoding Bael are known in the art and can be located, for example, in GenBank Accession No. ABS74060.1.

[0319] Exemplary nucleotide and amino acid sequences encoding Mmel are known in the art and can be located, for example, in GenBank Accession No. EU616582.1.

[0320] Any type IIS restriction enzyme nuclease domain with dimerization-dependent nuclease activity can be fused to a zinc finger domain and used to perform the methods described herein. In some embodiments, the nuclease domain is attached to the C-terminus of the zinc finger domain. In other embodiments, the nuclease domain is attached to the N-terminus of the zinc finger domain.

[0321] The dimerization-dependent nuclease domain and zinc finger domain of the zinc finger nuclease fusion protein can be joined together by an amino acid linker. The terms "linked," "joined," and "fused" are used interchangeably herein to refer to the means by which the two domains of the fusion protein are joined. The amino acid linker can comprise any sequence of at least one amino acid, and up to a maximum of 10 amino acids. In certain embodiments, the linker can comprise leucine, arginine, glycine, and serine (LRGS (SEQ ID NO: 50)); glycine, glycine, glycine, glycine, and serine (GGGGS (SEQ ID NO: 51)); or the non-standard amino acids threonine, glutamic acid, and asparagine (XTEN), as described by Shellenberger, et al. Nat Biotechnol. 2009 Dec;27(12):1186-90.

[0322] In some embodiments, the dimerization-dependent nuclease domain, zinc finger domain, TALE, and / or dCas9 domain can have an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of an exemplary dimerization-dependent nuclease domain, zinc finger domain, TALE, and / or dCas9 described herein.

[0323] Upon binding to the target site and forming a dimeric complex, the nuclease domain of the zinc finger nuclease fusion protein generates a 3' overhanging double-stranded break within the target site to induce homology-directed repair, thereby allowing the exogenous nucleic acid sequence, or a portion thereof, to be copied, incorporated, and / or integrated into the target site. If there is nucleotide sequence homology, the donor template oligonucleotide sequence (either single-stranded or double-stranded) can act as a template to repair the target DNA sequence that has experienced a double-stranded break, resulting in the transfer of genetic information from the donor to the target. Such transfer can involve mismatch correction of the heteroduplex DNA formed between the broken target and the donor, and / or synthesis-dependent strand annealing, in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Homologous recombination repair often results in an alteration of the sequence of the target nucleotide so that part or all of the sequence of the donor nucleotide sequence is copied and / or incorporated into the target nucleotide.

[0324] Zinc finger nuclease fusion proteins can create double-strand breaks in target sequences at predetermined sites, and exogenous nucleic acid sequences that act as donor templates and have homology with the nucleotide sequence of the break region can be copied, integrated, and / or introduced into genomic loci.The presence of double-strand breaks has been shown to significantly enhance the efficiency of these different repair outcomes.The donor sequence can be physically integrated, or the donor nucleotide can be used as a template for repairing the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence in the donor into the genomic locus.Therefore, the sequence in the genomic locus can be changed, and in certain embodiments, can be converted into the sequence present in the donor nucleotide.

[0325] Also described herein are dCas9 nuclease fusion proteins and methods for using them to enhance the frequency of homologous recombination repair at sites of nuclease-induced double-strand breaks. The dCas9 nuclease fusion protein comprises a catalytically inactive Cas9 carboxy- or amino-terminal domain linked to a dimerization-dependent nuclease domain that generates a 3' overhang double-strand break in DNA. The catalytically inactive Cas9 domain contains mutations (e.g., D10A and / or H841A) that result in the loss of native endonuclease activity (Qi et al., Cell (2013)). Instead, the endonuclease activity is provided by the linked dimerization-dependent nuclease domain to which it is fused. The monomeric forms of the dCas9 nuclease fusion protein join together to form a dimer, either before or upon binding to the dCas9 target site, thereby activating nuclease cleavage. Clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins comprise the CRISPR-Cas system. The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner (Gasiunas, G., et al., Proc Natl Acad Sci USA 109, E2579-E2586 (2012); Jinek, M., et al., Science 337, 816-821 (2012); Sternberg, SH, et al., Nature 507, 62 (2014); Deltcheva, E., et al., Nature 471, 602-607 (2011)), and has been widely used for programmable genome editing in various organisms and model systems (Cong, L., et al., Science 339, 819-823 (2013); Jiang, W., et al., Nat. Biotechnol 31, 233-239 (2013); Sander, JD & Joung, JK, Nature Biotechnol. 32, 347-355. (2014)).Cas9 is composed of two RNAs that are associated or covalently linked to create a guide RNA: CRISPR RNA (crRNA) and a transactivating RNA (tracrRNA). If the nucleotide sequence of a target genomic locus that requires a guide RNA is complementary to the guide RNA, Cas9 recognizes and cleaves the site. The ternary complex of Cas9 with crRNA and tracrRNA, or the binary complex of Cas9 with guide RNA, can bind to and cleave a dsDNA protospacer sequence that matches the crRNA spacer and is also adjacent to a short protospacer adjacent motif. Even if its native catalytic activity is inactivated, dCas9 can still associate with the crRNA / tracrRNA complex or guide RNA and then recognize and bind to the target site. The nucleotide and amino acid sequences encoding Cas9 are known in the art and can be located, for example, in GenBank accession number NC_002737.2.

[0326] The dCas9 nuclease fusion protein described herein can be used to induce homologous recombination repair events at target sites in a genomic locus of a cell. This method includes providing an exogenous nucleic acid sequence, a nucleic acid sequence encoding a dCas9 nuclease fusion protein, and one or more (for example, at least two) guide RNAs to the nucleus of a cell. The exogenous nucleic acid sequence includes terminal sequences homologous to sequences within the target site of the genomic locus. The guide RNAs are designed to guide two dCas9 nuclease fusions to predetermined target sites, where each dCas9 / gRNA complex binds to one of two "half-sites." The dCas9 domain recognizes and binds to those target sites that are complementary to the guide RNA and specifically adjacent PAM sequences. Upon binding to the target site, the linked nuclease domain of the fusion protein functions as a dimer to generate a 3' overhang double-strand break within the target site, thereby inducing homologous recombination repair between the sequence surrounding the break and the exogenous nucleic acid sequence, thereby copying, integrating, and / or inserting the exogenous nucleic acid sequence into the target site of the genomic locus of the cell. The nucleotide sequence and amino acid sequence encoding dCas9 are known in the art and can be located, for example, in GenBank Accession No. KR011748.1. dCas9 is also described in Zetsche et al., Nature Biotechnology 33, 139-142 (2015).

[0327] The dCas9 nuclease fusion protein can contain any nuclease domain capable of generating a 3' overhang double-strand break in DNA upon dimerization. The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain, including, but not limited to, Acul, Alol, Bpml, Bael, or Mmel nuclease domain. The dimerization-dependent nuclease domain and the dCas9 domain of the dCas9 nuclease fusion protein are joined together by any amino acid linker. The amino acid linker can contain any sequence of at least one amino acid, and up to a maximum of 10 amino acids. In certain embodiments, the amino acid linker can contain, for example, glycine, glycine, glycine, glycine, and serine (GGGGS (SEQ ID NO: 51)) or the non-standard amino acid threonine, glutamic acid, and asparagine (XTEN).

[0328] In any of the methods and compositions described herein, the exogenous nucleotide sequence acting as the donor can contain a sequence that is homologous but not identical to the genomic sequence at the target site, thereby stimulating homology-directed repair to copy, integrate, and / or insert the non-identical sequence into the target site. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the sequence at the region of interest exhibits approximately 80-99% sequence identity (or any integer therebetween) to the genomic sequence to be replaced. In other embodiments, the homology between the donor sequence and the genomic sequence is greater than 99%, for example, when only a single nucleotide differs, such as between the donor sequence and the genomic sequence of more than 100 consecutive base pairs. In certain cases, the non-homologous portion of the donor sequence can contain a sequence that is not present at the target site, such that a new sequence is introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50 to 1,000 base pairs (or any integer value therebetween), or any number of base pairs greater than 1,000, that are homologous or identical to the sequence of the target site.

[0329] In some embodiments, the entire donor template sequence or a portion of the donor template sequence is integrated into the target site. Any of the methods described herein can be used for the partial or complete inactivation of one or more genomic loci in cells by targeted integration of the donor sequence, which disrupts the expression of the gene(s) of interest. Any of the methods described herein can be used to replace the mutated sequence within the target site, thereby correcting the mutated gene or inducing the expression of a previously inactive gene. The nature of the exogenous nucleic acid sequence to be integrated depends on the therapeutic goal to be achieved and can range from inducing or inhibiting gene transcription, replacing the mutated sequence of a defective gene, or adding or deleting a sequence within a gene.

[0330] In other embodiments, a DBD (e.g., zinc finger or dCas9) nuclease fusion protein introduces variable-length insertion or deletion mutations that partially or completely overlap a nuclease target site in a cell's genomic locus via non-homologous end joining or microhomology-mediated end joining. In these embodiments, no exogenous donor sequence is provided. Rather, a nucleic acid sequence encoding a zinc finger nuclease fusion protein or an isolated zinc finger nuclease fusion protein is provided to the cell's nucleus, where the zinc finger nuclease fusion protein binds to the nuclease target site and generates a 3' overhanging double-stranded break within the nuclease target site, followed by repair of the break by non-homologous end joining or microhomology-mediated end joining. Both non-homologous end joining and microhomology-mediated end joining can result in insertions or deletions that disrupt or inhibit gene transcription at the nuclease target site.

[0331] Non-limiting examples of targetable 3' overhang nucleases (e.g., Type IIS restriction enzymes, e.g., DBD nuclease fusion proteins), nuclease-encoding sequences, compositions, methods of use, and systems include those described in WO2020160481, which is incorporated herein by reference in its entirety.

[0332] Base Editing In some cases, the cargo delivered by a delivery vehicle of the present disclosure comprises a nucleobase editor (also referred to as a "base editor") or one or more components of a nucleobase editing (also referred to as a "base editing") complex.

[0333] The term "base editor (BE)" or "nucleobase editor (NBE)," as used herein, can refer to an agent, including a polypeptide, that is capable of making modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, a base editor is capable of deaminating a base in a nucleic acid. In some embodiments, a base editor is capable of deaminating a base in a DNA molecule. In some embodiments, a base editor is capable of deaminating an adenosine (A) in DNA. In some embodiments, a base editor is capable of deaminating a cytosine (C) in DNA.

[0334] In some cases, the base editors disclosed herein comprise a deaminase or a functional domain thereof (a "deaminase domain") that catalyzes a deamination reaction.

[0335] The term "deaminase" or "deaminase domain" as used herein refers to a protein or enzyme that catalyzes deamination reaction.In some embodiments, the deaminase domain or deaminase domain is an adenosine deaminase, which catalyzes the deamination of adenosine and converts it into the nucleoside hypoxanthine.In some embodiments, the deaminase domain or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively.In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, which catalyzes the hydrolytic deamination of cytosine to uracil.In some embodiments, the deaminase or deaminase domain is a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from a non-naturally occurring organism. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from the organism.

[0336] As used herein, "adenosine deaminase" is an enzyme that catalyzes the deamination of adenosine, converting it to the nucleoside hypoxanthine. Under standard Watson-Crick hydrogen bond pairing, the adenosine base hydrogen bonds to the thymine base (or uracil in the case of RNA). When adenine is converted to hypoxanthine, hypoxanthine undergoes hydrogen bond pairing with cytosine. Thus, the conversion of "A" to hypoxanthine by adenosine deaminase results in the insertion of "C" instead of "T" during cellular repair and / or replication processes. Because cytosine "C" pairs with guanine "G," adenosine deaminase, in concert with DNA replication, causes the conversion of A·T pairings to C·G pairings in double-stranded DNA molecules.

[0337] In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable R / DNA binding protein (napR / DNAbp) fused to a deaminase (by way of example, a cytidine deaminase or an adenosine deaminase) domain. The term "nucleic acid programmable D / RNA binding protein (napR / DNAbp)" refers to any protein that may associate (by way of example, form a complex with) one or more nucleic acid molecules (i.e., which may be broadly referred to as "napR / DNAbp programming nucleic acid molecules," which, in the case of a Cas system, would include a guide RNA) that orients or otherwise programs the protein to localize to a specific target nucleotide sequence (by way of example, a genomic locus or an RNA molecule) that is complementary to one or more nucleic acid molecules (or portions or regions thereof) associated with the protein, thereby binding the protein to the nucleotide sequence at the specific target site. The term napR / DNAbp encompasses CRISPR Cas9 proteins, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), as well as Cas9 equivalents, homologs, orthologs, or paralogs, and may encompass Cas9 equivalents from any type of CRISPR system (e.g., Type II, Type V, Type VI), including Cpf1 (Type V CRISPR-Cas system), C2c1 (Type V CRISPR-Cas system), C2c2 (Type VI CRISPR-Cas system), and C2c3 (Type V CRISPR-Cas system). Additional Cas equivalents are described in Makarova et al., "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector," Science 2016;353(6299), the contents of which are incorporated herein by reference. However, the nucleic acid programmable R / DNA binding proteins (napR / DNAbp) that may be used in connection with the present invention are not limited to CRISPR-Cas systems.The present invention encompasses any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo), which may also be used for DNA-guided genome editing. The NgAgo-guided DNA system does not require a PAM sequence or guide RNA molecule, meaning that genome editing can be performed simply by expressing the generic NgAgo protein and introducing synthetic oligonucleotides into any genomic sequence. Gao F, Shen XZ, Jiang F, Wu Y, Han C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol 2016;34(7):768-73; which is incorporated herein by reference.

[0338] In some cases, napR / DNAbp is derived from a nuclease disclosed herein, such as Cas9 (by way of example, dCas9 and nCas9), CasX, CasY, Cas14, Cpfl, C2cl, C2c2, C2c3, an Argonaute protein, or a variant thereof. In some embodiments, the base editor comprises Cas9 (by way of example, dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or an Argonaute protein fused to a deaminase (by way of example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cas9 nickase (nCas9) fused to a deaminase (by way of example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a CasX protein fused to a deaminase (for example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase (for example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a CasY protein fused to a deaminase (for example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cas14 protein fused to a deaminase (for example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cpfl protein fused to a deaminase (for example, cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a C2c1 protein fused to a deaminase (by way of example, a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a C2c2 protein fused to a deaminase (by way of example, a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a C2c3 protein fused to a deaminase (by way of example, a cytidine deaminase or an adenosine deaminase).In some embodiments, the base editor comprises an Argonaute protein fused to a deaminase (by way of example, a cytidine deaminase or an adenosine deaminase).

[0339] In some embodiments, the adenosine deaminase provided herein is capable of deaminating adenosine. In some embodiments, the adenosine deaminase provided herein is capable of deaminating adenosine in deoxyadenosine residues in DNA. The adenosine deaminase may be derived from any suitable organism (for example, E. coli). In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (for example, the ecTadA mutation). Those skilled in the art will be able to identify any homologous proteins and corresponding residues in their encoding nucleic acids by methods well known in the art (for example, by aligning sequences and determining homologous residues). Thus, those skilled in the art will be able to generate mutations in any naturally occurring adenosine deaminase (for example, having homology with ecTadA) that correspond to any of the mutations described herein, for example, any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

[0340] In some cases, the deaminase domain of a base editor disclosed herein is derived from a cytidine deaminase. In some cases, the cytidine deaminase domain is derived from an apolipoprotein B mRNA editing complex (APOBEC) family deaminase, such as APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, or APOBEC3H deaminase.

[0341] In some embodiments, the base editor further comprises an inhibitor of base excision repair, e.g., fused to a uracil glycosylase inhibitor (UGI) domain or as part of a fusion protein.

[0342] In some cases, a base editor disclosed herein is a fusion protein that includes a structure such as: NH2-[deaminase domain]-[napR / DNAbp]-[UGI domain]-COOH; NH2-[deaminase domain]-[napR / DNAbp]-[UGI]-[UGI]-COOH; NH2-[deaminase domain]-[napR / DNAbp]-[UGI]-COOH; NH2-[UGI]-[deaminase domain]-[napR / DNAbp]-COOH; NH2-[deaminase domain]-[UGI]-[napR / DNAbp]-COOH; NH2-[napR / DNAbp]-[UGI]-[deaminase domain]-COOH; or NH2-[napR / DNAbp]-[deaminase domain]-[UGI]-COOH; where each instance of "-" includes an optional linker.

[0343] In some cases, the base editor is fused to a uracil-binding protein (UBP) or further comprises a uracil-binding protein as part of a fusion protein. The term "uracil-binding protein" or "UBP," as used herein, refers to a protein capable of binding to uracil. In some embodiments, the uracil-binding protein is a uracil-modifying enzyme. In some embodiments, the uracil-binding protein is a uracil-base-excising enzyme. In some embodiments, the uracil-binding protein is uracil-DNA glycosylase (UDG). In some embodiments, the uracil-binding protein binds to uracil with an affinity that is at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the affinity with which wild-type UDG (e.g., human UDG) binds to uracil. The term "base excision enzyme" or "BEE," as used herein, refers to a protein capable of removing a base (e.g., A, T, C, G, or U) from a nucleic acid molecule (e.g., DNA or RNA). In some embodiments, a BEE is capable of removing a cytosine from DNA. In some embodiments, a BEE is capable of removing a thymine from DNA. Exemplary BEEs include, but are not limited to, UDG Tyr147Ala and UDG Asn204Asp, as described in Sang et al., "A Unique Uracil-DNA binding protein of the uracil DNA glycosylase superfamily," Nucleic Acids Research, Vol. 43, No. 17 2015 (the entire contents of which are incorporated herein by reference).

[0344] In some embodiments, the UBP is a uracil-modifying enzyme. In some embodiments, the UBP is a uracil base-excising enzyme. In some embodiments, the UBP is a uracil DNA glycosylase. In some embodiments, the UBP is any of the uracil-binding proteins provided herein. For example, the UBP may be UDG, UdgX, UdgX*, UdgX_On, or SMUG1. In some embodiments, the UBP comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a uracil-binding protein, a uracil base-excising enzyme, or a uracil DNA glycosylase (UDG) enzyme.

[0345] In some cases, the base editor is fused to a nucleic acid polymerase domain (NAP) or includes a nucleic acid polymerase domain as part of a fusion protein. Illustratively, the nucleic acid polymerase domain is a eukaryotic nucleic acid polymerase domain. In some cases, the nucleic acid polymerase domain is a DNA polymerase domain. In some cases, the nucleic acid polymerase domain has translesion polymerase activity. In some cases, the nucleic acid polymerase domain is a translesion DNA polymerase. In some cases, the nucleic acid polymerase domain is from Rev7, Rev1 complex, polymerase iota, polymerase kappa, and polymerase eta. In some cases, the nucleic acid polymerase domain is selected from the group of eukaryotic polymerases consisting of alpha, beta, gamma, delta, epsilon, gamma, eta, iota, kappa, lambda, mu, and neu.

[0346] In some cases, a base editor disclosed herein is a fusion protein that includes a structure such as: NH2-[deaminase domain]-[napR / DNAbp domain]-[UBP]-[NAP]-COOH; NH2-[deaminase domain]-[napR / DNAbp]-[NAP]-[UBP]-COOH; NH2-[deaminase domain]-[NAP]-[napR / DNAbp]-[UBP]-COOH; or NH2-[NAP]-[deaminase domain]-[napR / DNAbp]-[UBP]-COOH; where each instance of "-" includes an optional linker.

[0347] In some cases, a base editor disclosed herein is complexed with a napR / DNAbp programming nucleic acid molecule. In some cases, a base editing system disclosed herein comprises a base editor and a napR / DNAbp programming nucleic acid molecule, for example, a base editor complexed with a napR / DNAbp programming nucleic acid molecule. In some cases, a delivery vehicle of the present disclosure delivers a base editing system comprising both a base editor and a napR / DNAbp programming nucleic acid molecule, for example, a base editor complexed with a napR / DNAbp programming nucleic acid molecule. In some cases, the base editor is delivered into a cell separately from the napR / DNAbp programming nucleic acid molecule, via a delivery vehicle disclosed herein or with other delivery methods.

[0348] The term "napR / DNAbp programming nucleic acid molecule" or, equivalently, "guide sequence" refers to one or more nucleic acid molecules that associate with, orient, or otherwise program a napR / DNAbp protein to localize to a specific target nucleotide sequence (e.g., a genomic locus) that is complementary to one or more nucleic acid molecules (or portions or regions thereof) associated with the protein, thereby binding the napR / DNAbp protein to the nucleotide sequence at the specific target site. A non-limiting example is the guide RNA for the Cas protein of a CRISPR-Cas genome editing system.

[0349] Exemplary configurations, sequences, and mutations thereof for the deaminase domain, napR / DNAbp domain, UGI domain, and entire base editor proteins, as well as exemplary configurations of base editing systems (including, by way of example, both base editors and napR / DNAbp programming nucleic acid molecules) that can be delivered by the delivery vehicles disclosed herein are described in U.S. Patent Application Publication Nos. US20170121693, US20180073012, US20180312828, US20180312828, US20180312829, US20180312829, US20180312829, US20180312829, US20180312829, US201803121693 ...1693, US20180312828, US20180312829, US20180312829, US20180312829, US20180312829, US20180312829, US20180312829, US201803121693, US201803121693, US20180073012, US20180312828, US201803 Nos. US20210230577, US20210198330, US20210277379, US2020399626, US2021371858, US2021380955, US2021277379, US2021301274; International Publication Nos. WO20051562, WO21041885, WO21050512, and WO21113494, each of which is incorporated herein by reference in its entirety.Exemplary configurations, sequences, and mutations thereof for deaminase domains, napR / DNAbp domains, UGI domains, and entire base editor proteins that can be delivered by the delivery vehicles disclosed herein are also described in Komor AC et al. Nature. 2016 May 19;533(7603):420-4; Kim YB et al. Nat Biotechnol. 2017 Apr;35(4):371-376; Rees HA et al. Nat Commun. 2017 Jun 6;8:15790; Newby GA et al. Mol Ther. 2021 Nov 3;29(11):3107-3124; Huang TP et al. Nat Protoc. 2021 Feb;16(2):1089-1128; Lapinite A et al. Science. 2020 Jul 31;369(6503):566-571; Anzalone AV et al. Nat Biotechnol. 2020 Jul;38(7):824-844; Rees HA et al. Nat Rev Genet. 2018 Dec;19(12):770-788; Koblan LW et al. Nat Biotechnol. 2018 Oct;36(9):843-846; and Gaudelli NM et al. Nature. 2017 Nov 23;551(7681):464-471, each of which is incorporated herein by reference in its entirety.

[0350] Prime Edit In some cases, the cargo delivered by the delivery vehicle of the present disclosure comprises one or more components of a prime editing system.

[0351] Prime editing is a "search-and-replace" genome editing technique in which the genome of a living organism may be modified. In some cases, the prime editing system delivered by the delivery vehicle of the present disclosure uses a fusion protein including a nucleic acid-programmable RNA or DNA-binding protein (napR / DNAbp) and a nucleic acid polymerase (e.g., reverse transcriptase or RNA-dependent RNA polymerase), and a napR / DNAbp programming nucleic acid molecule. In some cases, the fusion protein includes a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase. In some cases, the napR / DNAbp programming nucleic acid molecule includes a prime editing guide RNA (pegRNA) that can identify a target site and provide new genetic information to replace the target DNA nucleotide. 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.

[0352] In some cases, a prime editing system, for example, a napR / DNAbp programming nucleic acid molecule for a prime editing guide RNA (pegRNA), can (i) identify the target nucleotide sequence to be edited and (ii) encode new genetic information to replace the target sequence. In some cases, the pegRNA includes an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a template sequence for a nucleic acid polymerase (e.g., reverse transcriptase or RNA polymerase). In some cases, during genome editing, the primer binding site allows the 3' end of a nicked DNA strand to hybridize to the pegRNA, while the reverse transcriptase template serves as a template for synthesis of the edited genetic information.

[0353] One or more components of the prime editing system that can be delivered by the delivery vehicle of the present disclosure are described in International Publication Nos. WO2020191242, WO2020191234, WO2020086908, WO2021072328, WO2021226558, and WO2020191248, as well as 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. 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 by reference in its entirety.

[0354] Epigenetic editing In some cases, the cargo delivered by a delivery vehicle of the present disclosure comprises one or more components of an epigenetic editor or an epigenetic editing complex (which, by way of example, comprises an epigenetic editor and a nucleic acid molecule that guides the epigenetic editor to bind to and / or modify one or more specific target sequences).

[0355] In some cases, the epigenetic editor or epigenetic editing complex disclosed herein exhibits a methyltransferase activity, a demethylase activity, a dismutase activity, an alkylating activity, a depurinating activity, an oxidizing activity, a pyrimidine dimer forming activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity or a glycosylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phospholipase ... In some cases, the epigenetic editor or epigenetic editing complex disclosed herein has an epigenetic activity such as a chromosome-modifying enzyme, or a functional domain with functional activity equivalent to a chromosome-modifying enzyme, such as a methylase, demethylase, acetylase, deacetylase, deaminase, phosphorylase, dephosphorylase, histone-modifying enzyme, or nucleotide-modifying enzyme, such as a chromosome-modifying enzyme. In some cases, the epigenetic editors or epigenetic editing complexes disclosed herein comprise a histone-modifying enzyme or a functional domain with equivalent functional activity to a histone-modifying enzyme. In some cases, the epigenetic editors or epigenetic editing complexes disclosed herein comprise a nucleotide-modifying enzyme or a functional domain with equivalent functional activity to a nucleotide-modifying enzyme.

[0356] In some cases, the epigenetic editor or epigenetic editing system provides the effect of modulating the expression of the target gene without changing the DNA sequence of the target gene.For example, in some embodiments, the epigenetic editor or epigenetic editing system causes the suppression or silencing of the expression of the target gene.In some embodiments, the epigenetic editor or epigenetic editing system causes the activation or increased expression of the target gene.

[0357] In one aspect, the epigenetic editor or epigenetic editing system is not sequence-specific, and for example, the epigenetic modification achieved by the epigenetic editor or epigenetic editing system is not specific to one or more target sequences. In another aspect, the epigenetic editor or epigenetic editing system described herein is sequence-specific or allele-specific. For example, the epigenetic editor may specifically target a DNA sequence recognized by the DNA-binding domain of the epigenetic editor. In some embodiments, the target DNA sequence is specific to one copy of the target gene. In some embodiments, the target gene sequence is specific to one allele of the target gene. Thus, the epigenetic modification and the modulation of its expression may be specific to one copy or one allele of the target gene.

[0358] In some embodiments, the epigenetic editor or epigenetic editing system comprises a histone methyltransferase domain. In some embodiments, the histone methyltransferase domain is a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a / EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof.

[0359] In some embodiments, the epigenetic editor or epigenetic editing system comprises a DNA methyltransferase domain or a histone methyltransferase domain. The DNA methyltransferase domain may mediate methylation at any DNA nucleotide, for example, an A, T, G, or C nucleotide. In some embodiments, the methylated nucleotide is N6-methyladenosine (m6A). In some embodiments, the methylated nucleotide is 5-methylcytosine (5mC). In some embodiments, the methylation is at a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C.

[0360] In some embodiments, the epigenetic editor or epigenetic editing system comprises a DNA methyltransferase DNMT domain that catalyzes the transfer of a methyl group to cytosine, thereby repressing expression of a target gene through the recruitment of an inhibitory regulatory protein. In some embodiments, the epigenetic editor or epigenetic editing system comprises a DNA methyltransferase (DNMT) family protein domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises a DNMT1 domain, a TRDMT1 domain, a DNMT3 domain, a DNMT3A domain, a DNMT3B domain, a DNMT3C domain, a DNMT3L domain, a TRDMT1 (DNMT2) domain, a M.MpeI domain, a M.SssI domain, a M.HpaII domain, a M.AluI domain, a M.HaeIII domain, a M.HhaI domain, a M.MspI domain, a Masc1 domain, a MET1 domain, a Masc2 domain, a Dim-2 domain, a dDnmt2 domain, a Pmt1 domain, a DRM1 domain, a DRM2 domain, a CMT1 domain, a CMT2 domain, a CMT3 domain, a Rid domain, an hsdM gene domain, an hsdS gene domain, a M.TaqI domain, a M.EcoDam domain, a M.CcrMI domain, a CamA domain, or any combination thereof (by way of example only, a fusion protein comprising any combination thereof).

[0361] In some embodiments, the epigenetic editor or epigenetic editing system recruits one or more protein domains that suppress the expression of a target gene. In some embodiments, the epigenetic editor or epigenetic editing system interacts with a scaffold protein domain that recruits one or more protein domains that suppress the expression of a target gene. For example, the epigenetic editor or epigenetic editing system may recruit or interact with a scaffold protein domain that recruits a PRMT protein, an HDAC protein, a SETDB1 protein, or a NuRD protein domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises a Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, an FKHR (forkhead in rhabdomosarcoma gene) repressor domain, an aEGR-1 (early growth response gene product-1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3-interacting domain (SID), a WRPW motif of a hairy-related basic helix-loop-helix (bHLH) repressor protein; an HP1 alpha chromoshadow repression domain, or any combination thereof. In some embodiments, the epigenetic editor or epigenetic editing system comprises a KRAB domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises a tripartite motif containing 28 (TRIM28, TIF1-beta, or KAP1) protein.

[0362] In some embodiments, the epigenetic editor or epigenetic editing system comprises a protein domain that represses expression of a target gene. For example, the epigenetic editor or epigenetic editing system may comprise a functional domain derived from a zinc finger repressor protein. In some embodiments, the epigenetic editor or epigenetic editing system comprises a functional repression domain derived from a KOX1 / ZNF10 domain, a KOX8 / ZNF708 domain, a ZNF43 domain, a ZNF184 domain, a ZNF91 KRAB domain, a HPF4 domain, a HTF10 domain, or a HTF34 domain, or any combination thereof. In some embodiments, the epigenetic editor or epigenetic editing system is a ZIM3 protein domain, a ZNF436 domain, a ZNF257 domain, a ZNF675 domain, a ZNF490 domain, a ZNF320 domain, a ZNF331 domain, a ZNF816 domain, a ZNF680 domain, a ZNF41 domain, a ZNF189 domain, a ZNF528 domain, a ZNF543 domain, a ZNF554 domain, a ZNF140 domain, a ZNF610 domain, a ZNF264 domain, a ZNF350 domain, a ZNF8 domain, a ZNF582 domain, a ZNF30 domain, a ZNF324 domain, a ZNF98 domain, a ZNF669 domain, a ZNF677 domain, a ZNF596 domain, a ZNF214 domain, a ZNF37A ...41 domain, a ZNF189 domain, a ZNF528 domain, a ZNF543 domain, a ZNF554 domain, a ZNF140 domain, a ZNF610 domain, a ZNF264 domain, a ZNF350 domain, a ZNF8 domain, a ZNF582 domain, a ZNF30 domain, a ZNF324 domain, a ZNF98 domain, a ZNF669 domain, a ZNF677 domain, In addition, the invention also includes a functional inhibitory domain derived from a ZNF34 domain, a ZNF250 domain, a ZNF547 domain, a ZNF273 domain, a ZNF354A domain, a ZFP82 domain, a ZNF224 domain, a ZNF33A domain, a ZNF45 domain, a ZNF175 domain, a ZNF595 domain, a ZNF184 domain, a ZNF419 domain, a ZFP28-1 domain, a ZFP28-2 domain, a ZNF18 domain, a ZNF213 domain, a ZNF394 domain, a ZFP1 domain, a ZFP14 domain, a ZNF416 domain, a ZNF557 domain, a ZNF566 domain, a ZNF729 domain, a ZIM2 domain, a ZNF254 domain, a ZNF764 domain, a ZNF785 domain, or any combination thereof.In some embodiments, the domain is a ZIM3 domain, a ZNF554 domain, a ZNF264 domain, a ZNF324 domain, a ZNF354A domain, a ZNF189 domain, a ZNF543 domain, a ZFP82 domain, a ZNF669 domain, or a ZNF582 domain, or any combination thereof. In some embodiments, the domain is a ZIM3 domain, a ZNF554 domain, a ZNF264 domain, a ZNF324 domain, or a ZNF354A domain, or any combination thereof.

[0363] Exemplary functional domain sequences for epigenetic editors or epigenetic editing systems that may reduce or silence target gene expression are provided, and are described in PCT / US2021 / 030643 and Tycko et al. (Tycko J, DelRosso N, Hess GT, Aradhana, Banerjee A, Mukund A, Van MV, Ego BK, Yao D, Spees K, Suzuki P, Marinov GK, Kundaje A, Bassik MC, Bintu L. High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell. 2020 Dec 23;183(7):2020-2035.e16.doi:10.1016 / j.cell.2020.11.024. Epub 2020 Dec 15. PMID:33326746; PMCID:PMC8178797.), each of which is incorporated herein by reference in its entirety.

[0364] In some embodiments, the epigenetic editor or epigenetic editing system is selected from the group consisting of ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37A, ZNF34, ZNF250, ZNF547, ZNF273, ZNF354A, ZFP82, ZNF224, ZNF33A, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP2 8-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF764, ZNF785, ZNF10 (KOX1), CBX5 (chromoshadow domain), RYBP (YAF2_RYBP component of PRC1), YAF2 (YAF2_RYBP component of PRC1), MGA (component of PRC1.6), CBX1 (chromoshadow), SCMH1 (SAM_1 / SPM), MPP8 (chromodomain), SUMO3 (Rad60-SLD), HERC2 (Cyt-b5), BIN1 (SH3_9), PCGF2 (RING finger protein domain), TOX (HMG box), FOXA1 (HNF3A The protein comprises a functional repression domain derived from: FOXA2 (HNF3B C-terminal domain), IRF2BP1 (IRF-2BP1_2 N-terminal domain), IRF2BP2 (IRF-2BP1_2 N-terminal domain), IRF2BPL IRF-2BP1_2 N-terminal domain, HOXA13 (homeodomain), HOXB13 (homeodomain), HOXC13 (homeodomain), HOXA11 (homeodomain), HOXC11 (homeodomain), HOXC10 (homeodomain), HOXA10 (homeodomain), HOXB9 (homeodomain), HOXA9 (homeodomain), or any combination thereof.

[0365] In some embodiments, the epigenetic editor or epigenetic editing system comprises a histone deacetylase protein domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises an HDAC family protein domain, such as an HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In some embodiments, the epigenetic editor or epigenetic editing system removes acetyl groups from histones. In some embodiments, the epigenetic editor or epigenetic editing system comprises a nucleosome remodeling domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

[0366] In some embodiments, the epigenetic editor or epigenetic editing system comprises a tripartite motif containing 28 (TRIM28, TIF1-beta, or KAP1) protein. In some embodiments, the epigenetic editor or epigenetic editing system comprises one or more KAP1 proteins. The KAP1 protein in the epigenetic editor may form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulating gene expression in a cellular environment. For example, KAP1 may be recruited by the KRAB domain of a transcriptional repressor. In some embodiments, KAP1 interacts with or recruits histone deacetylase proteins, histone-lysine methyltransferase proteins (e.g., deposition of a methyl group on lysine 9 [K9] of histone H3 tail [H3K9]), chromatin remodeling proteins, and / or heterochromatin proteins. In some embodiments, the KAP1 protein interacts with or recruits one or more protein complexes that reduce or silence gene expression. In some embodiments, the KAP1 protein interacts with or recruits heterochromatin protein 1 (HP1) protein (for example, via the chromoshadow domain of the HP1 protein), SETDB1 protein, HDAC protein, and / or NuRD protein complex components. In some embodiments, the KAP1 protein recruits the CHD3 subunit of the nucleosome remodeling and deacetylation (NuRD) complex, thereby reducing or silencing the expression of target genes. In some embodiments, the KAP1 protein recruits SETDB1 protein (for example, to the promoter region of target genes), thereby reducing or silencing the expression of target genes, for example, through H3K9 methylation associated with the promoter region of target genes. In some embodiments, the recruitment of SETDB1 protein results in heterochromatinization of the chromosomal region carrying the target gene, thereby reducing or silencing the expression of target genes.In some embodiments, KAP1 protein interacts with or recruits HP1 protein, thereby reducing or silencing the expression of target genes through the reduction of the acetylation of H3K9 or H3K14 on the histone tails associated with target genes.The recruitment of SETDB1 induces heterochromatinization.In some embodiments, KAP1 protein interacts with or recruits ZFP90 protein (for example, ZFP90 isoform 2) and / or FOXP3 protein.

[0367] In some embodiments, the epigenetic editor or epigenetic editing system comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, the epigenetic editor or epigenetic editing system may comprise a methyl-CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides in a target gene. In some embodiments, the MECP2 protein interacts with methylated DNA nucleotides in a CpG island of the target gene. In some embodiments, the MECP2 protein interacts with methylated DNA nucleotides that are not in a CpG island of the target gene. In some embodiments, the MECP2 protein in the epigenetic editor creates a condensed chromatin structure, thereby reducing or silencing the expression of the target gene. In some embodiments, the MECP2 protein in the epigenetic editor interacts with histone deacetylases (for example, HDACs), thereby suppressing or silencing the expression of the target gene. In some embodiments, the MECP2 protein in the epigenetic editor blocks access of a transcription factor or transcription activator to the target gene, thereby repressing or silencing expression of the target gene.

[0368] In some embodiments, the epigenetic editor or epigenetic editing system comprises a chromoshadow domain, a ubiquitin-2-like Rad60 SUMO-like (Rad60-SLD / SUMO) domain, a chromatin organization modifier domain (chromo) domain, a Yaf2 / RYBP C-terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (zf-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a high mobility group box domain (HMG-box), a sterile alpha motif domain (SAM_1), a basic leucine zipper domain (bZIP_1), a Myb_DNA binding domain, a homeodomain, a MYM-type zinc finger with FCS sequence domain (zf-FCS), an interferon regulatory factor 2 binding protein zinc finger domain (IRF-2B P1_2), SSX repression region (SSXRD), B-box type zinc finger domain (zf-B_box), sterile alpha motif domain (SAM_2), CXXC zinc finger domain (zf-CXXC), regulator of chromosome condensation 1 domain (RCC1), SRC homology 3 domain (SH3_9), sterile alpha motif / Pointed domain (SAM_PNT), vestigial / Tondu family domain (Vg_Tdu), LIM domain, RNA recognition motif domain (RRM_1), basic leucine zipper domain (bZIP_2), paired amphipathic helix domain (PAH), proteasome ATPase OB C-terminal domain (Prot_ATP_ID_OB), nerve homology 2 domain (NHR2), helix-hairpin-helix motif domain (HHH_3), hinge domain 2 of cleavage stimulatory factor subunit (CSTF2_hinge), PPAR gamma N-terminal region domain (PPAR gamma_N), CDC48 N-terminal domain (CDC48_2), WD40 repeat domain (WD40), Fip1 motif domain (Fip1), PDZ domain (PDZ_6), von Willebrand factor type C domain (VWC), NAB conversion region 1 domain (NCD1), S1RNA binding domain (S1), HNF3 C-terminal domain (HNF_C), Tudor domain (Tudor_2), histone-like transcription factor (CBF / NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), zinc finger protein domain (DUF3669), EGF-like domain (cEGF), GATA zinc finger domain (GATA), TEA / ATTS domain (TEA), phorbol ester / diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), transactivation domain of FOXO protein family (FOXO-TAD), homeoboc The epigenetic editor or epigenetic editing system may comprise a protein domain comprising a YAF2_RYBP domain, a homeobox domain, or any combination thereof. In some embodiments, the homeobox domain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In some embodiments, the epigenetic editor or epigenetic editing system comprises a protein domain selected from the group consisting of a SUMO3 domain, a chromodomain from M-phase phosphoprotein 8 (MPP8), a chromoshadow domain from chromobox 1 (CBX1), and a SAM_1 / SPM domain from Scm polycomb group protein homolog 1 (SCMH1).In some embodiments, the epigenetic editor or epigenetic editing system comprises a C-terminal domain (HNF_C). In some embodiments, the HNF_C domain is derived from FOXA1 or FOXA2. In some embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif. In some embodiments, the epigenetic editor or epigenetic editing system comprises an interferon regulatory factor 2 binding protein zinc finger domain (IRF-2BP1_2). In some embodiments, the epigenetic editor or epigenetic editing system comprises a Cyt-b5 domain from the DNA repair factor HERC2 E3 ligase. In some embodiments, the epigenetic editor or epigenetic editing system comprises a variant SH3 domain (SH3_9) from bridging integrator 1 (BIN1). In some embodiments, the epigenetic editor or epigenetic editing system comprises an HMG-box domain from the transcription factor TOX, or a zf-C3HC4_2 RING finger domain from the Polycomb component PCGF2. In some em...

Claims

1. A lipid-containing particle comprising a lipid membrane encapsulating a protein core, wherein the protein core comprises a fusion protein, wherein the fusion protein comprises (a) a plasma membrane-localized protein, (b) a therapeutic cargo, (c) a cleavable linker, and (d) a nuclear export sequence (NES), wherein the NES is positioned between the plasma membrane-localized protein and the therapeutic cargo, and the cleavable linker is positioned between the therapeutic cargo and the NES.

2. 10. The lipid-containing particle of claim 1, wherein the protein core further comprises proteins including a group-specific antigen (gag) and a protease (pro).

3. The lipid-containing particle of claim 2 , wherein the protein comprises MMLV gag pro polyprotein or FMLV gag pro polyprotein.

4. 4. The lipid-containing particle of any one of claims 1 to 3, wherein the plasma membrane-localized protein comprises a retroviral gag protein, optionally wherein the retroviral gag protein comprises a gag nucleocapsid polyprotein, and / or wherein the plasma membrane-localized protein comprises an MMLV gag nucleocapsid protein or an FMLV gag nucleocapsid protein.

5. The lipid-containing particle of any one of claims 1 to 3, wherein the plasma membrane-localized protein comprises a human endogenous retrovirus (HERV) structural protein, a humanized viral structural protein, a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane recruiting protein.

6. 4. The lipid-containing particle of any one of claims 1 to 3, wherein the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease, a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, a small molecule compound, or any combination thereof.

7. 4. The lipid-containing particle of claim 1, wherein the protein core further comprises a cleavage product comprising a sequence of a plasma membrane-localized protein and an NES, and lacks a therapeutic cargo.

8. 4. The lipid-containing particle of claim 1, wherein the cleavable linker comprises a protease cleavage site, optionally wherein the protease cleavage site is a Moloney Murine Leukemia Virus (MMLV) protease cleavage site or a Friend Murine Leukemia Virus (FMLV) protease cleavage site.

9. The lipid-containing particle according to any one of claims 1 to 3, wherein the fusion protein comprises at least three NESs.

10. 4. The lipid-containing particle of any one of claims 1 to 3, wherein the fusion protein further comprises at least one nuclear localization sequence (NLS), optionally wherein the at least one NLS is located near the N-terminus and / or C-terminus of the therapeutic cargo.

11. The fusion protein contains, from the N-terminus to the C-terminus, [plasma membrane-localized protein]-[n ＊ NES]-[cleavable linker]-[m1 ＊ NLS]-[Therapeutic Cargo]-[m2 ＊ NLS], where n, m1, and m2 are integers ranging from 0 to 10 and represent the number of repeats of each element of the fusion protein to which they refer.

12. 11. The lipid-containing particle of claim 10, wherein the fusion protein comprises, from N-terminus to C-terminus, [plasma membrane-localized protein]-[3X NES]-[cleavable linker]-[NLS]-[therapeutic cargo]-[NLS].

13. The lipid-containing particle of any one of claims 1 to 3, wherein the fusion protein comprises, from N-terminus to C-terminus, a plasma membrane-localized protein, an NES, a cleavable linker and a therapeutic cargo.

14. 4. The lipid-containing particle of any one of claims 1 to 3, wherein the lipid-containing particle further comprises a viral envelope glycoprotein or a human endogenous retrovirus (HERV) envelope protein, optionally wherein the viral envelope glycoprotein is a retroviral envelope glycoprotein, and optionally wherein the retroviral envelope glycoprotein is a baboon retrovirus envelope glycoprotein.

15. The lipid-containing particle of any one of claims 1 to 3, further comprising a targeting moiety, optionally wherein the targeting moiety is an antibody or an antigen-binding fragment thereof.

16. 4. The lipid-containing particle of any one of claims 1 to 3, wherein the lipid-containing particle further comprises a therapeutic cargo cleaved from the fusion protein, and optionally wherein the amount of therapeutic cargo cleaved from the fusion protein is higher than the amount of fusion protein in the lipid-containing particle, and / or the ratio of the amount of therapeutic cargo cleaved from the fusion protein to the amount of fusion protein is at least 1.5.

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