Engineered extracellular vesicles and their uses

Abstract

Compositions and methods for making extracellular vesicles are provided herein.In some embodiments, the extracellular vesicles are used in methods for modifying cells and treating diseases of subjects.Provided herein are plasmid systems and extracellular vesicles for delivering nucleic acid sequences (e.g., mRNA sequences encoding heterologous polypeptides) and heterologous polypeptides to cells.Also provided are methods for modifying cells using the plasmid systems and extracellular vesicles described herein.

Description

[Technical field]

[0001] Prior Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 148,872, filed February 12, 2022, which is incorporated by reference herein in its entirety. Field

[0002] This disclosure describes compositions and methods of using same for eukaryotic gene editing. Reference to sequence listing submitted as a text file via EFS-WEB

[0003] An official copy of the sequence listing has been submitted electronically via EFS-Web as an ASCII format sequence listing with the file 1293190_seqlist.txt having a size of 121 KB, created on February 7, 2022, and filed concurrently with the specification. The sequence listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] background It is important that CRISPR-based genome editing effectors reduce off-target effects and immune responses. Recently, extracellular vesicles (EVs) have been investigated for Cas9 ribonucleoprotein (RNP) delivery. However, the efficiency of these EVs as RNP delivery vehicles has been limited. Therefore, compositions and methods for efficiently loading functional RNPs into EVs are needed. Summary of the Invention [Means for solving the problem]

[0005] Abstract Provided herein are plasmid systems and extracellular vesicles for delivering nucleic acid sequences (e.g., mRNA sequences encoding heterologous polypeptides) and heterologous polypeptides to cells. Also provided are methods for modifying cells using the plasmid systems and extracellular vesicles described herein.

[0006] Provided herein is a plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising (i) a nucleic acid sequence encoding a CRISPR-associated endonuclease, and (ii) a guide RNA (gRNA) coding sequence comprising at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, the second mammalian expression plasmid comprising an aptamer binding protein (ABP) binding to at least one aptamer coding sequence of the first mammalian expression plasmid. In some embodiments, the plasmid system further comprises an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

[0007] In some embodiments, the CRISPR-associated endonuclease is Cas9 protein, Cpf1 protein or their derivatives.In some embodiments, the CRISPR-associated endonuclease is catalytically impaired CRISPR-associated endonuclease.In some embodiments, the catalytically impaired CRISPR-associated endonuclease coding sequence codes for Cas9 D10A protein.

[0008] In some embodiments, the nucleic acid sequence of the first mammalian expression plasmid encodes a nucleic acid sequence encoding an adenosine base pair editor (ABE), where the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease. In some embodiments, the adenine base editor is ABE 7.10 or ABE8.

[0009] In some embodiments, at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein. In some embodiments, the aptamer sequence is an MS2 aptamer sequence, a PP7 aptamer sequence, a BoxB aptamer sequence, or a com aptamer sequence.

[0010] In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same. In some embodiments, the first and second ABPs are Com binding proteins.

[0011] In some embodiments, the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence. In some embodiments, the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence.

[0012] Also provided is an extracellular vesicle, comprising: (a) a ribonucleotide protein (RNP) complex comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer binding protein (ABP), wherein the ABP binds to the at least one aptamer coding sequence. In some embodiments, the extracellular vesicle further comprises VSV-G protein.

[0013] In some embodiments, the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein, or a derivative thereof. In some embodiments, the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease. In some embodiments, the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein. In some embodiments, the RNP comprises an adenine base pair editor (ABE), and the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease. In some embodiments, the adenine base editor is ABE 7.10 or ABE8.

[0014] In some embodiments, at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein. In some embodiments, the aptamer sequence is an MS2 aptamer sequence or a com aptamer sequence.

[0015] In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same. In some embodiments, the first and second ABPs are Com binding proteins.

[0016] In some embodiments, the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence. In some embodiments, the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence. In some embodiments, the extracellular vesicles are exosomes or microvesicles.

[0017] Further provided is a method for producing extracellular vesicles, comprising: (a) transfecting a plurality of eukaryotic cells with a first mammalian expression plasmid and a second mammalian expression plasmid of any of the plasmid systems described herein; and (b) culturing the transfected eukaryotic cells for a sufficient time to produce extracellular vesicles. In some embodiments, the method further comprises transfecting a plurality of eukaryotic cells with an envelope plasmid of any of the plasmid systems described herein.

[0018] In some embodiments, the extracellular vesicles comprise (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to at least one aptamer coding sequence. In some embodiments, the extracellular vesicles comprise (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the aptamer coding sequence; and (c) a VSV-G protein.

[0019] In some embodiments, the plurality of eukaryotic cells are mammalian cells. In some embodiments, the method further comprises isolating extracellular vesicles from the cultured transfected eukaryotic cells.

[0020] Also provided are extracellular vesicles produced by any of the methods provided herein.

[0021] Further provided is a method for modifying a genomic target sequence in a cell, comprising transducing a plurality of eukaryotic cells with a plurality of extracellular vesicles, wherein the plurality of extracellular vesicles comprises the extracellular vesicles described herein, and the RNP binds to a genomic target sequence in the genomic DNA of the cell, thereby modifying the genomic target sequence. In some embodiments, the plurality of eukaryotic cells are mammalian cells. In some embodiments, the plurality of eukaryotic cells are cells present in a subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is injected with a plurality of extracellular vesicles.

[0022] Also provided are cells containing any of the plasmid systems described herein. Also provided are cells modified using any of the methods for modifying cells described herein.

[0023] Further provided is a method of treating a disease in a subject, comprising: a) obtaining cells from the subject; b) modifying the cells of the subject using any of the methods for modifying cells described herein; and c) administering the modified cells to the subject. In some embodiments, the disease is cancer. In some embodiments, the disease is sickle cell anemia. In some embodiments, the cells are T cells.

[0024] Description of the drawings This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The drawings do not limit the scope of the compositions and methods, unless the written description expressly indicates otherwise. [Brief description of the drawings]

[0025] [Figure 1A]1A shows a fusion protein comprising Com and CD63 according to certain embodiments of the present disclosure. Com was fused to the N-terminus, C-terminus, or both termini of CD63 with a linker peptide in between. The sequences of linker 1 and linker 2 were "GGNHSGGGGQSPGPAA" (SEQ ID NO: 115) and "SGGGGSMASNFTQFVLVDNGGTGDV" (SEQ ID NO: 116), respectively.

[0026] [Figure 1B] FIG. 1B illustrates the recruitment of Cas9 or ABE RNP to exosomes according to certain embodiments of the present disclosure. RNP associates with Com-CD63-Com on the plasma membrane via com / Com interactions and then enters the endosomal system via endocytosis (step 1). Early endosomes become multivesicular bodies (MVBs) following inward budding of the outer endosomal membrane (step 2). When the membrane of the MVB fuses with the plasma membrane, the intraluminal vesicles are released into the medium as exosomes (step 3). Here, Com is depicted as a monomer, but can function as a homodimer.

[0027] [Figure 1C] FIG. 1C shows the recruitment of Cas9 RNP to microvesicles (step 4) according to certain embodiments of the present disclosure.

[0028] [Figure 2A] Figure 2A is a Western blot showing the expression of Com fusion protein and CD63 fusion protein according to a certain embodiment of the present disclosure. Plasmid DNA for CD63, CD63-Com, Com-CD63 and Com-CD63-Com expression was transfected into HEK293T cells, and the expression of CD63 or CD63 fusion protein was detected by anti-CD63 antibody. GAPDH was detected for loading control.

[0029] [Figure 2B]Figure 2B shows flow cytometry detection of gene editing activity according to certain embodiments of the present disclosure. 2.5x104 HBB-IL2RG GFP reporter cells were treated with RNP-enriched EVs secreted by 600,000 cells for 48 hours. RNPs were IL2RG-targeted SaCas9 RNPs (n=6). *** indicates p<0.0001 between the indicated conditions (ANOVA followed by Tukey post-hoc test).

[0030] [Figure 2C] FIG. 2C shows that vesicular stomatitis virus-G (VSV-G) improves the gene editing activity of EV-delivered RNPs according to certain embodiments of the present disclosure. IL2RG-targeted SaCas9 RNPs were packaged into EVs with or without VSV-G protein. RNP-enriched EVs were added to HBB-IL2RG GFP reporter cells and examined for GFP-positive cells by flow cytometry (n=3). ##, p<0.01 compared to the no VSV-G group; ***, p<0.0001 compared to all other conditions (ANOVA followed by Tukey post-hoc test).

[0031] [Figure 2D] FIG. 2D shows detection of gene editing activity of EV-delivered SpCas9 RNP by flow cytometry according to certain embodiments of the present disclosure. HBB-IL2RG GFP reporter cells (2.5×104) were treated with RNP-enriched EVs secreted by 600,000 cells for 48 hours. RNPs were IL2RG-targeted SpCas9 RNPs. *** indicates p<0.0001 between the indicated conditions (n=3, ANOVA followed by Tukey post-hoc test).

[0032] [Diagram 3]Figure 3 shows the optimization of DNA ratio for the most efficient production of RNP-loaded EVs according to certain embodiments of the present disclosure. The indicated amounts of pCom-CD63-Com and pX601-Tetra-com-IL2RG DNA were used for transfection to generate RNP-loaded EVs. The RNP-loaded EVs produced by 500,000 cells in 48 hours were concentrated and added to 2.5x104 HBB-IL2RG GFP reporter cells. GFP-positive cells were detected by flow cytometry. *** indicates p<0.0001 between the indicated conditions (n=3, ANOVA followed by Tukey post-hoc test).

[0033] [Figure 4] 4 is a Western blot showing detection of VSV-G and VSV-G-Com fusion proteins according to certain embodiments of the present disclosure. Control cells were mock transfected.

[0034] [Figure 5A] Figure 5A is a schematic diagram of sgRNAs driven by an RNA polymerase II promoter and flanked by a hammerhead (HH) ribozyme and a hepatitis delta virus (HDV) ribozyme. The sgRNAs were IL2RG-targeting sgRNAs for SaCas9 and SpCas9.

[0035] [Figure 5B] Figure 5B is a comparison of gene editing activity of EV-delivered SaCas9 or SpCas9 RNPs (targeting IL2RG and DMD exon 53, respectively) with or without extra sgRNA, according to certain embodiments of the present disclosure. RNP-loaded EVs were prepared with and without the respective plasmid DNA shown in Figure 5A. Gene editing activity was assayed by flow cytometry in HBB-IL2RG GFP reporter cells.

[0036] [Figure 6A]Figure 6A shows the enrichment of SaCas9 RNP in EVs according to certain embodiments of the present disclosure. Com-CD63-Com, SaCas9 and IL2RG sgRNA with and without com modification were co-expressed in 5x106 HEK293T cells, and EVs were collected for 48 hours. 1 / 5 of EVs were analyzed by Western blot. The number under the protein band was the estimated protein mass (ng) based on protein standard.

[0037] [Figure 6B] Figure 6B shows the enrichment of SpCas9 RNP and ABE in EVs according to certain embodiments of the present disclosure. IL2RG and site 5 targeted SpCas9 and ABE, respectively. The experimental conditions were the same as in Figure 6A.

[0038] [Figure 6C] Figure 6C shows Com- and com-dependent enrichment of sgRNA into EVs according to certain embodiments of the present disclosure. SaCas9 and IL2RG-targeting sgRNA were packaged into EVs (n=3). sgRNA was unmodified (com-) or com replacing the tetraloop (com+). CD63 was overexpressed instead in EVs without Com-CD63-Com. RNA was extracted from EVs and sgRNA was detected by RT-qPCR using primers Scid-g2F and sgRNA-R3. *** indicates p<0.0001 (ANOVA followed by Tukey post-hoc test) when compared to all other conditions.

[0039] [Figure 7A] 7A is a Western blot analysis of proteins in EVs according to certain embodiments of the present disclosure. EVs were collected from cells with or without IL2RG-targeted SaCas9 RNP. 1 / 20 of EVs were secreted and loaded by 1 million cells in 48 hours.

[0040] [Figure 7B] FIG. 7B shows transmission electron microscopy analysis of EVs according to certain embodiments of the present disclosure.

[0041] [Figure 7C] FIG. 7C shows nanoparticle tracking analysis of EV particle concentration according to certain embodiments of the present disclosure.

[0042] [Figure 7D] Figure 7D shows nanoparticle tracking analysis of EV size distribution according to certain embodiments of the present disclosure. EVs secreted by 5 million cells at 48 hours were resuspended in 500 μl of PBS for analysis in Figures 7B, 7C, and 7D.

[0043] [Figure 8A] 8A is a Western blot of the degradation of EV-delivered SpCas9 RNP according to certain embodiments of the present disclosure. The arrow indicates the position of the SpCas9 protein. The asterisk indicates a non-specific band indicating similar loading. EVs secreted by 200,000 cells for 48 hours were added to 2.5x104 HEK293T cells.

[0044] [Figure 8B] Figure 8B shows a densitometric analysis (IMAGEJ) of Cas9 protein levels at various time points according to certain embodiments of the present disclosure. Half-life was estimated using a single phase decay (GraphPad Prism 5).

[0045] [Figure 8C] Figure 8C is a Western blot of the degradation of EV-delivered SaCas9 RNP according to certain embodiments of the present disclosure. The arrow indicates the position of the SaCas9 protein. The asterisk indicates a non-specific band indicating similar loading. EVs secreted by 200,000 cells for 48 hours were added to 2.5x104 HEK293T cells.

[0046] [Figure 9A]9A shows an exemplary strategy for detecting simultaneous targeting of two loci according to certain embodiments of the present disclosure. Three sgRNAs, Sa-50, Sa-51 and Sp-53, target DMD intron 50, intron 51 and exon 53, respectively. Primers 50-F, 51-R and 53-R used for PCR detection of deletions are also shown. Solid boxes indicate hDMD exons 51-53. The distance between primers before sequence removal is listed.

[0047] [Figure 9B] FIG. 9B shows that, according to certain embodiments of the present disclosure, co-packaged SaCas9 RNPs targeting different loci were more efficient in multiplex genome editing.

[0048] [Figure 9C] FIG. 9C shows that SaCas9 RNP and SpCas9 RNP can be co-packaged into EVs for efficient multiplex genome editing, according to certain embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0050] Use of the terms "including," "comprising," or "having" and variations thereof herein is meant to encompass the subsequently listed elements and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising," or "having" specific elements are also considered to "consist essentially of," and "consist of," those specific elements. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0051] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be construed to include the recited materials or steps and "which do not materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ461,463 (CCPA 1976) (emphasis in original). See also MPEP §2111.03. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprising."

[0052] The term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (e.g., mRNA) and polymers thereof in single-stranded or double-stranded form. When RNA is described, its corresponding DNA is also described, and it is understood that uridine is represented as thymidine. Similarly, when DNA is described, its corresponding RNA is also described, and thymidine is represented by uridine. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Polynucleotides of the invention also encompass all forms of sequences, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0053] The term "gene" can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions before and after the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can refer to a segment of DNA involved in producing or encoding a non-translated RNA, such as rRNA, tRNA, guide RNA, or microRNA.

[0054] As used herein, the term "heterologous" refers to something that is not normally found in nature. The term "heterologous polypeptide" refers to a polypeptide that is not normally found in a given cell in nature. Thus, a heterologous polypeptide can be (a) foreign to its host cell (i.e., exogenous to the cell) or (b) naturally found in the host cell (i.e., endogenous) but present in the cell in an unnatural amount (i.e., greater or less than the amount naturally found in the host cell). The term "heterologous promoter" refers to a promoter sequence that is not normally found in a given cell in nature or that is not normally found operably linked to a polynucleotide that expresses a given protein.

[0055] "Treat" refers to any indication of success in treating or improving or preventing a disease, condition or disorder, including any objective or subjective parameter, such as reduction; remission; reducing symptoms or making disease symptoms more tolerable to the patient; slowing down the rate of degeneration or decline; or making the end point of degeneration less debilitating. Treating or improving symptoms can be based on objective or subjective parameters, including the results of a physician's examination. Thus, the term "treat" includes the administration of a compound or agent of the present disclosure to prevent or delay, alleviate, or stop or inhibit the onset of symptoms or symptoms associated with a disease, condition or disorder described herein. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject. "Treating" or "treatment" using the methods of the present disclosure includes preventing the onset of symptoms in a subject who may be at high risk for a disease or disorder associated with the disease, condition, or disorder described herein, but has not yet experienced or exhibited symptoms, inhibiting (delaying or halting) the symptoms of the disease or disorder, relieving (including palliative) symptoms or side effects of the disease, and relieving (causing regression) symptoms of the disease. Treatment can be prophylactic (to prevent or delay the onset of the disease or to prevent the onset of its clinical or subclinical symptoms), or therapeutic suppression or alleviation of symptoms after the onset of the disease or condition. As used herein, the term "treatment" includes preventative (e.g., prophylactic), curative, or palliative treatment.

[0056] "Promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, promoter includes the necessary nucleic acid sequence near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element. Promoter also includes, if necessary, distal enhancer or repressor elements that may be located several thousand base pairs from the start site of transcription.

[0057] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to natural and non-natural amino acid polymers. As used herein, the term encompasses full-length proteins, truncated proteins, and fragments thereof, as well as amino acid chains in which the amino acid residues are linked by covalent peptide bonds. The term "fusion polypeptide" or "fusion protein," as used throughout, is a polypeptide that includes two or more proteins or fragments thereof. In some embodiments, a linker containing about 3-10 amino acids can be placed between any two proteins or fragments thereof to help promote proper folding of the proteins upon expression.

[0058] The term "identity" or "substantial identity" as used in the context of polynucleotide or polypeptide sequences described herein refers to a sequence having at least 60% sequence identity with a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% as compared to a reference sequence using the programs described herein; preferably, BLAST using standard parameters as described below. It is understood that any nucleotide or polypeptide sequence described herein, such as a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 1-49, can be used in the compositions and methods provided herein. It is understood that a nucleic acid sequence can comprise, consist of, or consist essentially of any nucleic acid sequence described herein. Similarly, a polypeptide can comprise, consist of, or consist essentially of any polypeptide sequence described herein. For sequence comparison, typically one sequence acts as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0059] As used herein, a "comparison window" includes reference to any one segment of a number of consecutive positions selected from the group consisting of 20-600, about 20-50, about 20-100, about 50-200, or about 100-150, and a sequence may be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homologous sequence alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0060] Suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residuals; always >0) and N (penalty score for mismatched residuals; always <0). Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value. The cumulative score falls below 0 due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.

[0061] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0062] As used throughout, subject means an individual. For example, subject is a mammal, such as a primate, more specifically a human. Non-human primates are also subjects. The term subject includes domestic animals, such as cats, dogs, livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses as well as medical uses and formulations are contemplated herein. The term does not indicate a particular age or sex. Thus, living and newborn subjects are intended to be covered, whether male or female. As used herein, patient or subject can be used interchangeably and can refer to a subject suffering from a disease or disorder.

[0063] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct that has a set of specific nucleic acid elements that allow the transcription of a specific polynucleotide sequence in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette contains a polynucleotide to be transcribed that is operably linked to a promoter and is followed by a transcription termination signal sequence. An expression cassette may or may not contain specific regulatory sequences, such as 5' or 3' untranslated regions from human globin genes.

[0064] A "reporter gene" encodes a protein that is easily detectable due to a biochemical characteristic, such as an enzymatic activity or a chemiluminescent property. These reporter proteins can be used as selectable markers. One specific example of such a reporter is green fluorescent protein. The fluorescence generated from this protein can be detected with a variety of commercially available fluorescence detection systems. Other reporters can be detected by staining. A reporter can also be an enzyme that generates a detectable signal when contacted with an appropriate substrate. A reporter can be an enzyme that catalyzes the formation of a detectable product. Suitable enzymes include, but are not limited to, proteases, nucleases, lipases, phosphatases and hydrolases. A reporter can encode an enzyme whose substrate is substantially impermeable to the eukaryotic plasma membrane, thus allowing for tight control of signal formation. Specific examples of suitable reporter genes encoding enzymes include, but are not limited to, CAT (chloramphenicol acetyltransferase; Alton and Vapnek (1979) Nature 282:864-869); luciferase (lux); β-galactosidase; LacZ; β-glucuronidase; and alkaline phosphatase (Toh, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101), each of which is incorporated herein by reference in its entirety. Other suitable reporters include those that encode specific epitopes that can be detected with a labeled antibody that specifically recognizes the epitope.

[0065] "CRISPR / Cas" systems refer to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II and type III subtypes. The classification of CRISPR / Cas systems as described by Makarova, et al. (Nat Rev Microbiol. 2015 Nov;13(11):722-36) defines five types and 16 subtypes based on shared characteristics and evolutionary similarities. These are divided into two broad classes based on the structure of the effector complex that cleaves genomic DNA. Type II CRISPR / Cas systems were first used for genome engineering, followed by type V in 2015. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease Cas protein or homolog (referred to herein as "CRISPR-associated endonucleases") that form a complex with a guide RNA to recognize and cleave foreign nucleic acids. Cas9 protein also uses activating RNA (also called transactivating RNA or tracr RNA). Guide RNA or guide RNA with both guide RNA and activating RNA activity is also known in the art depending on the type of CRISPR-associated endonuclease used with it. In some cases, such dual-activity guide RNA is called single guide RNA (sgRNA). Synthetic guide RNA that does not contain activating RNA sequence may also be called sgRNA. In this disclosure, the terms sgRNA and gRNA are used interchangeably to refer to RNA molecules that form a complex with CRISPR-associated endonuclease and localize the ribonucleoprotein complex to the target DNA sequence.

[0066] In the compositions and methods provided herein, the CRISPR-associated endonuclease can be a catalytically impaired nuclease. "Catalytically impaired" as used throughout refers to the reduced CRISPR-associated endonuclease enzyme activity to cleave one or both strands of DNA. Examples of catalytically impaired CRISPR-associated endonucleases include, but are not limited to, catalytically impaired Cas9, catalytically impaired Cpf1 and catalytically impaired C2c2. In some examples, the catalytically impaired CRISPR-associated endonuclease is a catalytically impaired Cas9, such as Cas9 D10A, which only cleaves or nicks one strand of DNA. In some examples, the CRISPR-associated endonuclease can be a catalytically impaired CRISPR-associated endonuclease, and the endonuclease cannot cleave both strands of a double-stranded DNA molecule, i.e., cannot perform double-stranded cleavage. Modifications include, but are not limited to, one or more amino acid modifications to inactivate nuclease activity or nuclease domain. For example, but not limited to, D10A and / or H840A mutations can be made in Cas9 from Streptococcus pyogenes to reduce or inactivate Cas9 nuclease activity. Other modifications include removing all or part of the nuclease domain of Cas9 so that the sequence that exhibits nuclease activity is absent in Cas9. Thus, catalytically impaired Cas9 can include a polypeptide sequence that is modified to reduce nuclease activity, or the removal of a polypeptide sequence or sequences to reduce nuclease activity. Catalytically impaired Cas9 retains the ability to bind to DNA even though nuclease activity is inactivated. Thus, a catalytically impaired Cas9 contains the polypeptide sequence(s) necessary for DNA binding, but contains an altered nuclease sequence, or lacks the nuclease sequence responsible for nuclease activity.It is understood that similar modifications can be made to reduce nuclease activity in other site-specific nucleases, such as Cpf1 or C2c2. In some examples, the Cas9 protein is a full-length Cas9 sequence from S.pyogenes that lacks the polypeptide sequence of the RuvC nuclease domain and / or the HNH nuclease domain and retains DNA binding function. In other examples, the Cas9 protein sequence has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to a Cas9 polypeptide sequence that lacks the RuvC nuclease domain and / or the HNH nuclease domain and retains DNA binding function.

[0067] As used herein, "activity" in the context of sgRNA activity or RNP activity, i.e., the RNP activity of a complex of (1) gRNA and (2) CRISPR-associated endonuclease, refers to the ability of sgRNA to bind to a target genetic element. Typically, activity also refers to the ability of RNP (i.e., sgRNA complexed with and CRISPR-associated endonuclease) to edit the genome of a cell. In some examples, activity refers to the ability of ABE RNP (i.e., sgRNA complexed with ABE) to edit base pairs, i.e., to make an A to G change in one strand of DNA.

[0068] As used herein, the phrase "editing" in the context of editing the genome of a cell refers to inducing a structural change in a sequence of a genome at a target genomic region, e.g., cleaving a genomic sequence and inserting a donor sequence into the genome of the cell at the cleavage site via homology directed repair (HDR), or cleaving a sequence and allowing repair via non-homologous recombination end joining (NHEJ). In some instances, the editing is performed by ABE. For example, the editing can take the form of an A to G change in one strand of DNA (or a T to C change in the opposite strand of DNA) in the target genomic region. The nucleotide sequence can encode a polypeptide or a fragment thereof. See, e.g., Gaudelli et al., "Programmable base editing of AT to GC in genomic DNA without DNA cleavage," Nature 551:464-471 (2017).

[0069] As used herein, "adenine base editor" or "ABE" refers to a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease. In some cases, the adenosine deaminase is a tadA enzyme that deaminates adenine on a single strand of DNA to form inosine. See Gaudelli et al, (2017). In some examples, the ABE is a fusion protein comprising a catalytically impaired CRISPR-associated endonuclease and one or more copies, such as 2, 3, 4 copies, of adenosine deaminase. In some examples, the ABE comprises a fusion protein encoded by a nucleic acid sequence comprising SEQ ID NO:27. In some examples, the ABE comprises SEQ ID NO:28.

[0070] As used herein, the terms "ribonucleoprotein complex," "RNP," and the like refer to a complex between (1) a CRISPR-associated endonuclease and a crRNA (e.g., a guide RNA or a single guide RNA), (2) a CRISPR-associated endonuclease and a trans-activating crRNA (tracrRNA), (3) a CRISPR-associated endonuclease and a guide RNA, or (4) a combination thereof (e.g., a complex comprising a CRISPR-associated endonuclease and a catalytically impaired Cas9 protein, a tracrRNA, and a crRNA guide). In some embodiments, the CRISPR-associated endonuclease is catalytically impaired. In some embodiments, the catalytically impaired CRISPR-associated endonuclease is fused to an adenosine deaminase.

[0071] As used herein, a "cell" can be any eukaryotic cell, such as a human T cell, or a cell that can differentiate into a T cell (e.g., a T cell that expresses a TCR receptor molecule). These include hematopoietic stem cells and cells derived from hematopoietic stem cells. Also provided is a population of cells, such as a population of cells that comprises viral particles or genetically modified cells produced by any of the genome editing methods provided herein.

[0072] As used herein, the phrase "hematopoietic stem cell" refers to a type of stem cell that can give rise to blood cells. Hematopoietic stem cells can give rise to cells of myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are primarily found in bone marrow, but can be isolated from peripheral blood or a portion thereof. Various cell surface markers can be used to identify, select, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified as c-kit+ and lin-. In some cases, human hematopoietic stem cells are identified as CD34+, CD59+, Thy1 / CD90+, CD38lo / -, C-kit / CD117+, lin-. In some cases, human hematopoietic stem cells are identified as CD34-, CD59+, Thy1 / CD90+, CD38lo / -, C-kit / CD117+, lin-. In some cases, the human hematopoietic stem cells are identified as CD133+, CD59+, Thy1 / CD90+, CD38lo / -, C-kit / CD117+, lin-. In some cases, the mouse hematopoietic stem cells are identified as CD34lo / -, SCA-1+, Thy1+ / lo, CD38+, C-kit+, lin-. In some cases, the hematopoietic stem cells are CD150+CD48-CD244-.

[0073] As used herein, the phrase "hematopoietic cells" refers to cells derived from hematopoietic stem cells. Hematopoietic cells can be obtained or provided by isolation from an organism, system, organ or tissue (e.g., blood or a portion thereof). Alternatively, hematopoietic stem cells can be isolated and hematopoietic cells can be obtained or provided by differentiating the stem cells. Hematopoietic cells include cells that have limited potential to differentiate into additional cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include lymphoid and myeloid cells, such as lymphocytes, erythrocytes, granulocytes, monocytes, and platelets. In some embodiments, the hematopoietic cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells. In some embodiments, the cells are innate immune cells.

[0074] As used herein, the phrase "T cells" refers to lymphoid cells that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., not cultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells can be CD4+, CD8+, or CD4+ and CD8+. T cells can also be CD4-, CD8-, or CD4- and CD8-. T cells can be helper cells, such as TH1, TH2, TH3, TH9, TH17, or TFH types of helper cells. T cells can be cytotoxic T cells. Regulatory T cells can be FOXP3+ or FOXP3-. The T cells can be alpha / beta T cells or gamma / delta T cells. In some cases, the T cells are CD4+CD25hiCD127lo regulatory T cells. In some cases, the T cells are regulatory T cells selected from the group consisting of type 1 regulatory (Tr1), TH3, CD8+CD28-, Treg17, and Qa-1 restricted T cells, or a combination or subpopulation thereof. In some cases, the T cells are FOXP3+ T cells. In some cases, the T cells are CD4+CD25loCD127hi effector T cells. In some cases, the T cells are CD4+CD25loCD127hiCD45RAhiCD45RO- naive T cells. The T cells can be genetically engineered recombinant T cells.

[0075] As used herein, the term "primary" in the context of primary cells is a cell that has not been transformed or immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cultures). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from an organism, system, organ, or tissue, optionally sorted, and directly utilized without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contacting with (e.g., culturing in the presence of) CD3, CD28 agonist, IL-2, IFN-γ, or a combination thereof.

[0076] As used herein, the term "extracellular vesicles (EVs)" refers to membrane-bound vesicles that are naturally released from eukaryotic cells. Thus, EVs are cell-derived vesicles, i.e., lipid bilayer-bound particles that contain a membrane surrounding an internal space (lumen). Generally, EVs range in diameter from 20 nm to 1000 nm. EVs include, but are not limited to, exosomes and microvesicles. EVs are released by cells and are found in most biological fluids, including urine, plasma, cerebrospinal fluid, saliva, etc., as well as in tissue culture conditioned media. Detailed Description

[0077] The following description lists various aspects and embodiments of the present compositions and methods.Specific embodiments are not intended to define the scope of the compositions and methods.Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods.The description should be read from the perspective of those skilled in the art.Therefore, it does not necessarily include information that is well known to those skilled in the art.

[0078] Provided herein are compositions, systems, methods of manufacture, and methods for efficient delivery of nucleic acid sequences (e.g., mRNA sequences) and RNPs to eukaryotic cells using EVs. Using the compositions and methods described herein, nucleic acid sequences (e.g., mRNA sequences) and RNPs can be efficiently packaged into EVs and delivered to eukaryotic cells. For example, components, systems, methods of manufacture, and methods are provided for efficient delivery of RNPs, including (1) CRISPR-associated endonucleases and (2) sgRNAs, to cells via EVs. The EVs described herein have a limited half-life, reducing the risk of RNA and DNA off-target mediated mutagenesis. Delivery of RNPs to eukaryotic cells allows for efficient delivery in difficult to transfect cells, such as primary cells, while reducing off-target effects, for example. Plasmid Systems

[0079] Provided herein is a plasmid system used to deliver CRISPR component coding sequences, i.e., sgRNA and CRISPR-associated endonuclease, to mammalian cells used to generate the EVs of the present disclosure. For example, provided herein is a plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising (i) a nucleic acid sequence encoding a CRISPR-associated endonuclease, and (ii) a guide RNA (gRNA) coding sequence comprising at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, the second mammalian expression plasmid comprising an aptamer binding protein (ABP) binding to at least one aptamer coding sequence of the first mammalian expression plasmid. In some embodiments, the plasmid system further comprises an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

[0080] The first mammalian expression plasmid of the system provided herein includes a CRISPR component coding sequence, such as a coding sequence for a CRISPR-associated endonuclease and a gRNA. In some examples, the gRNA coding sequence includes at least one aptamer coding sequence. In some examples, the at least one aptamer coding sequence can be located at the 5' or 3' end of the gRNA. In some examples, the at least one aptamer coding sequence can be inserted at an internal location within the gRNA, such as one or more of the loops formed within the folded gRNA. For example, if the gRNA is for a Cas9 protein, the at least one aptamer coding sequence can be located in the tetraloop, stem loop 2 (ST2) or 3' end of the gRNA. In some examples, a spacer of 1 to 30 nucleotides can be located between the gRNA and the at least one aptamer coding sequence or adjacent to the at least one aptamer coding sequence. In some embodiments, the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence. In some embodiments, the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence.

[0081] In the systems provided herein, the first mammalian expression plasmid includes at least one aptamer coding sequence that encodes an aptamer sequence that is specifically bound by an aptamer binding protein (ABP) encoded by a second mammalian expression plasmid of the system. In the context of the present disclosure, an aptamer sequence is an RNA sequence that forms a tertiary loop structure that is specifically bound by the ABP. An ABP is an RNA binding protein or an RNA binding protein domain. Suitable aptamer coding sequences include polynucleotide sequences that encode known bacteriophage aptamer sequences. Exemplary aptamer coding sequences include those that encode the aptamer sequences shown in Table 1 above. In some instances, the aptamer is bound by a dimer of the ABP. These aptamer sequences are RNA sequences to which a bacteriophage protein is known to specifically bind. In some situations, the at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein. In some embodiments, at least one aptamer coding sequence is a com aptamer and the ABP is a Com protein. [Table 1]

[0082] In some examples, the first mammalian expression vector comprises an sgRNA that comprises one aptamer coding sequence downstream. In other examples, the gRNA can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 aptamer coding sequences. For example, in some examples, the gRNA can comprise two aptamer coding sequences in tandem. In some embodiments, the two or more tandem aptamer coding sequences are the same. In some embodiments, the two or more tandem aptamer coding sequences are different.

[0083] As used throughout, a sgRNA is a single guide RNA sequence that specifically binds or hybridizes to a target nucleic acid (genomic target sequence) in the genome of a cell such that it interacts with a CRISPR-associated endonuclease (CRISPR site-specific nuclease) and the sgRNA and CRISPR-associated endonuclease co-localize to the target nucleic acid in the genome of the cell. Each sgRNA contains a DNA targeting sequence or protospacer sequence, approximately 10-50 nucleotides in length, that specifically binds or hybridizes to a target DNA sequence in the genome. For example, the DNA targeting sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. For example, the DNA targeting sequence can be about 15-30 nucleotides, about 15-25 nucleotides, about 10-25 nucleotides, or about 18-23 nucleotides. In one example, the DNA targeting sequence is about 20 nucleotides. In some embodiments, the sgRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the sgRNA does not comprise a tracrRNA sequence.

[0084] Generally, the DNA targeting sequence is designed to be complementary (e.g., fully complementary) or substantially complementary (e.g., with 1-4 mismatches) to the target DNA sequence. In some cases, the DNA targeting sequence can incorporate wobble or degenerate bases to bind to multiple genetic elements. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are fully complementary to the target genetic element(s). In some cases, the binding region can be modified to increase stability. For example, non-natural nucleotides can be incorporated to increase RNA resistance to degradation. In some cases, the binding region can be modified or designed to avoid or reduce the formation of secondary structures in the binding region. In some cases, the binding region can be designed to optimize GC content. In some cases, the GC content is preferably about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some cases, the binding region can be selected to begin with a sequence that promotes efficient transcription of the sgRNA. For example, the binding region may begin at the 5' end with a G nucleotide. In some cases, the binding region may include modified nucleotides, such as, but not limited to, methylated or phosphorylated nucleotides.

[0085] As used herein, the term "complementary" or "complementarity" refers to base pairing between nucleotides or nucleic acids, such as, but not limited to, base pairing between an sgRNA and a target sequence. Complementary nucleotides are generally A and T (or A and U), and G and C. Guide RNAs described herein can include a DNA targeting sequence that is fully complementary or substantially complementary (e.g., with 1-4 mismatches) to a sequence, e.g., a genomic sequence.

[0086] The sgRNA comprises a sgRNA constant region that interacts with or binds to a CRISPR-associated endonuclease. In the constructs provided herein, the constant region of the sgRNA can be about 75-250 nucleotides in length. In some examples, the constant region is a modified constant region that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide substitutions in the stem, stem loop, hairpin, region between the hairpin, and / or nexus of the constant region. In some examples, modified constant regions that have at least 80%, 85%, 90% or 95% activity compared to the activity of the native or wild-type sgRNA constant region from which the modified constant region is derived can be used in the constructs described herein. In particular, modifications should not be made at nucleotides that directly interact with the CRISPR-associated endonuclease or that are critical to the secondary structure of the constant region.

[0087] The CRISPR-associated endonuclease encoded on the first mammalian expression plasmid of any of the systems described herein is an RNA-guided site-specific nuclease.For example, but not limited to, the CRISPR-associated endonuclease can be a Cas9 polypeptide (type II) or a Cpf1 polypeptide (type V).See, for example, Abudayyeh et al., Science 2016 August 5;353(6299):aaf5573; Fonfara et al. Nature 532:517-521(2016), and Zetsche et al., Cell 163(3):p.759-771,22 October 2015.The term "Cas9 polypeptide" used throughout refers to the Cas9 protein, or fragments or derivatives thereof, identified in any bacterial species that encodes a type II CRISPR / Cas system. See, for example, Makarova et al. Nature Reviews, Microbiology, 9:467-477 (2011) (including supplementary information), which is incorporated herein by reference in its entirety. CRISPR-related endonucleases such as Cas9 and Cas9 homologues are found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is Streptococcus pyogenes Cas9 protein (SpCas9). Another exemplary Cas9 protein is Staphylococcus aureus Cas9 protein (SaCas9).Additional Cas9 proteins and homologs thereof are described, for example, in Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep 24; 110(39): 15644-9; Sampson et al., Nature. 2013 May 9; 497(7448): 254-7; and Jinek, et al., Science. 2012 Aug 17; 337(6096): 816-21. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in the host cell. Other CRISPR-associated endonucleases include Cpf1 (see, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015) and its homologs.

[0088] Full-length Cas9 is an endonuclease that contains a recognition domain and two nuclease domains (HNH and RuvC, respectively) that generate double-stranded breaks in DNA sequences. In the amino acid sequence of Cas9, HNH is linearly continuous, while RuvC is separated into three regions, one to the left of the recognition domain and the other two to the right of the recognition domain adjacent to the HNH domain. Cas9 targets genomic sites in cells by interacting with guide RNAs that hybridize to a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by Cas9. This results in a double-stranded break in the genomic DNA of the cell. In some instances, Cas9 nucleases can be utilized that require an NGG protospacer adjacent motif (PAM) immediately 3' of the region targeted by the guide RNA. As another example, Cas9 proteins with orthogonal PAM motif requirements can be utilized to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Esvelt et al., Nature Methods 10:1116-1121 (2013). Various Cas9 nucleases can be utilized in the methods described herein. For example, a Cas9 nuclease (e.g., SpCas9) that requires a NGG protospacer adjacent motif (PAM) immediately 3' to the region targeted by the guide RNA can be utilized. Such a Cas9 nuclease can target any region of the genome that contains a NGG sequence. In another example, a Cas9 nuclease (e.g., SaCas9) that requires a NNGRRT (SEQ ID NO: 106) or NNGRR(N) (SEQ ID NO: 107) PAM immediately 3' to the region targeted by the guide RNA can be utilized. As another example, a Cas9 protein with orthogonal PAM motif requirement can be utilized to target a sequence that does not have an adjacent NGG PAM sequence.Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Nature Methods 10, 1116-1121 (2013) and those described in Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015.

[0089] Any of the CRISPR-associated endonucleases described herein can be catalytically impaired. In some cases, the catalytically impaired CRISPR-associated endonuclease is a Cas9 nickase, such as Cas9 D10A. In some examples, Cas9 10A is encoded by SEQ ID NO: 21. In some examples, Cas9 10A comprises SEQ ID NO: 22. Usually, when Cas9 nickase binds to a target nucleic acid as part of a complex with a guide RNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each of which is bound to a structurally different guide RNA, can be targeted to two adjacent sites of a target genome region. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations.

[0090] In some embodiments, the nucleic acid sequence of the first mammalian expression plasmid encodes a nucleic acid sequence encoding an adenosine base pair editor (ABE), where the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease. In some embodiments, the adenine base editor is ABE 7.10 or ABE8.

[0091] The plasmid system described herein includes a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the at least one aptamer coding sequence of the first mammalian expression plasmid.

[0092] In some embodiments, the fusion protein comprises an ABP fused to the N-terminus of CD63 and / or an ABP fused to the C-terminus of CD63. In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, where the first and second ABPs are the same. In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, where the first and second ABPs are different. In some embodiments, the first and second ABPs are Com that bind to the com aptamer in the first mammalian expression plasmid.

[0093] As used herein, CD63, CD63 antigen or fragment thereof is a member of the transmembrane 4 protein superfamily, also known as tetraspanin family.CD63 is a cell surface protein characterized by the presence of four hydrophobic domains found on the cell surface of extracellular vesicles.The exemplary amino acid sequence of CD63 is shown as SEQ ID NO: 43.SEQ ID NO: 43 is encoded by SEQ ID NO: 42.It is understood that any isoform of CD63 or fragment thereof can be used in the compositions and methods described herein. Exemplary CD63 isoform protein sequences are set forth in GenBank Accession Nos. NP_001244318.1, NP_001771.1, NP_001254627.1, NP_001244319.1, NP_001244320.1, NP_001244329.1, NP_001244330.1, NP_001244321.1, and XP_024305051.1.

[0094] The mammalian expression plasmid comprises a eukaryotic promoter operably linked to a nucleic acid sequence encoding a CRISPR-endonuclease, a sgRNA coding sequence, or a CD63-ABP fusion protein. The system described herein can also comprise an envelope plasmid having an envelope coding sequence encoding a viral envelope glycoprotein. For example, the Env nucleotide sequence can encode VSV-G. The envelope coding sequence is operably linked to a eukaryotic promoter. In some examples, the eukaryotic promoter is an RNA polymerase II promoter.

[0095] As used herein, "vesicular stomatitis virus G" or "VSV-G" is a viral envelope protein that can be used to facilitate transport or escape of any of the EVs described herein from the endosomal system in a recipient cell. An exemplary VSV-G amino acid sequence is shown as SEQ ID NO: 45. SEQ ID NO: 45 is encoded by SEQ ID NO: 44. Another exemplary VSV-G protein sequence is set forth in GenBank Accession No. CAC47944.1.

[0096] In some examples, an RNA polymerase II promoter is operably linked to the CRISPR-associated endonuclease coding sequence and an RNA polymerase III promoter is operably linked to the gRNA coding sequence.

[0097] The RNA polymerase II promoter sequence is selected from a mammalian species. The RNA polymerase III promoter sequence is selected from a mammalian species. For example, these promoter sequences can be selected from human, bovine, ovine, buffalo, porcine, or murine, to name a few. In some examples, the RNA polymerase II promoter sequence is a CMV, FE1α, or SV40 sequence. In some examples, the RNA polymerase III promoter sequence is a U6 or H1 sequence. In some examples, the RNA polymerase II sequence is a modified RNA polymerase II sequence. For example, an RNA polymerase II sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a wild-type RNA polymerase II promoter sequence from any mammalian species can be used in the constructs provided herein. In some examples, the RNA polymerase III sequence is a modified RNA polymerase III sequence. For example, RNA polymerase III sequences with at least 80%, 85%, 90%, 95% or 99% identity to wild-type RNA polymerase III promoter sequences from any mammalian species can be used in the constructs provided herein.Those skilled in the art will easily understand how to determine the identity of two polypeptides or nucleic acids.For example, identity can be calculated after aligning the two sequences so that identity is at its highest level, as described above.In some examples, the eukaryotic promoter is an inducible or regulatable promoter.

[0098] Coding sequences transcribed from RNA pol II promoters contain a poly(A) signal and a transcription terminator sequence downstream of the coding sequence. Commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) contain the sequence motif AAUAAA (SEQ ID NO: 108), which promotes both polyadenylation and termination. Coding sequences transcribed from RNA pol III promoters contain a simple run of T residues downstream of the coding sequence as a terminator sequence. The role of a terminator, a sequence-based element, is to define the end of a transcription unit (such as a gene) and initiate the process of release of newly synthesized RNA from the transcription machinery. Terminators are

[0099] They are found downstream of the transcribed gene, typically occurring immediately following any 3' regulatory elements such as a polyadenylation signal or poly(A) signal.

[0100] In some examples, the mammalian expression plasmid may also include at least one polynucleotide sequence encoding an RNA stabilizing sequence located downstream of the sequence encoding the CRISPR component, or an aptamer coding sequence if located downstream of the CRISPR component coding sequence. The polynucleotide sequence encoding the RNA stabilizing sequence is transcribed downstream of the CRISPR / Cas system component coding sequence and stabilizes the life span of the transcribed RNA sequence. In one example, the polynucleotide sequence encoding the RNA stabilizing sequence is located downstream of the catalytically impaired CRISPR-associated endonuclease coding sequence. In another example, the polynucleotide sequence encoding the RNA stabilizing sequence is located downstream of the gRNA coding sequence. An exemplary RNA stabilizing sequence is the sequence of the 3'UTR of the human beta globin gene shown in SEQ ID NO: 17 (DNA) and SEQ ID NO: 18 (RNA). In some embodiments, the RNA stabilizing sequence includes two or more copies of SEQ ID NO: 17. Other RNA stabilizing sequences are described in Hayashi, T. et al., Developmental Dynamics 239(7):2034-2040(2010) and Newbury, S. et al., Cell 48(2):297-310(1987). In some examples, a spacer of 1 to 30 nucleotides may be placed between the CRISPR component coding sequence and at least one polynucleotide sequence encoding an RNA stabilizing sequence.

[0101] In some examples, any of the mammalian expression plasmids described herein may comprise one or more expression cassettes.In some examples, the first mammalian expression plasmid comprises a first expression cassette that encodes a CRISPR-associated endonuclease and a second expression cassette that encodes a gRNA that comprises at least one aptamer.In some examples, the mammalian expression plasmid may also comprise a reporter gene.

[0102] Also provided herein are kits that contain components of the systems described in this disclosure. In some embodiments, the kits contain one or more of the plasmids described herein. Extracellular vesicles

[0103] In another aspect, EVs are provided, for example EVs produced by any of the systems and methods described herein. In some embodiments, the EVs are exosomes and / or microvesicles. Extracellular vesicles produced by any of the methods described herein are also provided. A plurality of EVs are also provided. The EVs comprise (a) a ribonucleotide protein (RNP) complex comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer binding protein (ABP), wherein the ABP binds to the at least one aptamer coding sequence. In some embodiments, the extracellular vesicles further comprise a VSV-G protein.

[0104] Any of the first mammalian expression plasmids described herein in which at least one aptamer is linked or inserted into a gRNA sequence can be used to generate EVs containing RNPs. These EVs are useful for transducing eukaryotic cells of interest.

[0105] In some embodiments, one or more first mammalian expression plasmids targeting different sites in the genome of a cell are used to generate EVs containing one or more RNPs targeting one or more sites in the genome of a cell. For example, but not limited to, a first mammalian expression plasmid containing an sgRNA targeting site A of the genome of a cell and a first mammalian expression plasmid containing an sgRNA targeting site B of the genome of a cell can be transduced into a cell together with a second mammalian expression plasmid encoding a CD63-ABP fusion protein to generate EVs containing an RNP targeting site A and an RNP targeting site B. EVs targeting three or more sites, four or more sites, five or more sites, or six or more sites in the genome can be generated using similar methods.

[0106] The EV may comprise a fusion protein comprising one or more ABPs. In some examples, the EV comprises CD63 or a fragment thereof fused with at least one ABP. In some embodiments, the fusion protein comprises an ABP fused to the N-terminus of CD63 and / or an ABP fused to the C-terminus of CD63. In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, where the first and second ABPs are the same. In some embodiments, the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, where the first and second ABPs are different. In some embodiments, the first and second ABPs are Com and bind to the com aptamer bound or inserted into the gRNA. In some embodiments, the fusion protein comprises two or more ABPs in tandem fused to the N-terminus of CD63 and / or the C-terminus of CD63.

[0107] ABPs are polypeptide sequences that bind to RNA aptamer sequences. As noted above, several ABPs are suitable for use in the present disclosure. In particular, suitable ABPs include bacteriophage RNA binding proteins that specifically bind to known RNA aptamer sequences, which are RNA sequences that form stem-loop structures. Exemplary non-viral aptamer binding proteins include MS2 coat protein, PP7 coat protein, lambda N peptide, and Com protein. The lambda N peptide can be amino acids 1-22 of the lambda N protein, which is the RNA binding domain of the protein. Information regarding these ABPs and the aptamer sequences to which they bind is provided in Table 1 above.

[0108] As mentioned above, gRNA generally comprises a DNA targeting sequence and a constant region that interacts with CRISPR-associated endonuclease. In some examples, gRNA may comprise a transactivating crRNA (tracrRNA) sequence. For example, gRNA may comprise a tracrRNA that is used with Cas9 protein or derivative. In other examples, gRNA does not comprise a tracrRNA sequence. For example, gRNA may not comprise a tracrRNA sequence that is used with Cpf1 protein or derivative.

[0109] In some examples, the gRNA comprises at least one aptamer sequence. In some examples, the at least one aptamer sequence may be placed at the 5' or 3' end of the gRNA. In some examples, the at least one aptamer sequence may be inserted at an internal location within the gRNA, for example, one or more of the loops formed within the folded gRNA. For example, if the gRNA is for a Cas9 protein, the at least one aptamer sequence may be placed in the tetraloop, stem loop 2 (ST2) or 3' end of the gRNA. In some examples, a spacer of 1-30 ribonucleotides may be placed between the gRNA and the at least one aptamer sequence or adjacent to the at least one aptamer sequence. How to make EVs

[0110] Methods for producing EVs using any of the plasmids and systems provided herein are described. For example, methods for producing extracellular vesicles are provided herein, comprising: (a) transfecting a plurality of eukaryotic cells with a first mammalian expression plasmid (i.e., a plasmid encoding a CRISPR-associated endonuclease and sgRNA) and a second mammalian expression plasmid (i.e., a plasmid encoding a CD63-ABP fusion protein) of any of the plasmid systems described herein; and (b) culturing the transfected eukaryotic cells for a sufficient time for extracellular vesicles to be produced. In some embodiments, the method further comprises transfecting a plurality of eukaryotic cells with an envelope plasmid (i.e., a plasmid encoding VSV-G) of any of the plasmid systems described herein.

[0111] In some embodiments, the extracellular vesicles comprise (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to at least one aptamer coding sequence. In some embodiments, the extracellular vesicles comprise (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the aptamer coding sequence; and (c) a VSV-G protein.

[0112] In some embodiments, the plurality of eukaryotic cells are mammalian cells. In some embodiments, the method further comprises isolating extracellular vesicles from the cultured transfected eukaryotic cells. Methods for isolating EVs are known in the art. See, for example, Konoshenko et al. "Isolation of Extracellular Vesicles: General Methodologies and Latest Trends", Hindawi BioMed Research International Vol. 2018, Article ID 8545347; and Furi et al. "Extracellular vesicle isolation: present and future", Ann Transl. Med. 5(12):263 (2017). In some embodiments, the eukaryotic cells are selected from the group consisting of HEK293T cells, C2C12 cells, primary myoblasts, neural stem cells, pluripotent stem cells, and mesenchymal stem cells. Gene editing methods

[0113] Described herein are methods of using the plasmids and systems provided in this disclosure in CRISPR / Cas systems for editing DNA targets, such as genes, in the genome of eukaryotic cells.

[0114] In the methods provided herein, a eukaryotic cell containing a target genomic sequence of interest to be modified is transduced with an EV comprising a fusion protein comprising CD63 or a fragment thereof fused to at least one aptamer-binding protein (ABP) and an RNP comprising (1) a gRNA and (2) a CRISPR-associated endonuclease. In some embodiments, the EV comprises (1) a fusion protein comprising CD63 or a fragment thereof fused to at least one aptamer-binding protein (ABP); (2) an RNP comprising (a) a gRNA and (b) a CRISPR-associated endonuclease; and (3) an envelope protein or a fragment thereof (e.g., VSV-G or a fragment thereof). In some embodiments, VSV-G facilitates the entry of the EV into the transduced cell. In some embodiments, the CRISPR-associated endonuclease is catalytically impaired. In some embodiments, the catalytically impaired CRISPR-associated endonuclease is fused to adenosine deaminase as part of the ABE. Examples of CRISPR-associated endonucleases, including catalytically impaired CRISPR-associated endonucleases, are described above.

[0115] In some embodiments, the gene editing method provided herein results in an increase in the editing efficiency by CRISPR-associated endonucleases or ABEs.For example, when using any of the EVs comprising RNPs described herein, the method provided herein can increase the editing efficiency by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to the editing efficiency when RNPs are delivered using non-EV delivery.In some examples, the increase in gene editing efficiency is 2-fold, 4-fold, 10-fold, 20-fold, 50-fold or more.In some embodiments, the increase is compared to lentiviral delivery of RNPs.

[0116] In some embodiments, the transduction efficiency of using the methods provided herein to deliver RNP to cells using EVs as described herein is increased compared to non-EV delivery. In some examples, the transduction efficiency is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to the transduction efficiency when RNP is delivered to cells using non-EV delivery. In some examples, the increase in transduction efficiency is 2-fold, 4-fold, 10-fold, 20-fold, 50-fold or more. In some embodiments, the increase is compared to lentiviral delivery of RNP.

[0117] In some embodiments, the gene editing method provided herein results in a reduction in guide-independent RNA off-target gene editing events, such as associated CRISPR-associated endonucleases or ABEs. For example, when ABEs are used in the methods provided herein, guide-independent RNA off-target activity can be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to the RNA off-target activity when RNP is delivered using non-EV delivery. In some examples, guide-independent DNA off-target gene editing events are also reduced. For example, when RNP is delivered using non-EV delivery, the methods provided herein can reduce guide-dependent DNA off-target activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, 99% or more.

[0118] In some examples, the transduced eukaryotic cells are mammalian cells. In some examples, the eukaryotic cells can be in vitro cultured cells. In some examples, the eukaryotic cells can be ex vivo cells obtained from a subject. In other examples, the eukaryotic cells are present within a subject. As used throughout, subject refers to an individual. For example, the subject is a mammal, such as a primate, more specifically a human. Non-human primates are also subjects. The term subject includes domestic animals, such as cats, dogs, livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary uses as well as medical uses and formulations are contemplated herein. The term does not indicate a particular age or sex. Thus, living and newborn subjects are intended to be covered, whether male or female. As used herein, patient or subject may be used interchangeably and may refer to a subject suffering from a disease or disorder. The EVs provided by the present disclosure can be injected into subjects according to known routine methods. In some examples, the viral particles of the system are injected intravenously (IV), intraperitoneally (IP), intramuscularly, or into specific organs. The EVs can also be implanted, for example, into tumors.

[0119] In some examples, a method is provided for modifying a target locus of interest, the method comprising transducing a plurality of EVs into a plurality of eukaryotic cells, the plurality of EVs comprising a fusion protein comprising CD63 or a fragment thereof fused to at least one ABP; and a ribonucleotide protein (RNP) complex comprising (1) a gRNA and (2) a CRISPR-associated endonuclease, the RNP binds to a genomic target sequence in the genomic DNA of the cell, and the CRISPR-associated endonuclease alters the genomic DNA of the cell. As described above, an EV comprising two or more RNPs, each RNP targeting a different locus, can be used to modify two or more loci of interest in a eukaryotic cell. As described above, the RNP is packaged into the EV via the interaction of an aptamer sequence bound to or inserted into a gRNA sequence that forms a complex with a catalytically impaired CRISPR-associated endonuclease.

[0120] The described method can be used with any CRISPR-associated endonuclease that requires the constant region of sgRNA for function. These include, but are not limited to, RNA-guided site-specific nucleases. Examples include nucleases present in any bacterial species that encodes a type II or type V CRISPR / Cas system. Suitable CRISPR-associated endonucleases are described throughout this disclosure. For example, but not limited to, the site-specific nuclease can be a catalytically impaired Cas9 polypeptide, a catalytically impaired Cpf1 polypeptide, a catalytically impaired Cas9 nickase, or a derivative thereof.

[0121] Generally, sgRNAs target specific regions at or near genes. In some instances, sgRNAs can target regions where a single base change is needed, for example, to correct a single base mutation in the human beta globin gene that causes sickle cell anemia. sgRNAs allow the RNPs described herein to specific sites in the genomic sequence of a cell. When the RNPs bind to a specific site in the genomic sequence, the CRISPR-associated endonuclease cleaves one or more strands of DNA at the specific site. When ABEs are used, the ABE catalyzes adenosine (A) on one strand to inosine formation, while a catalytically impaired endonuclease, e.g., Cas9 D10A, nicks the opposite, i.e., unedited, strand. Since inosine is read as guanosine by the polymerase enzyme, DNA repair and replication machinery replaces the original AT base pair with a GC base pair at the target site. See Gaudelli et al. (2017).

[0122] In some examples, modifications to the system components described in this disclosure do not impair how the system components function after transduction into eukaryotic cells. Rather, the components may function similarly or better than unmodified components when transduced into eukaryotic cells. For example, CD63-ABP fusion protein in EVs may not interfere with EV transduction of eukaryotic cells. Similarly, if the RNP packaged in EVs contains at least one aptamer sequence, the at least one aptamer sequence may not interfere with EV transduction of eukaryotic cells. In some examples, EVs containing CD63-ABP fusion protein may result in greater gene editing when transduced into eukaryotic cells compared to EVs that do not contain CD63-ABP fusion protein.

[0123] The eukaryotic cells can be in vitro, ex vivo or in vivo. In some embodiments, the cells are primary cells (isolated from a subject). As used herein, primary cells are cells that are not transformed or immortalized. Such primary cells can be cultured, subcultured or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cultures). In some cases, the primary cells are adapted to in vitro culture conditions. In some cases, the primary cells are isolated from an organism, system, organ or tissue, optionally sorted, and utilized directly without culture or subculture. In some cases, the primary cells are stimulated, activated, or differentiated. In some embodiments, the cells are cultured under conditions effective to expand a population of modified cells. In some embodiments, the cells modified by any of the methods provided herein are purified. In some cases, cells are removed from the subject, modified using any of the methods described herein, and readministered to the patient.

[0124] In some examples, once cells are transduced with the EVs described above, the cells are cultured for a sufficient time to allow gene editing to occur so that a pool of cells expressing a detectable phenotype can be selected from the multiple transduced cells. The phenotype can be, for example, cell growth, survival or proliferation. In some examples, the phenotype is cell growth, survival or proliferation in the presence of an agent such as a cytotoxic agent, an oncogene, a tumor suppressor, a transcription factor, a kinase (e.g., a receptor tyrosine kinase), a gene (e.g., an exogenous gene) under the control of a promoter (e.g., a heterologous promoter), a checkpoint gene or a cell cycle regulator, a growth factor, a hormone, a DNA damaging agent, a drug, or a chemotherapeutic agent. The phenotype can also be protein expression, RNA expression, protein activity, or cell motility, migration or invasion. In some examples, selecting cells based on phenotype includes fluorescence activated cell sorting, affinity purification of cells, or selection based on cell motility.

[0125] In some examples, selecting the cells may also include analyzing the genomic DNA of the cells by amplification, sequencing, SNP analysis, etc. Sequencing methods include, but are not limited to, shotgun sequencing, bridge PCR, Sanger sequencing (including microfluidic Sanger sequencing), pyrosequencing, massively parallel signature sequencing, nanopore DNA sequencing, single molecule real-time sequencing (SMRT) (Pacific Biosciences, Menlo Park, CA), ion semiconductor sequencing, ligation sequencing, sequencing by synthesis (Illumina, San Diego, Ca), polony sequencing, 454 sequencing, solid-phase sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, mass spectrometry sequencing, pyrosequencing, supported oligo-ligation detection (SOLiD) sequencing, DNA microarray sequencing, RNAP sequencing, tunneling current DNA sequencing, and any other DNA sequencing methods identified in the future. One or more of the sequencing methods described herein can be used in high-throughput sequencing methods. As used herein, the term "high-throughput sequencing" refers to any method related to sequencing of nucleic acids in which more than one nucleic acid sequence is sequenced at a given time. Treatment

[0126] Any of the methods and compositions described herein can be used to treat a disease in a subject, such as cancer, a blood disorder (e.g., sickle cell anemia or beta thalassemia), an infectious disease, an autoimmune disease, transplant rejection, graft-versus-host disease or other inflammatory disorder.

[0127] In some methods, the cancer to be treated is selected from cancer of B-cell origin, breast cancer, cancer of the stomach, neuroblastoma, osteosarcoma, lung cancer, colon cancer, chronic myeloid cancer, leukemia (e.g., acute myeloid leukemia, chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL)), prostate cancer, colon cancer, renal cell carcinoma, liver cancer, kidney cancer, ovarian cancer, stomach cancer, testicular cancer, rhabdomyosarcoma, and Hodgkin's lymphoma. In some embodiments, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0128] In some methods, cells of a subject are modified in vivo, for example, by delivering any of the EVs described herein to a subject. See, e.g., Murphy et al. Experimental and Molecular Medicine 51:1-12 (2019)). In some embodiments, EVs can be targeted to cells or tissues by modifying the EVs to include a binding moiety that binds to a target, for example, a tumor antigen on a tumor cell. In some embodiments, a desired ligand can be linked to the EVs via association with polyethylene glycol (PEG), such that PEGylated EVs coated with the desired ligand can be specifically targeted to cell surface targets. See, e.g., Rocco et al. Stem Cells International, vol. 2016, Article ID 5029619, 12 pages, 2016. In some embodiments, an effective amount of any of the EVs described herein is administered to a subject. As used herein, an "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. In some embodiments, the EVs are delivered in a pharmaceutical composition. Typically, such pharmaceutical compositions are formulated for in vivo, ex vivo or in vitro use using pharma- ceutically acceptable excipients.

[0129] The dosage of EVs administered to a subject depends on the disease or symptoms to be treated or alleviated, the route of administration, as well as various other relevant parameters known to those skilled in the art. The amount of EVs administered to a subject can be determined by quantifying EV proteins, such as CD63, with the bicinchoninic acid (BCA) method. The amount of protein delivered to a subject can be determined by Western blot detection of CRISPR-associated endonucleases, such as Cas9, in the EVs, respectively. It is understood that populations of EVs are also provided herein. The EV concentration in any of the compositions described herein can be expressed in many different ways, such as the amount of EV protein per unit (often by volume), or per dose, the number of EVs or particles per unit (often by volume, per subject, per kg of body weight, etc.). For example, but not limited to, about 10 6 ~about 10 25 A composition containing EVs can be administered to a subject in one or more doses. For example, 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 , 10 21 , 10 22 , 10 23 , 10 24 , 10 25 Or any other amount between these amounts, a composition containing EVs can be administered to a subject in one or more administrations.

[0130] In some methods, the method of treating a disease in a subject includes a) obtaining cells from a subject; b) modifying the cells using any of the methods provided herein; and c) administering the modified cells to a subject. In some methods, the modified cells are grown and / or differentiated before being administered to a subject. In some embodiments, the modification occurs by contacting a eukaryotic cell with any of the EVs described herein, and the RNP (i.e., a complex containing a CRISPR-associated endonuclease and a gRNA) delivered to the cell by the EV binds to a site in the genome targeted by the sgRNA and modifies the genome of the cell. In some embodiments, the cell is modified to, for example, correct a mutation, insert a functional copy of a gene, insert a nucleic acid sequence encoding a tumor antigen, edit bases, or otherwise change the genomic sequence of the cell to treat a disease.

[0131] Optionally, the disease is selected from the group consisting of cancer, blood disorder (e.g., sickle cell anemia or beta thalassemia), infectious disease, autoimmune disease, transplant rejection, graft-versus-host disease or other inflammatory disorder in the subject. In some methods for treating cancer, cells obtained from the subject are modified to express tumor-specific antigens. As used throughout, the phrase "tumor-specific antigen" refers to an antigen that is unique to cancer cells or that is more abundantly expressed in cancer cells than in non-cancerous cells. Optionally, the cells obtained from the subject are T cells. Optionally, the modified cells are expanded before administration to the subject.

[0132] All patents, patent publications, patent applications, journal articles, books, technical references, and the like, discussed in this disclosure are hereby incorporated by reference in their entirety for all purposes.

[0133] It is to be understood that the figures and descriptions of the present disclosure are simplified to show elements relevant to a clear understanding of the present disclosure. It is to be understood that the drawings are presented for illustrative purposes and are not presented as structural diagrams. The omitted details and modified or alternative embodiments are within the understanding of those skilled in the art.

[0134] It will be understood that in certain aspects of the disclosure, multiple components may be substituted for single components, and multiple components may be substituted for single components, to provide an element or structure or to perform a given function(s). Except where such substitution would not be operative to practice a particular embodiment of the disclosure, such substitution is deemed to be within the scope of the disclosure.

[0135] The examples presented herein are intended to illustrate potential and specific embodiments of the present disclosure. It will be understood that the examples are intended primarily to illustrate the present disclosure for those skilled in the art. There may be variations in these figures or the operations described herein without departing from the spirit of the present disclosure. For example, in certain cases, method steps or operations may be performed or executed in a different order, or operations may be added, deleted, or modified.

[0136] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed, to the smallest fraction of the unit of the lower limit, unless the context clearly dictates otherwise. Any smaller range between any stated or unstated intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either or both limits are included in the smaller range or neither limit is included, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0137] Different arrangements of the components shown in the drawings or described above, as well as components and steps not shown or described, are possible. Similarly, some features and subcombinations are useful and can be employed without reference to other features and subcombinations. The embodiments of the present disclosure have been described for purposes of illustration and not limitation, and alternative embodiments will become apparent to the reader of this patent. Thus, the present disclosure is not limited to the embodiments described above or shown in the drawings, and various embodiments and modifications can be made without departing from the scope of the following claims.

[0138] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties. Exemplary embodiments

[0139] Exemplary embodiments of the present invention include:

[0140] 1. A plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising: (i) a nucleic acid sequence encoding a CRISPR-associated endonuclease, and (ii) a guide RNA (gRNA) coding sequence comprising at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, wherein an aptamer binding protein (ABP) binds to the at least one aptamer coding sequence of the first mammalian expression plasmid.

[0141] 2. The plasmid system of embodiment 1, further comprising an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

[0142] 3. The plasmid system of embodiment 1 or 2, wherein the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein or a derivative thereof.

[0143] 4. The plasmid system of any one of embodiments 1 to 3, wherein the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease.

[0144] 5. The plasmid system of embodiment 4, wherein the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.

[0145] 6. The plasmid system of embodiment 4 or 5, wherein the nucleic acid sequence of the first mammalian expression plasmid encodes a nucleic acid sequence encoding an adenosine base pair editor (ABE), wherein the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease.

[0146] 7. The plasmid system of embodiment 6, wherein the adenine base editor is ABE 7.10 or ABE8.

[0147] 8. The plasmid system of any one of embodiments 1 to 7, wherein at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein.

[0148] 9. The plasmid system of embodiment 8, wherein the aptamer sequence is an MS2 aptamer sequence, a pp7 aptamer, a Box-B aptamer, or a com aptamer sequence.

[0149] 10. The plasmid system of any one of embodiments 1 to 9, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same.

[0150] 11. The plasmid system of embodiment 10, wherein the first and second ABPs are Com-binding proteins.

[0151] 12. The plasmid system of any one of embodiments 1-11, wherein the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence.

[0152] 13. The plasmid system of embodiment 12, wherein the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence.

[0153] 14. An extracellular vesicle comprising: (a) a ribonucleotide-protein (RNP) complex comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein (ABP), wherein the ABP binds to the at least one aptamer coding sequence.

[0154] 15. The extracellular vesicle of embodiment 14, further comprising a VSV-G protein.

[0155] 16. The extracellular vesicle of embodiment 14 or 15, wherein the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein or a derivative thereof.

[0156] 17. The extracellular vesicle of any one of embodiments 14 to 16, wherein the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease.

[0157] 18. The extracellular vesicle of embodiment 17, wherein the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.

[0158] 19. The extracellular vesicle of embodiment 17 or 18, wherein the RNP comprises an adenine base pair editor (ABE), and the ABE is a fusion protein comprising adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease.

[0159] 20. The extracellular vesicle of embodiment 19, wherein the adenine base editor is ABE 7.10 or ABE8.

[0160] 21. The extracellular vesicle according to any one of embodiments 14 to 20, wherein at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein.

[0161] 22. The extracellular vesicle of embodiment 21, wherein the aptamer sequence is an MS2 aptamer sequence or a com aptamer sequence.

[0162] 23. The extracellular vesicle according to any one of embodiments 14 to 22, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same.

[0163] 24. The extracellular vesicle of embodiment 23, wherein the first and second ABPs are Com-binding proteins.

[0164] 25. The extracellular vesicle according to any one of embodiments 14 to 24, wherein the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence.

[0165] 26. The extracellular vesicle of embodiment 25, wherein the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence.

[0166] 27. An extracellular vesicle according to any one of embodiments 14 to 26, wherein the extracellular vesicle is an exosome or a microvesicle.

[0167] 28. A method for producing extracellular vesicles, comprising: A method comprising: (a) transfecting a plurality of eukaryotic cells with a first mammalian expression plasmid and a second mammalian expression plasmid of the system according to any one of embodiments 1 to 13; and b) culturing the transfected eukaryotic cells for a sufficient period of time to produce extracellular vesicles.

[0168] 29. The method according to embodiment 28, further comprising transfecting a plurality of eukaryotic cells with an envelope plasmid of the system according to any one of embodiments 2 to 13.

[0169] 30. The method according to embodiment 28 or 29, wherein the extracellular vesicles comprise: (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein, wherein the aptamer-binding protein (ABP) binds to the at least one aptamer coding sequence.

[0170] 31. The method according to embodiment 29, wherein the extracellular vesicles are A method comprising: (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the aptamer coding sequence; and (c) a VSV-G protein.

[0171] 32. The method of any one of embodiments 28 to 31, wherein the plurality of eukaryotic cells is a mammalian cell.

[0172] 33. The method of any one of embodiments 28 to 32, further comprising isolating extracellular vesicles from the cultured transfected eukaryotic cells.

[0173] 34. Extracellular vesicles produced by the method according to any one of embodiments 28 to 33.

[0174] 35. A method for modifying a genomic target sequence in a cell, comprising transducing a plurality of eukaryotic cells with a plurality of extracellular vesicles, wherein the plurality of extracellular vesicles comprises an extracellular vesicle described in any one of embodiments 14 to 27, and wherein the RNP binds to a genomic target sequence in the genomic DNA of the cell, thereby modifying the genomic target sequence.

[0175] 36. The method of embodiment 35, wherein the plurality of eukaryotic cells are mammalian cells.

[0176] 37. The method of embodiment 35 or 36, wherein the plurality of eukaryotic cells are cells present within a subject.

[0177] 38. The method of embodiment 37, wherein the subject is a human subject.

[0178] 39. The method of embodiment 38, wherein the subject is injected with a plurality of extracellular vesicles.

[0179] 40. A cell comprising a plasmid system according to any one of embodiments 1 to 13.

[0180] 41. A cell modified using the method according to any one of embodiments 35 to 39.

[0181] 42. A method for treating a disease in a subject, comprising:

[0182] A method comprising: a) obtaining cells from a subject; b) modifying the cells of the subject using a method according to any one of embodiments 35 to 39; and c) administering the modified cells to the subject.

[0183] 43. The method of embodiment 42, wherein the disease is cancer.

[0184] 44. The method of embodiment 42, wherein the disease is sickle cell anemia.

[0185] 45. The method of any one of embodiments 42 to 44, wherein the cell is a T cell.

[0186] 46. ​​A plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, the nucleic acid sequence comprising (i) a nucleic acid sequence encoding a heterologous polypeptide, and (ii) at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, wherein an aptamer binding protein (ABP) binds to the at least one aptamer coding sequence of the first mammalian expression plasmid.

[0187] 47. The plasmid system of embodiment 46, wherein the nucleic acid sequence of the first mammalian expression plasmid encodes a CRISPR-associated endonuclease and further comprises a guide RNA (gRNA) coding sequence.

[0188] 48. The plasmid system of embodiment 46, wherein the nucleic acid sequence encoding the heterologous polypeptide comprises at least one aptamer coding sequence.

[0189] 49. The plasmid system of embodiment 47, wherein the gRNA coding sequence comprises at least one aptamer coding sequence.

[0190] 50. The plasmid system of any of embodiments 46 to 49, further comprising an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

[0191] 51. The plasmid system of embodiment 47, wherein the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein or a derivative thereof.

[0192] 52. The plasmid system of embodiment 47, wherein the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease.

[0193] 53. The plasmid system of embodiment 52, wherein the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.

[0194] 54. The plasmid system of embodiment 52 or 53, wherein the nucleic acid sequence of the first mammalian expression plasmid encodes a nucleic acid sequence encoding an adenosine base pair editor (ABE), and the ABE is a fusion protein comprising an adenosine deaminase and a catalytically impaired CRISPR-associated endonuclease.

[0195] 55. The plasmid system of embodiment 54, wherein the adenine base editor is ABE 7.10 or ABE8.

[0196] 56. A plasmid system according to any one of embodiments 46 to 55, wherein at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein.

[0197] 57. The plasmid system of embodiment 56, wherein the aptamer sequence is an MS2 aptamer sequence, a pp7 aptamer, a Box-B aptamer, or a com aptamer sequence.

[0198] 58. The plasmid system of any one of embodiments 46 to 57, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same.

[0199] 59. The plasmid system of embodiment 58, wherein the first and second ABPs are Com-binding proteins.

[0200] 60. The plasmid system of any one of embodiments 49 to 59, wherein the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into the tetraloop or ST2 loop of the sgRNA coding sequence.

[0201] 61. The plasmid system of embodiment 60, wherein the sgRNA coding sequence comprises at least one com aptamer sequence inserted into the tetraloop or ST2 loop of the gRNA coding sequence.

[0202] 62. An extracellular vesicle comprising: (a) an mRNA encoding a heterologous polypeptide and at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein (ABP), wherein the ABP binds to the at least one aptamer coding sequence.

[0203] 63. The extracellular vesicle of embodiment 62, further comprising a VSV-G protein.

[0204] 64. The extracellular vesicle of embodiment 62, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, wherein the first and second ABPs are the same.

[0205] 65. The extracellular vesicle of claim 64, wherein the first and second ABPs are Com-binding proteins.

[0206] 66. Extracellular vesicles according to any one of embodiments 62 to 65, wherein the extracellular vesicles are exosomes or microvesicles.

[0207] 67. A method for producing extracellular vesicles, comprising: A method comprising: (a) transfecting a plurality of eukaryotic cells with a first mammalian expression plasmid and a second mammalian expression plasmid of the system described in any one of embodiments 46 to 61; and b) culturing the transfected eukaryotic cells for a sufficient time to produce extracellular vesicles.

[0208] 68. The method of embodiment 67, wherein the nucleic acid sequence of the first mammalian expression plasmid encodes a CRISPR-associated endonuclease and further comprises a guide RNA (gRNA) coding sequence.

[0209] 69. The method of embodiment 67 or 68, wherein the nucleic acid sequence encoding the heterologous polypeptide comprises at least one aptamer coding sequence.

[0210] 70. The method of embodiment 68, wherein the gRNA coding sequence comprises at least one aptamer coding sequence.

[0211] 71. The method of any one of embodiments 67 to 70, further comprising transfecting a plurality of eukaryotic organisms with an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

[0212] 72. The method according to embodiment 71, wherein the extracellular vesicles are 1. A method comprising: (a) an mRNA encoding a heterologous polypeptide and at least one aptamer sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein, wherein the aptamer-binding protein (ABP) binds to the at least one aptamer-coding sequence.

[0213] 73. The method according to embodiment 71, wherein the extracellular vesicles are A method comprising: (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer coding sequence; (b) a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the aptamer coding sequence; and (c) a VSV-G protein.

[0214] 74. The method of any one of embodiments 67 to 73, wherein the plurality of eukaryotic cells is a mammalian cell.

[0215] 75. The method of embodiment 74, further comprising isolating extracellular vesicles from the cultured transfected eukaryotic cells.

[0216] 76. Extracellular vesicles produced by a method according to any one of embodiments 67 to 75.

[0217] 77. A method for modifying a genomic target sequence in a cell, comprising transducing a plurality of eukaryotic cells with a plurality of extracellular vesicles, wherein the plurality of extracellular vesicles comprises an extracellular vesicle according to any one of embodiments 62-66 and 76, and wherein the RNP binds to a genomic target sequence in the genomic DNA of the cell, thereby modifying the genomic target sequence.

[0218] 78. The method of embodiment 77, wherein the plurality of eukaryotic cells are mammalian cells.

[0219] 79. The method of embodiment 78, wherein the plurality of eukaryotic cells are cells present within a subject.

[0220] 80. The method of embodiment 79, wherein the subject is a human subject.

[0221] 81. The method of embodiment 80, wherein the subject is injected with a plurality of extracellular vesicles.

[0222] 82. A cell comprising a plasmid system according to any one of embodiments 46 to 61.

[0223] 84. A cell modified using the method according to any one of embodiments 77 to 81.

[0224] 85. A method of treating a disease in a subject, comprising: a) obtaining cells from a subject; b) modifying cells of a subject using a method according to any one of embodiments 77 to 81; and c) administering the modified cells to a subject.

[0225] 86. The method of embodiment 85, wherein the disease is cancer.

[0226] 87. The method of embodiment 85, wherein the disease is sickle cell anemia.

[0227] 88. The method of embodiment 86 or 87, wherein the cell is a T cell. EXAMPLES

[0228] Example 1. Materials and Methods Plasmids. CD63-pEGFP-C2 (Addgene#62964) and pMD2.G (Addgene#12259) were purchased from Addgene. Plasmids generated for the studies described herein are provided in Table 2. Gene synthesis was performed by GenScript Inc. (Piscataway NJ). All generated constructs were confirmed by Sanger sequencing. Sequence information for primers, oligos and synthetic DNA fragments is provided in Table 3. Target sequences for sgRNAs are listed in Table 4. All generated constructs were confirmed by Sanger sequencing. Sequence information for primers and oligonucleotides is listed in Tables 3 and 4. It is understood that the sequences of the components of the plasmids listed in Table 2 can be separated by a nucleic acid linker, e.g., a linker of about 2-100 bases. If desired, any of the constructs described herein can include one or more introns, e.g., between the promoter sequence and the nucleic acid encoding the polypeptide sequence (e.g., CD63, ABE, etc.), to facilitate expression of one or more polypeptide sequences in the construct. [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0229] GFP reporter assay for gene editing activity. HEK293T-derived HBB-IL2RG EGFP reporter cells with target sequences for human beta-hemoglobin (HBB) sickle cell mutant and human IL2RG (Javidi-Parsijani et al. PLoS One 12, e0177444 (2017)), as well as DMD reporter cells with target sequences from human DMD exon 53 (Lyu et al., PLoS One 15, e0239468 (2020)) were used to detect the gene editing activity of Cas9 RNPs targeting HBB, IL2RG and DMD exon 53, respectively. The GFP reporter cells did not express EGFP due to the disruption of the EGFP reading frame by inserting the respective target sequences immediately after the start codon of the EGFP coding sequence. INDELs formed after gene editing could restore the EGFP reading frame, resulting in EGFP expression. GFP-positive cells were analyzed by fluorescence microscopy or flow cytometry (BD Biosciences, Accuri C6, San Jose, Calif.).

[0230] Production of Cas9 or ABE RNP-enriched extracellular vesicles (EVs). RNP-enriched EVs were produced by co-transfection of three plasmids: plasmid DNA expressing a fusion protein between ABP Com and CD63, pDM2.G expressing VSV-G, and target plasmids expressing gene editing effectors (SaCas9, SpCas9 or ABE) and their respective gene-specific sgRNAs into HEK293T cells (see Table 2 for various target plasmids). Briefly, 5 million actively growing HEK293T cells grown in 10 cm dishes were incubated with 5 ml of Opti-MEM® medium. 3 μg of Com-CD63-Com fusion protein expression plasmid, 3 μg of pMD2.G and 12 μg of target plasmid DNA were mixed in 0.5 ml of Opti-MEM® medium. 54 μl of Fugene HD (Promega, Madison, WI) or 54 μg of polyethyleneimine (Synchembio, Cat. No. SH-35421, Chicago, IL) were mixed in 0.5 ml of Opti-MEM® medium. The DNA mixture and transfection reagent mixture were then mixed and incubated at room temperature for 15 minutes before being added to the cells in Opti-MEM® medium. 24 hours after transfection, the medium was replaced with 10 ml of Opti-MEM® medium, and RNP-enriched EVs were harvested 72 hours after transfection. For transfection of cells grown in other tissue culture vessels, the amount of DNA and transfection reagent was scaled based on the tissue culture surface area.

[0231] EV concentration. Ultracentrifugation was used to enrich EVs from tissue culture medium following the procedure described by Lu et al. (PloS One 12,e e0185992(2017)). Briefly, cell culture medium was centrifuged at 1000 × g for 30 min at 4 °C to remove cellular debris. The supernatant was centrifuged at 120,000 × g for 70 min at 4 °C. The pellet was washed once with PBS and centrifuged again under the same conditions. The resulting pellet containing EVs was resuspended in PBS. Typically, for in vitro experiments, EVs from 10 ml of supernatant were resuspended in 500 μl (20X concentration). These EVs can be stored at -80 °C or used immediately.

[0232] Nanoparticle tracking analysis of EVs. Hydrodynamic diameter and concentration of EVs were measured using a Nanosight NS500 instrument (Malvern Instruments, UK) using the instrument's software (version NTA3.2). The instrument was primed with phosphate-buffered saline (PBS), pH 7.4, and the temperature was maintained at 25 °C. Accurate particle tracking was verified using 50 nm and 100 nm polystyrene nanoparticle standards (Malvern Instruments) before samples were examined. Concentrated samples containing EVs were serially diluted 1000-fold in PBS. The linear range for quantification of EV concentration in each sample was between 10 and 40,000-fold dilution. Therefore, all samples were diluted 1,000-fold in PBS. Five independent measurements (60 s each) were obtained in triplicate for each sample. Data are reported as the mean of these measurements (multiplied by the dilution factor for concentration determination) ± standard error of the mean.

[0233] EV-mediated RNP delivery. RNP-enriched EVs enriched from the supernatant of 6–20 million cells were cultured in 2.5x10 cells grown in Opti-MEM (Cat. No. 11058021, ThermoFisher, Waltham, MA) in 24-well plates. 4EVs were added to the cells. It is important that the medium is low in serum, as the presence of FBS in the medium inhibits EV-mediated RNP delivery. After 12–24 h of incubation, the medium was replaced with DMEM medium containing 10% FBS. 36 h after EV treatment, gene editing was analyzed by flow cytometry, fluorescence microscopy or next-generation sequencing (NGS).

[0234] Investigating the degradation of EV-delivered Cas9. HEK293T cells (2.5x10 4 EVs (approximately 8x10 cells) were grown in RMPI1640 medium containing 0.5% FBS in 24-well plates to limit proliferation. Cells were treated with RNP-loaded EVs at different times but harvested at the same time. EVs secreted by 200,000 cells in 48 h (approximately 8x10 cells) were 9 vesicles) were added to each well. Just before EV treatment, the medium was replaced with Opti-MEM® medium. Cells were harvested 6, 12, 18, 24, 36 and 48 hours after EV addition, washed twice with PBS buffer and dissolved in Laemmli buffer for Western blot analysis.

[0235] Western blotting analysis of EVs. 5x10 at 48 hours 6EVs secreted by cells were concentrated and resuspended in 500 μl of PBS. 60 μl of EV solution was mixed with an equal volume of 2× Laemmli buffer and boiled at 95°C for 5 min. 10 μl of each sample was loaded into each lane of a 10% SDS-PAGE gel for Western blot analysis. The antibodies used included mouse monoclonal anti-SaCas9 antibody (Sigma, Cat. MAB131872, clone 6F7, 1:1000 (St. Louis, MO)), GAPDH antibody (CST, 1:1000), RAB5B antibody (Abcam, Cat. ab230020, 1:1000 (Cambridge, UK)), CD9 antibody SBI, Cat. EXOAB-CD9A-1, 1:1000 (Palo Alto, CA)), VSV-G antibody (Sigma, Cat. V4888, 1:1000), CD63 antibody (Abcam, Cat. ab68418, 1:1000), GRP94 antibody (CST, Cat. 20292T, 1:1000 (Danvers, MA)), and anti-rabbit IgG (H+L) (Cat. No. 31460, 1:5000) secondary antibody was included. A chemiluminescence reagent (Pierce ECL Western Blotting Substrate, Cat. 32106 (Waltham, MA)) was used to visualize protein signals under a LAS-3000 system (Fujifilm, Valhalla, NY). Densitometry (ImageJ software (version 1.49), National Institutes of Health, imagej.nih.gov / ij / index.html) was used to quantify protein amounts based on the Western blotting images.

[0236] RT-qPCR and qPCR analysis. RNA was isolated from collected EVs using RNeasy® Plus Mini Kit (QIAGEN Cat No. 74136 (Hilden, Germany)). RNA was reverse transcribed into cDNA using QuantiTect® Reverse Transcription Kit (QIAGEN). For HBB sgRNA1 and HBB sgRNA1 Tetra-com detection, sgRNA-F1 and sgRNA-R3 were used as primers in SYBR™ Green-based RT-qPCR. ABE-g5-onF and g5-ABE-R were used as qPCR primers to detect base editing at site 5. PCR was performed on an ABI 7500 instrument. Primer information is included in Table 3.

[0237] Transmission electron microscopy. Transmission electron microscopy was performed at the Cellular Imaging Shared Resource at Wake Forest Baptist Health Center (Winston-Salem, NC). The collected EVs (approximately 6.0 × 10 11 Vesicles (0.01 vesicles / ml) were stained with uranyl acetate. Particles were absorbed onto flat carbon grids, dried, and viewed under an FEI Tecnai G2 30 electron microscope (FEI, Hillsboro, OR). Particle diameters were measured using NIH ImageJ software (version 1.49).

[0238] Next-generation sequencing and data analysis. Genomic DNA was isolated from cultured cells using the DNeasy Blood & Tissue Kit (Qiagen). DNA regions containing target sequences were amplified by proofreading HotStart® ReadyMix from KAPA Biosystems (Wilmington, MA). PCR primers used to amplify each target sequence are listed in Table 3. Purified PCR products were shipped to Genewiz Inc. (Morrisville, NC) for next-generation sequencing using Amplicon EZ service. Typically, 50,000 reads / amplicon were obtained. Analysis of insertions and deletions (INDELs) was performed using the online Cas-Analyzer software28 and CRISPRESSO229, with very similar results.

[0239] Intramuscular injection of RNP-loaded EVs into mouse tibialis anterior (TA) muscles. Five-month-old female del52hDMD / mdx mice were used for injections. These mice carry a copy of the human DMD gene with an exon 52 deletion in the mdx background (Veltrop, M. et al., PLoS One 13, e0193289 (2018)). EVs secreted by 5 million cells (low dose) or 40 million cells (high dose) in 48 hours were injected into each TA muscle using a Hamilton syringe. Mice were sacrificed 7 days after injection. TA muscles were removed for cryosectioning. Tissues were mounted in OCT embedding compound and frozen in liquid nitrogen. Six or seven blocks of three serial 10 μm thick sections 200 μm apart from each block were collected for histological analysis. The remaining tissue was used to purify genomic DNA for NGS analysis.

[0240] Immunohistochemistry assay. Sections were fixed in 4% paraformaldehyde for 10 min. After three PBS washes, sections were incubated in 0.2% Triton® X-100 PBS buffer for 10 min and Protein Block (Cat.X090930-2, Agilent) for 30 min. Sections were then incubated with anti-dystrophin primary antibody (Abcam, ab3068050, 1:10000) for 1 h. Alexa Fluor® 633 secondary antibody (A-21052, Thermo Fisher, 1:5000) was then incubated with the tissue for 45 min after three PBS washes. Slides were then washed six times with PBS and mounted in DAPI-containing mounting medium for 5 min. Slides were observed under a fluorescent microscope.

[0241] Statistical analysis. GraphPad Prism software (version 5.0) was used for statistical analysis. T-tests were used to compare the means of two groups. Analysis of variance (ANOVA) was performed followed by Tukey's post-hoc test to analyze data from more than two groups. p<0.05 was considered statistically significant. Example 2. CD63, aptamer and ABP-mediated RNP enrichment in extracellular vesicles (EVs)

[0242] The above experiments were used to determine whether specific interactions between Com and Com can enrich RNPs into EVs. CD63 is a tetraspan transmembrane protein with N- and C-termini in the cytoplasm. Com was fused to the N-, C- or both termini of CD63 so that Com faces the cytoplasmic side of the plasma membrane (Figure 1A). During exosome generation, Cas9 or ABE RNPs were hypothesized to be enriched in exosomes through interactions between CD63-Com, com-sgRNA and Cas9 (Figure 1B). Alternatively, RNPs could also be enriched into microvesicles through outward budding and fission of membrane vesicles from the cell surface (Figure 1C). In these studies, it is understood that all vesicular structures produced, including exosomes and microvesicles, are referred to as EVs regardless of the mechanism of vesicle generation.

[0243] We investigated whether fusion of Com affects CD63 expression and found that fusion of Com to either end of CD63 did not impair CD63 expression. Indeed, fusion of Com to the C-terminus significantly increased CD63 expression (Figure 2A). We also tested whether Com fusion to CD63 allows packaging of SaCas9 RNP into EVs. Because EV escape from cells can be limited, we expressed VSV-G in EV-producing cells so that EVs have the VSV-G protein to facilitate escape from the endosomal system in recipient cells. Com-CD63, CD63-Com or Com-CD63-Com were co-expressed with VSV-G, SaCas9 and com-modified IL2RG-targeting sgRNA 12 in HEK293T cells. EVs were collected from the supernatant of transfected cells and concentrated by ultracentrifugation. Resuspended EVs were added to the medium of HBB-IL2RG GFP reporter cells as previously described (Javidi-Parsijani 2017). These cells did not express EGFP due to the disruption of the EGFP reading frame by the insertion of the HBB sickle mutation and the IL2RG targeting sequence 119 nt immediately after the start codon of the EGFP coding sequence. The INDELs formed after gene editing could restore the EGFP reading frame and result in EGFP expression.

[0244] Flow cytometry analysis revealed that without the aptamer-binding protein Com or aptamer com, few GFP-positive cells could be observed, indicating random packaging of RNPs into EVs. When sgRNAs were modified with aptamer com, Com-CD63-Com generated the most GFP-positive reporter cells, followed by CD63-Com and Com-CD63 (Figure 2B). CD63-Com had a stronger expression level than Com-CD63, and EV-delivered RNPs also generated more GFP-positive cells than Com-CD63. Although the expression of Com-CD63-Com was weaker than that of CD63-Com, its EV-associated RNPs generated significantly more GFP-positive cells. The data suggested that Com at both termini had a synergistic effect on the recruitment of RNPs. The data indicated that both ABP(Com) and aptamer(com) were necessary to generate EV-associated RNPs with high gene editing activity.

[0245] Com-CD63-Com produced EV-associated RNPs with the highest gene editing activity and was therefore used in further experiments. Different ratios of Com-CD63-Com and plasmid DNA expressing Cas9 RNP were tested, and the best activity was obtained when they were in a mass ratio of 1:4 (Figure 3).

[0246] VSV-G was co-expressed in EV-producing cells to help EVs escape from the endosomal system of recipient cells. To confirm whether VSV-G is required for functional delivery of EV-associated RNPs, we generated EV-associated RNPs in the absence of VSV-G. These RNPs produced background levels of GFP-positive reporter cells (Figure 2C). We tested whether fusing Com to the C-terminus of VSV-G could further increase gene editing activity. This fusion significantly reduced the gene editing activity of EV-associated RNPs. Two possibilities could underlie this observation: 1) fusing Com to the C-terminus of VSV-G reduced the expression of VSV-G by more than 50% (Figure 4); 2) doing so could interfere with the fusion activity of VSV-G.

[0247] The data showed that RNPs are enriched in EVs and can be functionally delivered by EVs. Furthermore, aptamer Com, ABP Com, and optionally VSV-G protein can be used for the functional delivery of RNPs by EVs.

[0248] We also tested whether SpCas9 RNP could be packaged and delivered by EVs. Replacing the ST2 loop with the com aptamer best preserved the function of the SpCas9 sgRNA and allowed efficient delivery of SpCas9 RNP by lentivirus-like particles. Thus, a similarly modified sgRNA was used to package SpCas9 RNP into EVs. Various Com-CD63 fusion proteins, VSV-G, SpCas9, and ST2-com modified IL2RG targeting sgRNA were co-expressed in HEK293T cells. The resulting EVs were applied to HBB-IL2RG GFP reporter cells. SpCas9 RNP was efficiently packaged by Com-CD63-Com, and both the ABP and the aptamer were required for the best gene editing activity (Figure 2D). Thus, the ABP / aptamer interaction can be used to package SpCas9 RNP into EVs.

[0249] Adenine base editor (ABE) RNPs targeting ABE site 5 (GRCh38.p13, chromosome 20, 32752960-32752979) were packaged into EVs. qPCR showed that EV-delivered site 5 ABE RNPs produced 24.3 ± 0.7-fold (n = 3) more base-edited products compared to EV-delivered non-targeted ABE RNPs. The data indicate that EVs can also concentrate and deliver ABE RNPs.

[0250] Nuclear export of sgRNA driven by the U6 promoter can be inefficient. An RNA polymerase II promoter-driven sgRNA flanked by hammerhead (HH) ribozymes and hepatitis delta virus (HDV) ribozymes was used in several studies. This design has been shown to generate mature sgRNA after ribozyme cleavage (Yoshioka et al., Sci Rep 5, 18341 (2015)). This design only slightly increased the gene editing activity of EV-delivered RNPs (Figure 5). This suggested that sgRNA nuclear export was not a limiting factor in these studies. It is possible that aptamer-modified sgRNAs provide an active concentration mechanism for recruiting sgRNAs to membrane vesicles. Example 3. Com / com interactions enriched RNPs in EVs

[0251] We investigated whether Com / com interactions enriched RNPs in EVs by Western blotting. EVs loaded with sgRNA-containing RNPs were prepared with and without the com aptamer. SaCas9, SpCas9 and ABE content were detected in each EV. Two to five times more Cas9 or ABE protein was detected in RNPs with com-modified sgRNA (com+) compared to RNPs with unmodified sgRNA (com-), while the amount of Com-CD63-Com protein was similar (Figure 6A-6B). Consistent with these observations, the presence of Com and com packaged the most sgRNA in EVs (Figure 6C). The data showed that Com / com interactions enriched RNPs in EVs, but there was random packaging of RNPs. Using the Pierce™ BCA Protein Assay Kit, we found that one million transfected cells secreted approximately 625 ng of total EV protein in 48 hours. Among them, 65 ng of SaCas9, 80 ng of SpCas9 and 35 ng of ABE protein were detected based on Western blotting of known concentrations of Cas9 protein (Figures 6A-6B). Example 4. EV-delivered RNPs achieved efficient gene editing on multiple targets in different cells

[0252] Genome editing activity of EV-delivered RNPs targeting various targets in different cells was confirmed by NGS. As shown in Table 5, INDELs were observed at different lentiviral integration targets in GFP reporter cells, HEK293T cells where EVs were generated, and MDA-MB-231 cells, which are different from EV source cells and difficult to transfect. A to G change at site 5 in different cells treated with EV-delivered ABE RNP was also detected (Table 5). [Table 5]

[0253] Based on the estimation of the production rate of EV-associated RNPs (Figure 6A-B), the amount of EV-delivered RNP used in these experiments ranged from 0.13 to 6.4 µg per 10 cells. The highest dose was lower than the dose (10 µg) used in typical electroporation experiments. Example 5. Characterization of EVs by RNP enrichment

[0254] We investigated whether overexpressed Com-CD63-Com affected EV biogenesis by isolating EVs from cells with and without Com-CD63-Com overexpression and examining protein expression by Western blotting. The expression of exosome marker proteins CD9 and RAB5B was found not to be reduced from EVs isolated from cells with Com-CD63-Com overexpression (Figure 7A), suggesting that Com-CD63-Com overexpression did not reduce overall EV biogenesis. Furthermore, overexpression of Cas9 RNP also did not impair EV biogenesis. Com-CD63-Com overexpression significantly increased the detection of CD63-reactive antigen in EVs. The observed CD63 size was much larger than expected, and the band appeared diffuse. Both observations were the result of heterogeneous glycosylation of CD63. For the same reason, the size difference between endogenous CD63 and overexpressed Com-CD63-Com was not obvious. Compared with cellular Com-CD63-Com (Figure 2A), Com-CD63-Com in EVs appeared obviously larger and more heterogeneous in size. CD63 protein in EVs appeared to have a high degree of glycosylation. As expected, VSV-G and Cas9 were observed only in EVs from cells overexpressing the respective proteins. GRP94, a protein not involved in the endosomal pathway, was not observed in EV preparations, suggesting minimal cellular protein contamination.

[0255] Transmission electron microscopy was performed to examine the morphology of EVs isolated from cells without CD63 overexpression, with CD63 or Com-CD63-Com overexpression (all cells overexpressed VSV-G and Cas9 RNP). No differences were observed in the morphology of EVs isolated from these cells (Figure 7B). We performed nanoparticle tracking analysis (Nanosight) to examine the particle number and size of EVs isolated from cells and found that overexpressing Com-CD63-Com and Cas9 RNP slightly reduced the total number of EV particles generated (Figure 7C). Furthermore, overexpressing Com-CD63-Com and Cas9 RNP altered the EV size distribution, decreasing EVs with a diameter of 100 nm and increasing EVs with a diameter of 200 nm (Figure 7D). The right shift in size distribution explained why the total EV number was reduced with Com-CD63-Com overexpression, but CD9 and RAB5B expression in EVs was not reduced. Example 6. Short half-life of Cas9 RNP delivered by EVs

[0256] To investigate the fate of EV-delivered RNP in human cells, EV-delivered, SpCas9 RNP targeting DMD exon 53 was added to HEK293T cultures and cells were harvested at different times after EV addition. Western blotting revealed that 6 h after delivery, SpCas9 protein levels were highest, after which SpCas9 protein levels rapidly decreased (Figure 8A). Densitometry analysis revealed that 18 h after delivery, Cas9 levels were only 10% of the Cas9 levels 6 h after delivery (Figure 8B). The half-life of EV-delivered SpCas9 protein in human cells was estimated to be 3 h.

[0257] Similar experiments were performed using EV-delivered IL2RG-targeted SaCas9 RNP, and similar rapid degradation of the RNP (Figure 5C) was observed. Thus, the Cas9 RNP delivered by EVs had a very short half-life. This short half-life ensures the specificity and safety of the EV-delivered RNP. Example 7. Single preparation of RNP-enriched EVs for multiplex gene editing

[0258] Because each CD63 molecule is fused to two Com molecules and each EV can have more than one Com-CD63-Com molecule, it was reasoned that EVs may be ideal for delivery of RNPs to target multiple loci simultaneously. To remove the 2361 bp between the two target sites (Figure 9A), two SaCas9 RNPs were tested, targeting DMD intron 50 (Sa-50) and intron 51 (Sa-51), respectively. EVs loaded with an RNP targeting DMD intron 50, an RNP targeting DMD intron 51, and an RNP targeting both introns were prepared. EVs loaded with RNPs targeting both introns were prepared in a single EV preparation using only half of each target plasmid DNA. Two individually packaged RNPs were used together to compare with copackaged RNPs for exon 51 removal. All RNP-loaded EVs were prepared in parallel and similarly concentrated. Exon 51 removal was detected using PCR. Without exon 51 removal: 2645 bp is amplified with primers DMD50-F and DMD51-R2 (Figure 9A), otherwise a 284 bp product is amplified. The 284 bp amplicon was observed in cells treated with copackaged RNPs, but not in cells treated with two individually packaged RNPs (Figure 9B). DNA sequencing confirmed that the approximately 284 bp amplicon was the result of deleting the sequence between the two sgRNAs. This experiment showed that one single preparation can produce RNP-loaded EVs targeting more loci than one and doing so is more efficient for multiplex gene targeting.

[0259] We also tested the packaging of SaCas9 and SpCas9 RNPs in one single preparation. In this case, the SaCas9 RNP targeting DMD intron 50 and the SpCas9 RNP targeting DMD exon 53 were individually packaged or copackaged into EVs. To remove the sequence between the two target sites, which are 96 kb apart, two individually packaged RNPs were used together and compared to the copackaged RNP (Figure 9A). PCR showed that the expected 263 bp DNA amplicon, which indicated the 96 kb deletion and was confirmed by DNA sequencing, was only observed in cells treated with the copackaged RNP, but not in cells treated with the two individually packaged RNPs (Figure 9C). Thus, RNPs of Cas9 from different species can also be copackaged into EVs in a single preparation for efficient multiplex gene editing. Example 8. In vivo activity of EV-delivered RNPs

[0260] The in vivo activity of EV-delivered DMD exon 53-targeted RNP was examined. RNP-loaded EVs produced by 40 million cells in 48 hours were concentrated and injected into each TA muscle of del52hDMD / mdx mice. After one week, mice were sacrificed and TA muscles were harvested to examine targeted gene editing. Genomic DNA was isolated from TA muscles and the targeted DNA region was amplified for NGS analysis. A maximum of 0.2% INDEL rate (316 out of 157982 reads) was observed in RNP-injected muscles and 0% INDEL rate was observed in PBS-injected muscles (0 out of 51302 reads by chi-square test, p<0.0001). All INDELs were around the predicted cleavage site.

[0261] Immunostaining was performed to examine dystrophin expression in RNP-injected TA muscles. These mice have a low background of dystrophin expression in skeletal muscles due to spontaneous exon 53 skipping, which restores the dystrophin reading frame. Areas with stronger dystrophin expression were observed in RNP-injected TA muscles, which was not observed in PBS-injected muscles.

[0262] The work described herein showed that a specific interaction between an aptamer-binding protein (fused to CD63) and an aptamer (inserted into sgRNA) can be used to actively enrich Cas9 and ABE RNPs in EVs. In this example, the com / Com interaction associates RNPs with the N- and C-termini of CD63, which is in the cytoplasm of cells and in the lumen of exosomes. Because CD63 is abundant in exosomes, this method specifically enriched Cas9 and ABE RNPs into EVs. Indeed, up to 10-fold higher gene editing activity was observed compared to EVs without an RNP enrichment mechanism (e.g., without aptamer Com or aptamer-binding protein Com). Because Cas9- and sgRNA-expressing plasmid DNA was also present in conditions without an RNP enrichment mechanism, the low gene editing activity observed in these conditions excluded a major contribution of EV-transferred plasmid DNA.

[0263] It is interesting to note that the addition of the aptamer-binding protein Com to both the N- and C-termini of CD63 showed a synergistic effect on gene editing activity. There could be several explanations for this observation: 1) Com can function as a homodimer, and fusing Com to both the N- and C-termini of CD63 increases the likelihood of creating a functional unit, or 2) a threshold of Cas9 molecules needs to be functional, and fusing Com to both the N- and C-termini of CD63 increases the likelihood of reaching that threshold.

[0264] Another interesting finding is that VSV-G may be important for the genome editing activity of EV-delivered RNPs. The most likely explanation is that VSV-G helps RNPs escape from the endosomal system in recipient cells. Based on the studies described herein, it is likely that an intact and free C-terminus is important for VSV-G to induce endosomal escape.

[0265] The advantage of the EV-mediated RNP delivery system is that RNPs targeting more than one locus and RNPs with Cas9 proteins from different species can be concentrated into EVs in a single RNP preparation. As demonstrated herein, copackaging of SaCas9 and SpCas9 RNPs is possible. It is expected that Cas9 proteins from other species can also be copackaged as long as com aptamers can be inserted into their sgRNAs. Furthermore, the copackaged RNPs are more active than a combination of individually packaged RNPs for multiplex genome editing. These features make EVs an ideal delivery tool for multiplex genome editing, which is required in many situations, including knocking out antigens to reduce the risk of immune rejection, eradicating HIV proviral DNA from the genome, and enhancing responses in cancer therapy.

[0266] In addition to these advantages, EVs may be more suitable for systemic delivery compared to, for example, virus-like particles, which tend to be inactivated by the complement system in the circulation. Also, EVs have the ability to cross the blood-brain barrier, thus expanding their therapeutic potential. In summary, RNPs can be efficiently and functionally packaged into EVs. EV-delivered RNPs exhibit high on-target gene editing activity. [Table 6-1] [Table 6-2] [Table 6-3]

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Claims

1. 1. A plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, said nucleic acid sequence comprising: (i) a nucleic acid sequence encoding a CRISPR-associated endonuclease; and (ii) a guide RNA (gRNA) coding sequence that includes at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the at least one aptamer coding sequence of the first mammalian expression plasmid.

2. 2. The plasmid system of claim 1, further comprising an envelope plasmid comprising a nucleic acid sequence encoding a vesicular stomatitis virus G (VSV G) protein.

3. 2. The plasmid system of claim 1, wherein the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein, or a derivative thereof.

4. 2. The plasmid system of claim 1, wherein the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease.

5. 5. The plasmid system of claim 4, wherein the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.

6. 5. The plasmid system of claim 4, wherein the nucleic acid sequence of the first mammalian expression plasmid encodes a nucleic acid sequence encoding an adenosine base pair editor (ABE), the ABE being a fusion protein comprising an adenosine deaminase and the catalytically impaired CRISPR-associated endonuclease.

7. 7. The plasmid system of claim 6, wherein the adenine base editor is ABE 7.10 or ABE8.

8. 2. The plasmid system of claim 1, wherein the at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein.

9. The plasmid system of claim 8 , wherein the aptamer sequence is an MS2 aptamer sequence, a pp7 aptamer, a Box-B aptamer, or a com aptamer sequence.

10. 2. The plasmid system of claim 1, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, and the first and second ABPs are the same.

11. The plasmid system of claim 10 , wherein the first and second ABPs are Com-binding proteins.

12. 2. The plasmid system of claim 1, wherein the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into a tetraloop or ST2 loop of the sgRNA coding sequence.

13. 13. The plasmid system of claim 12, wherein the sgRNA coding sequence comprises at least one com aptamer sequence inserted into a tetraloop or ST2 loop of the gRNA coding sequence.

14. An extracellular vesicle comprising: (a) a ribonucleotide protein (RNP) complex comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer-encoding sequence; and (b) an extracellular vesicle comprising a fusion protein comprising CD63 and at least one aptamer-binding protein (ABP), wherein the ABP binds to the at least one aptamer-encoding sequence.

15. The extracellular vesicle of claim 14, further comprising a VSV-G protein.

16. The extracellular vesicle of claim 14, wherein the CRISPR-associated endonuclease is a Cas9 protein, a Cpf1 protein, or a derivative thereof.

17. The extracellular vesicle of claim 14, wherein the CRISPR-associated endonuclease is a catalytically impaired CRISPR-associated endonuclease.

18. The extracellular vesicle of claim 17, wherein the catalytically impaired CRISPR-associated endonuclease coding sequence encodes a Cas9 D10A protein.

19. The extracellular vesicle of claim 17, wherein the RNP comprises an adenine base pair editor (ABE), the ABE being a fusion protein comprising adenosine deaminase and the catalytically impaired CRISPR-associated endonuclease.

20. The extracellular vesicle of claim 19, wherein the adenine base editor is ABE 7.10 or ABE8.

21. The extracellular vesicle of claim 14, wherein the at least one aptamer coding sequence encodes an aptamer sequence that is specifically bound by an ABP selected from the group consisting of MS2 coat protein, PP7 coat protein, lambda N RNA binding domain, or Com protein.

22. The extracellular vesicle of claim 21, wherein the aptamer sequence is an MS2 aptamer sequence or a com aptamer sequence.

23. The extracellular vesicle of claim 14, wherein the fusion protein comprises a first ABP fused to the N-terminus of CD63 and a second ABP fused to the C-terminus of CD63, and the first and second ABPs are the same.

24. The extracellular vesicle of claim 23, wherein the first and second ABPs are Com-binding proteins.

25. The extracellular vesicle of claim 14, wherein the sgRNA coding sequence comprises at least one aptamer coding sequence inserted into a tetraloop or ST2 loop of the sgRNA coding sequence.

26. The extracellular vesicle of claim 25, wherein the sgRNA coding sequence comprises at least one com aptamer sequence inserted into a tetraloop or ST2 loop of the gRNA coding sequence.

27. The extracellular vesicle of claim 14, wherein the extracellular vesicle is an exosome or a microvesicle.

28. A method for producing extracellular vesicles, comprising: (a) transfecting a plurality of eukaryotic cells with the first mammalian expression plasmid and the second mammalian expression plasmid of the system of claim 1; and b) culturing the transfected eukaryotic cells for a period of time sufficient to produce extracellular vesicles.

29. 29. The method of claim 28, further comprising transfecting the plurality of eukaryotic cells with the envelope plasmid of the system of claim 2.

30. 29. The method of claim 28, wherein the extracellular vesicles comprise: (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer-encoding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein, wherein the aptamer-binding protein (ABP) binds to the at least one aptamer-coding sequence.

31. 30. The method of claim 29, wherein the extracellular vesicles comprise: (a) an RNP comprising (i) a CRISPR-associated endonuclease and (ii) a gRNA comprising at least one aptamer-encoding sequence; (b) a fusion protein comprising CD63 and at least one aptamer-binding protein, wherein the aptamer-binding protein (ABP) binds to the aptamer-coding sequence; and (c) a VSV-G protein.

32. 30. The method of claim 28, wherein the plurality of eukaryotic cells are mammalian cells.

33. 30. The method of claim 28, further comprising isolating the extracellular vesicles from the cultured transfected eukaryotic cells.

34. 29. An extracellular vesicle produced by the method of claim 28.

35. 15. A composition comprising a plurality of extracellular vesicles for use in a method of modifying a genomic target sequence in a cell, the method comprising transducing a plurality of eukaryotic cells with the composition, the plurality of extracellular vesicles comprising the extracellular vesicles of claim 14, and wherein the RNPs bind to the genomic target sequence in genomic DNA of the cells, thereby modifying the genomic target sequence.

36. 36. The composition of claim 35, wherein the plurality of eukaryotic cells are mammalian cells.

37. The composition of claim 35 , wherein the plurality of eukaryotic cells are cells present within a subject.

38. 38. The composition of claim 37, wherein the subject is a human subject.

39. 39. The composition of claim 38, wherein the subject is injected with the plurality of extracellular vesicles.

40. A cell comprising the plasmid system of claim 1.

41. A cell modified using the composition of claim 35.

42. 1. A composition comprising modified cells for use in a method of treating a disease in a subject, the method comprising: a) obtaining cells from said subject; b) modifying the cells of the subject using the composition of claim 35; and c) administering said composition to said subject.

43. 43. The composition of claim 42, wherein the disease is cancer.

44. 43. The composition of claim 42, wherein the disease is sickle cell anemia.

45. The composition of claim 42, wherein the cell is a T cell.

46. 1. A plasmid system comprising: (a) a first mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence, said nucleic acid sequence comprising: (i) a nucleic acid sequence encoding a heterologous polypeptide; and (ii) a first mammalian expression plasmid comprising at least one aptamer coding sequence; and (b) a second mammalian expression plasmid comprising a eukaryotic promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising CD63 and at least one aptamer binding protein, wherein the aptamer binding protein (ABP) binds to the at least one aptamer coding sequence of the first mammalian expression plasmid.

47. 47. The plasmid system of claim 46, wherein the nucleic acid sequence encoding the heterologous polypeptide comprises the at least one aptamer coding sequence.

48. An extracellular vesicle comprising:

1. An extracellular vesicle comprising: (a) an mRNA encoding a heterologous polypeptide and at least one aptamer coding sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein (ABP), wherein the ABP binds to the at least one aptamer coding sequence.

49. A method for producing extracellular vesicles, comprising: (a) transfecting a plurality of eukaryotic cells with the first mammalian expression plasmid and the second mammalian expression plasmid of the system of claim 46; and b) culturing the transfected eukaryotic cells for a period of time sufficient to produce extracellular vesicles.

50. 50. The method of claim 49, wherein the extracellular vesicles comprise: (a) an mRNA encoding said heterologous polypeptide and said at least one aptamer sequence; and (b) a fusion protein comprising CD63 and at least one aptamer-binding protein, wherein the aptamer-binding protein (ABP) binds to the at least one aptamer-coding sequence.

51. 47. A cell comprising the plasmid system of claim 46.

52. A method for delivering an mRNA encoding a heterologous polypeptide to a cell, comprising contacting the cell with an extracellular vesicle described in claim 48.

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