Expression of FOXP3 in gene-edited CD34+ cells

Abstract

To provide a novel approach for the expression or regulation of FOXP3 in a primary human lymphocyte.SOLUTION: Aspects of the invention described herein concern the incorporation of a FOXP3 cDNA (e.g., full-length of a human codon-optimized FOXP3 cDNA) into a FOXP3 gene or a non-FOXP3 locus so as to provide constitutive or regulated FOXP3 expression in a primary human CD34+ cells or cells derived from edited CD34+ cells. In some embodiments, guide RNA sequences that are directed to FOXP3, AAVS1, or other candidate loci are used for CRISPR / Cas9-mediated gene regulation, and gene delivery cassettes for homology-directed repair (HDR)-based gene-modification are provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 663,545, entitled “EXPRESSION OF MRNA ENCODING HUMAN FOXP3 FULL LENGTH PROTEIN FROM CANIDATE GENETIC LOCI IN GENE EDITED CD34 CELLS AND CELLS DERIVED FROM EDITED CD34 CELLS,” filed April 27, 2018, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which has been provided as an approximately 430 kb file created on April 24, 2019 under the file name SCRI188WOSEQLISTING. The information set forth in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Aspects of the invention described herein include CD34 + Gene-edited CD34 by integrating the coding sequence of FOXP3 into the FOXP3 gene or a non-FOXP3 locus in cells + The present invention relates to constitutive or regulated expression of FOXP3 in a cell or a cell derived from this cell (such as a T cell). [Background technology]

[0004] Lentiviral gene transfer of FOXP3 (also known as forkhead box protein P3, forkhead box P3, AAID, DIETER, IPEX, JM2, PIDX, XPID or scurfin) has been previously reported in Chen, C. et al. (2011). Transplant. Proc. 43(5):2031-2048, Passerini, L. et al. (2013). Sci. Transl. Med. , 5(215):215ra174 and Passerini, L. et al. (2017). Front. Immunol. 8:1282, all of which are expressly incorporated herein by reference in their entireties. Passerini et al. (2017) also demonstrated T cell proliferation and regrowth in T lymphocytes from patients with FOXP3 mutations. reg We have previously reported the development of a method to restore CD4 function. As reported by Passerini et al. (2017), + Lentivirus-mediated gene transfer is used in T cells and effector T cells, which converts these T cells into effector T cells and reg These cells exhibited CD4+-like characteristics and conferred potent in vitro and in vivo suppressive activity. Furthermore, Passerini demonstrated that lentivirus-mediated transfer of the FOXP3 gene inhibited CD4+ + T cells are T reg We have demonstrated that this T reg The cells were shown to be stable under inflammatory conditions (Passerini et al. (2013)). Also, Chen et al. (2011) reported adoptive transfer of recombinant T cells infected with a lentiviral vector encoding a FOXP3-IRES-GFP fragment. These cells were shown to prevent GVHD in recipient mouse models. New approaches to express and control FOXP3 in primary human lymphocytes are needed.

[0005] Regulatory T cells have the potential to induce antigen-specific immune tolerance, and many researchers are interested in using them to treat autoimmune diseases. reg ") come in various forms, and according to current nomenclature, they are classified as "thymus-derived regulatory T cells" or "tT reg ") and peripherally derived regulatory T cells (peripherally derived regulatory T cells or "pT reg ") are classified into two types.

[0006] An important aspect of regulatory T cell biology is the expression of the transcription factor FOXP3, which is thought to be required for the induction of differentiation of the regulatory T cell lineage. This concept is based on the observation that humans lacking FOXP3 develop severe autoimmune diseases during the neonatal period. FOXP3 expression is thought to be epigenetically regulated, which may provide a valuable tool in the treatment of autoimmune diseases. reg or pT reg Using tT may not be the best solution. reg In this study, the upstream region of the FOXP3 gene, known as the "thymus-specific demethylation region," is demethylated, which is thought to result in stable expression of FOXP3. reg FOXP3 is also expressed in pT cells under inflammatory conditions. reg It is thought to be epigenetically silenced in reg It is also epigenetically silenced in tT reg Some researchers believe that pT is completely stable. reg It is believed that pT cells can transform into pro-inflammatory CD4 T cells. reg Infusion of pT reg The potential lack of stability is a significant issue. Summary of the Invention [Means for solving the problem]

[0007] In this specification, a deoxyribonucleic acid (DNA) endonuclease, or a nucleic acid encoding said DNA endonuclease; CD34 + A guide RNA (gRNA) comprising a spacer sequence complementary to a sequence in the FOXP3 gene, AAVS1 locus, or TRA gene of the cell, or a nucleic acid encoding the gRNA; and A donor template comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof. This paper describes a system including: In some embodiments, the gRNA comprises: i) a spacer sequence shown in any one of SEQ ID NOs: 1 to 7, 15 to 20, and 27 to 29, or a variant of the spacer sequence having three or less mismatches compared to any one of SEQ ID NOs: 1 to 7, 15 to 20, and 27 to 29; ii) a spacer sequence as set forth in any one of SEQ ID NOs: 1 to 7, or a variant of said spacer sequence having 3 or less mismatches compared to any one of SEQ ID NOs: 1 to 7; or iii) a spacer sequence as set forth in any of SEQ ID NOs: 2, 3 and 5, or a variant of said spacer sequence having 3 or fewer mismatches compared to any of SEQ ID NOs: 2, 3 and 5; Includes. In some embodiments, the FOXP3 or functional derivative thereof is human wild-type FOXP3. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the DNA endonuclease or the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in a liposome or lipid nanoparticle.

[0008] Also, as used herein, CD34 + A method for editing the genome of a cell, comprising administering any one of the systems described herein to a subject infected with CD34 + In some embodiments, the method includes providing a cell with CD34. + The cells are not germ cells.

[0009] Further, the present disclosure provides a method for the detection of CD34 by any one of the methods described herein. + Genome-edited recombinant CD34 + Cells and the recombinant CD34 + In some embodiments, the present invention relates to a composition comprising a recombinant CD34 cell. + The cell is not a germ cell.

[0010] Further provided herein is a method of treating a FOXP3-associated disease or condition in a subject, comprising administering any one of the systems described herein to a subject's CD34 + The method includes providing a recombinant CD34 cell to a patient. The disease or condition may be an inflammatory disease or an autoimmune disease, such as IPEX syndrome or graft-versus-host disease (GVHD). In some embodiments, the recombinant CD34 cell is + The cell is not a germ cell. [Brief description of the drawings]

[0011] [Figure 1] Schematic diagram showing two different AAV donor template designs engineered to integrate a donor cassette into the FOXP3 gene: one in which FOXP3 is expressed from a heterologous FOXP3 cDNA under the control of the endogenous FOXP3 promoter (upper diagram), and one in which GFP is expressed under the heterologous MND promoter (lower diagram). HA: homology arms; MND: MND promoter; pA: SV40 polyadenylation signal.

[0012] [Diagram 2] Results show the viability of CD34+ cells treated with AAV donor template (#3037 or #3008) alone, treated with Cas9 / gRNA RNP (T3 gRNA or T9 gRNA) + AAV donor template (#3037 or #3008), and mock-treated CD34+ cells on days 1 (D1), 2 (D2), and 5 (D5) after treatment.

[0013] [Diagram 3] FIG. 2 shows the homologous recombination rate in CD34+ cells edited with Cas9 / gRNA RNP (T3 gRNA or T9 gRNA) and the AAV donor template shown in FIG. 1.

[0014] [Figure 4] FIG. 1 is a bar graph comparing cell viability of CD34+ cells treated with RNPs containing Cas9 obtained from two different sources (IDT Alt-R Sp Cas9 Nuclease V3 or Aldevron SpyFi Cas9) and two different FOXP3-targeting gRNAs (T3 or T9).

[0015] [Diagram 5] FIG. 1 is a bar graph comparing cell viability of CD34+ cells edited with RNP + AAV donor templates containing Cas9 from two different sources (IDT Alt-R Sp Cas9 Nuclease V3 or Aldevron SpyFi Cas9) and two different FOXP3-targeting gRNAs (T3 or T9).

[0016] [Figure 6] 1 shows exemplary results showing the percentage of GFP+ cells among total hCD45+ cells recovered from the spleens of NSGW41 mice transplanted with mock-treated or SpyFi Cas9 / gRNA RNP (T3)-edited cells targeting FOXP3. Mean values ​​± SEM are displayed on the graph.

[0017] [Figure 7] 1 shows exemplary results showing the percentage of GFP+ cells among hCD19+ cells recovered from the spleens of NSGW41 mice transplanted with mock-treated or SpyFi Cas9 / gRNA RNP (T3)-edited cells targeting FOXP3. Mean values ​​± SEM are displayed on the graph.

[0018] [Figure 8] 1 shows exemplary results showing the percentage of GFP+ cells among hCD33+ cells recovered from the spleens of NSGW41 mice transplanted with mock-treated or SpyFi Cas9 / gRNA RNP (T3)-edited cells targeting FOXP3. Mean values ​​± SEM are displayed on the graph. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Herein, we describe the expression of FOXP3 from a DNA sequence (e.g., a codon-optimized DNA sequence, e.g., a DNA sequence that is codon-optimized for expression in human cells) integrated into a FOXP3 gene or non-FOXP3 locus. Guide RNA is used to target a FOXP3 gene or non-FOXP3 locus (e.g., mouse, human or non-human primate) and perform genome editing via CRISPR / Cas. Thus, an aspect of the present invention relates to using novel guide RNA in combination with Cas protein to cleave DNA in a FOXP3 gene or non-FOXP3 target locus and promote integration of the coding sequence of FOXP3. In some embodiments, this integration is performed by non-homologous end joining (NHEJ) or homology-directed repair (HDR) in association with a donor template that contains the coding sequence of FOXP3. Some embodiments described herein can be used in combination with broad range selectable markers such as LNG FR, RQR8, CISC / DISC / μDISC, and can be multiplexed in combination with editing of additional target loci or co-expression of additional gene products (such as cytokines).

[0020] As described in more detail below, the applicants have demonstrated that the expression of primary human CD34+ / -CD ... + We identified guide RNAs that induce on-target cleavage in cells and can integrate the gene delivery cassette into the FOXP3 gene. + The cells were able to engraft persistently and express a GFP reporter construct integrated into the FOXP3 gene for long periods. These findings demonstrate that genome editing systems, such as the CRISPR / Cas system described herein, can efficiently edit human hematopoietic stem cells to express human wild-type FOXP3 and sustain engraftment at levels predicted to provide clinical benefit for diseases or disorders with abnormal FOXP3 function after administration, such as autologous adoptive cell therapy, to subjects with IPEX syndrome. Previous studies have suggested that subjects with IPEX syndrome with donor chimerism as low as 5% may also see clinical benefit after allogeneic stem cell transplantation. See Seidel, MG et al. (2009). Blood, 113(22):5689-5691.

[0021] The use of a CRISPR / Cas system with gRNA and a donor template configured to insert the cDNA of the FOXP3 gene into the endogenous FOXP3 gene is promising as a treatment for inflammatory diseases such as the autoimmune disease IPEX syndrome. For the treatment of IPEX syndrome, the disease may result from a variety of mutations spread throughout the gene, and therefore insertion of the entire FOXP3 cDNA (e.g., the entire human codon-optimized FOXP3 cDNA) into the start codon may be desirable. CD34 + It is anticipated that utilization of the endogenous FOXP3 promoter following differentiation of cells from these blastocysts will provide the transcriptional signals required to obtain optimal FOXP3 expression levels.

[0022] Definition of Terms As used herein, "nucleic acid" and "nucleic acid molecule" include, for example, polynucleotides or oligonucleotides, including, but not limited to, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease action, exonuclease action, and synthesis. Nucleic acid molecules may be composed of natural nucleotide monomers (e.g., DNA, RNA), or monomers consisting of analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety and / or pyrimidine or purine base moieties. Modifications in the sugar moiety include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Additionally, the entire sugar moiety may be replaced with conformationally or electronically similar structures, such as, for example, azasugars or carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Linkages similar to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoroamidate bonds. "Nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded.

[0023] As used herein, the term "coding strand" includes, but is not limited to, a DNA strand having the same base sequence as the RNA transcript produced, except that thymines are replaced by uracils. The coding strand contains codons, and the non-coding strand contains anticodons.

[0024] As used herein, "regulatory element" includes, for example, a segment of a nucleic acid molecule capable of increasing or decreasing the expression of a particular gene in an organism, including, but not limited to, a segment of a nucleic acid molecule capable of affecting the transcription and / or translation of an operably linked transcribable DNA molecule. Regulatory elements, such as promoters (e.g., MND promoter), leaders, introns, transcription termination regions, and the like, are DNA molecules that have gene regulatory activity and play an essential role in the overall expression of genes in living cells. Thus, isolated regulatory elements (such as promoters) that function in plants are useful for modifying plant phenotypes by genetic engineering methods. Control of gene expression is a fundamental function of all organisms and viruses. Regulatory elements include, but are not limited to, CAAT boxes, CCAAT boxes, Pribnow boxes, TATA boxes, SECIS elements, mRNA polyadenylation signals, A boxes, Z boxes, C boxes, E boxes, G boxes, hormone response elements such as insulin gene regulatory sequences, DNA binding regions, activation regions, and / or enhancer regions.

[0025] In some embodiments, the guide RNA comprises an additional segment at either the 5' or 3' end that provides any of the above features. For example, suitable third segments can include a 5' cap (e.g., a 7-methylguanylic acid cap (m7G)); a 3' polyadenylation tail (e.g., a 3' poly(A) tail); a riboswitch sequence (e.g., one that provides controlled stabilization and / or allows controlled access by a protein or protein complex); a stability control sequence; a sequence that forms a dsRNA duplex (e.g., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that allows tracking (e.g., direct binding to a fluorescent molecule, binding to a moiety that facilitates fluorescent detection, a sequence that allows fluorescent detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, such as a transcription activator, a transcription repressor, a DNA methyltransferase, a DNA methylase, a histone acetyltransferase, a histone deacetylase, etc.); and combinations thereof.

[0026] The guide RNA and the Cas endonuclease (e.g., Cas9 endonuclease) may form a ribonucleoprotein complex (e.g., may be associated via non-covalent interactions). The guide RNA provides target specificity to the ribonucleoprotein complex by including a nucleotide sequence complementary to the sequence of the target DNA. The site-specific modifying enzyme of the ribonucleoprotein complex provides the endonuclease activity. In other words, the site-specific modifying enzyme is guided to the target DNA sequence (e.g., a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid (e.g., an episomal nucleic acid, a minicircle, etc.); a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid, etc.) by association with the protein-binding segment of the guide RNA.

[0027] As used herein, "FOXP3" includes, but is not limited to, proteins involved in immune system responses. The FOXP3 gene (also known as forkhead box protein P3, forkhead box P3, AAID, DIETER, IPEX, JM2, PIDX, XPID or scurfin) contains 11 coding exons. Foxp3 is involved in the regulation of endogenous regulatory T cells (nT reg (T cell line)) and adoptively transferred / inducible regulatory T cells (a / iT reg ) is a specific marker for inflammatory bowel disease (AIDS). In animal studies, induction or administration of FOXP3-positive T cells significantly reduces the severity of disease (autoimmune disease) in models of diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis, or kidney disease. However, previous studies have reported that T cells exhibit plasticity. Thus, there is a risk in the therapeutic use of regulatory T cells, since regulatory T cells transferred into a subject may change into proinflammatory helper T17 (Th17) cells rather than regulatory cells. In light of this, a method for avoiding the risks resulting from the change of regulatory cells into proinflammatory cells is provided herein. For example, iT reg FOXP3 expressed in T cells serves as a master regulator of the immune system and is also involved in immune tolerance and immune suppression. reg It is believed to play a crucial role in various autoimmune diseases, such as IPEX syndrome, type 1 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. reg Various approaches to enhance the number or function of cells are currently in clinical trials, for example, the use of low-dose IL-2 and expanded autologous T cells. reg Therapies combining IL-2 with adoptive transfer of T cells are currently in clinical trials. The efficacy of IL-2 therapy is limited due to its pleiotropic activity and potential "off-target" effects that may enhance inflammation. reg Therapy also involves the expansion of cultured T reg are likely to be of limited use due to problems with in vivo stability and viability and a lack of therapeutically useful antigen specificity.

[0028] As used herein, "nuclease" includes, but is not limited to, a protein or enzyme that can cleave, for example, a phosphodiester bond between nucleotide subunits of a nucleic acid. The nucleases described herein are used in "gene editing". Gene editing is a type of genetic engineering technique that uses a nuclease or recombinant nuclease or multiple nucleases to insert, delete, or replace DNA in the genome of an organism. Nucleases include, but are not limited to, nucleases used in CRISPR / Cas systems (e.g., CRISPR / Cas9 systems), zinc finger nucleases, and TALEN nucleases. Nucleases can be used to target loci, for example, target loci in a nucleic acid sequence.

[0029] As used herein, "coding exon" includes, but is not limited to, a portion of a gene that codes for a portion of the final mature RNA produced by the gene after introns have been removed by RNA splicing. "Exon" refers to both the DNA sequence within a gene and the corresponding RNA transcript sequence. When introns are removed by RNA splicing, the remaining exons are covalently linked to each other and form part of the mature messenger RNA.

[0030] As used herein, "Cas endonuclease" or "Cas nuclease" includes, but is not limited to, RNA-guided DNA endonuclease enzymes associated with, for example, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. As used herein, "Cas endonuclease" refers to both native and recombinant Cas endonucleases.

[0031] As used herein, "Cas9" or "CAS9" (also known as Csn1 and Csx12) includes, but is not limited to, an RNA-guided DNA endonuclease enzyme associated with, for example, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. As used herein, "Cas9" refers to both native and recombinant Cas9.

[0032] As used herein, "zinc finger nuclease" includes, but is not limited to, artificial restriction enzymes created by fusing a zinc finger DNA binding domain with a DNA degradation domain. The zinc finger domain can be engineered to target a specific DNA sequence of interest, allowing the zinc finger nuclease to target unique sequences within a complex genome.

[0033] As used herein, "TALEN" or "transcription activator-like effector nuclease" includes, but is not limited to, restriction enzymes that can be modified to cleave specific DNA sequences. TALENs are generated by fusing a TAL effector DNA binding domain to a DNA degradation domain (a nuclease that cleaves DNA strands). Transcription activator-like effectors (TALEs) can be modified to bind to virtually any desired DNA sequence, and thus can be combined with a nuclease to cleave DNA at a specific location. Restriction enzymes can be introduced into cells for the purpose of in situ gene or genome editing, a technique known as genome editing using recombinant nucleases. TALENs are one of the tools in the genome editing field, along with zinc finger nucleases and CRISPR / Cas.

[0034] As used herein, "knock-in" includes, but is not limited to, for example, genetic engineering methods for one-to-one replacement of DNA sequence information with a wild-type copy within a gene locus or for inserting sequence information not present within a gene locus.

[0035] As used herein, the term "promoter" includes, but is not limited to, a nucleotide sequence that induces transcription of a structural gene. In some embodiments, the promoter is located in the non-coding region at the 5' end of the gene, near the transcription start site of the structural gene. The elements of the promoter sequence that initiate transcription are often characterized by consensus nucleotide sequences. A promoter is a DNA region that initiates transcription of a particular gene. A promoter is located upstream (towards the 5' region of the sense strand) in the same DNA strand, near the transcription start site of the gene. The length of the promoter may be 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, 700 base pairs, 800 base pairs, or 1000 base pairs, or about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, about 600 base pairs, about 700 base pairs, about 800 base pairs, or about 1000 base pairs, or a length within a range defined by any two of these lengths. As used herein, a promoter may be a constitutively active promoter, a repressible promoter, or an inducible promoter. When a promoter is an inducible promoter, the transcription rate increases in response to an inducing agent. In contrast, when a promoter is a constitutive promoter, the transcription rate is not controlled by an inducing agent. Repressible promoters are also known. Examples of promoters include, but are not limited to, constitutive promoters, weak heterologous promoters (e.g., promoters that express less than endogenous promoters and / or constitutive promoters), or inducible promoters.Furthermore, examples of promoters include EF1α promoters, PGK promoters, MND promoters, KI promoters, Ki-67 gene promoters, or promoters that can be induced by drugs such as tamoxifen and / or its metabolites.Commonly used constitutive promoters include, but are not limited to, SV40, CMV, UBC, EF1A, PGK or CAGG used in mammalian systems.

[0036] As used herein, a "transcription enhancer domain" includes, but is not limited to, a short DNA region (50-1500 bp) to which a protein (activator) can bind, which can increase, promote, or enhance the likelihood or amount of transcription of a particular gene by binding the activator to the transcription enhancer domain. Such activator proteins are typically referred to as transcription factors. Enhancers are typically cis-acting, located up to 1 Mbp (1,000,000 bp) away from the target gene, and can be upstream or downstream of the transcription start site, in either forward or reverse orientation. Enhancers may be located upstream or downstream of the gene they control. In some embodiments, multiple enhancer domains may be used to increase the amount of transcription, for example, multimerized activation binding domains may be used to further enhance or increase the amount of transcription. Furthermore, enhancers do not need to be located in the vicinity of the transcription start site to affect transcription, as some researchers have found that enhancers can be located hundreds of thousands of base pairs upstream or downstream from the transcription start site. Enhancers do not act on the promoter region itself, but bind to activator proteins. The activator proteins interact with the mediator complex, recruit polymerase II and general transcription factors, and initiate gene transcription. Enhancers can also be present within introns. The orientation of the enhancer can be reversed, and this does not affect its function. Furthermore, enhancers can be cut or inserted at any position in the chromosome, and such treatments can still have an effect on gene transcription. In some embodiments, enhancers are used to silence repression mechanisms that block transcription of the FOXP3 gene. An example of an enhancer binding domain is the TCR alpha enhancer. In some embodiments, the enhancer domain is the TCR alpha enhancer. In some embodiments, the enhancer binding domain is placed upstream of the promoter, thereby activating the promoter and increasing protein transcription.In some embodiments, an enhancer binding domain is placed upstream of the promoter, thereby activating the promoter and increasing transcription of the FOXP3 gene.

[0037] As used herein, a "transcription activator domain" or "transcription activation domain" includes, but is not limited to, a specific DNA sequence to which a transcription factor can bind, and which can control the rate of transcription of genetic information from DNA to messenger RNA by binding to the transcription activation region. Specific examples of transcription factors include, but are not limited to, SP1, AP1, C / EBP, heat shock factor, ATF / CREB, c-Myc, Oct-1, and NF-1. In some embodiments, an activator domain is utilized to silence a repression mechanism that prevents transcription of the FOXP3 gene.

[0038] As used herein, "ubiquitous chromatin opening elements (UCOEs)" include, but are not limited to, elements that are characterized by dual promoters that are bidirectionally transcribed, e.g., in unmethylated CpG islands derived from housekeeping genes. UCOEs are promising tools for preventing silencing and maintaining expression of exogenous genes in various cell models, such as cell lines, pluripotent hematopoietic stem cells, PSCs, and their differentiated progeny.

[0039] As used herein, "operably linked" includes, but is not limited to, for example, functional linkage between a regulatory sequence and a heterologous nucleic acid sequence to express the heterologous nucleic acid sequence. In some embodiments, a first molecule is linked to a second molecule, and the molecules are arranged such that the first molecule affects the function of the second molecule. The two molecules may be part of a single continuous molecule or may be adjacent to each other. For example, if a promoter controls the transcription of a transcribable DNA molecule of interest in a cell, the promoter is operably linked to the transcribable DNA molecule.

[0040] The term "concentration" as used in describing a molecule, such as a peptide fragment, refers to the amount of the molecule present in a given volume of solution, e.g., moles of the molecule.

[0041] The terms "individual", "subject" and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient. In some embodiments, the subject has a disorder or condition associated with FOXP3 or is suspected of having a disorder or condition associated with FOXP3. In some embodiments, the subject is a human diagnosed at or after diagnosis as being at risk for a disorder or condition associated with FOXP3. In some cases, the diagnosis of having a risk for a disorder or condition associated with FOXP3 can be determined based on the presence of one or more mutations in the endogenous gene encoding FOXP3 or in nearby genomic sequences that may affect the expression of FOXP3. For example, in some embodiments, the subject has an autoimmune disorder, is suspected of having an autoimmune disorder, and / or has one or more symptoms of an autoimmune disorder. In some embodiments, the subject is a human diagnosed at or after diagnosis as being at risk for an autoimmune disorder. In some cases, a diagnosis of being at risk for an autoimmune disorder can be determined based on the presence of one or more mutations in the endogenous FOXP3 gene or nearby genomic sequences that may affect expression of the FOXP3 gene.

[0042] The term "treatment", when used in reference to a disease or condition, means that the symptoms associated with the condition from which an individual suffers are at least alleviated, where "alleviation" is used in a broad sense to refer to at least a reduction in the severity of a parameter (e.g., a symptom) associated with the condition being treated (e.g., an autoimmune disease). Treatment therefore also includes a condition in which the pathological condition or at least a symptom associated therewith is completely suppressed, e.g., the onset of the pathological condition or its symptoms is prevented, or a condition in which the pathological condition or its symptoms is completely eliminated such that the host is no longer afflicted by the pathological condition or at least a symptom characterizing the pathological condition. Treatment therefore includes (i) prevention, i.e., reducing the risk of developing clinical symptoms, e.g., preventing the onset of clinical symptoms, e.g., preventing disease progression, and (ii) suppression, i.e., preventing the onset or further onset of clinical symptoms, e.g., reducing or completely suppressing active disease.

[0043] As used herein, "effective amount", "pharmaceutical effective amount" or "therapeutically effective amount" refers to an amount of a composition sufficient to provide a desired benefit when administered to a subject having a particular condition. The term "effective amount" as used in the description of ex vivo treatment of an autoimmune disorder refers to the amount of a population of therapeutic cells or their progeny required to prevent or alleviate at least one or more signs or symptoms of an autoimmune disorder, and relates to an amount of a composition comprising therapeutic cells or their progeny sufficient to provide a desired effect, e.g., to treat a symptom of an autoimmune disorder in a subject. Thus, the term "therapeutically effective amount" refers to a number of therapeutic cells or an amount of a composition comprising therapeutic cells sufficient to promote a particular effect when administered to a subject in need of treatment, such as a subject having or at risk of having an autoimmune disorder. An "effective amount" can also include an amount sufficient to prevent or delay the onset of a disease symptom, an amount sufficient to alter the course of a disease symptom (e.g., but not limited to, an amount sufficient to slow the progression of a disease symptom), or an amount sufficient to ameliorate a disease symptom. In the context of in vivo treatment of an autoimmune disorder in a subject (e.g., a patient) or genome editing in in vitro cultured cells, an "effective amount" refers to the amount of components used in genome editing, such as gRNA, donor template and / or site-specific polypeptide (e.g., DNA endonuclease) required to edit the genome of a cell in the subject or in vitro cultured cells. In any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using no more than routine experimentation.

[0044] As used herein, "autoimmune disease" includes, but is not limited to, conditions in which the immune system is abnormally underactive or overactivated. When the immune system is overactivated, the body's own tissues are attacked and damaged (autoimmune disease). In immunodeficiency diseases, the body's ability to fight off invaders is weakened, resulting in low resistance to infection. Examples of autoimmune disorders or diseases that may be inhibited, alleviated or treated using the compositions and methods described herein include, but are not limited to, systemic lupus erythematosus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes mellitus, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion deficiency, NF-κB essential modulator (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disease, IPEX, immune dysregulation, polyendocrine disorders, enteropathy, X-linked (IPEX) syndromes (disorders characterized by immune dysregulation, polyendocrine disorders, enteropathy and X-linked inheritance), and ataxia-telangiectasia. Immune diseases can be analyzed, for example, by examining the profile of neurospecific autoantibodies or other biomarkers, if these are detected in the serum or cerebrospinal fluid of a subject. In some of the exemplary methods provided herein, these methods are methods of treating, alleviating or suppressing an autoimmune disorder.In some embodiments, the autoimmune disorder is systemic lupus erythematosus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes mellitus, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome, myopathy, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, common variable immunodeficiency, chronic granulomatous disease, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion deficiency, NF-κB essential modulator (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndromes (disorders characterized by immune dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance), and ataxia-telangiectasia, or any combination thereof.

[0045] "IPEX syndrome" refers to a syndrome characterized by immune dysregulation, polyendocrinopathy, enteropathy, and X-linkage, a rare disorder associated with dysfunction of the transcription factor FOXP3, widely believed to be the master regulator of the regulatory T cell lineage. Subjects affected by IPEX syndrome may exhibit symptoms such as autoimmune enteropathy, psoriatic or eczematous dermatitis, nail dystrophy, autoimmune endocrinopathy, and autoimmune skin diseases, including alopecia universalis and bullous pemphigoid. IPEX syndrome is an autoimmune disease in which the immune system attacks the body's own tissues and organs. IPEX syndrome is characterized by a CD4+ immune deficiency. + CD25 + This leads to a deficiency in T regulatory cells and expression of the transcription factor FOXP3. The reduction in FOXP3 is thought to result in the loss of regulatory T cells, followed by unchecked T cell activation.

[0046] As used herein, "organ transplantation" includes, but is not limited to, for example, transferring an organ from one body to another, or from a donor site to another site in the patient's own body to replace a damaged organ in the recipient, or transferring an organ not present in the recipient's body. Transplantation of organs and / or tissues within the same individual's living body is called autotransplantation. Transplantation between two subjects belonging to the same species, as is currently practiced, is called allotransplantation. In allotransplantation, an organ or tissue obtained from a living or cadaveric body is transplanted. In some of the embodiments described herein, methods are provided for treating, inhibiting, or mitigating side effects of organ transplantation, such as organ rejection in a subject.

[0047] Transplantable organs include, for example, the heart, kidney, liver, lung, pancreas, intestine, and thymus. Transplantable tissues include, for example, bone and tendons (called musculoskeletal grafts), cornea, skin, heart valves, nerves, and veins. Kidney, liver, and heart are the most commonly transplanted organs. Cornea and musculoskeletal grafts are the most commonly transplanted tissues.

[0048] In some embodiments described herein, a method is provided for treating, inhibiting or ameliorating a side effect of organ transplantation, such as organ rejection, in a subject. In some embodiments, the subject is a subject selected for administration of an anti-rejection drug. In some embodiments, the anti-rejection drug comprises prednisone, Imuran (azathioprine), Cellcept (mycophenolate mofetil (MMF)), Myfortic (mycophenolic acid), Rapamune (sirolimus), Neoral (cyclosporine), or Prograf (tacrolimus).

[0049] In some embodiments, the subject is a subject selected for performing the suppression, alleviation, or treatment using the genetically modified cells described in the embodiments of the present invention. In some embodiments, the subject is experiencing side effects to anti-inflammatory or anti-rejection drugs. Thus, the representative cells or compositions provided herein are provided to the selected subject. Side effects of anti-rejection drugs include increased or decreased blood levels of tacrolimus due to interactions with other drugs, nephrotoxicity, hypertension, neurotoxicity (tremors, headaches, tingling, and insomnia), diabetes (hyperglycemia), diarrhea, nausea, hair loss, or hyperkalemia, or any combination thereof. Thus, the subject is selected for performing the treatment, suppression, or alleviation methods described herein. Such selection or identification can be performed by clinical or diagnostic evaluation.

[0050] As used herein, "organ rejection" or "transplant rejection" includes, but is not limited to, the rejection and destruction of transplanted tissue by the recipient's immune system.

[0051] As used herein, "graft-versus-host disease (GVHD or GvHD)" includes, but is not limited to, medical complications that occur after receiving tissue transplants from genetically dissimilar humans. GVHD often accompanies stem cell or bone marrow transplants, but the term GVHD is also used to refer to complications from other forms of transplanted tissue. Immune cells in the donor tissue recognize the recipient as foreign, not "self." In some embodiments of the invention, the methods of the invention can be used to prevent or alleviate complications resulting from GVHD.

[0052] As used herein, the term "pharmaceutical additive" includes, but is not limited to, an inactive substance to which cells are added to obtain a composition.

[0053] As used herein, "chimeric antigen receptor (CAR)" includes, but is not limited to, an artificial T cell receptor or genetically engineered receptor, also known as, for example, a chimeric T cell receptor, that can graft a desired specificity onto an effector immune cell. A CAR may be a synthetically engineered receptor that includes a ligand binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder, and the ligand binding domain is linked via a spacer domain to one or more intracellular signaling domains (e.g., costimulatory domains) from a T cell receptor or other receptor. In some embodiments, a cell (e.g., a mammalian cell) is produced that includes a nucleic acid encoding a fusion protein and includes a chimeric antigen receptor. The chimeric antigen receptor can be used to graft, for example, the specificity of a monoclonal antibody or binding portion thereof onto a T cell. In some embodiments of the invention, the genetically engineered cell further includes a sequence encoding a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is specific for a molecule on a tumor cell. A genetically engineered cell expressing a T cell receptor or a chimeric antigen receptor can be used to target a specific tissue that requires expression of FOXP3. Some embodiments include a method of providing and delivering FOXP3 by targeting a specific tissue. In some embodiments, the tissue is a transplant tissue, hi some embodiments, the chimeric antigen receptor is specific for a target molecule on the transplant tissue.

[0054] As described herein, the genetically modified cells of the present invention are cells genetically modified to express FOXP3, and therefore, in embodiments of the present invention, these cells are referred to as "T reg These cells are also referred to as "CD34 phenotype" cells. + Cells, such as CD34 + It may be a hematopoietic stem cell.

[0055] As used herein, "protein sequence" includes, but is not limited to, a polypeptide sequence of amino acids that is the primary structure of a protein. As used herein, "upstream" refers to a 5' position on a polynucleotide and toward the N-terminus on a polypeptide. As used herein, "downstream" refers to a 3' position on a nucleotide and toward the C-terminus on a polypeptide. Thus, "N-terminus" refers to the position of an element or a specific position on a polynucleotide toward the N-terminus on a polypeptide.

[0056] As used herein, the term "expression" or "protein expression" refers to the translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental or morphological characteristics, and quantitative or qualitative indicators. In some embodiments, the protein or proteins are expressed in a configuration that allows them to dimerize in the presence of a ligand.

[0057] A "functional equivalent" or "fragment of a functional equivalent" in the context of describing a protein may have one or more conservative amino acid substitutions. A "conservative amino acid substitution" refers to the substitution of an amino acid with another amino acid that has similar properties to the original amino acid. Conservative amino acid groups are shown below. [Table 1]

[0058] Conservative substitutions may be introduced at any position of a given peptide or fragment thereof. However, non-conservative substitutions may be desirable, particularly, but not limited to, at any one or more positions. Non-conservative substitutions may form functionally equivalent fragments of peptides, for example, substantially different in polarity, charge, and / or steric bulk, but still maintain the functionality of the derivative or variant fragment.

[0059] "Sequence identity (%)" is determined by comparing two sequences in optimal alignment over a comparison window. A portion of a polynucleotide or polypeptide sequence over the comparison window may have additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (not including additions or deletions). In some cases, sequence identity (%) can be calculated by measuring the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, calculating the number of positions where nucleic acid bases or amino acid residues match, dividing the number of matched positions by the total number of positions over the comparison window, and multiplying the result by 100.

[0060] "Identity" or "percent identity" with respect to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are determined to be identical when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the sequence comparison algorithms described below or by manual alignment and visual inspection, or to two or more sequences or subsequences that have a specified percentage of identical amino acid residues or nucleotides (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region, e.g., an entire polypeptide sequence or an individual domain of a polypeptide). Such sequences are referred to as "substantially identical." This definition also applies to the complement of a test sequence.

[0061] The terms "complementary" and "substantially complementary" are used interchangeably herein to mean that a particular nucleic acid (e.g., DNA or RNA) has a nucleotide sequence capable of non-covalently binding to another nucleic acid in a sequence-specific and antiparallel manner (i.e., the particular nucleic acid specifically binds to the complementary nucleic acid), i.e., forming Watson-Crick base pairs or G / U base pairs. As known in the art, standard Watson-Crick base pairings include thymidine (T) and adenine (A), uracil (U) and adenine (A), and cytosine (C) and guanine (G).

[0062] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that can be transcribed into RNA. A DNA polynucleotide can code for an RNA that is translated into a protein (mRNA) or an RNA that is not translated into a protein (e.g., tRNA, rRNA, or guide RNA; also referred to herein as "non-coding" RNA or "ncRNA"). A "protein coding sequence" or a "sequence that codes for a particular protein or polypeptide" is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) when placed under the control of appropriate regulatory sequences in vitro or in vivo.

[0063] As used herein, a "codon" refers to a single genetic code unit formed by a sequence of three nucleotides in a DNA or RNA molecule. As used herein, "codon degeneracy" refers to the property of the genetic code that allows for variation in the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.

[0064] "Codon-optimized" or "codon optimization" refers to genes or coding regions of a nucleic acid molecule for transformation of various hosts, and refers to the modification of codons in the genes or coding regions of a nucleic acid molecule to reflect the codons typically used in the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one codon, multiple codons, or a very large number of codons with one or more codons that are more frequently used in the genes of that organism. Tables showing the frequency of codon usage are readily available, for example, from the "Codon Usage Database." Using knowledge of the frequency of codon usage or codon preference in each organism, one skilled in the art can apply the frequency of codons to a given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that encodes a polypeptide and in which the optimal codons for a given organism are used. Codon-optimized coding regions can be designed by various methods known to those skilled in the art.

[0065] For example, the term "recombinant" or "engineered" when used with respect to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by or is the result of a laboratory method. Thus, for example, a recombinant or engineered protein includes a protein produced by a laboratory method. A recombinant or engineered protein may contain amino acid residues that are not found in the native (non-recombinant or wild-type) protein, or may contain amino acid residues that have been modified (e.g., labeled). The term "recombinant" or "engineered" may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include chemical modification of a peptide, protein, or nucleic acid sequence (including one or more amino acids, deoxyribonucleotides, or ribonucleotides); addition, deletion, or substitution of one or more amino acids in a peptide or protein; or addition, deletion, or substitution of one or more nucleic acids in a nucleic acid sequence.

[0066] "Genomic DNA" or "genomic sequence" refers to the genomic DNA of an organism, including, but not limited to, the genomic DNA of a bacterium, a fungus, an archaea, a plant or an animal.

[0067] As used herein, "transgene," "exogenous gene" or "exogenous sequence" with respect to a nucleic acid refers to a nucleic acid sequence or gene that is not present in the genome of a cell, but that has been artificially introduced into the genome, e.g., via genome editing.

[0068] As used herein, "endogenous gene" or "endogenous sequence" with respect to a nucleic acid refers to a nucleic acid sequence or gene that is naturally present in the genome of a cell, without being introduced through artificial means.

[0069] A "vector", "expression vector" or "construct" is a nucleic acid used to introduce heterologous nucleic acid into a cell, and contains various regulatory elements, allowing the expression of the heterologous nucleic acid in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some embodiments, the vector is a plasmid, minicircle, yeast, or viral genome. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a vector for protein expression in bacterial systems, such as E. coli. As used herein, "expression" or "protein expression" refers to the translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental or morphological characteristics, and quantitative or qualitative indicators. In some embodiments, the protein or proteins are expressed in an arrangement that allows them to dimerize in the presence of a ligand. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some embodiments, the vector is an adeno-associated virus (AAV) vector (e.g., but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, etc.).

[0070] As used herein, "fusion protein" or "chimeric protein" includes, but is not limited to, a protein created by, for example, joining two or more genes that originally encoded separate proteins or portions of separate proteins. Fusion proteins can also be created from specific protein domains from two or more separate proteins. Such fusion genes can be translated to yield a single polypeptide or multiple polypeptides with functional properties from each of the original proteins. Recombinant fusion proteins can be artificially created by recombinant DNA techniques used in biological research or therapy. Methods for creating such fusion proteins are known to those skilled in the art. Some fusion proteins combine entire peptides and therefore may contain all domains, and in particular all functional domains, of the original proteins. However, other fusion proteins, particularly non-naturally occurring proteins, combine only portions of the coding sequences and therefore do not maintain the original functions of the parent genes from which such proteins are derived. In some embodiments, a fusion protein is provided that includes an interferon or PD-1 protein or both.

[0071] As used herein, a "conditional" promoter or an "inducible" promoter refers to a nucleic acid construct comprising a promoter that causes a gene to be expressed in the presence of an inducer, but does not substantially cause the gene to be expressed in the absence of the inducer.

[0072] As used herein, "constitutive" refers to a nucleic acid construct that contains a constitutive promoter and thus directs expression of a polypeptide that is produced continuously.

[0073] In some embodiments, the inducible promoter has low basal activity. In some embodiments, when using lentiviral vectors, the basal activity in uninduced cells is 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less (but not 0%), or a percentage within a range defined by any two of these values, of the basal activity in cells in which expression of the gene is induced. Basal activity can be determined by measuring the expression of a transgene (e.g., a marker gene) in the absence of an inducer (e.g., a drug) using flow cytometry. In some of the embodiments described herein, expression is measured using a marker protein such as Akt.

[0074] In some embodiments, the inducible promoter is capable of inducing higher activity when expressed than when expression is not induced or at basal levels. In some embodiments, the level of activity when expression is induced is 2-fold, 4-fold, 6-fold, 8-fold, 9-fold, 10-fold or more than when expression is not induced, or within a range defined by any two of these values. In some embodiments, the transgene under the control of the inducible promoter is turned off in the absence of a transactivator for less than 10 days, less than 8 days, less than 6 days, less than 4 days, less than 2 days, or less than 1 day, or within a range defined by any two of these periods, but not at 0 days.

[0075] In some embodiments, inducible promoters are designed or engineered to have low basal activity, be induced to produce high levels of expression, and / or be capable of being switched on and off for short periods of time.

[0076] "Woodchuck Hepatitis Virus (WHP) Post-transcriptional Regulatory Element (WPRE)" is a DNA sequence that, when transcribed, forms a tertiary structure that enhances expression. WPRE may be used to increase expression of a gene delivered by a viral vector. In the embodiments described herein, a WPRE3 element is used to enhance expression of a delivered nucleic acid (such as a delivered cDNA).

[0077] In some embodiments, the immunomodulatory imide drug used in the methods described herein may include thalidomide (including its analogs, derivatives, or pharmaceutically acceptable salts). Thalidomide may include Immunoprin, Thalomid, Talidex, Talizer, Neurosedyn, α-(N-phthalimido)glutarimide, 2-(2,6-dioxopiperidin-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione; or pomalidomide (including its analogs, derivatives, or pharmaceutically acceptable salts). Pomalidomide may include Pomalyst, Imnovid, (RS)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindole-1,3-dione; or lenalidomide (including its analogs, derivatives, or pharmaceutically acceptable salts). Lenalidomide can include Revlimid, (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione; or Apremilast (including analogs, derivatives, or pharma- ceutically acceptable salts thereof). Apremilast can include Otezla, CC-10004, N-{2-[(1S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide; or any combination thereof.

[0078] As used herein, the term "extracellular binding domain" refers to one of the domains constituting a complex, configured to bind to a specific atom or a specific molecule, and present outside a cell. In some embodiments, the extracellular binding domain of CISC is an FKBP domain or a portion thereof. In some embodiments, the extracellular binding domain is an FRB domain or a portion thereof. In some embodiments, the extracellular binding domain is configured to stimulate dimerization of two CISC components by binding to a ligand or a drug. In some embodiments, the extracellular binding domain is configured to bind to a cytokine receptor modulator.

[0079] CISC (chemically induced signaling complex) is a synthetic protein complex composed of multiple components that is configured to be co-expressed as two chimeric proteins in a host cell, as described in International Patent Application PCT / US2017 / 065746, which is expressly incorporated herein by reference in its entirety. CISC contains two chimeric protein components, one of which is an extracellular domain that constitutes one half of a rapamycin-binding complex, which is fused to an intracellular signaling complex that constitutes the other half of the rapamycin-binding complex. By delivering a nucleic acid encoding CISC to a host cell, intracellular signaling that can be controlled by the presence of rapamycin or a rapamycin-related compound can be generated in the host cell.

[0080] Although rapamycin-induced CISC dimerization can induce intracellular signaling, the presence of rapamycin inhibits host cell proliferation and viability, limiting its usefulness in therapeutic and research applications.Therefore, there is a need for novel compositions and methods that can utilize rapamycin-mediated intracellular signaling by CISC and mitigate the adverse effects of rapamycin or rapamycin-related compounds on host cell proliferation and viability.

[0081] As used herein, "dimeric chemical-induced signaling complex," "dimerized CISC," or "dimer" refers to two components that make up CISC, which may or may not bind to each other to form a fusion protein complex. "Dimerization" refers to the process by which two separate entities bind to each other to become a single entity, such as in response to binding of a ligand (e.g., rapamycin). In some embodiments, dimerization is stimulated by a ligand or drug. In some embodiments, "dimerization" refers to homodimerization, i.e., two identical entities bind and dimerize, e.g., two identical CISC components bind and dimerize. In some embodiments, "dimerization" refers to heterodimerization, i.e., two different entities bind and dimerize, e.g., two different and separate CISC components bind and dimerize. In some embodiments, dimerization of CISC components forms a cell signaling pathway. In some embodiments, dimerization of CISC components allows selective expansion of cells or cell populations. Additional CISC systems may include CISC and gibberellin dimerization systems or CISC dimerization systems using SLF-TMP. Other chemically inducible dimerization (CID) systems and their components may also be used.

[0082] As used herein, "chemical-induced signaling complex" or "CISC" refers to a genetically engineered complex that initiates a signal inside a cell and, as a direct result, undergoes ligand-induced dimerization. CISCs can be homodimers (dimerization of two identical components) or heterodimers (dimerization of two different components). Thus, as used herein, the term "homodimer" refers to a dimer consisting of two protein components described herein that have identical amino acid sequences. Additionally, the term "heterodimer" refers to a dimer consisting of two protein components described herein that do not have identical amino acid sequences.

[0083] CISC may be a synthetic complex as described in further detail herein. As used herein, "synthetic" refers to a complex, protein, dimer, or composition described herein that is not naturally occurring or found in nature. In some embodiments, "IL2R-CISC" refers to a signaling complex that includes an interleukin 2 receptor component. In some embodiments, "IL2 / 15-CISC" refers to a signaling complex that includes a receptor signaling subunit shared by interleukin 2 and interleukin 15. In some embodiments, "IL7-CISC" refers to a signaling complex that includes an interleukin 7 receptor component. Thus, CISC may be named according to the component parts that make up a particular CISC component. One of skill in the art will recognize that the component parts of a chemically induced signaling complex may be composed of natural or synthetic components that are useful for incorporation into a CISC. Thus, these examples provided herein are not intended to limit the invention.

[0084] As used herein, "FKBP" refers to an FK506 binding protein domain. FKBP refers to a family of proteins with prolyl isomerase activity, which are related in function to cyclophilin but are not similar in amino acid sequence. FKBP has been identified in many eukaryotic organisms, including yeast and humans, and functions as a protein folding chaperone for proteins containing proline residues. FKBP, along with cyclophilin, belongs to the immunophilin family. FKBP includes, for example, FKBP12, as well as proteins encoded by the AIP gene, AIPL1 gene, FKBP1A gene, FKBP1B gene, FKBP2 gene, FKBP3 gene, FKBP5 gene, FKBP6 gene, FKBP7 gene, FKBP8 gene, FKBP9 gene, FKBP9L gene, FKBP10 gene, FKBP11 gene, FKBP14 gene, FKBP15 gene, FKBP52 gene, or LOC541473 gene; homologs and functional protein fragments thereof.

[0085] As used herein, "FRB" refers to the FKBP rapamycin binding domain. The FRB domain is a polypeptide region (protein "domain") that is configured to form a ternary complex with the FKBP protein and rapamycin or its rapalog. The FRB domain is present in a variety of natural proteins, including the mTOR protein (also referred to herein as FRAP, RAPT1 or RAFT) from humans and other species; yeast proteins, including Tor1 or Tor2; and the FRAP homologue from Candida. Both FKBP and FRB are key components of mammalian target of rapamycin (mTOR) signal transduction.

[0086] "Naked FKBP rapamycin binding domain polypeptide" or "naked FRB domain polypeptide" (also referred to as "FKBP rapamycin binding domain polypeptide" or "FRB domain polypeptide") refers to a polypeptide that contains only the amino acid sequence of the FRB domain, or to a protein in which 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of the protein consists of the amino acid sequence of the FRB domain. The FRB domain can be expressed as a soluble protein of 12 kDa (Chen, J. et al. (1995). Proc. Natl. Acad. Sci. USA, 92(11):4947-4951). The FRB domain forms a four-helix bundle, a structural motif commonly found in globular proteins. The overall dimensions of the FRB domain are 30 Å × 45 Å × 30 Å, and the four helices are connected by short lower loops that resemble the fold of cytochrome b562 (Choi, J. et al. (1996). Science, 273(5272):239-242). In some embodiments, the naked FRB domain includes the amino acids set forth in SEQ ID NO: 37 (MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK; SEQ ID NO: 37) or SEQ ID NO: 38 (MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK; SEQ ID NO: 38).

[0087] As used herein, "activate" refers to the enhancement of at least one biological activity of a protein of interest. Similarly, "activation" refers to the state of a protein of interest in an enhanced state of activity. "Activatable" refers to the protein of interest being activatable in the presence of a signal, drug, ligand, compound, or stimulus. In some embodiments, the dimers described herein are activated in the presence of a signal, drug, ligand, compound, or stimulus to become signal transduction-competent dimers. As used herein, "signal transduction-competent" refers to the ability of a dimer or the configuration thereof to initiate or sustain a downstream signal transduction pathway.

[0088] As used herein, a "signaling domain" refers to a domain of a fusion protein or a component of a CISC that is involved in a signaling cascade inside a cell (such as a mammalian cell). A "signaling domain" refers to a signaling moiety that provides a signal to a cell (such as a T cell) that mediates a cellular response (such as a T cell response) in addition to a first signal provided by, for example, the CD3ζ chain of the TCR / CD3 complex, including, but not limited to, activation, proliferation, differentiation, or cytokine secretion, or any combination thereof. In some embodiments, the signaling domain is N-terminal to the transmembrane domain, hinge domain, and extracellular domain. In some embodiments, the signaling domain is a synthetic or naturally occurring domain. In some embodiments, the signaling domain is a linked cytoplasmic signaling domain. In some embodiments, the signaling domain is a cytokine signaling domain. In some embodiments, the signaling domain is an antigen signaling domain. In some embodiments, the signaling domain is an interleukin 2 receptor gamma subunit (IL2Rγ or IL2Rg) domain. In some embodiments, the signaling domain is an interleukin 2 receptor beta subunit (IL2Rβ or IL2Rb) domain, or a truncated IL2Rβ domain (e.g., a truncated IL2Rβ domain comprising the amino acid sequence of SEQ ID NO:5). In some embodiments, binding of an agent or ligand to the extracellular binding domain results in dimerization of the CISC components, which in turn activates a signaling pathway and causes signal transduction via the signaling domain. As used herein, "signal transduction" refers to activation of a signaling pathway by binding of a ligand or agent to the extracellular domain. Binding of the extracellular domain to a ligand or agent results in dimerization of the CISC components and activation of a signal.

[0089] As used herein, "IL2Rb" or "IL2Rβ" refers to the interleukin 2 receptor β subunit. Similarly, "IL2Rg" or "IL2Rγ" refers to the interleukin 2 receptor γ subunit, and "IL2Ra" or "IL2Rα" refers to the interleukin 2 receptor α subunit. There are three forms of IL-2 receptor, namely, the α chain, the β chain, and the γ chain, which are also subunits of the receptors for other cytokines. IL2Rβ and IL2Rγ are members of the type I cytokine receptor family. As used herein, "IL2R" refers to the interleukin 2 receptor, which is involved in T-cell-mediated immune responses. IL2R is involved in receptor-mediated endocytosis and the transmission of mitogenic signals from interleukin 2.

[0090] Similarly, "IL-2 / 15R" refers to the receptor signaling subunit shared by IL-2 and IL-15 and can include the α subunit (IL2 / 15Ra or IL2 / 15Rα), β subunit (IL2 / 15Rb or IL2 / 15Rβ) or γ subunit (IL2 / 15Rg or IL2 / 15Rγ).

[0091] In some embodiments, the chemical-induced signaling complex is a heterodimerization-activated signaling complex that includes two components. In some embodiments, the first component includes an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracytoplasmic signaling domains that are one member of a heterodimerization pair. In some embodiments, the second component includes an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracytoplasmic signaling domains that are the other member of a heterodimerization pair. Thus, in some embodiments, two genetic recombination events occur. In some embodiments, the two CISC components are expressed in a cell, such as a mammalian cell. In some embodiments, a cell, such as a mammalian cell, or a cell population, such as a mammalian cell population, is contacted with a ligand or factor that causes heterodimerization, thereby initiating signaling. In some embodiments, the homodimerization pair dimerizes, thereby resulting in a single CISC component being expressed in a cell, such as a mammalian cell, and the homodimerized CISC component initiates signaling.

[0092] As used herein, "selective expansion" refers to the expansion potential of a desired cell, such as a mammalian cell, or a desired cell population, such as a mammalian cell population. In some embodiments, selective expansion refers to the generation or expansion of a population of pure cells (such as mammalian cells) in which two genes are recombined. One component of the dimerized CISC is involved in one genetic modification, and the other component is involved in the other genetic modification. Thus, each component of the heterodimerized CISC is associated with each genetic modification. By exposing the cells to a ligand, it becomes possible to selectively expand only cells (such as mammalian cells) that have both desired modifications. Thus, in some embodiments, the only cells (e.g., mammalian cells) that can respond to contact with the ligand are those that express both components of the heterodimerized CISC.

[0093] As used herein, a "cytokine receptor modulator" refers to an agent that modulates phosphorylation of downstream targets of a cytokine receptor, activation of a signaling pathway associated with a cytokine receptor, and / or expression of a specific protein, such as a cytokine. Such agents may directly or indirectly modulate phosphorylation of downstream targets of a cytokine receptor, activation of a signaling pathway associated with a cytokine receptor, and / or expression of a specific protein, such as a cytokine. Thus, examples of cytokine receptor modulators include, but are not limited to, cytokines; fragments of cytokines; fusion proteins; or antibodies or binding portions thereof that immunospecifically bind to a cytokine receptor or a fragment thereof. Additionally, examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides (e.g., soluble cytokine receptors), fusion proteins, or antibodies or binding portions thereof that immunospecifically bind to a cytokine or a fragment thereof.

[0094] As used herein, "hinge domain" refers to a domain that may link the extracellular binding domain to the transmembrane domain, thereby providing flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain close to the cell membrane, minimizing the possibility of recognition by an antibody or binding fragment thereof. In some embodiments, the extracellular binding domain is located N-terminal to the hinge domain. In some embodiments, the hinge domain may be natural or synthetic.

[0095] As used herein, "transmembrane domain" or "TM domain" refers to a domain that is stable inside a membrane, such as a cell membrane. The terms "transmembrane span", "integral membrane protein" and "integral membrane domain" are also used herein. In some embodiments, the hinge domain and the extracellular domain are located N-terminal to the transmembrane domain. In some embodiments, the transmembrane domain is a natural domain or a synthetic domain. In some embodiments, the transmembrane domain is an IL-2 transmembrane domain.

[0096] As used herein, a "host cell" includes any type of cell (e.g., a mammalian cell) that is susceptible to transformation, transfection, or transduction with a nucleic acid construct or vector. In some embodiments, the host cell, such as a mammalian cell, is a T cell or a regulatory T cell (referred to herein as "Treg" or "T reg In some embodiments, the host cell, such as a mammalian cell, is a hematopoietic stem cell. In some embodiments, the host cell is a CD34 + Cells, such as CD34 + Hematopoietic stem cells. As used herein, a "cell population" refers to a cell population (such as a mammalian cell population) that includes two or more types of cells. In some embodiments, cells (such as mammalian cells) that include the protein sequences described herein or expression vectors that encode the protein sequences are produced.

[0097] As used herein, "transformed" or "transfected" refers to a cell (such as a mammalian cell), tissue, organ, or organism into which an exogenous polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule may be integrated into the genomic DNA of the recipient cell (such as a mammalian cell), tissue, organ, or organism, thereby allowing the introduced polynucleotide molecule to be passed on to subsequent progeny. Furthermore, a "transgenic" cell (such as a mammalian cell) or "transgenic" organism, or a "transfected" cell (such as a mammalian cell) or "transfected" organism, includes the progeny of a transgenic cell or organism or a transfected cell or organism, including progeny produced by a breeding program using such transgenic organisms as parents in matings, and including those that exhibit an altered phenotype resulting from the presence of the exogenous polynucleotide molecule. "Transgenic" also refers to a bacterium, fungus, or plant that contains one or more heterologous polynucleic acid molecules. "Transduction" refers to the virus-mediated transfer of genes into a cell, such as a mammalian cell.

[0098] As used herein, "mammal" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates (monkeys, chimpanzees, or apes), and in particular humans. In some embodiments, the subject is a human.

[0099] A "marker sequence" as described herein encodes a protein that is used to select or track cells (such as mammalian cells) that have a protein of interest or a protein of interest. In embodiments described herein, fusion proteins are provided that may include a selectable marker sequence in experiments such as flow cytometry.

[0100] As used herein, an "epitope" refers to a portion of an antigen or molecule that is recognized by the immune system, including antibodies, T cells, or B cells. An epitope typically has at least seven amino acids and may be a linear epitope or a conformational epitope. In some embodiments, a cell (such as a mammalian cell) expressing a fusion protein is provided, the cell further comprising a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor comprises an scFv capable of recognizing an epitope on a cancer cell. The terms "isolated" or "purified" as used in describing various polypeptides or nucleic acids disclosed herein refer to a polypeptide or nucleic acid that has been identified, separated, and / or recovered from other components in the natural environment of the polypeptide or nucleic acid. In some embodiments, an isolated polypeptide or nucleic acid is free of other components with which it is naturally associated. Impurities in the natural environment of an isolated polypeptide or nucleic acid are typically substances that would interfere with diagnostic or therapeutic uses of the polypeptide or nucleic acid, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, a method is provided that includes delivering a nucleic acid according to any one of the embodiments described herein or an expression vector according to any one of the embodiments described herein to a bacterial cell, a mammalian cell or an insect cell, growing the cell in culture, inducing expression of a fusion protein, and purifying and processing the fusion protein.

[0101] The "percent amino acid sequence identity" with respect to the CISC sequences described herein is defined as the percentage of amino acid residues in each of the extracellular binding domain, hinge domain, transmembrane domain and / or signaling domain in the candidate sequence that match the amino acid residues in each of the domains in the reference sequence, and this amino acid sequence identity is calculated after aligning the candidate sequence with the reference sequence and inserting gaps as necessary to calculate the maximum sequence identity percentage, and conservative substitutions are not considered as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to maximize alignment over the entire length of the sequences being compared. For example, percent amino acid sequence identity is calculated using the WU-BLAST-2 computer program (Altschul, SF et al. (1996). Methods Enzymol., 266:460-480) using several search parameters, most of which are set to default values. Parameters that are not set to default values ​​(e.g., adjustable parameters) are set as follows: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. In some embodiments of CISCs, the CISCs include an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, each of which includes a naturally occurring or synthetic domain, or a mutated or truncated version of a naturally occurring domain (e.g., a truncated interleukin-2 receptor beta signaling domain).In some embodiments, variants or truncations of a given domain comprise amino acid sequences having 100%, 95%, 90% or 85% sequence identity, or a percentage of sequence identity within a range defined by any two of the foregoing percentages, with a sequence set forth in the sequences provided herein.

[0102] As used herein, "T cells" or "T lymphocytes" may be from any mammal, e.g., primates or other species, including monkeys, dogs, and humans. In some embodiments, the T cells are allogeneic (same species but from a different donor) to the recipient subject. In some embodiments, the T cells are autologous (donor and recipient are the same). In some embodiments, the T cells are syngeneic (donor and recipient are different but identical twins).

[0103] As used herein, the terms "comprise(s)" and "comprising," whether in the transitional or main body of a claim, are to be construed as having an open-ended meaning; that is, these terms are to be construed as synonymous with the expressions "having at least" or "comprising at least." When the term "comprise" is used in the context of a process, it means that the process includes at least the specified steps and may include additional steps. When the term "comprise" is used in the context of a compound, composition, or device, it means that the compound, composition, or device includes at least the specified features or components and may include additional features or components.

[0104] Genome editing system The present invention relates to a method for the treatment of a cell (e.g., a cell line comprising a human CD34 cell) that is capable of regulating FOXP3 expression, function or activity, such as by targeting and integrating a nucleic acid encoding FOXP3 or a functional derivative thereof into the genome of the cell. +In some embodiments, the subject has or is suspected of having an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from an organ transplant (e.g., graft-versus-host disease (GVHD)). The disclosure also provides systems for genome editing of a subject (e.g., a mammalian cell) using ex vivo and / or in vivo genome editing to provide treatment to a subject having or suspected of having a FOXP3-associated disorder or condition. In some embodiments, the subject has or is suspected of having an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from an organ transplant (e.g., graft-versus-host disease (GVHD)).

[0105] Some embodiments include (a) a DNA endonuclease, or a nucleic acid encoding the DNA endonuclease; (b) a gRNA (e.g., sgRNA) or a nucleic acid encoding the gRNA capable of targeting the DNA endonuclease to the FOXP3 gene or a non-FOXP3 locus (e.g., AAVS1 (i.e., an adeno-associated virus integration site in the genome of a cell)); and (c) Donor template containing the coding sequence of FOXP3. The present invention relates to a system including: In some embodiments, the DNA endonuclease is Cas1 endonuclease, Cas1B endonuclease, Cas2 endonuclease, Cas3 endonuclease, Cas4 endonuclease, Cas5 endonuclease, Cas6 endonuclease, Cas7 endonuclease, Cas8 endonuclease, Cas9 endonuclease (also known as Csn1 and Csx12), Cas100 endonuclease, Csy1 endonuclease, Csy2 endonuclease, Csy3 endonuclease, Cse1 endonuclease, Cse2 endonuclease, Csc1 endonuclease, Csc2 endonuclease, Csa5 endonuclease, Csn2 endonuclease, Csm2 endonuclease, Csm3 endonuclease, Cs m4 endonuclease, Csm5 endonuclease, Csm6 endonuclease, Cmr1 endonuclease, Cmr3 endonuclease, Cmr4 endonuclease, Cmr5 endonuclease, Cmr6 endonuclease, Csb1 endonuclease, Csb2 endonuclease, Csb3 endonuclease, Csx17 endonuclease, Csx14 endonuclease, Cs The DNA endonuclease is selected from the group consisting of x10 endonuclease, Csx16 endonuclease, CsaX endonuclease, Csx3 endonuclease, Csx1 endonuclease, Csx15 endonuclease, Csf1 endonuclease, Csf2 endonuclease, Csf3 endonuclease, Csf4 endonuclease and Cpf1 endonuclease, and functional derivatives thereof. In some embodiments, the DNA endonuclease is a Cas endonuclease, such as Cas9 endonuclease (e.g., Cas9 endonuclease from Streptococcus pyogenes). In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in the FOXP3 gene. In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in exon 1 of the FOXP3 gene.In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7 and 27-29, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7 and 27-29. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 2, 3, and 5. In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in a non-FOXP3 locus (e.g., AAVS1). In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 15-20, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 15-20. In some embodiments, the coding sequence for FOXP3 encodes FOXP3 or a functional derivative thereof. In some embodiments, the coding sequence for FOXP3 is a FOXP3 cDNA. An exemplary FOXP3 cDNA sequence may be included in an AAV donor template having the nucleotide sequence of SEQ ID NO:34.In some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof has at least 70% or at least about 70% sequence identity, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least more, or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least more, sequence identity to the sequence shown in SEQ ID NO: 110 or 111. In some embodiments, the system comprises a DNA endonuclease. In some embodiments, the system comprises a nucleic acid encoding a DNA endonuclease. In some embodiments, the system comprises a gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the system comprises a nucleic acid encoding a gRNA, hi some embodiments, the system further comprises one or more additional gRNAs or a nucleic acid encoding the one or more additional gRNAs.

[0106] In some embodiments, according to any of the systems described herein, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7, 15-20, and 27-29, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7, 15-20, and 27-29. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 2, 3, and 5. In some embodiments, the gRNA comprises a spacer sequence as set forth in SEQ ID NO: 2, or a variant of the spacer sequence having no more than three mismatches compared to SEQ ID NO: 2. In some embodiments, the gRNA comprises a spacer sequence as set forth in SEQ ID NO: 3, or a variant of the spacer sequence having no more than three mismatches compared to SEQ ID NO: 3. In some embodiments, the gRNA comprises a spacer sequence as shown in SEQ ID NO:5, or a variant of said spacer sequence having 3 or fewer mismatches compared to SEQ ID NO:5.

[0107] In some embodiments, according to any of the systems described herein, the Cas DNA endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease is a Cas9 endonuclease from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 is a Cas9 from Staphylococcus lugdunensis (SluCas9).

[0108] In some embodiments, according to any of the systems described herein, the system includes a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding a DNA endonuclease is codon-optimized for expression in a host cell. In some embodiments, the nucleic acid encoding a DNA endonuclease is codon-optimized for expression in a human cell. In some embodiments, the nucleic acid encoding a DNA endonuclease is DNA (such as a DNA plasmid). In some embodiments, the nucleic acid encoding a DNA endonuclease is RNA (such as an mRNA).

[0109] In some embodiments, according to any of the systems described herein, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in a host cell, hi some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in a human cell.

[0110] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof and a promoter configured to express FOXP3 or a functional derivative thereof. Examples of promoters include MND promoter, PGK promoter, and EF1 promoter. In some embodiments, the promoter has a sequence as set forth in any of SEQ ID NOs: 147-149, or a variant having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 147-149. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0111] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette, the donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, but does not comprise an exogenous promoter configured to express FOXP3 or a functional derivative thereof. + In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0112] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, and the donor template is configured to integrate the donor cassette into a genomic locus targeted by a gRNA included in the system by homology directed repair (HDR). In some embodiments, the donor cassette is flanked by homology arms corresponding to the sequence of the targeted genomic locus. In some embodiments, the length of the homology arms is at least 0.2 kb or at least about 0.2 kb (e.g., at least 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb or more, or at least about 0.3 kb, about 0.4 kb, about 0.5 kb, about 0.6 kb, about 0.7 kb, about 0.8 kb, about 0.9 kb, about 1 kb or more). In some embodiments, the length of the homologous arm is at least 0.6 kb or at least about 0.6 kb. Exemplary homologous arms include those contained in a donor template having the sequence of SEQ ID NO: 34 or 161. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0113] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template being configured to integrate the donor cassette into a genomic locus targeted by a gRNA included in the system by non-homologous end joining (NHEJ). In some embodiments, the donor cassette is flanked on one or both sides by a gRNA target site. In some embodiments, the donor cassette is flanked on both sides by a gRNA target site. In some embodiments, the gRNA target site is a target site of a gRNA included in the system. In some embodiments, the gRNA target site of the donor template is the reverse complement of a cellular genomic gRNA target site targeted by a gRNA included in the system. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0114] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or its functional derivative, and the donor template further comprises a regulatory element that enhances stable expression. Exemplary regulatory elements that enhance stable expression include WPRE and UCOE. In some embodiments, the WPRE is a full-length WPRE. In some embodiments, the WPRE is a truncated WPRE. Exemplary WPREs include a WPRE contained in a donor template having a sequence as set forth in any of SEQ ID NOs: 33, 34, and 161. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0115] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, and the donor template further comprises a nucleic acid encoding a selection marker. In some embodiments, the selection marker is a surface marker that allows for the selection of cells expressing the selection marker. In some embodiments, the selection marker is a low affinity nerve growth factor receptor (LNGFR) polypeptide, a green fluorescent protein (GFP), or a functional derivative thereof. In some embodiments, the LNGFR polypeptide or a functional derivative thereof comprises an amino acid sequence of SEQ ID NO: 144 or a variant thereof having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the nucleic acid encoding GFP or a functional derivative thereof has a nucleic acid sequence of a GFP coding region as set forth in any of SEQ ID NOs: 33, 35, and 36. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0116] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template further comprising a nucleic acid encoding a 2A self-cleaving peptide between adjacent nucleic acids encoding components of the system. In some embodiments, the donor template comprises a nucleic acid encoding a 2A self-cleaving peptide between each adjacent nucleic acid encoding a component of the system. In some embodiments, each 2A self-cleaving peptide is independently a T2A self-cleaving peptide or a P2A self-cleaving peptide. For example, in some embodiments, the donor template comprises, in the direction from the 5' end to the 3' end, a nucleic acid encoding expression of FOXP3 or a functional variant thereof, a nucleic acid encoding a 2A self-cleaving peptide, and a nucleic acid encoding a selection marker, in that order. In some embodiments, the donor template comprises a nucleic acid as set forth in SEQ ID NO: 72 or 73, or a nucleic acid variant having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 72 or 73. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0117] Exemplary donor templates include donor templates having any one of the sequences of SEQ ID NOs: 33-36 and 161. In some embodiments, the donor template comprises the sequence of SEQ ID NO: 34 or 161. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0118] In some embodiments, according to any of the systems described herein, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated by encapsulation in a liposome or lipid nanoparticle. In some embodiments, the liposome or the lipid nanoparticle further comprises a gRNA. In some embodiments, the liposome or the lipid nanoparticle is a lipid nanoparticle. In some embodiments, the system comprises a lipid nanoparticle comprising a nucleic acid encoding a DNA endonuclease and a gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is an mRNA encoding a DNA endonuclease.

[0119] In some embodiments, according to any of the systems described herein, the DNA endonuclease complexes with a gRNA to form a ribonucleoprotein (RNP) complex.

[0120] nucleic acid Genome-targeting nucleic acids or guide RNAs The present disclosure provides a genome-targeting nucleic acid that can direct the activity of an associated polypeptide (e.g., a site-directed polypeptide or a DNA endonuclease) to a specific target sequence within a target nucleic acid. In some embodiments, the genome-targeting nucleic acid is an RNA. Herein, the genome-targeting RNA is referred to as a "guide RNA" or "gRNA." The guide RNA has at least a spacer sequence and a CRISPR repeat sequence that can hybridize to a target nucleic acid sequence of interest. In a type II system, the gRNA further has a second RNA called a tracrRNA sequence. In a type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a double strand. In a type V guide RNA (gRNA), the crRNA forms a double strand. In both systems, the double strand binds to a site-directed polypeptide to form a complex of the guide RNA and the site-directed polypeptide. The genome-targeting nucleic acid confers target specificity to the complex formed by associating with the site-directed polypeptide. Thus, the genome-targeting nucleic acid confers directionality to the activity of the site-directed polypeptide.

[0121] In some embodiments, the genome-targeting nucleic acid is a bimolecular guide RNA. In some embodiments, the genome-targeting nucleic acid is a single-molecule guide RNA. A bimolecular guide RNA has two RNA strands. The first strand has, from the 5' to 3' end, an optional spacer extension sequence, a spacer sequence, and a CRISPR minimal repeat sequence. The second strand has a tracrRNA minimal sequence (complementary to the CRISPR minimal repeat sequence), a 3'tracrRNA sequence, and an optional tracrRNA extension sequence. A single-molecule guide RNA (sgRNA) in the type II system has, from the 5' to 3' end, an optional spacer extension sequence, a spacer sequence, a CRISPR minimal repeat sequence, a single-molecule guide linker, a tracrRNA minimal sequence, a 3'tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension sequence may have elements that confer additional functions (e.g., stability) to the guide RNA. The single-molecule guide linker links the CRISPR minimal repeat sequence and the tracrRNA minimal sequence to form a hairpin structure. The optional tracrRNA extension sequence has one or more hairpins. The single-molecule guide RNA (sgRNA) of the V-type system has the CRISPR minimal repeat sequence and a spacer sequence from the 5' end to the 3' end.

[0122] As an example, guide RNAs used in the CRISPR / Cas / Cpf1 system, or other smaller RNAs, can be easily synthesized by chemical means known in the art, as described below. Although chemical synthesis procedures are continually expanding, the length of polynucleotides significantly exceeds about 100 nucleotides in length, making purification of said RNAs by procedures such as high performance liquid chromatography (HPLC without gels such as PAGE) difficult. One method used to generate long RNAs is to generate two or more molecules and then link them together. Very long RNAs, such as RNAs encoding Cas endonucleases (e.g., Cas9 or Cpf1 endonucleases), are easily generated using enzymes. Various RNA modifications can be introduced during or after the process of chemical synthesis and / or enzymatic generation of RNA, including modifications that increase stability, reduce the likelihood or magnitude of innate immune response, and / or enhance other attributes, as reported in the art.

[0123] In some embodiments, a guide RNA (gRNA) is provided that includes a spacer sequence complementary to a genomic sequence in or near a FOXP3 gene of a cell. In some embodiments, the gRNA includes a spacer sequence as set forth in any of SEQ ID NOs: 1-7 and 27-29, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 1-7 and 27-29. In some embodiments, the gRNA includes a spacer sequence as set forth in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA includes a spacer sequence as set forth in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 2, 3, and 5.

[0124] In some embodiments, a guide RNA (gRNA) is provided that includes a spacer sequence complementary to a genomic sequence in the AAVS1 gene of a cell or a nearby genomic sequence in the AAVS1 gene. In some embodiments, the gRNA includes a spacer sequence set forth in any of SEQ ID NOs: 15 to 20, or a variant of the spacer sequence having 3 or fewer mismatches compared to any of SEQ ID NOs: 15 to 20.

[0125] Guide RNAs generated by in vitro transcription may contain a mixture of full length and partial guide RNA molecules. Chemically synthesized guide RNA molecules typically contain more than 75% full length guide molecules and may further contain chemically modified bases, such as chemically modified bases to increase the resistance of the guide RNA to cleavage by nucleases in cells.

[0126] Spacer extension sequence In some embodiments of genome-targeted nucleic acids, the spacer extension sequence can modulate activity, confer stability, or provide a location for modifying the genome-targeted nucleic acid. The spacer extension sequence can also modulate on-target or off-target activity or specificity. In some embodiments, a spacer extension sequence is provided. The length of the spacer extension sequence can be greater than 1 nucleotide, greater than 5 nucleotides, greater than 10 nucleotides, greater than 15 nucleotides, greater than 20 nucleotides, greater than 25 nucleotides, greater than 30 nucleotides, greater than 35 nucleotides, greater than 40 nucleotides, greater than 45 nucleotides, greater than 50 nucleotides, greater than 60 nucleotides, greater than 70 nucleotides, greater than 80 nucleotides, greater than 90 nucleotides, greater than 100 nucleotides, greater than 120 nucleotides, greater than 140 nucleotides, greater than 160 nucleotides, greater than 180 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 350 ...350 nucleotides, greater than 40 nucleotides, greater than 450 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 9 The nucleic acid sequence may be greater than 1 nucleotide, greater than 220 nucleotides, greater than 240 nucleotides, greater than 260 nucleotides, greater than 280 nucleotides, greater than 300 nucleotides, greater than 320 nucleotides, greater than 340 nucleotides, greater than 360 nucleotides, greater than 380 nucleotides, greater than 400 nucleotides, greater than 1000 nucleotides, greater than 2000 nucleotides, greater than 3000 nucleotides, greater than 4000 nucleotides, greater than 5000 nucleotides, greater than 6000 nucleotides, or greater than 7000 nucleotides, or any of these nucleotide lengths.The length of the spacer extension sequence may be 1 nucleotide, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 120 nucleotides, 140 nucleotides, 160 nucleotides, 180 nucleotides, 200 nucleotides, 220 nucleotides, 240 nucleotides, 260 nucleotides, 280 nucleotides, 300 nucleotides, 320 nucleotides, 340 nucleotides, 360 nucleotides, 380 nucleotides, 400 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, 5000 nucleotides, 6000 nucleotides, or 7000 nucleotides, or about 1 nucleotide, about 5 nucleotides, about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 2 nucleotide length, about 15 nucleotide length, about 20 nucleotide length, about 25 nucleotide length, about 30 nucleotide length, about 35 nucleotide length, about 40 nucleotide length, about 45 nucleotide length, about 50 nucleotide length, about 60 nucleotide length, about 70 nucleotide length, about 80 nucleotide length, about 90 nucleotide length, about 100 nucleotide length, about 120 nucleotide length, about 140 nucleotide length, about 160 nucleotide length, about 180 nucleotide length, about 200 nucleotide length, about 220 nucleotide length, about The length may be 40 nucleotides, about 260 nucleotides, about 280 nucleotides, about 300 nucleotides, about 320 nucleotides, about 340 nucleotides, about 360 nucleotides, about 380 nucleotides, about 400 nucleotides, about 1000 nucleotides, about 2000 nucleotides, about 3000 nucleotides, about 4000 nucleotides, about 5000 nucleotides, about 6000 nucleotides, or about 7000 nucleotides, or more than these nucleotide lengths.The length of the spacer extension sequence may be less than 1 nucleotide, less than 5 nucleotides, less than 10 nucleotides, less than 15 nucleotides, less than 20 nucleotides, less than 25 nucleotides, less than 30 nucleotides, less than 35 nucleotides, less than 40 nucleotides, less than 45 nucleotides, less than 50 nucleotides, less than 60 nucleotides, less than 70 nucleotides, less than 80 nucleotides, less than 90 nucleotides, less than 100 nucleotides, less than 120 nucleotides, less than 140 nucleotides, less than 160 nucleotides, less than 180 nucleotides, less than 200 nucleotides, The length of the spacer extension sequence may be less than 1 nucleotide, less than 220 nucleotides, less than 240 nucleotides, less than 260 nucleotides, less than 280 nucleotides, less than 300 nucleotides, less than 320 nucleotides, less than 340 nucleotides, less than 360 nucleotides, less than 380 nucleotides, less than 400 nucleotides, less than 1000 nucleotides, less than 2000 nucleotides, less than 3000 nucleotides, less than 4000 nucleotides, less than 5000 nucleotides, less than 6000 nucleotides, less than 7000 nucleotides, or more than these nucleotide lengths. In some embodiments, the length of the spacer extension sequence is less than 10 nucleotides. In some embodiments, the length of the spacer extension sequence is 10 to 30 nucleotides. In some embodiments, the length of the spacer extension sequence is 30 to 70 nucleotides.

[0127] In some embodiments, the spacer extension sequence has another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). In some embodiments, the another moiety decreases or increases the stability of the nucleic acid targeting nucleic acid. In some embodiments, the another moiety is a transcription terminator segment (i.e., a transcription termination sequence). In some embodiments, the another moiety functions in eukaryotic cells. In some embodiments, the another moiety functions in prokaryotic cells. In some embodiments, the another moiety functions in both eukaryotic and prokaryotic cells. Suitable moieties include, but are not limited to, a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a riboswitch sequence (e.g., one that provides controlled stabilization and / or allows controlled access by a protein or protein complex); a sequence that forms a dsRNA duplex (i.e., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that allows for tracking (e.g., direct attachment to a fluorescent molecule, attachment to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.); or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, such as a transcriptional activator, transcriptional repressor, DNA methyltransferase, DNA methylase, histone acetyltransferase, histone deacetylase, etc.).

[0128] Spacer sequence The spacer sequence hybridizes to a sequence in the target nucleic acid of interest. The spacer of the genome targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the nucleotide sequence of the spacer varies according to the sequence of the target nucleic acid of interest.

[0129] In the CRISPR / Cas system described herein, the spacer sequence is designed to hybridize to the target nucleic acid located at the 5' end side of the PAM in the Cas endonuclease used in this system. The spacer may perfectly match the target sequence or may have a mismatch. Each Cas endonuclease has a specific PAM sequence that recognizes the target DNA. For example, Cas9 from Streptococcus pyogenes recognizes a PAM with the sequence 5'-NRG-3' (R represents A or G, and N is any nucleotide adjacent to the 3' end of the target nucleic acid sequence targeted by the spacer sequence) in the target nucleic acid.

[0130] In some embodiments, the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has 1 or more and less than 20 nucleotides. In some embodiments, the target nucleic acid has more than 20 nucleotides. In some embodiments, the target nucleic acid has at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, or at least more nucleotides. In some embodiments, the target nucleic acid has at most 5, at most 10, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 30, or at most more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases adjacent to the 5' end of the first nucleotide of the PAM. In some embodiments, the PAM sequence used in the disclosed compositions and methods as a sequence recognized by Cas9 from S. pyogenes is NGG.

[0131] In some embodiments, the length of the spacer sequence that hybridizes to the target nucleic acid is at least 6 nucleotides (nt) or at least about 6 nucleotides (nt) in length.The spacer sequence may be at least 6 nt or at least about 6 nt, 10 nt or about 10 nt, 15 nt or about 15 nt, 18 nt or about 18 nt, 19 nt or about 19 nt, 20 nt or about 20 nt, 25 nt or about 25 nt, 30 nt or about 30 nt, 35 nt or about 35 nt, or 40 nt or about 40 nt, or 6 nt to 80 nt or about 6 nt to about 80 nt, 6 nt to 50 nt or about 6 nt to about 50 nt, 6 nt to 45 nt or about 6 nt to about 45 nt, 6nt-40nt or about 6nt-about 40nt, 6nt-35nt or about 6nt-about 35nt, 6nt-30nt or about 6nt-about 30nt, 6nt-25nt or about 6nt-about 25nt, 6nt-20nt or about 6nt-about 20nt, 6nt-19nt or about 6nt-about 19nt, 10nt-50nt or about 10nt-about 50nt, 10nt-45nt or about 10nt-about 45nt, 10nt-40nt or about 10nt-about 40nt, 10nt-35nt or about 10nt-about 35nt, 10nt-30nt or about 10nt-about 30nt, 10nt-25nt or about 10nt-about 25nt, 10nt-20nt or about 10nt-about 20nt, 10nt-19nt or about 10nt-about 19nt, 19nt-25nt or about 19nt-about 25nt, 19nt-30nt or about 19nt-about 30nt, 19nt-35nt or about 19nt-about 35nt, 19nt-40nt or about 19nt-about 40nt, 19nt-45nt or about 19nt-about 45nt, 19nt It may be t to 50nt or about 19nt to about 50nt, 19nt to 60nt or about 19nt to about 60nt, 20nt to 25nt or about 20nt to about 25nt, 20nt to 30nt or about 20nt to about 30nt, 20nt to 35nt or about 20nt to about 35nt, 20nt to 40nt or about 20nt to about 40nt, 20nt to 45nt or about 20nt to about 45nt, 20nt to 50nt or about 20nt to about 50nt, or 20nt to 60nt or about 20nt to about 60nt. In some embodiments, the spacer sequence is 20 nucleotides long. In some embodiments, the spacer is 19 nucleotides long.In some embodiments, the spacer is 18 nucleotides in length. In some embodiments, the spacer is 17 nucleotides in length. In some embodiments, the spacer is 16 nucleotides in length. In some embodiments, the spacer is 15 nucleotides in length.

[0132] In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at most or at most about 30%, at most or at most about 40%, at most or at most about 40%, at most or at most about 50%, at most or at most about 60%, at most or at most about 65%, at most or at most about 70%, at most or at most about 75%, at most or at most about 80%, at most or at most about 80%, at most or at most about 85%, at most or at most about 90%, at most or at most about 90%, at most or at most about 95%, at most or at most about 97%, at most or at most about 97%, at most or at most about 98%, at most or at most about 98%, at most or at most about 99%, or at most about 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the 6 contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over 20 or about 20 contiguous nucleotides. In some embodiments, the length of the spacer sequence and the length of the target nucleic acid may differ by 1-6 nucleotides, which may be considered as one or more bulges.

[0133] In some embodiments, the spacer sequence is designed or selected using a computer program.The computer program can use variables, such as predicted melting temperature, formation of secondary structure, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC ratio (%), genomic occurrence frequency (e.g., changes at one or more positions in the same or similar sequence caused by mismatch, insertion or deletion, etc.), methylation status, the presence of SNPs, etc.

[0134] CRISPR minimal repeat sequence In some embodiments, a minimal repeat sequence of a CRISPR is a sequence having at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

[0135] In some embodiments, the CRISPR minimal repeat sequence has nucleotides that can hybridize with the tracrRNA minimal sequence in cells.The CRISPR minimal repeat sequence and the tracrRNA minimal sequence form a double strand, i.e., form a base pair double strand structure.The CRISPR minimal repeat sequence and the tracrRNA minimal sequence are combined and bind to the site-directed polypeptide.At least a part of the CRISPR minimal repeat sequence hybridizes with the tracrRNA minimal sequence. In some embodiments, at least a portion of the minimal repeat sequence of CRISPR has at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% complementarity to the minimal sequence of tracrRNA. In some embodiments, at least a portion of the minimal repeat sequence of CRISPR has at most 30% or at most about 30%, at most 40% or at most about 40%, 50% or at most about 50%, at most 60% or at most about 60%, at most 65% or at most about 65%, at most 70% or at most about 70%, at most 75% or at most about 75%, at most 80% or at most about 80%, at most 85% or at most about 85%, 90% or at most about 90%, at most 95% or at most about 95%, or at most 100% complementarity to the minimal sequence of tracrRNA.

[0136] The length of the minimum repeat sequence of CRISPR is 7 nucleotides to 100 nucleotides or about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum repeat sequence of CRISPR is 7 nucleotides (nt) to 50 nt or about 7 nt to about 50 nt, 7 nt to 40 nt or about 7 nt to about 40 nt, 7 nt to 30 nt or about 7 nt to about 30 nt, 7 nt to 25 nt or about 7 nt to about 25 nt, 7 nt to 20 nt or about 7 nt to about 20 nt, 7 nt to 15 nt or about 7 nt to about 15 nt, 8 nt to 40 nt or about 8 nt to about 40 nt, 8 nt to 30 nt or about 8 nt to about 30 nt, 8 nt to 2 5nt or about 8nt to about 25nt, 8nt to 20nt or about 8nt to about 20nt, 8nt to 15nt or about 8nt to about 15nt, 15nt to 100nt or about 15nt to about 100nt, 15nt to 80nt or about 15nt to about 80nt, 15nt to 50nt or about 15nt to about 50nt, 15nt to 40nt or about 15nt to about 40nt, 15nt to 30nt or about 15nt to about 30nt, or 15nt to 25nt or about 15nt to about 25nt. In some embodiments, the length of the minimum repeat sequence of CRISPR is about 9 nucleotides. In some embodiments, the length of the minimum repeat sequence of CRISPR is about 12 nucleotides.

[0137] In some embodiments, the CRISPR minimal repeat sequence has at least 60% or at least about 60% identity with a reference CRISPR minimal repeat sequence (e.g., wild-type crRNA from S. pyogenes) over a sequence of at least 6, 7, or 8 consecutive nucleotides. For example, the CRISPR minimal repeat sequence has at least 65% or at least about 65% identity, at least 70% or at least about 70% identity, at least 75% or at least about 75% identity, at least 80% or at least about 80% identity, at least 85% or at least about 85% identity, at least 90% or at least about 90% identity, at least 95% or at least about 95% identity, at least 98% or at least about 98% identity, at least 99% or at least about 99% identity, or at least 100% identity with a reference CRISPR minimal repeat sequence over a sequence of at least 6, 7, or 8 consecutive nucleotides.

[0138] Minimal sequence of tracrRNA In some embodiments, the minimal sequence of a tracrRNA is a sequence that has at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from S. pyogenes).

[0139] In some embodiments, the minimal sequence of tracrRNA has nucleotides that can hybridize to the minimal repeat sequence of CRISPR in cells.The minimal sequence of tracrRNA and the minimal repeat sequence of CRISPR form a double strand, i.e., form a base pair double strand structure.The minimal sequence of tracrRNA and the minimal repeat sequence of CRISPR are combined to bind to site-directed polypeptide.At least a part of the minimal sequence of tracrRNA can hybridize to the minimal repeat sequence of CRISPR. In some embodiments, the minimal sequence of tracrRNA has at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 95%, or at least 100% complementarity to the minimal repeat sequence of CRISPR.

[0140] The length of the minimum sequence of tracrRNA is 7 nucleotides to 100 nucleotides or about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum sequence of tracrRNA is 7 nucleotides (nt) to 50 nt or about 7 nt to about 50 nt, 7 nt to 40 nt or about 7 nt to about 40 nt, 7 nt to 30 nt or about 7 nt to about 30 nt, 7 nt to 25 nt or about 7 nt to about 25 nt, 7 nt to 20 nt or about 7 nt to about 20 nt, 7 nt to 15 nt or about 7 nt to about 15 nt, 8 nt to 40 nt or about 8 nt to about 40 nt, 8 nt to 30 nt or about 8 nt to about 30 nt, 8 nt to 25 nt, or about 8nt to about 25nt, 8nt to 20nt or about 8nt to about 20nt, 8nt to 15nt or about 8nt to about 15nt, 15nt to 100nt or about 15nt to about 100nt, 15nt to 80nt or about 15nt to about 80nt, 15nt to 50nt or about 15nt to about 50nt, 15nt to 40nt or about 15nt to about 40nt, 15nt to 30nt or about 15nt to about 30nt, or 15nt to 25nt or about 15nt to about 25nt. In some embodiments, the minimum sequence length of the tracrRNA is about 9 nucleotides in length. In some embodiments, the minimum sequence length of the tracrRNA is about 12 nucleotides in length. In some embodiments, the minimal sequence of the tracrRNA consists of 23-48 nt of the tracrRNA described in Jinek, M. et al. (2012). Science, 337(6096):816-821.

[0141] In some embodiments, the minimal sequence of tracrRNA has at least 60% or at least about 60% identity over a sequence of at least 6, 7, or 8 consecutive nucleotides to a reference minimal sequence of tracrRNA (e.g., wild-type tracrRNA from S. pyogenes). For example, the minimal sequence of tracrRNA has at least 65% or at least about 65% identity, at least 70% or at least about 70% identity, at least 75% or at least about 75% identity, at least 80% or at least about 80% identity, at least 85% or at least about 85% identity, at least 90% or at least about 90% identity, at least 95% or at least about 95% identity, at least 98% or at least about 98% identity, at least 99% or at least about 99% identity, or at least 100% identity over a sequence of at least 6, 7, or 8 consecutive nucleotides to a reference minimal sequence of tracrRNA.

[0142] In some embodiments, the duplex formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has a double helix structure. In some embodiments, the duplex formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least more than 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or at least more than 10 nucleotides. In some embodiments, the duplex formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or more nucleotides, or at most about 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or more nucleotides.

[0143] In some embodiments, the duplex has mismatches (i.e., the two strands in the duplex do not have 100% complementarity). In some embodiments, the duplex has at least 1, at least 2, at least 3, at least 4, or at least 5, or at least about 1, at least about 2, at least about 3, at least about 4, or at least about 5 mismatches. In some embodiments, the duplex has at most 1, at most 2, at most 3, at most 4, or at most 5 mismatches, or at most about 1, at most about 2, at most about 3, at most about 4, or at most about 5 mismatches. In some embodiments, the duplex has no more than 2 mismatches.

[0144] Bulge In some embodiments, a "bulge" is present in the duplex formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence. A bulge is a region of nucleotide unpairing within the duplex. In some embodiments, the bulge contributes to the binding of the duplex to a site-directed polypeptide. A bulge has an unpaired sequence 5'-XXXY-3' (where X is any purine base and Y has a nucleotide that can form a wobble base pair with a nucleotide in the opposite strand) on one strand of the duplex and an unpaired nucleotide region on the other strand of the duplex. The number of unpaired nucleotides on each strand of the duplex can be different.

[0145] In one example, the bulge has an unpaired purine base (e.g., adenine) on the CRISPR minimal repeat strand that forms the bulge. In some embodiments, the bulge has an unpaired sequence 5'-AAGY-3' on the tracrRNA minimal sequence strand that forms the bulge (wherein Y has a nucleotide that can wobble base pair with a nucleotide on the CRISPR minimal repeat strand).

[0146] In some embodiments, the bulge of the CRISPR minimal repeat strand that forms a double strand has at least 1, at least 2, at least 3, at least 4, at least 5, or at least more than 1 unpaired nucleotide.In some embodiments, the bulge of the CRISPR minimal repeat strand that forms a double strand has at most 1, at most 2, at most 3, at most 4, at most 5, or at most more than 1 unpaired nucleotide.In some embodiments, the bulge of the CRISPR minimal repeat strand that forms a double strand has 1 unpaired nucleotide.

[0147] In some embodiments, the bulge on the duplex-forming tracrRNA minimal sequence side has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least more unpaired nucleotides. In some embodiments, the bulge on the duplex-forming tracrRNA minimal sequence side has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10, or at most more unpaired nucleotides. In some embodiments, the bulge on the duplex-forming second strand (e.g., on the duplex-forming tracrRNA minimal sequence side) has 4 unpaired nucleotides.

[0148] In some embodiments, the bulge has at least one wobble base pair. In some embodiments, the bulge has no more than one wobble base pair. In some embodiments, the bulge has at least one purine nucleotide. In some embodiments, the bulge has at least three purine nucleotides. In some embodiments, the bulge sequence has at least five purine nucleotides. In some embodiments, the bulge sequence has at least one guanine nucleotide. In some embodiments, the bulge sequence has at least one adenine nucleotide.

[0149] hairpin In various embodiments, there are one or more hairpins in the 3' tracrRNA sequence at the 3' end of the tracrRNA minimal sequence.

[0150] In some embodiments, the hairpin begins at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more nucleotides, or at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20 or more nucleotides 3' from the last paired nucleotide of the duplex consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence. In some embodiments, the hairpin may begin at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 or more nucleotides, or at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10 or more nucleotides 3' from the last paired nucleotide of the duplex consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence.

[0151] In some embodiments, the hairpin has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more contiguous nucleotides, or at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20 or more contiguous nucleotides. In some embodiments, the hairpin has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, or at most 15 or more consecutive nucleotides, or at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15 or more consecutive nucleotides.

[0152] In some embodiments, the hairpin has a CC dinucleotide (ie, two consecutive cytosine nucleotides).

[0153] In some embodiments, the hairpin has double-stranded nucleotides (e.g., hairpin nucleotides that hybridize to each other). For example, the hairpin has a CC dinucleotide that hybridizes to a GG dinucleotide in the hairpin duplex of the 3'tracrRNA sequence.

[0154] The one or more hairpins can interact with a guide RNA interacting region of the site-directed polypeptide.

[0155] In some embodiments, there is more than one hairpin, and in some embodiments, there is more than one hairpin.

[0156] 3'tracrRNA sequence In some embodiments, the 3'tracrRNA sequence has at least or at least about 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

[0157] In some embodiments, the length of the 3'tracrRNA sequence is 6 nucleotides to 100 nucleotides or about 6 nucleotides to about 100 nucleotides. For example, the length of the 3'tracrRNA sequence is 6 nucleotides (nt) to 50 nt or about 6 nt to about 50 nt, 6 nt to 40 nt or about 6 nt to about 40 nt, 6 nt to 30 nt or about 6 nt to about 30 nt, 6 nt to 25 nt or about 6 nt to about 25 nt, 6 nt to 20 nt or about 6 nt to about 20 nt, 6 nt to 15 nt or about 6 nt to about 15 nt, 8 nt to 40 nt or about 8 nt to about 40 nt, 8 nt to 30 nt or about 8 nt to about 30 nt, 8 nt to 25 nt or about 8nt to about 25nt, 8nt to 20nt or about 8nt to about 20nt, 8nt to 15nt or about 8nt to about 15nt, 15nt to 100nt or about 15nt to about 100nt, 15nt to 80nt or about 15nt to about 80nt, 15nt to 50nt or about 15nt to about 50nt, 15nt to 40nt or about 15nt to about 40nt, 15nt to 30nt or about 15nt to about 30nt, or 15nt to 25nt or about 15nt to about 25nt. In some embodiments, the length of the 3'tracrRNA sequence is about 14 nucleotides in length.

[0158] In some embodiments, the 3'tracrRNA sequence has at least 60% or at least about 60% identity to a reference 3'tracrRNA sequence (e.g., a wild-type 3'tracrRNA sequence from S. pyogenes) over a sequence of at least 6, 7, or 8 consecutive nucleotides. For example, the 3'tracrRNA sequence has at least 60% or at least about 60% identity, at least 65% or at least about 65% identity, at least 70% or at least about 70% identity, at least 75% or at least about 75% identity, at least 80% or at least about 80% identity, at least 85% or at least about 85% identity, at least 90% or at least about 90% identity, at least 95% or at least about 95% identity, at least 98% or at least about 98% identity, at least 99% or at least about 99% identity, or at least 100% identity over a sequence of at least 6, 7, or 8 contiguous nucleotides to a reference 3'tracrRNA sequence (e.g., a wild-type 3'tracrRNA sequence from S. pyogenes).

[0159] In some embodiments, the 3'tracrRNA sequence has two or more double-stranded regions (e.g., hairpins, hybridized regions). In some embodiments, the 3'tracrRNA sequence has two double-stranded regions.

[0160] In some embodiments, the 3'tracrRNA sequence has a stem-loop structure. In some embodiments, the stem-loop structure of the 3'tracrRNA has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or at least more nucleotides. In some embodiments, the stem-loop structure of the 3'tracrRNA has at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 or at most more nucleotides. In some embodiments, the stem-loop structure has a functional moiety. For example, the stem-loop structure may have an aptamer, a ribozyme, a hairpin that interacts with a protein, a CRISPR array, an intron, or an exon. In some embodiments, the stem loop structure has at least 1, at least 2, at least 3, at least 4, at least 5 or more functional moieties, or at least about 1, at least about 2, at least about 3, at least about 4, at least about 5 or more functional moieties. In some embodiments, the stem loop structure has at most 1, at most 2, at most 3, at most 4, at most 5 or more functional moieties, or at most about 1, at most 2, at most 3, at most about 4, at most about 5 or more functional moieties.

[0161] In some embodiments, the hairpin of the 3'tracrRNA sequence has a P domain. In some embodiments, the P domain on the hairpin has a double-stranded region.

[0162] tracrRNA extension sequence In some embodiments, when the guide RNA is a unimolecular guide or a bimolecular guide, a tracrRNA extension sequence is provided. In some embodiments, the length of the tracrRNA extension sequence is 1 nucleotide to 400 nucleotides or about 1 nucleotide to about 400 nucleotides. In some embodiments, the length of the tracrRNA extension sequence is more than 1 nucleotide, more than 5 nucleotides, more than 10 nucleotides, more than 15 nucleotides, more than 20 nucleotides, more than 25 nucleotides, more than 30 nucleotides, more than 35 nucleotides, more than 40 nucleotides, more than 45 nucleotides, more than 50 nucleotides, more than 60 nucleotides, more than 70 nucleotides, more than 80 nucleotides, more than 90 nucleotides, more than 100 nucleotides, more than 120 nucleotides, more than 140 nucleotides, more than 160 nucleotides, more than 180 nucleotides, more than 200 nucleotides, more than 220 nucleotides, more than 240 nucleotides, more than 260 nucleotides, more than 280 nucleotides, more than 300 nucleotides, more than 320 nucleotides, more than 340 nucleotides, more than 360 nucleotides, more than 380 nucleotides, or more than 400 nucleotides long. In some embodiments, the length of the tracrRNA extension sequence is from 20 nucleotides to more than 5000 nucleotides in length or from about 20 nucleotides to more than about 5000 nucleotides in length. In some embodiments, the length of the tracrRNA extension sequence is more than 1000 nucleotides in length.In some embodiments, the length of the tracrRNA extension sequence is 1 nucleotide or more and is less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides in length. In some embodiments, the length of the tracrRNA extension sequence may be 1 nucleotide or more and less than 1000 nucleotides in length. In some embodiments, the length of the tracrRNA extension sequence is 1 nucleotide or more and less than 10 nucleotides in length. In some embodiments, the length of the tracrRNA extension sequence is 10-30 nucleotides in length. In some embodiments, the length of the tracrRNA extension sequence is 30-70 nucleotides in length.

[0163] In some embodiments, the tracrRNA extension sequence has a functional moiety (e.g., a stability control sequence, a ribozyme, or an endoribonuclease binding sequence). In some embodiments, the functional moiety is a transcription terminator segment (e.g., a transcription termination sequence). In some embodiments, the total length of the functional moiety is 10 nucleotides (nt) to 100 nucleotides in length or about 10 nt to about 100 nt, 10 nt to 20 nt or about 10 nt to about 20 nt, 20 nt to 30 nt or about 20 nt to about 30 nt, 30 nt to 40 nt or about 30 nt to about 40 nt, 40 nt to 50 nt or about 40 nt to about 50 nt, 50 nt to 60 nt or about 50 nt to about 60 nt, 60 nt to 70 nt or about 60 nt to about 70nt, 70nt-80nt or about 70nt-about 80nt, 80nt-90nt or about 80nt-about 90nt, 90nt-100nt or about 90nt-about 100nt, 15nt-80nt or about 15nt-about 80nt, 15nt-50nt or about 15nt-about 50nt, 15nt-40nt or about 15nt-about 40nt, 15nt-30nt or about 15nt-about 30nt, or 15nt-25nt or about 15nt-about 25nt. In some embodiments, the functional moiety functions in eukaryotic cells. In some embodiments, the functional moiety functions in prokaryotic cells. In some embodiments, the functional moiety functions in both eukaryotic and prokaryotic cells.

[0164] Examples of suitable functional portions of the tracrRNA extension sequence include, but are not limited to, a 3'-terminal polyadenylation tail; a riboswitch sequence (e.g., one that provides controlled stabilization and / or allows controlled access by a protein or protein complex); a sequence that forms a dsRNA duplex (i.e., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that allows tracking (e.g., direct attachment to a fluorescent molecule, attachment to a moiety that facilitates fluorescent detection, a sequence that allows fluorescent detection, etc.); and a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, such as a transcription activator, transcription repressor, DNA methyltransferase, DNA methylase, histone acetyltransferase, histone deacetylase, etc.). In some embodiments, the tracrRNA extension sequence has a primer binding site or a molecular index (e.g., a barcode sequence). In some embodiments, the tracrRNA extension sequence has one or more affinity tags.

[0165] Linker sequences of single molecule guides In some embodiments, the length of the linker sequence of the single molecule guide nucleic acid is 3 nucleotides to 100 nucleotides or about 3 nucleotides to about 100 nucleotides. Jinek, M. et al. (2012). Science, 337(6096):816-821, for example, used a simple "tetraloop" (-GAAA-) consisting of four nucleotides. The length of the linker is, for example, 3 nucleotides (nt) to 90 nt or about 3 nt to about 90 nt, 3 nt to 80 nt or about 3 nt to about 80 nt, 3 nt to 70 nt or about 3 nt to about 70 nt, 3 nt to 60 nt or about 3 nt to about 60 nt, 3 nt to 50 nt or about 3 nt to about 50 nt, 3 nt to 40 nt or about 3 nt to about 40 nt, 3 nt to 30 nt or about 3 nt to about 30 nt, 3 nt to 20 nt or about 3 nt to about 20 nt, or 3 nt to 10 nt or about 3 nt to about 10 nt. For example, the length of the linker is 3 nt to 5 nt or about 3 nt to about 5 nt, 5 nt to 10 nt or about 5 nt to about 10 nt, 10 nt to 15 nt or about 10 nt to about 15 nt, 15 nt to 20 nt or about 15 nt to about 20 nt, 20 nt to 25 nt or about 20 nt to about 25 nt, 25 nt to 30 nt or about 25 nt to about 30 nt, 30 nt to 35 nt or about 30 nt to about 35 nt, 3 It may be 5 nt to 40 nt or about 35 nt to about 40 nt, 40 nt to 50 nt or about 40 nt to about 50 nt, 50 nt to 60 nt or about 50 nt to about 60 nt, 60 nt to 70 nt or about 60 nt to about 70 nt, 70 nt to 80 nt or about 70 nt to about 80 nt, 80 nt to 90 nt or about 80 nt to about 90 nt, or 90 nt to 100 nt or about 90 nt to about 100 nt. In some embodiments, the length of the linker of the single molecule guide nucleic acid is 4 to 40 nucleotides in length.In some embodiments, the length of the linker is at least 100 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 3500 nucleotides, at least 4000 nucleotides, at least 4500 nucleotides, at least 5000 nucleotides, at least 5500 nucleotides, at least 6000 nucleotides, at least 6500 nucleotides, or at least 7000 nucleotides. or at least about 100 nucleotides long, at least about 500 nucleotides long, at least about 1000 nucleotides long, at least about 1500 nucleotides long, at least about 2000 nucleotides long, at least about 2500 nucleotides long, at least about 3000 nucleotides long, at least about 3500 nucleotides long, at least about 4000 nucleotides long, at least about 4500 nucleotides long, at least about 5000 nucleotides long, at least about 5500 nucleotides long, at least about 6000 nucleotides long, at least about 6500 nucleotides long, or at least about 7000 nucleotides long.In some embodiments, the length of the linker is at most 100 nucleotides, at most 500 nucleotides, at most 1000 nucleotides, at most 1500 nucleotides, at most 2000 nucleotides, at most 2500 nucleotides, at most 3000 nucleotides, at most 3500 nucleotides, at most 4000 nucleotides, at most 4500 nucleotides, at most 5000 nucleotides, at most 5500 nucleotides, at most 6000 nucleotides, at most 6500 nucleotides, or at most 7000 nucleotides. or at most about 100 nucleotides long, at most about 500 nucleotides long, at most about 1000 nucleotides long, at most about 1500 nucleotides long, at most about 2000 nucleotides long, at most about 2500 nucleotides long, at most about 3000 nucleotides long, at most about 3500 nucleotides long, at most about 4000 nucleotides long, at most about 4500 nucleotides long, at most about 5000 nucleotides long, at most about 5500 nucleotides long, at most about 6000 nucleotides long, at most about 6500 nucleotides long, or at most about 7000 nucleotides long.

[0166] The linker can have a variety of sequences, but in some embodiments, the linker does not have sequences with extensive regions of homology with other parts of the guide RNA, because such extensive regions of homology may result in intramolecular binding that may interfere with other functional regions of the guide RNA. In Jinek, M. et al. (2012). Science, 337(6096):816-821, a simple sequence of four nucleotides -GAAA- was used, but other various sequences, such as longer sequences, can be used as well.

[0167] In some embodiments, the linker sequence has a functional moiety. For example, the linker sequence may have one or more features, such as an aptamer, a ribozyme, a hairpin that interacts with a protein, a protein binding site, a CRISPR array, an intron, an exon, etc. In some embodiments, the linker sequence has at least 1, at least 2, at least 3, at least 4, at least 5, or at least more than 1, or at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or more than 1, or at most about 1, at most about 2, at most about 3, at most about 4, at most ...

[0168] In some embodiments, the genomic site targeted by the gRNA according to the present disclosure may be present at the FOXP3 gene in the genome (e.g., human genome), within the FOXP3 gene in the genome, or adjacent to the FOXP3 gene in the genome. Typical guide RNAs targeting such sites include the spacer sequences shown in SEQ ID NOs: 1-7, 15-20, and 27-29. For example, a gRNA containing a spacer sequence shown in SEQ ID NO: 1 may have a spacer sequence including i) the sequence of SEQ ID NO: 1, ii) the sequence consisting of the 2nd to 20th positions of SEQ ID NO: 1, iii) the sequence consisting of the 3rd to 20th positions of SEQ ID NO: 1, iv) the sequence consisting of the 4th to 20th positions of SEQ ID NO: 1, etc. As will be understood by those skilled in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. For example, each spacer sequence shown in SEQ ID NOs: 1-7, 15-20, and 27-29 can be incorporated into a single RNA chimera or into a crRNA (with the corresponding tracrRNA). See Jinek, M. et al. (2012). Science, 337(6096):816-821 and Deltcheva, E. et al. (2011). Nature, 471:602-607.

[0169] Donor DNA or donor template Site-directed polypeptides such as DNA endonucleases can introduce double-stranded or single-stranded breaks in nucleic acids (e.g., genomic DNA). The double-stranded breaks can stimulate endogenous DNA repair pathways in cells (e.g., homology-dependent repair (HDR), non-homologous end joining, alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining (MMEJ). NHEJ can repair the cut target nucleic acid without the need for a homologous template. This can result in small deletions or insertions (indels) in the target nucleic acid at the cut site, which can lead to disruption or alteration of gene expression. Homology-dependent repair (HDR), also known as homologous recombination (HR), can occur when a homologous repair template or donor is available.

[0170] A homologous donor template has a sequence that is homologous to the sequence adjacent to the cut site of the target nucleic acid. Generally, sister chromatids are used by cells as repair templates. Meanwhile, repair templates for genome editing are often provided as exogenous nucleic acids, such as plasmids, double-stranded oligonucleotides, single-stranded oligonucleotides, double-stranded oligonucleotides, or viral nucleic acids. Exogenous donor templates generally introduce additional nucleic acid sequences (such as transgenes) or modifications (such as one or more base changes or deletions) between adjacent homologous regions, allowing the additional or modified nucleic acid sequences to be integrated into the target locus. MMEJ achieves genetic outcomes similar to NHEJ in that small deletions and insertions can occur at the cut site. MMEJ utilizes homologous sequences consisting of several base pairs adjacent to the cut site to achieve the desired end-joining DNA repair results. In some cases, it may be possible to predict the likely repair results based on the analysis of short homologous sequences (microhomology) predicted in the target region of the nuclease.

[0171] Thus, in some cases, exogenous polynucleotide sequence is inserted into the cleavage site of target nucleic acid using homologous recombination.Herein, exogenous polynucleotide sequence is referred to as donor polynucleotide (or donor, donor sequence or polynucleotide donor template).In some embodiments, donor polynucleotide, a part of donor polynucleotide, a copy of donor polynucleotide, or a part of a copy of donor polynucleotide is inserted into the cleavage site of target nucleic acid.In some embodiments, donor polynucleotide is an exogenous polynucleotide sequence, i.e., a sequence that does not naturally exist at the cleavage site of target nucleic acid.

[0172] When a sufficient concentration of exogenous DNA molecules is provided in the nucleus of a cell where double-strand breaks occur, the exogenous DNA can be inserted into the double-strand break site during the NHEJ repair process, and thus become permanently added to the genome. Such exogenous DNA molecules are referred to as donor templates in some embodiments. When donor templates contain the coding sequence of a gene of interest, such as the FOXP3 gene, optionally with associated regulatory sequences, such as promoters, enhancers, polyA sequences and / or splice acceptor sequences (also referred to herein as "donor cassettes"), the gene of interest can be expressed from the copy integrated into the genome, and the gene can be permanently expressed during the life of the cell. Furthermore, the copy integrated from donor DNA templates can be transmitted to daughter cells when the cell divides.

[0173] If a donor DNA template containing adjacent DNA sequences (called homology arms) that are homologous to the DNA sequences on either side of the double-strand break is present in sufficient concentration, the donor DNA template can be integrated via the HDR pathway. The homology arms act as substrates for homologous recombination between the donor template and the sequences on either side of the double-strand break. This allows for error-free insertion of a donor template in which the sequences on either side of the double-strand break are unchanged from those of the unmodified genome.

[0174] Donors provided for editing by HDR are highly variable, but generally contain the intended sequence for editing flanked by short or long homologous arms that allow annealing to genomic DNA. The homologous regions flanking the introduced gene change site may be 30 bp or less, or as large as a multi-kilobase cassette that may contain promoters, cDNA, etc. Both single-stranded and double-stranded oligonucleotide donors can be used. These oligonucleotides range in size from less than 100 nt to several kb or more, although longer ssDNA can be generated and used. Double-stranded donors such as PCR amplicons, plasmids, and minicircles are often used. In general, AAV vectors have proven to be a very effective means of delivering donor templates, but the limit for packaging in individual donors is less than 5 kb. Active transcription of the donor has been shown to increase HDR rates by 3-fold, indicating that inclusion of a promoter can increase conversion. Conversely, CpG methylation of the donor may reduce gene expression and HDR rates.

[0175] In some embodiments, donor DNA can be introduced alone or together with nuclease by various methods, such as transfection, nanoparticle, microinjection, viral transduction, etc. In some embodiments, various methods of linking donor DNA and nuclease can be used to increase the availability of donor in HDR. Examples of such methods include binding donor to nuclease, binding to DNA binding protein that binds to the vicinity of donor and nuclease, or binding to protein involved in DNA end binding or DNA repair, etc.

[0176] In addition to genome editing by NHEJ or HDR, site-directed gene insertion using both the NHEJ pathway and HR can be performed. Such combined approaches are applicable in certain situations, which may involve intron / exon boundaries. NHEJ can prove effective for ligation at introns, whereas error-free HDR may be more suitable for coding regions.

[0177] In some embodiments, the exogenous sequence intended for insertion into the genome is a nucleotide sequence encoding FOXP3 or a functional derivative thereof. A functional derivative of FOXP3 includes a FOXP3 derivative having substantially the same activity as wild-type FOXP3 (such as wild-type human FOXP3), such as a FOXP3 derivative exhibiting at least or at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least about 95% or at least about 100% of the activity of wild-type FOXP3. In some embodiments, a functional derivative of FOXP3 may have at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, a person skilled in the art can test the functionality or activity of a compound (e.g., a peptide, a protein, etc.) using various methods known in the art. A functional derivative of FOXP3 can further include a fragment of modified FOXP3 or a fragment of wild-type FOXP3 having conservative modifications to one or more amino acid residues of the full-length wild-type FOXP3.Thus, in some embodiments, a nucleic acid sequence encoding a functional derivative of FOXP3 may have at least or at least about 30%, 40% or at least about 40%, 50% or at least about 50%, 60% or at least about 60%, 70% or at least about 70%, 80% or at least about 80%, 85% or at least about 85%, 90% or at least about 90%, 95% or at least about 95%, 96% or at least about 96%, 97% or at least about 97%, 98% or at least about 98%, or 99% or at least about 99% nucleic acid sequence identity to a nucleic acid sequence encoding FOXP3 (e.g. wild-type FOXP3). In some embodiments, the FOXP3 is human wild-type FOXP3.

[0178] In some embodiments in which a nucleic acid encoding FOXP3 or a functional derivative thereof is inserted, a cDNA of the FOXP3 gene or a functional derivative thereof can be inserted into the genome of a subject having an abnormal FOXP3 gene or a regulatory sequence thereof, In such cases, the donor DNA or donor template can be an expression cassette or vector construct that includes a sequence (e.g., a cDNA sequence) encoding FOXP3 or a functional derivative thereof.

[0179] In some embodiments of the donor template described herein that includes a donor cassette, the donor cassette is adjacent to a gRNA target site at one end or adjacent to a gRNA target site at both ends. For example, such a donor template may include a donor cassette that has a gRNA target site at the 5' end and / or a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette that has a gRNA target site at the 5' end. In some embodiments, the donor template includes a donor cassette that has a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette that has a gRNA target site at the 5' end and a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette that has a gRNA target site at the 5' end and a gRNA target site at the 3' end, where the two gRNA target sites include the same sequence. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises a reverse complement of a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises a donor cassette having a gRNA target site at the 5' end and a gRNA target site at the 3' end, and these two gRNA target sites in the donor template comprise the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is integrated. In some embodiments, the donor template comprises a donor cassette having a gRNA target site at its 5' end and a gRNA target site at its 3' end, wherein these two gRNA target sites in the donor template comprise the reverse complement of a gRNA target site in the target locus into which the donor cassette of the donor template will be integrated.

[0180] In some embodiments, a donor template is provided that includes a nucleotide sequence encoding FOXP3 or a functional derivative thereof for targeted integration into the FOXP3 gene, the donor template including, from the 5' to 3' end, i) a first gRNA target site; ii) a splice acceptor; iii) a nucleotide sequence encoding FOXP3 or a functional derivative thereof; and iv) a polyadenylation signal. In some embodiments, the donor template further includes iv) a second gRNA target site downstream of the polyadenylation signal. In some embodiments, the first gRNA target site and the second gRNA target site are the same. In some embodiments, the donor template further includes a polynucleotide spacer between i) the first gRNA target site and ii) the splice acceptor. In some embodiments, the polynucleotide spacer is 18 nucleotides long. In some embodiments, the donor template is adjacent to a first AAV ITR on one end and / or adjacent to a second AAV ITR on the other end. In some embodiments, the first AAV ITR is an AAV2 ITR and / or the second AAV ITR is an AAV2 ITR. In some embodiments, the FOXP3 is human wild-type FOXP3.

[0181] Nucleic acid encoding a site-directed polypeptide or a DNA endonuclease In some embodiments, in light of the above, the genome editing method and the composition can use a nucleic acid sequence (or oligonucleotide) encoding a site-directed polypeptide or a DNA endonuclease. The nucleic acid sequence encoding the site-directed polypeptide can be DNA or RNA. If the nucleic acid sequence encoding the site-directed polypeptide is RNA, the RNA can be covalently linked to the gRNA sequence or can be present as a separate sequence. In some embodiments, the peptide sequence of the site-directed polypeptide or DNA endonuclease can be used instead of these nucleic acid sequences.

[0182] vector In another aspect, the present disclosure provides a nucleic acid having a nucleotide sequence encoding a genome-targeting nucleic acid of the present disclosure, a site-directed polypeptide of the present disclosure, and / or any nucleic acid or protein molecule necessary to carry out an embodiment of a method of the present disclosure. In some embodiments, such a nucleic acid is a vector (e.g., a recombinant expression vector).

[0183] Expression vectors contemplated in the present invention include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, simian virus 40 (SV40), herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., murine leukemia virus; spleen necrosis virus; or vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, mammary tumor virus), or other recombinant vectors. Other vectors contemplated for use with eukaryotic target cells include, but are not limited to, pXT1 vector, pSG5 vector, pSVK3 vector, pBPV vector, pMSG vector, and pSVLSV40 vector (Pharmacia). Additional vectors contemplated for use with eukaryotic target cells include, but are not limited to, pCTx-1 vector, pCTx-2 vector, and pCTx-3 vector. Other vectors can be used as long as they are compatible with the host cell.

[0184] In some embodiments, vector has one or more transcriptional and / or translational control elements.Depending on the host / vector system used, various suitable transcriptional and translational control elements can be used in expression vector, such as constitutive promoter, inducible promoter, transcriptional enhancer element, transcriptional terminator, etc.In some embodiments, vector is a self-inactivating vector that inactivates viral sequence or CRISPR mechanism components or other elements.

[0185] Examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include, but are not limited to, the cytomegalovirus (CMV) promoter, the immediate early thymidine kinase promoter of herpes simplex virus (HSV), early or late SV40, long terminal repeats (LTRs) from retroviruses, the human elongation factor 1 promoter (EF1), a hybrid construct of the chicken beta actin promoter (CAG) fused to the cytomegalovirus (CMV) enhancer, the mouse stem cell virus promoter (MSCV), the phosphoglycerate kinase 1 locus promoter (PGK), or mouse metallothionein I.

[0186] A variety of promoters may be useful for expressing small RNAs, such as guide RNAs used with Cas endonucleases, for example RNA polymerase III promoters such as U6 and H1. Descriptions and parameters facilitating the use of such promoters are known in the art, and new information and approaches are constantly being reported. See, for example, Ma, H. et al. (2014). Molecular Therapy - Nucleic Acids 3, e161, doi:10.1038 / mtna.2014.12.

[0187] The expression vector may further include ribosome binding sites for translation initiation and transcription termination. The expression vector may also include appropriate sequences for amplifying expression. In addition, the expression vector may include a nucleotide sequence encoding a non-native tag (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that is fused to the site-directed polypeptide and thus expressed as part of the fusion protein. In some embodiments, the promoter is an inducible promoter (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., CMV promoter, or UBC promoter). In some embodiments, the promoter is a spatially restricted promoter or a temporally restricted promoter (e.g., tissue-specific promoter, cell type-specific promoter, etc.). In some embodiments, the vector does not include a promoter for at least one gene to be expressed in the host cell, if the gene is expressed under the control of an endogenous promoter present in the genome after insertion into the genome.

[0188] Site-directed polypeptide or DNA endonuclease Modification of the target DNA by NHEJ and / or HDR can result in, for example, mutations, deletions, alterations, integrations, gene corrections, gene replacements, gene tagging, transgene insertions, nucleotide deletions, gene disruptions, translocations, and / or gene mutations. The integration of non-natural nucleic acids into genomic DNA is an example of genome editing.

[0189] The site-directed polypeptide is a nuclease used in genome editing to cut DNA. The site-directed polypeptide can be administered to cells or subjects as one or more polypeptides or one or more mRNAs encoding the polypeptide.

[0190] In the context of the CRISPR / Cas or CRISPR / Cpf1 system, the site-directed polypeptide binds to the guide RNA, whereby the guide RNA identifies the site of the target DNA to which the site-directed polypeptide is directed. In embodiments of the CRISPR / Cas or CRISPR / Cpf1 system described herein, the site-directed polypeptide is an endonuclease, such as a DNA endonuclease.

[0191] In some embodiments, the site-directed polypeptide has multiple nucleic acid cleavage domains (e.g., nuclease sites). Two or more nucleic acid cleavage domains can be linked via a linker. In some embodiments, the linker has a flexible linker. The length of the linker can be 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, or more.

[0192] The native wild-type Cas9 enzyme has two nuclease domains, an HNH nuclease domain and a RuvC domain. The Cas9 enzymes contemplated herein have an HNH nuclease domain or an HNH-like nuclease domain, and / or a RuvC nuclease domain or an RuvC-like nuclease domain.

[0193] The HNH domain or HNH-like domain has an McrA-like fold. The HNH domain or HNH-like domain has two antiparallel β-strands and one α-helix. The HNH domain or HNH-like domain has a metal binding site (e.g., a divalent cation binding site). The HNH domain or HNH-like domain can cleave one strand of a target nucleic acid (e.g., the complementary strand of the target strand of crRNA).

[0194] The RuvC domain or RuvC-like domain has an RNaseH fold or an RNaseH-like fold. The RuvC domain or RNaseH domain is involved in a diverse set of nucleic acid-based functions, including acting on both RNA and DNA. The RNaseH domain has five β-strands surrounded by multiple α-helices. The RuvC domain or RNaseH domain or RuvC-like domain or RNaseH-like domain has a metal binding site (e.g., a divalent cation binding site). The RuvC domain or RNaseH domain or RuvC-like domain or RNaseH-like domain can cleave a single strand of a target nucleic acid (e.g., a non-complementary strand of a double-stranded target DNA).

[0195] In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% amino acid sequence identity to a typical wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8 described in US2014 / 0068797, or Cas9 described in Sapranauskas, R. et al. (2011). Nucleic Acids Res, 39(21): 9275-9282, and various other site-directed polypeptides).

[0196] In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% amino acid sequence identity to the nuclease domain of a typical wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra).

[0197] In some embodiments, the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 contiguous amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, the site-directed polypeptide has at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 contiguous amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, the HNH nuclease domain of the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 consecutive amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes as described above). In some embodiments, the HNH nuclease domain of the site-directed polypeptide has at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 consecutive amino acids to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes as described above). In some embodiments, the RuvC nuclease domain of the site-directing polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 contiguous amino acids to a wild-type site-directing polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, the RuvC nuclease domain of the site-directing polypeptide has at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity over 10 contiguous amino acids to a wild-type site-directing polypeptide (e.g., Cas9 from S. pyogenes, supra).

[0198] In some embodiments, the site-directed polypeptide has a modified form of a typical wild-type site-directed polypeptide. The modified form of a typical wild-type site-directed polypeptide has a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. In some embodiments, the modified form of a typical wild-type site-directed polypeptide has a nucleic acid cleavage activity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% (but not zero) of the nucleic acid cleavage activity of a typical wild-type site-directed polypeptide (e.g., Cas9 from S.pyogenes as described above). In addition, the modified form of the site-directed polypeptide may have substantially no nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that has substantially no nucleic acid cleavage activity, such a modified form is referred to herein as "enzymatically inactive".

[0199] In some embodiments, the modified form of the site-directed polypeptide has a mutation that allows it to induce a single-strand break (SSB) on the target nucleic acid (e.g., by cleaving only one of the sugar-phosphate backbones of a double-stranded target nucleic acid). In some embodiments, the mutation results in a nucleic acid cleavage activity that is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% (but not zero) of the nucleic acid cleavage activity of one or more of the multiple nucleic acid cleavage domains of the wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, as described above). In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave a non-complementary strand of the target nucleic acid while retaining the ability to cleave a complementary strand of the target nucleic acid. In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave a complementary strand of the target nucleic acid while retaining the ability to cleave a non-complementary strand of the target nucleic acid. For example, mutations are made in residues of a typical wild-type S. pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854, and Asn856, that inactivate one or more of the nucleic acid cleavage domains (e.g., nuclease domains). In some embodiments, the residues at which mutations are introduced correspond to the Asp10, His840, Asn854, and Asn856 residues of a typical wild-type S. pyogenes-derived Cas9 polypeptide (e.g., as determined by sequence and / or structure alignment). Examples of such mutations include, but are not limited to, D10A, H840A, N854A, or N856A. One of skill in the art will appreciate that mutations other than alanine substitutions are suitable.

[0200] In some embodiments, the D10A mutation, in combination with one or more of the H840A mutation, the N854A mutation, and the N856A mutation, generates a site-directed polypeptide that is substantially devoid of DNA cleavage activity. In some embodiments, the H840A mutation, in combination with one or more of the D10A mutation, the N854A mutation, and the N856A mutation, generates a site-directed polypeptide that is substantially devoid of DNA cleavage activity. In some embodiments, the N854A mutation, in combination with one or more of the H840A mutation, the D10A mutation, and the N856A mutation, generates a site-directed polypeptide that is substantially devoid of DNA cleavage activity. In some embodiments, the N856A mutation, in combination with one or more of the H840A mutation, the N854A mutation, and the D10A mutation, generates a site-directed polypeptide that is substantially devoid of DNA cleavage activity. Site-directed polypeptides with one substantially inactive nuclease domain are referred to as "nickases."

[0201] In some embodiments, variants of RNA-guided endonucleases (e.g., Cas9) can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas endonucleases are generally guided by a single-stranded guide RNA designed to hybridize with a specific sequence of about 20 nucleotides in the target sequence (e.g., an endogenous genomic locus). However, a few mismatches can be tolerated between the guide RNA and the target locus, effectively shortening the length of homologous sequence required at the target site to, for example, as little as 13 nt of homologous sequence, which may increase the likelihood that the CRISPR / Cas complex will bind elsewhere in the target genome and cleave the double-stranded nucleic acid. This is also known as off-target cleavage. On the other hand, since each nickase variant of Cas endonuclease cleaves only one strand, a pair of nickases must bind in close proximity on opposite strands of the target nucleic acid to create a pair of nicks that create a double-stranded break. This requires that two separate guide RNAs (one for each nickase) bind in close proximity on opposite strands of the target nucleic acid. Following this requirement, the minimum length of homologous sequence required to create a double-stranded break is effectively doubled, and as a result, the two guide RNA sites (if they are present) are unlikely to be close enough to each other to create a double-stranded break, thereby reducing the likelihood that a double-stranded break will occur elsewhere in the genome. As reported in the art, nickases can also be used to promote HDR over NHEJ. By performing HDR using a specific donor sequence that effectively mediates the desired change, the selected change can be introduced into the target site of the genome. Descriptions of various CRISPR / Cas systems for use in gene editing can be found, for example, in WO2013 / 176772 and Sander, JD et al. (2014). Nature Biotechnology, 32(4):347-355, and references cited therein.

[0202] In some embodiments, the site-directed polypeptide (e.g., a variant of a site-directed polypeptide, a mutant of a site-directed polypeptide, an enzymatically inactive site-directed polypeptide, or a site-directed polypeptide that is enzymatically inactive under certain conditions) targets a nucleic acid. In some embodiments, the site-directed polypeptide (e.g., a variant of an endoribonuclease, a mutant of an endoribonuclease, an enzymatically inactive endoribonuclease, or an endoribonuclease that is enzymatically inactive under certain conditions) targets a DNA. In some embodiments, the site-directed polypeptide (e.g., a variant of an endoribonuclease, a mutant of an endoribonuclease, an enzymatically inactive endoribonuclease, or an endoribonuclease that is enzymatically inactive under certain conditions) targets an RNA.

[0203] In some embodiments, the site-directed polypeptide has one or more non-native sequences (eg, the site-directed polypeptide is a fusion protein).

[0204] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain).

[0205] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes) and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain).

[0206] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains, one or both of which have at least 50% amino acid identity to a nuclease domain of the Cas endonuclease derived from a bacterium (e.g., S. pyogenes).

[0207] In some embodiments, the site-directing polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes), two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or a linker that links the site-directing polypeptide to the non-native sequence.

[0208] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), one or both of which have a mutation that reduces the cleavage activity of these nuclease domains by at least 50%.

[0209] In some embodiments, the site-directed polypeptide has an amino acid sequence that has at least 15% amino acid identity to a Cas endonuclease derived from a bacterium (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), and has a mutation at position 10 aspartic acid of one of the nuclease domains and / or a mutation at position 840 histidine of one of the nuclease domains, which mutation reduces the cleavage activity of the nuclease domain by at least 50%.

[0210] In some embodiments, one or more site-directed polypeptides (e.g., DNA endonucleases) comprise two nickases that cooperate to create one double-stranded break at a specific locus in the genome, or one or more site-directed polypeptides comprise four nickases that cooperate to create two double-stranded breaks at a specific locus in the genome, or one site-directed polypeptide (e.g., DNA endonuclease) acts on one double-stranded break at a specific locus in the genome.

[0211] In some embodiments, the polynucleotide encoding the site-directed polypeptide can be used for genome editing. In some of these embodiments, the polynucleotide encoding the site-directed polypeptide is codon-optimized for expression in cells that contain the target DNA of interest according to methods known in the art. For example, when the target nucleic acid of interest is in a human cell, it is envisioned that the polynucleotide encoding the Cas endonuclease (e.g., Cas9) is optimized for human codons and used to generate the Cas endonuclease polypeptide.

[0212] Provided below are some examples of site-directed polypeptides that can be used in various embodiments of the present disclosure.

[0213] CRISPR endonuclease system Genomic loci of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a type of immune system useful in the defense of prokaryotes from foreign invaders such as viruses and phages. The function of a CRISPR locus has three stages, consisting of the integration of new sequences into the CRISPR locus, the expression of CRISPR RNA (crRNA), and the silencing of foreign invading nucleic acids. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

[0214] CRISPR loci contain many short repeating sequences called "repeats". When expressed, the repeats can form secondary hairpin structures (e.g. hairpins) and / or unstructured single-stranded sequences. The repeats usually occur in clusters and vary widely between species. The repeats are spaced apart by unique intervening sequences called "spacers", resulting in a repeat-spacer-repeat structured locus. The spacers are identical or highly homologous to a known foreign invader sequence. The spacer-repeat unit encodes a crisprRNA (crRNA), which is processed to yield the mature form of the spacer-repeat unit. The crRNA has a "seed" or spacer sequence that is responsible for targeting the target nucleic acid (in the prokaryotic native form, the spacer sequence targets the foreign invader nucleic acid). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0215] CRISPR locus further has a polynucleotide sequence encoding CRISPR-associated (Cas) gene. Cas gene encodes an endonuclease involved in the biosynthesis and interference stages of crRNA function in prokaryotes. Some Cas genes have homologous secondary and / or tertiary structures.

[0216] Type II CRISPR systems The biogenesis of crRNA in natural type II CRISPR systems requires trans-activated CRISPR RNA (tracrRNA). tracrRNA is modified by endogenous RNaseIII and then hybridizes to the crRNA repeat sequence of the pre-crRNA array. Endogenous RNaseIII is recruited to cleave the pre-crRNA. The cleaved crRNA is trimmed (e.g., trimming of the 5' end) by an exoribonuclease to generate the mature form of crRNA. tracrRNA remains hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-directed polypeptide (e.g., a Cas endonuclease such as Cas9). In the crRNA-tracrRNA-Cas complex, the crRNA guides the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid activates the Cas endonuclease to cleave the target nucleic acid. The target nucleic acid in type II CRISPR systems is called the protospacer adjacent motif (PAM). In fact, the PAM is essential to facilitate the binding of site-directed polypeptides (e.g., Cas9) to the target nucleic acid. Type II systems (also called Nmeni or CASS4) are further subdivided into type II-A (CASS4) and type II-B (CASS4a). Jinek, M. et al. (2012). Science, 337(6096):816-821 show the utility of the CRISPR / Cas9 system for RNA-programmable genome editing, and WO 2013 / 176772 further describes numerous examples and applications of the CRISPR / Cas endonuclease system for site-directed gene editing.

[0217] V-type CRISPR system Type V CRISPR systems have some important differences from type II systems. For example, Cpf1 is a single-stranded RNA-guided endonuclease, but unlike type II systems, it lacks tracrRNA. Indeed, Cpf1-associated CRISPR arrays are processed into mature crRNAs without the further need for trans-activated tracrRNA. Type V CRISPR arrays are processed into short mature crRNAs of 42-44 nucleotides in length, and each mature crRNA begins with a 19-nucleotide-long direct repeat sequence followed by a 23-25-nucleotide-long spacer sequence. In contrast, mature crRNAs in type II systems begin with a 20-24-nucleotide-long spacer sequence followed by a direct repeat sequence of approximately 22 nucleotides in length. Moreover, Cpf1 utilizes a T-rich protospacer adjacent motif, allowing the target DNA following this short T-rich PAM to be efficiently cleaved by the Cpf1-crRNA complex. This is in contrast to type II systems, which utilize a G-rich PAM following the target DNA. Thus, type V systems cleave targets at a position distant from the PAM, whereas type II systems cleave targets adjacent to the PAM. Furthermore, in contrast to type II systems, Cpf1 cleaves the DNA double strand at a staggered position, resulting in the formation of 4- or 5-nucleotide long 5'-end overhangs. In contrast, type II double strand breaks result in the formation of blunt ends. Like type II systems, Cpf1 is predicted to contain a RuvC-like endonuclease domain, but unlike type II systems, it does not possess a second HNH endonuclease domain.

[0218] Cas genes / polypeptides and protospacer adjacent motifs Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptide shown in Figure 1 of Fonfara, I. et al. (2014). Nucleic Acids Res., 42(4):2577-2590. The nomenclature system for CRISPR / Cas genes has undergone significant revision since the discovery of Cas genes. Figure 5 by Fonfara et al., supra, shows the PAM sequences for Cas9 polypeptides from various species.

[0219] Complex of genome-targeting nucleic acid and site-directed polypeptide The genome-targeting nucleic acid interacts with a site-directed polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9) to form a complex. The genome-targeting nucleic acid (e.g., a gRNA) guides the site-directed polypeptide to the target nucleic acid.

[0220] As mentioned above, in some embodiments, the site-directed polypeptide and the genome-targeting nucleic acid can be administered separately to a cell or a subject. Meanwhile, in some other embodiments, the site-directed polypeptide can be pre-complexed with one or more guideRNAs, or the site-directed polypeptide can be pre-complexed with a tracrRNA and one or more crRNAs. Pre-complexed materials can be administered to a cell or a subject. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs).

[0221] How to edit a genome One method of expressing a FOXP3 protein or a functional derivative thereof in an organism requiring expression of the FOXP3 protein or a functional derivative thereof is to target an endogenous FOXP3 gene or a non-FOXP3 gene that is sufficiently expressed in a relevant cell type (e.g., T cells) by genome editing to integrate a nucleic acid comprising a coding sequence encoding a FOXP3 protein, whereby expression of the integrated coding sequence is driven by the endogenous promoter of the endogenous FOXP3 gene or non-FOXP3 gene. In some embodiments where a non-FOXP3 gene is targeted, expression of the non-FOXP3 gene is controlled by a promoter specific for the targeted cell type (e.g., CD34) to prevent expression in unrelated cell types. + CD34 in hematopoietic stem cells + It is desirable for the antibody to be specific for the cell, or a cell derived therefrom (eg, a T cell).

[0222] In some embodiments, the knock-in method involves knocking in a sequence encoding FOXP3 or a functional derivative thereof, such as a wild-type FOXP3 gene (e.g., a wild-type human FOXP3 gene), a FOXP3 cDNA, or a FOXP3 minigene (with a natural or synthetic enhancer and promoter, one or more exons, a natural or synthetic intron, and a natural or synthetic 3'UTR and polyadenylation signal) into a genomic sequence. In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located in, within, or adjacent to the FOXP3 gene. In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located in, within, or adjacent to exon 1 of the FOXP3 gene.

[0223] In some of the embodiments provided herein, a method is provided for knocking in a sequence encoding FOXP3 or its functional derivative into a genome. In one aspect, the disclosure provides for inserting a nucleic acid comprising a sequence encoding FOXP3 or its functional derivative into a cell genome. In some embodiments, the sequence encoding FOXP3 encodes wild-type FOXP3. The functional derivative of FOXP3 includes a FOXP3 derivative having substantially the same activity as wild-type FOXP3 (e.g., wild-type human FOXP3), such as a FOXP3 derivative that exhibits at least or at least about 30%, 40%, or at least about 40%, 50%, or at least about 50%, 60%, or at least about 60%, 70%, or at least about 70%, 80%, or at least about 80%, 90%, or at least about 90%, 95%, or at least about 95%, or at least about 100% of the activity of wild-type FOXP3. In some embodiments, a functional derivative of FOXP3 has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 is encoded by a nucleotide sequence that lacks an intron (e.g., FOXP3 cDNA). One of skill in the art can test the functionality or activity of a FOXP3 derivative using methods known in the art. A functional derivative of FOXP3 can include a fragment of wild-type FOXP3 having conservative modifications of one or more amino acid residues of full-length wild-type FOXP3.Thus, in some embodiments, a nucleic acid sequence encoding a functional derivative of FOXP3 may have at least or at least about 30%, 40% or at least about 40%, 50% or at least about 50%, 60% or at least about 60%, 70% or at least about 70%, 80% or at least about 80%, 85% or at least about 85%, 90% or at least about 90%, 95% or at least about 95%, 96% or at least about 96%, 97% or at least about 97%, 98% or at least about 98%, or 99% or at least about 99% nucleic acid sequence identity to a nucleic acid sequence encoding FOXP3 (e.g. wild-type FOXP3). In some embodiments, FOXP3 or a functional variant thereof is human wild-type FOXP3.

[0224] In some embodiments, genome editing methods utilize DNA endonucleases, such as CRISPR / Cas endonucleases, to genetically introduce (knock-in) a sequence encoding FOXP3 or a functional derivative thereof. In some embodiments, the DNA endonuclease is Cas1 endonuclease, Cas1B endonuclease, Cas2 endonuclease, Cas3 endonuclease, Cas4 endonuclease, Cas5 endonuclease, Cas6 endonuclease, Cas7 endonuclease, Cas8 endonuclease, Cas9 endonuclease (also known as Csn1 and Csx12), Cas100 endonuclease, Csy1 endonuclease, Csy2 endonuclease, Csy3 endonuclease, Cse1 endonuclease, Cse2 endonuclease, Csc1 endonuclease, Csc2 endonuclease, Csa5 endonuclease, Csn2 endonuclease, Csm2 endonuclease, Csm3 endonuclease, Csm4 endonuclease, Csm5 endonuclease, endonuclease, Csm6 endonuclease, Cmr1 endonuclease, Cmr3 endonuclease, Cmr4 endonuclease, Cmr5 endonuclease, Cmr6 endonuclease, Csb1 endonuclease, Csb2 endonuclease, Csb3 endonuclease, Csx17 endonuclease, Csx14 endonuclease, Csx10 endonuclease, Csx16 endonuclease In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is Cas9 from Streptococcus pyogenes (spCas9).In some embodiments, the Cas9 is Cas9 from Staphylococcus lugdunensis (SluCas9).

[0225] In some embodiments, the cell to be subjected to genome editing has one or more mutations in its genome that reduce the expression of the endogenous FOXP3 gene compared to the expression in a normal cell that does not have the mutation. The normal cell may be a healthy cell or a control cell derived from (isolated from) another subject that does not have a FOXP3 gene abnormality. In some embodiments, the cell to be subjected to genome editing may be a cell derived from (isolated from) a subject in need of treatment for a FOXP3 gene-associated condition or disorder (e.g., a subject suffering from an autoimmune disorder (e.g., IPEX syndrome)). Thus, in some embodiments, expression of the endogenous FOXP3 gene in such cells is reduced by at or about 10%, 20% or about 20%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or about 100% compared to expression of the endogenous FOXP3 gene in a normal cell.

[0226] In some embodiments, CD34 + 1. A method for editing the genome of a cell, comprising: (a) a Cas DNA endonuclease (e.g., a Cas9 endonuclease) or a nucleic acid encoding the Cas DNA endonuclease; (b) a gRNA (e.g., sgRNA) or a nucleic acid encoding the gRNA capable of targeting the Cas DNA endonuclease to a FOXP3 gene or a non-FOXP3 locus (e.g., AAVS1) in the genome of the cell; and (c) Donor template containing the coding sequence of FOXP3. CD34 + The present invention provides a method comprising the step of providing to a cell. In some embodiments, the Cas DNA endonuclease is a Cas9 endonuclease (e.g., a Cas9 endonuclease from Streptococcus pyogenes). In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in the FOXP3 gene. In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in exon 1 of the FOXP3 gene. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7 and 27-29, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7 and 27-29. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 2, 3, and 5. In some embodiments, the gRNA comprises a spacer sequence complementary to a target sequence in a non-FOXP3 locus (e.g., AAVS1). In some embodiments, the gRNA comprises a spacer sequence as set forth in any of SEQ ID NOs: 15-20, or a variant of said spacer sequence having no more than three mismatches compared to any of SEQ ID NOs: 15-20. In some embodiments, the coding sequence for FOXP3 encodes FOXP3 or a functional derivative thereof. In some embodiments, the coding sequence for FOXP3 is a FOXP3 cDNA. An exemplary FOXP3 cDNA sequence may be included in an AAV donor template having the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the method further comprises: + In some embodiments, the method further comprises providing a nucleic acid encoding the Cas DNA endonuclease to the CD34 cell. +In some embodiments, the method further comprises providing the gRNA to the CD34 cell. + In some embodiments, the gRNA is an sgRNA. In some embodiments, the method comprises providing a nucleic acid encoding the gRNA to the CD34 cell. + In some embodiments, the method comprises providing one or more additional gRNAs or nucleic acids encoding said one or more additional gRNAs to said CD34 cell. + The method further comprises the step of providing to the cell.

[0227] In some embodiments, according to any of the methods of editing the genome of a cell described herein, the DNA endonuclease is Cas9. In some embodiments, the Cas9 is Cas9 from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 is Cas9 from Staphylococcus lugdunensis (SluCas9).

[0228] In some embodiments, according to any of the methods of editing the genome of a cell described herein, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in said cell, in some embodiments, said cell is a human cell.

[0229] In some embodiments, according to any of the methods of editing the genome of a cell described herein, the method uses a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding a DNA endonuclease is codon-optimized for expression in the cell. In some embodiments, the cell is a human cell, such as a human CD34 cell. +In some embodiments, the nucleic acid encoding the DNA endonuclease is a cell. In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA (such as a DNA plasmid). In some embodiments, the nucleic acid encoding the DNA endonuclease is RNA (such as an mRNA).

[0230] In some embodiments, according to any of the methods for editing the genome of a cell described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, and the donor template is configured such that the donor cassette is integrated into the genomic locus targeted by the gRNA of (b) by homology directed repair (HDR). In some embodiments, the donor cassette is flanked on either side by homology arms corresponding to the sequence of the targeted genomic locus. In some embodiments, the length of the homologous arms is at least 0.2 kb or at least about 0.2 kb (e.g., at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb or at least more, or at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb or at least more). In some embodiments, the length of the homologous arms is at least 0.8 kb or at least about 0.8 kb. Exemplary homologous arms include those contained in a donor template having the sequence of SEQ ID NO: 34 or 161. Exemplary donor templates include donor templates having the sequence of SEQ ID NO: 34 or 161. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0231] In some embodiments, according to any of the methods for editing the genome of a cell described herein, the donor template comprises a donor cassette comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, and the donor template is configured to integrate the donor cassette into the genomic locus targeted by the gRNA of (b) by non-homologous end joining (NHEJ). In some embodiments, the donor cassette is flanked on one or both sides by a gRNA target site. In some embodiments, the donor cassette is flanked on both sides by a gRNA target site. In some embodiments, the gRNA target site is a target site of a gRNA included in the system. In some embodiments, the gRNA target site of the donor template is the reverse complement of a cell genome gRNA target site targeted by a gRNA included in the system. In some embodiments, the donor template is encoded by an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0232] In some embodiments, according to any of the methods of editing the genome of a cell described herein, the DNA endonuclease or a nucleic acid encoding the DNA endonuclease is formulated by encapsulation in a liposome or lipid nanoparticle. In some embodiments, the liposome or the lipid nanoparticle further comprises a gRNA. In some embodiments, the liposome or the lipid nanoparticle is a lipid nanoparticle. In some embodiments, the method uses a lipid nanoparticle comprising a nucleic acid encoding a DNA endonuclease and a gRNA. In some embodiments, the nucleic acid encoding a DNA endonuclease is an mRNA encoding a DNA endonuclease.

[0233] In some embodiments, according to any of the methods for editing the genome of a cell described herein, the DNA endonuclease is pre-complexed with a gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex is provided to the cell by electroporation. In some embodiments, the donor template is an AAV donor template encoded by an AAV vector (e.g., an AAV6 vector). In some embodiments, the AAV donor template is provided to the cell at or near the same time that the RNP complex is provided to the cell. For example, in some embodiments, the RNP complex is electroporated into the cell and transduced with the AAV donor template on the same day. In some embodiments, the cells are electroporated with the RNP complex and transduced with the AAV donor template within a time interval of at or about 12 hours (e.g., within or about 11 hours, within or about 10 hours, within or about 9 hours, within or about 8 hours, within or about 7 hours, within or about 6 hours, within or about 5 hours, within or about 4 hours, within or about 3 hours, within or about 2 hours, within or about 1 hour, or less). In some embodiments, the RNP complex is electroporated into the cells, followed by plating the cells and transducing the cells with the AAV donor template. In some embodiments, the cells are pre-stimulated in the presence of cytokines (e.g., TPO, SCF, FLT3L or IL6 or any combination thereof) and / or small molecules capable of promoting proliferation or self-renewal of HSCs (e.g., UM171 or StemRegenin (SR1)) prior to providing the RNP and AAV donor template.In some embodiments, the pre-stimulation is performed for at least or at least about 12 hours (e.g., for at or about 16 hours, at or about 20 hours, at or about 24 hours, at or about 36 hours, at or about 48 hours, or more). In some embodiments, the pre-stimulation is performed for at least or at least about 48 hours. In some embodiments, the pre-stimulation is performed in a cell composition comprising the cells, and the concentration of the cells in the cell composition and / or medium is such that at least or at least about 10% (e.g., at or about 20%, at or about 30%, at or about 40%, or at or about 50%) of the cells in the cell composition can remain quiescent after the end of the pre-stimulation. In some embodiments, 10%-60% or about 10%-60% (e.g., 10%-50% or about 10%-50%, 10%-40% or about 10%-40%, or 10%-30% or about 10%-30%) of the cells in the cell composition remain quiescent following termination of pre-stimulation. In some embodiments, the concentration of the cells in the cell composition is 5×10. 5 / ml or less or about 5 x 10 5 / ml or less (e.g., 4 × 10 5 / ml or less or about 4 x 10 5 pcs or less, 3×10 5 / ml or less or about 3 x 10 5 pcs or less, 2.5×10 5 / ml or less or about 2.5 x 10 5 pcs or less, 2×10 5 / ml or less or about 2 x 10 5 pcs or less, 1×10 5 / ml or less or about 1 x 10 5 pcs or less, 0.5×10 5 / ml or less or about 0.5 x 10 5In some embodiments, the concentration of the cells in the cell composition is 2.5×10 5 / ml or less or about 2.5 x 10 5 cells / ml or less.

[0234] In some embodiments, according to any of the methods of editing the genome of a cell described herein, the frequency of targeted integration of the donor template into the FOXP3 gene in the genome of the cell is between 0.1% and 99%, or between about 0.1% and about 99%. In some embodiments, the frequency of targeted integration is between 2% and 70%, or between about 2% and about 70% (e.g., between 2% and 65%, or between about 2% and about 65%, between 2% and 55%, between 3% and 70%, between 5% and 70%, between 5% and 60%, between 5% and 50%, between 5% and 50%, between 10% and 60%, or between 10% and 50%, or between 10% and 50%). In some embodiments, the cell is a cell of a subject (e.g., a human subject).

[0235] Selection of target sequence In some embodiments, the position of the 5' end boundary or the position of the 3' end boundary, or both, relative to a particular reference locus can be shifted to facilitate or enhance a particular application of gene editing, which will depend in part on the endonuclease system selected to perform the gene editing, as further described and illustrated herein.

[0236] In a first, but not limiting, embodiment of such target sequence selection, many endonuclease systems have rules or criteria for the initial selection of a cleavable target site, e.g., CRISPR type II or type V endonucleases have specific requirements for PAM sequence motifs at specific positions adjacent to the DNA cleavage site.

[0237] In another aspect related to the selection or optimization of a target sequence, but not limited thereto, the frequency of "off-target" activity (e.g., frequency of double-stranded breaks occurring at sites other than the selected target sequence) of a particular combination of target sequence and gene editing endonuclease is evaluated against the frequency of on-target activity. In some cases, cells precisely edited at a desired locus may have a selective advantage over other cells. Specific examples of selective advantages include, but are not limited to, gaining various attributes such as improved replication rate, persistence, resistance to certain conditions, improved engraftment success or persistence after in vivo introduction into a subject, and other attributes associated with maintaining or improving the number or viability of such cells. In other cases, cells precisely edited at a desired locus may be selected as positive cells by one or more screening methods used to identify, sort or otherwise select precisely edited cells. The selective advantage and directional selection methods may take advantage of the phenotype associated with the modification. In some embodiments, cells may be edited more than once to create a second modification to create a new phenotype used to select or purify the intended cell population. Such a second modification can be made by adding a second gRNA for a selectable or screenable marker. In some cases, a DNA fragment containing the cDNA and a selectable marker can be used to precisely edit cells at the desired locus.

[0238] In embodiments, regardless of whether selective advantage or directed selection is applicable in a particular case, target sequences are selected with consideration of off-target frequency to improve the effectiveness of selective advantage or directed selection application and / or reduce the possibility of undesired modifications at sites other than the desired target. As described in more detail and illustrated in the present specification and the art, off-target activity occurs under the influence of various factors, including the similarities and differences between the target site and various off-target sites, and the particular endonuclease used. Bioinformatics tools are available to assist in predicting off-target activity, and such tools can often be used to identify sites where off-target activity is most likely to occur, and such predictions or identifications can be evaluated experimentally to estimate the relative frequency of off-target activity to on-target activity, thereby allowing the selection of sequences with high relative on-target activity. Specific examples of such techniques are provided herein, and others are known in the art.

[0239] Another aspect of target sequence selection is related to homologous recombination events. Sequences that share homologous regions can play a central role in homologous recombination events that delete intervening sequences. Such recombination events occur during the normal replication of chromosomes and other DNA sequences, as well as at other times when DNA sequences are synthesized, such as during double-strand break repair, which occurs periodically in the normal cell replication cycle, but can also be enhanced by the occurrence of various events (such as UV light and other DNA break inducers) or the presence of certain agents (such as various chemical inducers). Many of these inducers indiscriminately generate double-strand breaks (DSBs) in the genome, and DSBs are regularly induced and repaired in normal cells. During double-strand break repair, the original sequence can be reconstructed with full fidelity, but in some cases, short insertions or deletions (called "indels") may be introduced at the DSB site.

[0240] Also, as in the case of using the endonuclease system described herein, double-strand breaks (DSBs) can be specifically induced at specific locations, which can be used to cause directed or selective recombination events at selected chromosomal locations.The tendency of homologous sequences to recombine during DNA repair (and replication) can be exploited in a variety of situations, and is one of the foundations for the application of gene editing systems such as CRISPR, in which the sequence of interest provided by using a "donor" polynucleotide is inserted into a specific location of desired chromosomes by homologous recombination repair.

[0241] Regions of homology between specific sequences can be used to create the desired deletions, which can be short "microhomology" regions that can be as little as 10 base pairs or less. For example, a single double-strand break (DSB) is introduced at a site that shows microhomology with a nearby sequence. Normal repair of such DSBs frequently results in deletion of the intervening sequence, which occurs as a result of recombination facilitated by the DSB and the associated cellular repair processes.

[0242] However, in some circumstances, selecting a target sequence within the region of homology can result in much larger deletions, such as gene fusions (deletions within the coding region), which may or may not be desirable in certain circumstances.

[0243] The examples provided herein further describe the selection of various target regions for creating DSBs designed to insert genes encoding FOXP3, and the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events.In some embodiments, the target locus is selected from FOXP3 gene, AAVS1 locus and TRA gene.

[0244] Nucleic acid modification In some embodiments, as described in detail herein and known in the art, the polynucleotide introduced into the cell has one or more modifications that can be used individually or in combination, e.g., to enhance activity, stability or specificity, alter delivery, reduce or otherwise enhance the innate immune response of the host cell.

[0245] In certain embodiments, modified polynucleotides are used in CRISPR / Cas systems (e.g., CRISPR / Cas9 systems), where the guide RNA (single-molecule guide or bi-molecule guide) and / or DNA or RNA encoding the Cas endonuclease introduced into a cell can be modified as described and illustrated below. Such modified polynucleotides can be used in CRISPR / Cas systems to edit one or more genomic loci.

[0246] When using the CRISPR / Cas system for such exemplary (but not limited to) applications, the guide RNA can be modified to improve the formation or stability of the CRISPR / Cas genome editing complex comprising the guide RNA, which may be formed from a single-molecule guide or a bimolecular guide and a Cas endonuclease. Alternatively, the guide RNA can be modified to improve the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, which can be used, for example, to enhance on-target activity. Alternatively, the guide RNA can be modified to improve specificity, for example, to improve the relative genome editing rate at the on-target site compared to the effect at other sites (off-target).

[0247] Alternatively or additionally, the stability of the guide RNA can be improved, for example by modifying it to make it more resistant to degradation by ribonucleases (RNases) present in the cell, thereby increasing the half-life of the guide RNA in the cell. Modifications that increase the half-life of the guide RNA can be particularly useful in embodiments in which editing is performed by introducing a Cas endonuclease into the cell using an RNA that needs to be translated to produce the endonuclease, since the increased half-life of the guide RNA introduced at the same time as the RNA encoding the endonuclease can be used to increase the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist in the cell.

[0248] Alternatively, or in addition, modifications can be used to reduce the likelihood or extent to which RNA introduced into cells induces natural immune responses. As described herein below and known in the art, such immune responses have been well-characterized with respect to RNA interference (RNAi), such as small interfering RNA (siRNA), and tend to be associated with shortening the half-life of RNA and / or induction of cytokines or other factors associated with immune responses.

[0249] The RNA encoding the endonuclease that is introduced into a cell can also be modified in one or more ways, including, but not limited to, a modification that improves the stability of the RNA (such as increasing its degradation by RNAses present in the cell), enhances translation of the product (e.g., the endonuclease), and / or reduces the likelihood or extent to which the RNA introduced into a cell will induce an innate immune response.

[0250] Various modifications, such as those mentioned above or other modifications, can be used in combination as well, for example, in the case of CRISPR / Cas, one or more modifications can be made to the guide RNA (including those exemplified above) and / or one or more modifications can be made to the RNA encoding the Cas endonuclease (including those exemplified above).

[0251] delivery In some embodiments, the nucleic acid molecule used in the methods provided herein, for example, the genome targeting nucleic acid of the present disclosure or the nucleic acid encoding the site-directed polypeptide, is packaged inside or on the surface of a delivery carrier for delivery to cells.Conceivable delivery carriers include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, or micelles.As reported in the art, various targeting moieties can be used to enhance the selective interaction of such carriers with desired cell types or locations.

[0252] Introduction of the complexes, polypeptides or nucleic acids of the present disclosure into cells can be achieved by virus or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, or nanoparticle-mediated nucleic acid delivery, and the like.

[0253] In embodiments, the guide RNA polynucleotide (RNA or DNA) and / or the endonuclease polynucleotide (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, single or multiple endonuclease polypeptides can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In some embodiments, the DNA endonuclease can be delivered as one or more polypeptides alone, or pre-complexed with one or more guide RNAs, or pre-complexed with tracrRNA and one or more crRNAs.

[0254] In embodiments, polynucleotide can be delivered by non-viral delivery carriers, such as non-viral delivery carriers include but are not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes.Some specific examples of non-viral delivery carriers are described in Peer, D. et al. (2011). Gene Therapy, 18: 1127-1133 (this document focuses on the non-viral delivery carriers for siRNA, which are also useful for the delivery of other polynucleotides).

[0255] In embodiments, polynucleotides such as guide RNAs, sgRNAs, or mRNAs encoding endonucleases can be delivered to cells or subjects by lipid nanoparticles (LNPs).

[0256] Although non-viral nucleic acid delivery methods have been tested in both animal models and humans, the most well-developed system is the lipid nanoparticle. Lipid nanoparticles (LNPs) are generally composed of ionizable cationic lipids and three or more additional components, usually cholesterol, DOPE, and lipid-containing polyethylene glycol (PEG) (see, for example, Example 2). The cationic lipids can bind to positively charged nucleic acids to form dense complexes that protect the nucleic acid from degradation. During passage through a microfluidic system, the components self-assemble to form particles of 50-150 nM size, with the nucleic acid encapsulated in the cationic lipid-complexed core and surrounded by a lipid bilayer-like structure. After injection into the circulatory system of a subject, these particles can bind apolipoprotein E (apoE). ApoE is a ligand for the LDL receptor and mediates uptake into hepatocytes in the liver by receptor-mediated endocytosis. This type of LNP has been shown to be capable of efficiently delivering mRNA and siRNA to hepatocytes in the liver of rodents, primates, or humans. After endocytosis, the LNPs reside in endosomes. The encapsulated nucleic acid escapes the endosome due to the ionizable nature of the cationic lipid. This delivers the nucleic acid to the cytoplasm and allows translation of the mRNA into the encoded protein. After escape from the endosome, the Cas mRNA (e.g., Cas9 mRNA) can be translated into a Cas protein and complexed with the gRNA. In some embodiments, the inclusion of a nuclear localization signal in the Cas protein sequence facilitates nuclear translocation of the Cas protein / gRNA complex. Alternatively, the small gRNA passes through the nuclear pore complex and complexes with the Cas protein in the nucleus. Once inside the nucleus, the gRNA / Cas complex scans the genome for homologous target sites and selectively generates double-strand breaks at the desired target site in the genome. The half-life of RNA molecules in vivo is generally short, on the order of hours to days. Similarly, the half-life of proteins also tends to be short, on the order of hours to days.Thus, in some embodiments, delivery of gRNA and Cas mRNA using LNPs can result in only transient expression and activity of gRNA / Cas complexes. This can provide the advantage of reducing the frequency of off-target cleavage, and therefore, in some embodiments, can minimize the risk of genotoxicity. LNPs are generally less immunogenic than viral particles. Many humans already have immunity to AAV, but there is no pre-existing immunity to LNPs. Furthermore, adaptive immune responses to LNPs are unlikely to occur, allowing repeated administration of LNPs.

[0257] Several types of ionizable cationic lipids have been developed for use in LNPs. These include C12-200 (Love, KT et al. (2010). Proc. Natl. Acad. Sci. USA, 107(5):1864-1869), MC3, LN16, MD1, and others. In one type of LNP, a GalNac moiety is added to the outside of the LNP, which serves as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids are used to formulate LNPs for delivery of gRNA and Cas mRNA to the liver.

[0258] In some embodiments, LNP refers to particles having a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. Alternatively, the size of the nanoparticles may range from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0259] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids such as DOPE, a fusogenic phospholipid, and cholesterol, a membrane component, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. The limitations of cationic lipids include low efficacy due to poor stability and rapid clearance, and the generation of inflammatory or anti-inflammatory responses. LNPs can also have hydrophobic, hydrophilic, or both hydrophobic and hydrophilic lipids.

[0260] Any lipid or combination thereof known in the art can be used to make LNPs. Examples of lipids used to make LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 and 7C1. Examples of neutral lipids include DPSC, DPPC, POPC, DOPE and SM. Examples of PEG-modified lipids include PEG-DMG, PEG-CerC14 and PEG-CerC20.

[0261] In embodiments, LNPs can be made by combining multiple lipids in any molar ratio, and further, LNPs can be made by combining single or multiple polynucleotides with single or multiple lipids in various molar ratios.

[0262] In embodiments, the site-directed polypeptide and the genome-targeted nucleic acid can be administered separately to cells or subjects. Alternatively, the site-directed polypeptide can be pre-complexed with one or more guideRNAs, or pre-complexed with tracrRNA and one or more crRNAs. Pre-complexed materials can be administered to cells or subjects. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs).

[0263] RNA can form specific interactions with other RNA or DNA. This property is exploited in many biological processes, but it also carries the risk of promiscuous interactions in the nucleic acid-rich cellular environment. One solution to this problem is to form ribonucleoprotein particles (RNPs) in which RNA is precomplexed with endonucleases. Another advantage of RNPs is the protection of RNA from degradation.

[0264] In some embodiments, the endonuclease contained in RNP can be modified or unmodified.Similarly, gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.Various modifications are known in the art and can be used.

[0265] The endonuclease and sgRNA can be combined in a molar ratio of usually 1:1. Alternatively, the endonuclease, crRNA and tracrRNA can be combined in a molar ratio of usually 1:1:1. However, various molar ratios can be used to generate RNPs.

[0266] In some embodiments, delivery can be achieved using recombinant adeno-associated virus (AAV) vectors. Techniques are known in the art for producing rAAV particles to provide cells with the polynucleotide to be delivered, the AAV genome packaged to include the rep and cap genes, and the function of a helper virus. The production of rAAV requires that the rAAV genome, the AAV rep and cap genes separated from (e.g., not contained within) the rAAV genome, and the function of a helper virus are present in a single cell (herein referred to as a packaging cell). The AAV rep and cap genes may be from any serotype of AAV capable of producing recombinant virus, or from an AAV of a different serotype than the ITRs in the genome of the rAAV, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. The production of pseudotyped rAAV is disclosed, for example, in International Patent Application WO01 / 83692. Table 1 shows the AAV serotypes and Genbank accession numbers of some selected AAVs. [Table 2]

[0267] In some embodiments, the method of producing packaging cells involves generating a cell line that stably expresses all the components necessary for the production of AAV particles, for example by integrating into the genome of the cell a single plasmid (or multiple plasmids) that contains a rAAV genome lacking the AAV rep and cap genes, a separate AAV rep and cap gene, and a selectable marker, such as a neomycin resistance gene. The AAV genome is introduced into a bacterial plasmid by techniques such as GC tailing (Samulski, RJ et al. (1982). Proc. Natl. Acad. Sci. USA, 79(6):2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin, CA et al. (1983). Gene, 23(1):65-73), or direct blunt-end ligation (Senapathy, P. et al. (1984). J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. Advantages of this method include that the cells are selectable and are suitable for large-scale production of rAAV. An example of a suitable alternative method is to use adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cell.

[0268] General principles of rAAV production are reviewed in, for example, Carter, BJ (1992). Curr. Opin. Biotechnol., 3(5):533-539; and Muzyczka, M. (1992). Curr. Top. Microbiol. Immunol., 158:97-129.Various approaches are described in Tratschin, JD et al. (1984). Mol. Cell. Biol., 4(10):2072-2081; Hermonat, PL et al. (1984). Proc. Natl. Acad. Sci. USA, 81(20):6466-6470; Tratschin, JD et al. (1985). Mo1. Cell. Biol., 5(11):3251-3260;McLaughlin, SK et al. (1988). J. Virol. , 62(6):1963-1973;Lebkowski, JS et al. (1988). Mol. Cell. Biol., 8(10):3988-3996.;Samulski, RJ et al. (1989), J. Virol. 63(9):3822-3828; U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin, P. et al. (1995). Vaccine, 13(13):1244-1250; Paul, RW et al. (1993). Hum. Gene Ther., 4(5):609-615; Clark, KR et al. (1996). Gene Ther. 3(12):1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

[0269] The serotype of AAV vector can be matched to the target cell type.For example, the typical cell types described below can be transduced with the AAV serotypes described herein.For example, the serotypes of AAV vectors suitable for hematopoietic stem cells include, but are not limited to, AAV2 and AAV6.In some embodiments, the serotype of AAV vector is AAV6.

[0270] In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to any one of SEQ ID NOs: 33-36 and 161. In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to SEQ ID NO: 33. In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to SEQ ID NO: 34. In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to SEQ ID NO: 35. In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to SEQ ID NO: 36. In some embodiments, the AAV vector comprises a nucleic acid sequence having at least 90% or at least about 90% (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more) sequence identity to SEQ ID NO: 161.

[0271] In addition to adeno-associated viral vectors, other viral vectors can be used, including, but not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpes viruses, Epstein-Barr viruses, papovaviruses, pox viruses, vaccinia viruses, and herpes simplex viruses.

[0272] In some embodiments, the Cas mRNA (e.g., Cas9 mRNA), the sgRNA targeting one or two loci of the FOXP3 gene, and the donor DNA are each formulated separately by encapsulation in a lipid nanoparticle, all of them are formulated together by encapsulation in a single lipid nanoparticle, or all of them are formulated together by encapsulation in two or more lipid nanoparticles.

[0273] In some embodiments, the Cas mRNA (e.g., Cas9 mRNA) is formulated by encapsulation in a lipid nanoparticle, while the sgRNA and donor DNA are delivered in an AAV vector. In some embodiments, the Cas mRNA and sgRNA are formulated together by encapsulation in a lipid nanoparticle, while the donor DNA is delivered in an AAV vector.

[0274] Cas endonucleases (e.g., Cas9 endonuclease) can also be delivered as DNA plasmids, as mRNA, or as proteins. Guide RNA can be expressed from the same DNA or can be delivered as RNA. The RNA can be chemically modified to alter or improve its half-life and / or reduce the likelihood or severity of an immune response. Endonuclease proteins can also be complexed with gRNAs prior to delivery. Viral vectors allow for efficient delivery. Split Cas endonucleases and smaller orthologs of Cas endonucleases can be packaged into AAVs, as can donors for HDR. There are also various non-viral delivery methods that can deliver each of these components, and non-viral and viral delivery methods can be used in combination. For example, nanoparticles can be used to deliver proteins and guide RNAs, and AAVs can be used to deliver donor DNA.

[0275] In some embodiments related to delivery of genome editing components for therapeutic treatment, at least two components, a sequence-specific nuclease and a DNA donor template, are expressed in a cell to be transformed (e.g., CD34 + In some embodiments, the AAV is selected from the serotypes AAV2 or AAV6. In some embodiments, the AAV-packaged DNA donor template is first administered to a subject (e.g., a patient) by peripheral intravenous injection, followed by administration of a sequence-specific nuclease. The advantage of first delivering the AAV-packaged donor DNA template is that the delivered donor DNA template binds to the transduced CD34 +The sequence-specific nuclease is stable in the nucleus of the cell, which allows the next administration of sequence-specific nuclease to create double-strand breaks in the genome and integrate the DNA donor by HDR or NHEJ. In some embodiments, it is desirable that the sequence-specific nuclease maintains its activity in the target cell for the time required to promote the targeted integration of the transgene at a level sufficient to exert the desired therapeutic effect. If the sequence-specific nuclease maintains its activity in the cell for a long period of time, this will increase the frequency of double-strand breaks at off-target sites. Specifically, the frequency of off-target cleavage is a function of the off-target cleavage efficiency multiplied by the time the nuclease is active. Since mRNA and the proteins translated from it are short-lived in the cell, the delivery of the sequence-specific nuclease in the form of mRNA results in a short duration of nuclease activity, ranging from a few hours to a few days. Therefore, it is expected that the delivery of the sequence-specific nuclease to the cell that already contains the donor template can maximize the ratio of targeted integration to off-target integration.

[0276] In some embodiments, the sequence-specific nuclease is a Cas endonuclease (e.g., Cas9 endonuclease) used in a CRISPR / Cas system composed of an sgRNA directed against the FOXP3 gene and a Cas endonuclease. In some embodiments, the Cas endonuclease is delivered as an mRNA encoding a Cas protein operably fused to one or more nuclear localization signals (NLS). In some embodiments, the sgRNA and Cas mRNA are packaged into lipid nanoparticles to deliver the sgRNA to CD34 + Cells (e.g., CD34 + Hematopoietic stem cells (HSCs) are delivered to the host.

[0277] In some embodiments, to facilitate nuclear localization of the donor template, a DNA sequence capable of facilitating nuclear localization of the plasmid, for example, a 366 bp region consisting of the Simian Virus 40 (SV40) origin of replication and early promoter, can be added to the donor template. Other DNA sequences that bind to cellular proteins can also be used to enhance nuclear import of DNA.

[0278] Transgenic Cells and Transgenic Cell Populations In one aspect, the disclosure herein provides a method of editing the genome of a cell to generate a genetically modified cell. In some aspects, a genetically modified cell population is provided. Thus, a genetically modified cell refers to a cell having at least one genetic modification introduced by genome editing (e.g., genome editing using the CRISPR / Cas system). In some embodiments, the genetically modified cell is a genetically modified hematopoietic stem cell, such as a genetically modified CD34 + Genetically modified CD34 in hematopoietic stem cells + A genetically modified cell is a cell that has an integrated coding sequence for FOXP3. In some embodiments, the genetically modified cell is not a germline cell.

[0279] In the embodiment described herein, gene editing nuclease is used to modify the regulatory elements of FOXP3 gene to stably express FOXP3, to generate therapeutic cells stably expressing FOXP3. In the representative data provided herein, a promoter (examples of constitutive promoters include EF1α promoter, PGK promoter, or MND promoter) is placed upstream of the coding exon of FOXP3 to induce FOXP3 expression, but it is envisioned that other approaches can be used to modify regulatory elements to stably express FOXP3. By modifying endogenous regulatory elements using several approaches, the endogenous FOXP3 gene can be constitutively expressed in therapeutic cells of the present invention, which is insensitive to the control that causes FOXP3 gene silencing or conversion to a non-repressed cell phenotype. Thus, the exemplary method described herein solves the problem of loss of FOXP3 expression due to epigenetic effects on endogenous regulatory sequences and endogenous promoters.

[0280] CD34 + It is also envisioned to propose a method for enhancing the expression of FOXP3 in a bulk population of cells. In autoimmune disease subjects or subjects with organ graft rejection, the endogenous TCR repertoire of the inflammatory T cell population contains TCRs with specificity to recognize and precisely bind to inflamed or foreign tissues in the organ. Such T cells are believed to mediate the autoinflammatory response or organ rejection. By converting a portion of the bulk T cell population to a regulatory phenotype, the TCR specificity of the proinflammatory population can be utilized in a therapeutic cell population. In this respect, the method of the present invention is superior to other therapies that utilize thymus-derived regulatory T cells (which are believed to have a different TCR repertoire that does not overlap with inflammatory T cells). Furthermore, in autoimmune disease subjects or subjects with organ rejection, it is likely that the in vivo tT regThe population appears to fail to induce the immune tolerance necessary to avoid inflammation. The methods described herein can be used to treat autoimmune diseases and to induce immune tolerance to transplanted organs.

[0281] A notable drawback is that it requires the use of gene editing tools that can efficiently recombine in FOXP3 gene.In the method provided herein, it has been shown that this reaction can be efficiently carried out using TALEN nuclease, but in principle, it is believed that any nuclease platform can recombine equally well.

[0282] Regulatory T cell therapy can be used to induce immune tolerance in transplantation and autoimmune diseases. reg The infusions have been expanded ex vivo. Phase 1 trials have shown limited efficacy in type 1 diabetes (T1D), but some benefit in GvHD after transplantation. In some embodiments, the next generation of genetically engineered regulatory T cells are targeted to endogenous T cells by a chimeric antigen receptor (CAR). reg By expressing FOXP3, effector T cells may be reg It can also be converted to.

[0283] However, the endogenous T reg and recombinant T reg So, there seems to be some difference. reg The therapy appears to be safe, but endogenous T reg Too few T reg plays an important role in various autoimmune diseases (IPEX, T1D, SLE, RA, EAE, etc.). reg Various approaches to enhance the number or function of cells are currently in clinical trials, for example, the use of low-dose IL-2 and expanded autologous T cells. regTherapies combining IL-2 with adoptive transfer of T cells are currently in clinical trials. The efficacy of IL-2 therapy is limited due to its pleiotropic activity and potential "off-target" effects that may enhance inflammation. reg Therapy also involves the expansion of cultured T reg are likely to be of limited use due to problems with in vivo stability and viability and a lack of therapeutically useful antigen specificity.

[0284] endogenous T reg (nT reg There are also potential drawbacks to the use of T. reg For example, some individuals may have a genetic predisposition to instability in expressing CAR. reg In addition, nT reg may be subject to epigenetic regulation of FOXP3, thus downregulating the desired induction of FOXP3. reg may not have the exact specificity for the TCR (T cell receptor). reg The function of the marker is also related to the selection marker, and the expanded endogenous T reg It is believed that inflammatory cells are always present in the cell population. Therefore, the method provided herein uses recombinant cells, and thus can induce the function of regulatory T cells by expressing CARs, and can also inhibit the engraftment of CAR T cells. reg This avoids the possibility of natural T cells being converted into pro-inflammatory CAR T cells, and in this respect reg This is an improved method compared to transplanting the

[0285] In some embodiments, the genome of a cell can be edited by inserting a nucleic acid sequence encoding FOXP3 or its functional derivative into the genome sequence of the cell. In some embodiments, the cell that is the subject of genome editing has one or more mutations in its genome that reduce the expression of endogenous FOXP3 gene compared to the expression in normal cells that do not have the mutation. The normal cell can be a healthy cell or a control cell derived from (isolated from) another subject that does not have FOXP3 gene abnormality. In some embodiments, the cell that is the subject of genome editing can be a cell derived from (or isolated from) a subject that needs treatment for a condition or disorder related to FOXP3 gene. Thus, in some embodiments, expression of the endogenous FOXP3 gene in such cells is increased by at or about 10%, 20% or about 20%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or about 100% compared to expression of the endogenous FOXP3 gene in a normal cell.

[0286] For example, upon successful insertion of a transgene, such as a nucleic acid encoding FOXP3 or a functional derivative thereof, expression in the cell of the introduced nucleic acid encoding FOXP3 or a functional derivative thereof can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200% or more of the expression of the endogenous FOXP3 gene in the cell. or at least about 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 2,000%, at least 3,000%, at least 5,000%, at least 10,000%, or more.In some embodiments, the activity of the introduced FOXP3 coding sequence product (including a functional derivative of FOXP3) in the genome edited cell is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200% or at least about the activity of the endogenous FOXP3 gene in the cell. It may be 200%, at least or at least about 300%, at least or at least about 400%, at least or at least about 500%, at least or at least about 600%, at least or at least about 700%, at least or at least about 800%, at least or at least about 900%, at least or at least about 1,000%, at least or at least about 2,000%, at least or at least about 3,000%, at least or at least about 5,000%, at least or at least about 10,000%, or more.In some embodiments, expression of the introduced FOXP3 coding sequence in the cell is at least or at least about 2-fold, at least or at least about 3-fold, at least or at least about 4-fold, at least or at least about 5-fold, at least or at least about 6-fold, at least or at least about 7-fold, at least or at least about 8-fold, at least or at least about 9-fold, at least or at least about 10-fold, at least or at least about 15-fold, at least or at least about 20-fold, at least or at least about 30-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 1000-fold, or more, than expression of the endogenous FOXP3 gene in the cell. Furthermore, in some embodiments, the activity of the introduced FOXP3 coding sequence product (including functional derivatives of FOXP3) in a genome-edited cell may be similar to or greater than the activity of the endogenous FOXP3 gene product in a normal healthy cell.

[0287] In one embodiment, ex vivo CD34 + The cells are genetically modified and then reintroduced into the subject, resulting in genetically modified T cells in the subject that express the inserted FOXP3 gene.

[0288] Method of preparation In some embodiments, there is provided a method of producing a genetically modified cell, comprising: CD34 comprising a first nucleic acid comprising at least one locus + providing a cell; providing a Cas endonuclease (e.g., a Cas9 endonuclease) or a second nucleic acid encoding the Cas endonuclease; The Cas endonuclease or the second nucleic acid is linked to the CD34 + introducing into a cell; introducing a third nucleic acid encoding at least one gRNA configured to hybridize to at least one locus, or a set of nucleic acids encoding said at least one gRNA; and A fourth nucleic acid comprising a gene delivery cassette is added to the CD34 + Step of introducing into cells The present invention provides a method comprising:

[0289] In some embodiments, the method for producing a genetically modified cell provided herein further comprises activating the CD34+ cells, the activation being performed prior to introducing a second nucleic acid into the CD34+ cells. In some embodiments, the activation comprises activating the CD34+ cells with a cytokine selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), FLT3L, and IL-6. + This is done by contacting the cells with the cytokines. These cytokines may be supported on beads.

[0290] In some embodiments, the methods of producing a genetically modified cell provided herein, wherein the at least one locus is a FOXP3 gene, an AAVS1 locus, or a TRA gene.

[0291] In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are provided in one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is a self-complementary vector. In some embodiments, the AAV vector is a single-stranded vector. In some embodiments, the AAV vector is a combination of a self-complementary vector and a single-stranded vector.

[0292] In some embodiments, the second nucleic acid encoding the Cas endonuclease is an mRNA. In some embodiments, the at least one gRNA comprises a spacer sequence comprising a sequence as set forth in SEQ ID NO: 2, 3 or 5. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are codon-optimized for expression in eukaryotic cells, such as human cells. In some embodiments, the fourth nucleic acid comprises a sequence encoding a human codon-optimized FOXP3 cDNA sequence. In some embodiments, the fourth nucleic acid further comprises a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter, or an E2F promoter. In some embodiments, the fourth nucleic acid further comprises a sequence encoding a low affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB or LNGFR (a coding sequence for the LNGFR epitope). In some embodiments, the fourth nucleic acid further comprises a coding sequence for the low affinity nerve growth factor receptor (LNGFR) or a coding sequence for LNGFRe (a coding sequence for an LNGFR epitope).

[0293] In some embodiments, the method further comprises: +The method further comprises a step of introducing the nucleic acid into a cell. In some embodiments, the fifth nucleic acid is included in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid comprises a sequence encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid comprises a sequence encoding a marker protein. In some embodiments, the fourth sequence and / or the fifth sequence further comprises a sequence encoding a P2A self-cleaving peptide. In some embodiments, the fourth sequence and / or the fifth sequence further comprises a sequence encoding a polyA sequence. In some embodiments, the polyA sequence comprises SV40 polyA or the 3'UTR of FOXP3. In some embodiments, the fourth sequence comprises a sequence as set forth in any of SEQ ID NOs: 37-42.

[0294] In some embodiments, the fourth sequence and the fifth sequence are + The fourth and fifth sequences are introduced into a cell, and comprise sequences encoding expression cassettes configured to express FOXP3 cDNA-LNGFR and DISC, FOXP3 cDNA-LNGFR and μ DISC, LNGFR-FOXP3 cDNA and DISC, LNGFR-FOXP3 cDNA and μ DISC, CISCβ-DN and CISCγ-FOXP3 cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3 cDNA.

[0295] In some embodiments, the fourth nucleic acid comprises at least one homologous arm having a sequence specific to a locus, and the length of the homologous arm is configured to achieve efficient packaging into an AAV vector.

[0296] In some embodiments, the length of at least one homology arm is 0.25 kb, 0.3 kb, 0.45 kb, 0.6 kb or 0.8 kb, or a length within a range defined by any two of these numbers.

[0297] In some embodiments, the marker is LNGF, RQR8 or EGFRt.

[0298] In some embodiments, the method further comprises adding a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3 to the CD34 + The method further comprises the step of introducing the vector into a cell.

[0299] In some embodiments, the method comprises increasing the concentration of the marker, thereby increasing the CD34 + The method further comprises the step of selecting the cells.

[0300] In some embodiments, the CD34 + The cells are contacted.

[0301] In some embodiments, the CD34 for FOXP3 expression produced by the method according to any one of the embodiments described herein. + A cell is provided. In some embodiments, the FOXP3 is constitutively expressed or regulated.

[0302] In some embodiments, a CD34 molecule for expressing FOXP3 comprises a nucleic acid encoding a gene encoding FOXP3. + In some embodiments, the cell is provided. In some embodiments, the gene encoding FOXP3 is introduced into the FOXP3 gene or a non-FOXP3 locus. In some embodiments, the non-FOXP3 locus is the AAVS1 locus or the TRA gene.

[0303] In some embodiments, the CD34 + The cells express CISCβ:FRB-IL2Rβ, DISC, CISC-FRB, μDISC, μCISC-FRB, FRB, LNGFR, or LNGFRe. + The cells are T regIncludes phenotype.

[0304] In some embodiments, the CD34 of any one of the preceding embodiments + A composition comprising the cells is provided.

[0305] In some embodiments, a method of treating, alleviating and / or suppressing a disease and / or condition in a subject comprises administering to a subject having the disease and / or condition a CD34 antibody according to any one of the embodiments described herein. + The method includes providing a cell or composition. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome. In some embodiments, the condition is graft-versus-host disease (GVHD).

[0306] In some of the embodiments described herein, there is provided a method of producing a genetically modified cell, comprising: CD34 comprising a first nucleic acid comprising at least one locus + providing a cell; providing a Cas endonuclease (e.g., a Cas9 endonuclease) or a second nucleic acid encoding the Cas endonuclease; The Cas endonuclease or the second nucleic acid is linked to the CD34 + introducing into a cell; introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one locus, or a set of nucleic acids encoding the at least one CRISPR spacer sequence; and A fourth nucleic acid comprising a gene delivery cassette is added to the CD34 + Step of introducing into cells The present invention provides a method comprising: In some embodiments, the fourth nucleic acid further comprises a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter, or an E2F promoter. In some embodiments, the promoter is an MND promoter. As described in the embodiments described herein, the MND promoter is provided in vector #3008 (pAAV_FoxP3.0.6kb.MND.GFP.WPRE3.pA) (SEQ ID NO: 33).

[0307] In some embodiments, the cells differentiate into T cells, and the T cells express FOXP3. In some embodiments, the endogenous FOXP3 promoter drives expression of an introduced FOXP3 cDNA.

[0308] When the same coding sequence is expressed by a weak promoter and a strong promoter, the weak promoter will result in lower mRNA expression than the strong promoter. This can be compared, for example, by analyzing in an agarose gel. An example of a promoter that is controlled by nearby chromatin is the short EF1α promoter, which is highly active at some loci but largely inactive at others (Eyquem, J. et al. (2013). Biotechnol. Bioeng., 110(8):2225-2235).

[0309] Therapeutic approach One aspect provided herein is a gene therapy approach for providing treatment to a subject having or suspected of having a disorder or condition associated with FOXP3 protein by editing the genome of the subject. For example, in some embodiments, the disorder or condition is an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from organ transplantation (e.g., GVHD). In some embodiments, the gene therapy approach integrates a nucleic acid comprising a sequence encoding a functional FOXP3 gene into the genome of a relevant cell type of the subject, thereby curing the disorder or condition. In some embodiments, the cell type targeted by the gene therapy approach to integrate a sequence encoding FOXP3 is CD34. + Cells, such as CD34 + These are hematopoietic stem cells because they can efficiently differentiate into T cells in the subject.

[0310] In another aspect, the present invention provides an ex vivo and in vivo method for generating permanent changes in the genome of a cell by using genome engineering tools to knock-in a coding sequence encoding FOXP3 or its functional derivatives into a locus of the cell genome and restore FOXP3 activity. In such a method, endonucleases such as CRISPR-related (CRISPR / Cas9, Cpf1, etc.) nucleases are used to permanently delete, insert, edit, modify or replace any sequence from the cell genome or to insert an exogenous sequence (e.g., a sequence encoding FOXP3) into the cell genome locus. In this way, the exemplary embodiment described in this disclosure restores FOXP3 activity in a single therapeutic step (without the need to deliver replacement therapy throughout the subject's life).

[0311] In some embodiments, ex vivo cell therapy involves the use of CD34 cells isolated from a subject. + Cells, such as CD34 cells derived from umbilical cord blood +The method is carried out using cells. The chromosomal DNA of the cells is then edited using the systems, compositions and methods described herein. Finally, the edited cells are transplanted into the subject.

[0312] One advantage of ex vivo cell therapy approach is that it allows comprehensive analysis of therapeutic agents before administration. Nuclease-based therapy always has some degree of off-target effects. Ex vivo gene correction allows comprehensive characterization of corrected cell population before transplantation. Aspects of the present disclosure include sequencing the entire genome of corrected cells to confirm that off-target breaks, if any, are in genomic locations that pose minimal risk to the subject. Furthermore, specific populations of cells, such as clonal populations, can be isolated before transplantation.

[0313] Another embodiment of such a method is an in vivo therapeutic method, in which the chromosomal DNA of cells in a subject is modified using the systems, compositions and methods described herein. In some embodiments, the cells are CD34 + It is a cell.

[0314] The advantage of in vivo gene therapy is that the therapeutic agent is easy to manufacture and administer.The same therapeutic approach and treatment method can be used to treat more than one subject, for example, multiple subjects with the same or similar genotype or allele.In contrast, ex vivo cell therapy usually uses the subject's own cells, which are isolated, genetically engineered, and then returned to the same subject.

[0315] In some embodiments, the subject in need of a treatment according to the disclosure herein is a subject with symptoms of a FOXP3-associated disease or condition. For example, in some embodiments, the subject has symptoms of an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from an organ transplant (e.g., GVHD). In some embodiments, the subject may be a human suspected of having the disease or condition. Alternatively, the subject may be a human diagnosed as being at risk for the disease or condition. In some embodiments, the subject in need of a treatment is a subject with one or more genetic abnormalities (e.g., deletions, insertions, and / or mutations) in the endogenous FOXP3 gene or its regulatory sequences, resulting in a significantly reduced expression or functionality of FOXP3 compared to normal healthy subjects.

[0316] In some embodiments, a method of treating or inhibiting a FOXP3-associated disease or condition (e.g., an autoimmune disease) in a subject includes the steps of: (a) Guide RNA (gRNA) targeting the FOXP3 gene in the cell genome; (b) a DNA endonuclease, or a nucleic acid encoding the DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof; providing to a cell of a subject. In some embodiments, the gRNA targets the FOXP3 gene, the AAVS1 locus, or the TRA gene. In some embodiments, the gRNA includes a spacer sequence shown in any one of SEQ ID NOs: 1 to 7, 15 to 20, and 27 to 29.

[0317] In some embodiments, a method of treating or inhibiting a FOXP3-associated disease or condition (e.g., an autoimmune disease) in a subject includes the steps of: (a) a gRNA comprising a spacer sequence complementary to a genomic sequence within or adjacent to an endogenous FOXP3 gene of a cell; (b) a DNA endonuclease, or a nucleic acid encoding the DNA endonuclease; and (c) a donor template comprising a nucleic acid sequence encoding FOXP3 or a functional derivative thereof; providing to a cell of a subject. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 1-7 and 27-29, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 1-7 and 27-29. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 1-7, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 1-7. In some embodiments, the gRNA comprises a spacer sequence shown in any of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having 3 or less mismatches compared to any of SEQ ID NOs: 2, 3, and 5. In some embodiments, the gRNA comprises a spacer sequence shown in SEQ ID NO: 2, or a variant of the spacer sequence having 3 or less mismatches compared to SEQ ID NO: 2. In some embodiments, the gRNA comprises a spacer sequence shown in SEQ ID NO: 5, or a variant of the spacer sequence having 3 or less mismatches compared to SEQ ID NO: 5. In some embodiments, the cell is a human cell, such as a human stem cell, such as a human CD34 + In some embodiments, the subject is a patient with or suspected of having an autoimmune disease (e.g., IPEX syndrome) or graft-versus-host disease (GVHD). In some embodiments, the subject is a subject diagnosed as being at risk for an autoimmune disease (e.g., IPEX syndrome) or graft-versus-host disease (GVHD).

[0318] In some embodiments, a method of treating or suppressing a FOXP3-associated disease or condition (e.g., an autoimmune disease) in a subject is provided, comprising providing to the subject a genetically modified cell prepared by any of the methods for editing a cell's genome described herein. In some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof is expressed under the control of an endogenous FOXP3 promoter. In some embodiments, the nucleic acid sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in the cell. In some embodiments, the cell is CD34 + In some embodiments, the genetically modified cells are autologous cells obtained from the subject. In some embodiments, the method further comprises obtaining a biological sample from the subject, the biological sample comprising input cells, and preparing the genetically modified cells from the input cells. In some embodiments, the input cells are CD34 + It is a cell.

[0319] Some embodiments include a pharmaceutical product for use in treating or suppressing a FOXP3-associated disease or condition (e.g., an autoimmune disease) in a subject. Another embodiment includes a genetically modified CD34 gene edited by any one of the methods described herein for use in suppressing or treating a FOXP3-associated disease or condition, such as an inflammatory disease or an autoimmune disease. + Further embodiments relate to cells that have been genome edited by any one of the methods described herein. + The use of cells as a pharmaceutical.

[0320] Transplantation of cells into a subject In some embodiments, the ex vivo methods of the present disclosure include transplanting genome-edited cells into a subject in need of such methods. This transplantation step can be performed using any transplantation method known in the art. For example, the genetically modified cells can be directly injected into the blood of the subject or can be administered to the subject in other ways.

[0321] In some embodiments, the methods disclosed herein include "administration," which may be used interchangeably with "introduction" and "implantation," and the methods disclosed herein include administering genetically modified therapeutic cells to a subject by a method or route that allows at least a portion of the introduced cells to localize to a desired site so as to achieve a desired effect or effects. The therapeutic cells or their differentiated progeny can be administered via a suitable route that allows delivery to a desired site in the subject's body where at least a portion of the transplanted cells or at least a portion of its components can remain viable. The viability of the cells after administration to a subject can be as short as a few hours (e.g., 24 hours to a few days) to several years or even the lifetime of the subject (e.g., long-term engraftment).

[0322] When the therapeutic cells described herein are provided for prophylactic purposes, the therapeutic cells can be administered to a subject prior to the onset of any symptoms of a FOXP3-associated disease or condition (e.g., an autoimmune disease such as IPEX syndrome). Thus, in some embodiments, prophylactic administration of a genetically modified stem cell population is used to prevent the onset of symptoms of the disease or condition.

[0323] In some embodiments, where the genetically modified stem cells are provided for therapeutic purposes, the genetically modified stem cells are provided upon (or after) the onset of a symptom or sign of a FOXP3-associated disease or FOXP3-associated condition (e.g., an autoimmune disease such as IPEX syndrome), e.g., at the onset of the disease or condition.

[0324] An effective amount of therapeutic cells (e.g., genome-edited stem cells) used in various embodiments described herein is at least 10 2 Pieces, at least 5 × 10 2 Pieces, at least 10 3 Pieces, at least 5 × 10 3 Pieces, at least 10 4 Pieces, at least 5 × 10 4 Pieces, at least 10 5 Pieces, at least 2 x 10 5 Pieces, at least 3 × 10 5 Pieces, at least 4 × 10 5 Pieces, at least 5 × 10 5 Pieces, at least 6 x 10 5 Pieces, at least 7 x 10 5 Pieces, at least 8 x 10 5 Pieces, at least 9 x 10 5 Pieces, at least 1 × 10 6 Pieces, at least 2 x 10 6 Pieces, at least 3 × 10 6 Pieces, at least 4 × 10 6 Pieces, at least 5 × 10 6 Pieces, at least 6 x 10 6 Pieces, at least 7 x 10 6 Pieces, at least 8 x 10 6 Pieces, at least 9 x 10 6 The therapeutic cells may be from one or more donors, or may be autologous. In some of the embodiments described herein, the therapeutic cells are expanded in culture prior to administration to a subject in need thereof.

[0325] In some embodiments, a moderate and gradual increase in the expression of functional FOXP3 in cells of a subject having a FOXP3-associated disease or condition (e.g., IPEX syndrome) may be beneficial in alleviating one or more symptoms of the disease or condition, improving long-term survival, and / or reducing side effects associated with other treatments. Administering such cells to a human subject is beneficial because it results in the presence of therapeutic cells that increase the production of functional FOXP3. In some embodiments, administering an effective treatment to a subject results in at least 1%, 3%, 5%, or 7% or at least about 1%, 3%, 5%, or 7% functional FOXP3 relative to the total amount of FOXP3 in the treated subject. In some embodiments, functional FOXP3 accounts for at least 10% or at least about 10% of the total amount of FOXP3. In some embodiments, functional FOXP3 comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the total amount of FOXP3, or at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, or at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, or at most 100%. Similarly, introduction of even relatively small numbers of subpopulations of cells with significantly elevated amounts of functional FOXP3 may be beneficial in various subjects, as in some circumstances normalized cells will have a selective advantage over diseased cells. However, even modest numbers of therapeutic cells with increased amounts of functional FOXP3 may be beneficial in alleviating one or more aspects of the disease or condition in a subject.In some embodiments, at or about 10%, 20% or about 20%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90% or more of the therapeutic cells in a subject to which such cells are administered have increased production of functional FOXP3.

[0326] In embodiments, a therapeutic cell composition (e.g., a composition comprising a plurality of cells, any of the cells described herein) can be delivered to a subject by a particular method or route, thereby allowing at least a portion of the cell composition to localize to a desired site. The cell composition can be administered by any suitable route that results in an effective treatment in the subject, for example, administration of the cell composition can result in at least a portion of the composition (e.g., at least 1×10 4 The cells can be delivered to a desired site in the subject over a period of time. Methods of administration include injection, infusion, infusion, and oral ingestion. "Injection" includes, but is not limited to, intravenous injection, intramuscular injection, intraarterial injection, intrathecal injection, intraventricular injection, intracapsular injection, intraorbital injection, intracardiac injection, intradermal injection, intraperitoneal injection, transtracheal injection, subcutaneous injection, subcuticular injection, intraarticular injection, subcapsular injection, subarachnoid injection, intraspinal injection, intracerebrospinal injection, and intrasternal injection and infusion. In some embodiments, the route of administration is intravenous. Delivery of cells can be by injection or infusion.

[0327] In one embodiment, the cells are administered systemically, in other words, the therapeutic cell population is not administered directly to a target site, tissue or organ, but rather enters the subject's circulatory system and undergoes metabolic and other similar processes.

[0328] The efficacy of a treatment using a composition for treating or suppressing a FOXP3-related disease or condition (e.g., IPEX syndrome) can be determined by a skilled clinician. However, a treatment is considered effective if any one or all of the signs or symptoms of the disease (e.g., the amount of functional FOXP3, for example) are beneficially altered (e.g., increased by at least 10%), or other clinically recognized symptoms or markers are improved or alleviated. Efficacy can also be measured by determining that an individual has stopped worsening (e.g., disease progression is halted or at least slowed), as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. A treatment includes any treatment or suppression of a disease in an individual or animal (e.g., but not limited to, a human or mammal), including (1) suppressing the disease, e.g., stopping or slowing the progression of a symptom, or (2) alleviating the disease, e.g., inducing regression of a symptom, and (3) preventing the onset of a symptom or reducing the likelihood of the onset of a symptom.

[0329] composition In one embodiment, the present disclosure provides a composition for carrying out the methods disclosed herein.The composition can include one or more of a genome-targeting nucleic acid (e.g., gRNA); a site-directed polypeptide (e.g., DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide; and a polynucleotide (e.g., donor template) that is inserted to perform the desired genetic recombination according to the methods disclosed herein.

[0330] In some embodiments, the composition comprises a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA).

[0331] In some embodiments, the composition comprises a site-directed polypeptide (e.g., a DNA endonuclease). In some embodiments, the composition comprises a nucleotide sequence encoding the site-directed polypeptide.

[0332] In some embodiments, the composition includes a polynucleotide (eg, a donor template) to be inserted into the genome.

[0333] In some embodiments, the composition comprises (i) a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA), and (ii) a site-directed polypeptide (e.g., a DNA endonuclease), or a nucleotide sequence encoding the site-directed polypeptide.

[0334] In some embodiments, the composition includes (i) a nucleotide sequence encoding a nucleic acid (e.g., a gRNA) that targets a genome, and (ii) a polynucleotide (e.g., a donor template) to be inserted into the genome.

[0335] In some embodiments, the composition comprises (i) a site-directed polypeptide (e.g., a DNA endonuclease), or a nucleotide sequence encoding the site-directed polypeptide, and (ii) a polynucleotide to be inserted into the genome (e.g., a donor template).

[0336] In some embodiments, the composition includes (i) a nucleotide sequence encoding a genome-targeting nucleic acid (e.g., a gRNA), (ii) a site-directed polypeptide (e.g., a DNA endonuclease) or a nucleotide sequence encoding the site-directed polypeptide, and (iii) a polynucleotide to be inserted into the genome (e.g., a donor template).

[0337] In some of the embodiments of any of the compositions, the composition comprises a genome-targeting nucleic acid using a single molecular guide. In some of the embodiments of any of the compositions, the composition comprises a genome-targeting nucleic acid using a bimolecular guide. In some of the embodiments of any of the compositions, the composition comprises two or more bimolecular or single molecular guides. In some embodiments, the composition comprises a vector encoding a nucleic acid that targets a nucleic acid. In some embodiments, the genome-targeting nucleic acid is configured for use in combination with a DNA endonuclease, particularly a Cas endonuclease (e.g., Cas9 endonuclease).

[0338] In some embodiments, the composition can comprise one or more gRNAs that can be used for genome editing, particularly for inserting sequences encoding FOXP3 or its derivatives into cell genome.The one or more gRNAs can target genomic sites on endogenous FOXP3 gene, genomic sites in endogenous FOXP3 gene, or genomic sites near endogenous FOXP3 gene.Thus, in some embodiments, the one or more gRNAs can have a spacer sequence that is complementary to the genomic sequence on FOXP3 gene, a spacer sequence that is complementary to the genomic sequence in FOXP3 gene, or a spacer sequence that is complementary to the genomic sequence near FOXP3 gene.

[0339] In some embodiments, the gRNA for the composition comprises a spacer sequence selected from any one of the spacer sequences set forth in SEQ ID NOs: 1-7, 15-20, and 27-29, and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity or homology to any one of SEQ ID NOs: 1-7, 15-20, and 27-29. In some embodiments, the gRNA variant for the kit comprises a spacer sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1-7, 15-20, and 27-29.

[0340] In some embodiments, the gRNA for the composition has a spacer sequence that is complementary to a target site in the genome. In some embodiments, the spacer sequence is 15-20 bases in length. In some embodiments, the complementarity between the spacer sequence and the genome sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0341] In some embodiments, the composition may have a donor template having a DNA endonuclease or a nucleic acid encoding the DNA endonuclease, and / or a nucleic acid sequence encoding FOXP3 or a functional derivative thereof. In some embodiments, the DNA endonuclease is a Cas endonuclease (e.g., Cas9 endonuclease). In some embodiments, the nucleic acid encoding the DNA endonuclease is DNA or RNA.

[0342] In some embodiments, one or more of the nucleic acids for the kit can be encoded in an adeno-associated virus (AAV) vector. Thus, in some embodiments, the gRNA can be encoded in an AAV vector. In some embodiments, the nucleic acid encoding the DNA endonuclease can be encoded in an AAV vector. In some embodiments, the donor template can be encoded in an AAV vector. In some embodiments, two or more nucleic acids can be encoded in a single AAV vector. Thus, in some embodiments, the nucleic acid encoding the DNA endonuclease and the gRNA sequence can be encoded in a single AAV vector.

[0343] In some embodiments, the composition may have liposomes or lipid nanoparticles. Thus, in some embodiments, any compound of the composition (e.g., DNA endonuclease or nucleic acid encoding the DNA endonuclease, gRNA and donor template) may be formulated to be encapsulated in liposomes or lipid nanoparticles. In some embodiments, one or more of such compounds are bound to liposomes or lipid nanoparticles via covalent or non-covalent bonds. In some embodiments, any of the compounds may be encapsulated in liposomes or lipid nanoparticles separately or together. Thus, in some embodiments, each of the DNA endonuclease or nucleic acid encoding the DNA endonuclease, gRNA and donor template is formulated to be encapsulated in liposomes or lipid nanoparticles separately. In some embodiments, the DNA endonuclease is formulated to be encapsulated in liposomes or lipid nanoparticles together with gRNA. In some embodiments, the DNA endonuclease or nucleic acid encoding the DNA endonuclease, gRNA and donor template are formulated to be encapsulated in liposomes or lipid nanoparticles together.

[0344] In some embodiments, the composition further comprises one or more additional reagents, such additional reagents being selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for generating polypeptides from DNA in vitro, adaptors for sequencing, etc. The buffers may be stabilization buffers, reconstitution buffers, dilution buffers, etc. In some embodiments, the composition may further comprise one or more components used to promote or enhance on-target binding, cleave DNA with an endonuclease, or improve targeting specificity.

[0345] In some embodiments, any of the components of the composition are formulated with pharma- ceutically acceptable additives, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the particular method of administration and dosage form. In embodiments, the guide RNA composition is typically formulated to achieve a physiologically compatible pH, which may range from 3 to 11 or about 3 to about 11, or from 3 to 7 or about 3 to about 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range of pH 5.0 to pH 8 or a range of about pH 5.0 to about pH 8. In some embodiments, the composition has a therapeutically effective amount of at least one compound described herein and one or more pharma- ceutically acceptable additives. In some cases, the composition can have a combination of compounds described herein, or can include a second active ingredient useful for treating or preventing bacterial growth (e.g., but not limited to, an antibacterial or antimicrobial agent), or can include a combination of reagents of the present disclosure. In some embodiments, the gRNA is formulated with one or more additional nucleic acids, such as a nucleic acid encoding a DNA endonuclease and / or a donor template. Alternatively, the nucleic acid encoding a DNA endonuclease and the donor template are each formulated separately or in combination with another nucleic acid, as described above for gRNA formulations.

[0346] Suitable additives include, for example, carrier molecules including slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, inactive virus particles, etc. Other typical additives include antioxidants (such as, but not limited to, ascorbic acid), chelating agents (such as, but not limited to, EDTA), carbohydrates (such as, but not limited to, dextrin, hydroxyalkylcellulose, or hydroxyalkylmethylcellulose), stearic acid, liquids (such as, but not limited to, oils, water, saline, glycerol, or ethanol), wetting or emulsifying agents, or pH buffering substances, etc.

[0347] In some embodiments, any of the components of the composition (e.g., the DNA endonuclease or a nucleic acid encoding the DNA endonuclease, the gRNA, and the donor template) can be delivered to the cell by transfection, such as chemical transfection (e.g., lipofection) or electroporation. In some embodiments, the DNA endonuclease can be pre-complexed with the gRNA to form a ribonucleoprotein (RNP) complex prior to providing to the cell. In some embodiments, the RNP complex is delivered to the cell by transfection. In such embodiments, the donor template is delivered to the cell by transfection.

[0348] In some embodiments, "composition" refers to a therapeutic composition comprising therapeutic cells for use in an ex vivo therapy.

[0349] In embodiments, a therapeutic composition comprises a cell composition and a physiologically acceptable carrier, and optionally at least one additional bioactive agent as described herein dissolved or dispersed in the composition as an active ingredient. In some embodiments, a therapeutic composition is substantially non-immunogenic when administered to a mammalian or human subject for therapeutic purposes, except where immunogenicity is desired.

[0350] Typically, the genetically modified therapeutic cells described herein are administered as a suspension comprising a pharma- ceutically acceptable carrier. One skilled in the art will understand that the pharma- ceutical acceptable carrier used in the cell composition does not contain an amount of buffers, compounds, cryopreservatives, preservatives or other agents that would substantially inhibit the viability of the cells delivered to the subject. The cell-containing formulation may, for example, comprise an osmotic buffer that maintains the integrity of the cell membrane, and may optionally comprise nutrients to maintain cell viability or enhance cell engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with progenitor cells as described herein using routine experimentation.

[0351] In some embodiments, the cell compositions can be emulsified or provided as liposomal compositions, so long as the emulsification procedure does not adversely affect the viability of the cells. The cells and other active ingredients can be mixed with one or more additives that are pharma- ceutically acceptable and compatible with the active ingredients, in amounts suitable for use in the therapeutic methods described herein.

[0352] Additional agents contained in the cell composition include pharmaceutically acceptable salts of the components in the cell composition.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as hydrochloric acid and phosphoric acid, or organic acids such as acetic acid, tartaric acid or mandelic acid.Also, salts formed with free carboxyl groups can be derived from inorganic bases such as sodium, potassium, ammonium, calcium, and iron hydroxides, or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0353] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no other material than the active ingredient and water, or sterile aqueous solutions that contain a buffer such as sodium phosphate or saline at a physiological pH value, or both (such as phosphate buffered saline). Additionally, aqueous carriers can contain one or more buffer salts, salts such as sodium chloride or potassium chloride, dextrose, or polyethylene glycol, or other solutes. Liquid compositions can also contain other liquid phases in addition to water, or to the exclusion of water. Examples of such other liquid phases include glycerin, vegetable oils such as cottonseed oil, or water-oil emulsions. The amount of active compound used in a cell composition that is effective in treating a particular disorder or condition will vary depending on the characteristics of the disorder or condition, and can be determined by known clinical techniques.

[0354] kit Some embodiments provide kits comprising any of the above compositions, e.g., a composition for genome editing or a cellular composition (e.g., a therapeutic cellular composition), and one or more additional components.

[0355] In some embodiments, the kit may comprise one or more additional therapeutic agents that can be administered simultaneously or sequentially with the composition for a desired purpose, such as, for example, genome editing or cell therapy.

[0356] In some embodiments, the kit may further include instructions for carrying out the method using each of the components of the kit. The instructions for carrying out the method are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be included in the kit as a package insert on the label of the container (i.e., a label affixed to the packaging or subpackaging) of the kit or its components. The instructions may also be included as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, diskette, flash drive, etc.). In some cases, the actual instructions are not included in the kit, and a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, the means for obtaining the instructions may be recorded on a suitable substrate.

[0357] Further embodiments In some embodiments, there is provided a method of producing a genetically modified cell, comprising: CD34 comprising a first nucleic acid comprising at least one locus + providing a cell; providing a CAS9 protein or a second nucleic acid sequence encoding said CAS9 protein; The CAS9 protein or the second nucleic acid is + introducing into a cell; introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one locus, or a set of nucleic acids encoding the at least one CRISPR spacer sequence; and A fourth nucleic acid comprising a gene delivery cassette is added to the CD34 + Step of introducing into cells The present invention provides a method comprising:

[0358] In some embodiments, the method further comprises administering to said subject CD34 + The method further comprises the step of activating the cells, said activation comprising activating said CD34 + In some embodiments, activation is performed prior to introducing a second nucleic acid into the cells. In some embodiments, activation is achieved by inducing CD34 to a cytokine selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), FLT3L, and IL-6. + The method is carried out by contacting the cell. In some embodiments, the at least one locus is FOXP3 gene, AAVS1 locus or TRA gene. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are provided in one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0359] In some embodiments, the AAV vector is a self-complementary vector. In some embodiments, the AAV vector is a single-stranded vector. In some embodiments, the AAV vector is a combination of a self-complementary vector and a single-stranded vector. In some embodiments, the second nucleic acid encoding the CAS9 protein is an mRNA. In some embodiments, the at least one spacer sequence comprises a sequence as set forth in SEQ ID NO: 2, 3 or 5. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are codon-optimized for expression in eukaryotic cells, such as human cells. In some embodiments, the fourth nucleic acid comprises a sequence encoding a human codon-optimized FOXP3 cDNA sequence. In some embodiments, the fourth nucleic acid further comprises a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter or an E2F promoter. In some embodiments, the fourth nucleic acid further comprises a coding sequence for the low affinity nerve growth factor receptor (LNGFR), a sequence encoding μCISC, CISCγ, FRB and / or LNGFRe (a coding sequence for the LNGFR epitope). In some embodiments, the fourth nucleic acid further comprises a coding sequence for the low affinity nerve growth factor receptor (LNGFR) and / or a sequence encoding LNGFRe (a coding sequence for the LNGFR epitope).

[0360] In some embodiments, the method further comprises: +The method further comprises a step of introducing into a cell. In some embodiments, the fifth nucleic acid is comprised in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid comprises a sequence encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid comprises a sequence encoding a marker protein. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a P2A self-cleaving peptide. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a polyA sequence. In some embodiments, the polyA sequence comprises SV40 polyA or the 3′UTR of FOXP3. In some embodiments, the fourth nucleic acid comprises a WPRE3 element. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid comprises the CD34 + In some embodiments, the fourth and / or fifth nucleic acid is introduced into a cell and comprises a sequence encoding an expression cassette that expresses FOXP3 cDNA-LNGFR and DISC, FOXP3 cDNA-LNGFR and μDISC, LNGFR-FOXP3 cDNA and DISC, LNGFR-FOXP3 cDNA and μDISC, CISCβ-DN and CISCγ-FOXP3 cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3 cDNA. +A fourth nucleic acid and / or a fifth nucleic acid is introduced into the cell, the fourth nucleic acid and / or the fifth nucleic acid comprises a sequence encoding an expression cassette. In some embodiments, the fourth nucleic acid comprises at least one homologous arm having a sequence specific to a locus, and the length of the homologous arm is configured to achieve efficient packaging into the AAV vector. In some embodiments, the length of the at least one homologous arm is 0.25 kb, 0.3 kb, 0.45 kb, 0.6 kb, 0.8 kb, or 1 kb, or a length within a range defined by any two of these numbers. In some embodiments, the marker is LNGF, RQR8, or EGFRt. In some embodiments, the method further comprises adding a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3 to the CD34 vector. + In some embodiments, the method further comprises introducing into the cell the protein or cytokine. In some embodiments, the protein or cytokine is a T cell receptor, a chimeric antigen receptor, or IL10. In some embodiments, the fourth nucleic acid comprises a sequence as set forth in SEQ ID NO: 34 or 36. In some embodiments, the method further comprises increasing the concentration of the marker, thereby increasing the concentration of the marker, + In some embodiments, the CD34 cells are selected in a medium containing hTPO, hFlt3, hSCF, and / or hIL6. + The cells are contacted.

[0361] In some embodiments, the CD34 for FOXP3 expression produced by the method according to any one of the embodiments described herein. + In some embodiments, the FOXP3 is constitutively expressed or regulated. CD34 comprising a first nucleic acid comprising at least one locus + providing a cell; providing a CAS9 protein or a second nucleic acid sequence encoding said CAS9 protein; The CAS9 protein or the second nucleic acid is + introducing into a cell; introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one locus, or a set of nucleic acids encoding the at least one CRISPR spacer sequence; and A fourth nucleic acid comprising a gene delivery cassette is added to the CD34 + Step of introducing into cells Includes. In some embodiments, the method further comprises administering to said subject CD34 + The method further comprises the step of activating the cells, said activation comprising activating said CD34 + In some embodiments, the activation is performed by inducing CD34 to a cytokine selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), FLT3L, and IL-6. +The method is carried out by contacting a cell with a nucleic acid sequence of at least one of the following: In some embodiments, the at least one locus is a FOXP3 gene, an AAVS1 locus, or a TRA gene. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids, and / or the fourth nucleic acid are provided in one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is a self-complementary vector. In some embodiments, the AAV vector is a single-stranded vector. In some embodiments, the AAV vector is a combination of a self-complementary vector and a single-stranded vector. In some embodiments, the second nucleic acid encoding the CAS9 protein is an mRNA. In some embodiments, the at least one spacer sequence comprises a sequence as set forth in SEQ ID NO: 2, 3, or 5. In some embodiments, the second nucleic acid, the third nucleic acid, the set of nucleic acids, and / or the fourth nucleic acid are codon-optimized for expression in eukaryotic cells (such as human cells). In some embodiments, the fourth nucleic acid comprises a sequence encoding a human codon-optimized FOXP3 cDNA sequence. In some embodiments, the fourth nucleic acid further comprises a promoter. In some embodiments, the promoter is an MND promoter, a PGK promoter, or an E2F promoter. In some embodiments, the fourth nucleic acid further comprises a sequence encoding a low affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB, and / or LNGFRe (a coding sequence for an LNGFR epitope). In some embodiments, the fourth nucleic acid further comprises a coding sequence for a low affinity nerve growth factor receptor (LNGFR) and / or a sequence encoding LNGFRe (a coding sequence for an LNGFR epitope). In some embodiments, the method further comprises administering a fifth nucleic acid comprising a second gene delivery cassette to the CD34 +The method further comprises a step of introducing into a cell. In some embodiments, the fifth nucleic acid is comprised in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid comprises a sequence encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid comprises a sequence encoding a marker protein. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a P2A self-cleaving peptide. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a polyA sequence. In some embodiments, the polyA sequence comprises SV40 polyA or the 3′UTR of FOXP3. In some embodiments, the fourth nucleic acid comprises a WPRE3 element. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid comprises the CD34 + In some embodiments, the fourth and / or fifth nucleic acid is introduced into a cell and comprises a sequence encoding an expression cassette that expresses FOXP3 cDNA-LNGFR and DISC, FOXP3 cDNA-LNGFR and μDISC, LNGFR-FOXP3 cDNA and DISC, LNGFR-FOXP3 cDNA and μDISC, CISCβ-DN and CISCγ-FOXP3 cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3 cDNA. +A fourth nucleic acid and / or a fifth nucleic acid is introduced into the cell, the fourth nucleic acid and / or the fifth nucleic acid comprises a sequence encoding an expression cassette. In some embodiments, the fourth nucleic acid comprises at least one homologous arm having a sequence specific to a locus, and the length of the homologous arm is configured to achieve efficient packaging into the AAV vector. In some embodiments, the length of the at least one homologous arm is 0.25 kb, 0.3 kb, 0.45 kb, 0.6 kb, 0.8 kb, or 1 kb, or a length within a range defined by any two of these numbers. In some embodiments, the marker is LNGF, RQR8, or EGFRt. In some embodiments, the method further comprises adding a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3 to the CD34 vector. + In some embodiments, the method further comprises introducing into the cell the protein or cytokine. In some embodiments, the protein or cytokine is a T cell receptor, a chimeric antigen receptor, or IL10. In some embodiments, the fourth nucleic acid comprises a sequence as set forth in SEQ ID NO: 34 or 36. In some embodiments, the method further comprises increasing the concentration of the marker, thereby increasing the concentration of the marker, + In some embodiments, the CD34 cells are selected in a medium containing hTPO, hFlt3, hSCF, and / or hIL6. + The cells are contacted.

[0362] In some embodiments, a CD34 molecule for expressing FOXP3 comprises a nucleic acid encoding a gene encoding FOXP3. + In some embodiments, the gene encoding FOXP3 is introduced into the FOXP3 gene or a non-FOXP3 locus. In some embodiments, the non-FOXP3 locus is the AAVS1 locus or the TRA gene. In some embodiments, the CD34 + The cells express CISCβ:FRB-IL2Rβ, DISC, CISC-FRB, μDISC, μCISC-FRB, FRB, LNGFR and / or LNGFRe.+ These cells differentiate in the thymus to become T reg A precursor cell that gives rise to T cells with the phenotype

[0363] In some embodiments, CD34 according to any one of the embodiments described herein. + A composition comprising the cells is provided.

[0364] In some embodiments, a method of treating, alleviating and / or suppressing a disease and / or condition in a subject comprises administering to a subject having the disease and / or condition a CD34 antibody according to any one of the embodiments described herein. + The present invention provides a method comprising providing a cell or composition. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is IPEX syndrome. In some embodiments, the condition is graft-versus-host disease (GVHD).

[0365] Representative Embodiments Embodiment 1. A method for producing a genetically modified cell, comprising: CD34 containing a first nucleic acid comprising at least one target locus + providing a cell; providing a CAS9 protein or a second nucleic acid sequence encoding said CAS9 protein; The CAS9 protein or the second nucleic acid is + introducing into a cell; Introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one target locus, or a set of nucleic acids encoding the at least one CRISPR spacer sequence; and A fourth nucleic acid comprising a gene delivery cassette is added to the CD34 + Step of introducing into cells The method includes:

[0366] Embodiment 2. The CD34 +The method further comprises the step of activating the cells, said activation comprising activating said CD34 + 2. The method of embodiment 1, which is performed prior to introducing a second nucleic acid into the cell.

[0367] Embodiment 3. The activation is + 3. The method of embodiment 2, performed by contacting the cell with CD3 and / or CD28.

[0368] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the at least one target locus is the FOXP3 gene, the AAVS1 locus, or the TRA gene.

[0369] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are provided in one or more vectors.

[0370] Embodiment 6 The method of embodiment 5, wherein the one or more vectors are viral vectors.

[0371] Embodiment 7. The method of embodiment 6, wherein the viral vector is an adeno-associated viral (AAV) vector.

[0372] Embodiment 8. The method of embodiment 7, wherein the AAV vector is a self-complementary vector.

[0373] Embodiment 9. The method of embodiment 7 or 8, wherein the AAV vector is a single-stranded vector.

[0374] Embodiment 10. The method of any one of embodiments 7 to 9, wherein the AAV vector is a combination of a self-complementary vector and a single-stranded vector.

[0375] Embodiment 11 The method of any one of embodiments 1 to 4, wherein the second nucleic acid encoding the CAS9 protein is mRNA.

[0376] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the at least one spacer sequence comprises a sequence as set forth in SEQ ID NO: 2, 3 or 5.

[0377] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the second nucleic acid, the third nucleic acid, the set of nucleic acids and / or the fourth nucleic acid are codon-optimized for expression in a eukaryotic cell (such as a human cell).

[0378] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the fourth nucleic acid comprises a sequence encoding a human codon-optimized FOXP3 cDNA sequence.

[0379] Embodiment 15 The method of embodiment 13, wherein the fourth nucleic acid further comprises a promoter.

[0380] Embodiment 16 The method of embodiment 15, wherein the promoter is an MND promoter, a PGK promoter or an E2F promoter.

[0381] Embodiment 17. The method of any one of embodiments 14 to 16, wherein the fourth nucleic acid further comprises a coding sequence for a low affinity nerve growth factor receptor (LNGFR) and / or a coding sequence for a LNGFRe (a coding sequence for an LNGFR epitope).

[0382] Embodiment 18. A fifth nucleic acid comprising a second gene delivery cassette is administered to said CD34 + 18. The method according to any one of embodiments 1 to 17, further comprising the step of introducing into a cell.

[0383] Embodiment 19. The method of embodiment 18, wherein the fifth nucleic acid is contained in a vector.

[0384] Embodiment 20 The method of embodiment 18, wherein the vector is an AAV vector.

[0385] Embodiment 21 The method of any one of embodiments 18 to 20, wherein the fifth nucleic acid comprises a sequence encoding a marker protein.

[0386] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a P2A self-cleaving peptide.

[0387] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the fourth nucleic acid and / or the fifth nucleic acid further comprises a sequence encoding a polyA sequence.

[0388] Embodiment 24. The method of embodiment 23, wherein the polyA sequence comprises SV40 polyA or the 3'UTR of FOXP3.

[0389] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the fourth nucleic acid comprises a WPRE3 element.

[0390] Embodiment 26. The fourth and / or fifth nucleic acid is a nucleic acid encoding the CD34 + 26. The method according to any one of the preceding embodiments, wherein the fourth nucleic acid and / or the fifth nucleic acid introduced into the cell comprises a sequence encoding an expression cassette.

[0391] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the fourth nucleic acid comprises at least one homologous arm having a sequence specific to a locus, and the length of the homologous arm is configured to achieve efficient packaging into an AAV vector.

[0392] Embodiment 28. The method of embodiment 27, wherein the length of at least one homology arm is 0.25 kb, 0.3 kb, 0.45 kb, 0.6 kb, 0.8 kb or 1 kb, or a length within a range defined by any two of these numbers.

[0393] Embodiment 29. The method of any one of embodiments 21 to 28, wherein the marker is LNGF, RQR8 or EGFRt.

[0394] Embodiment 30. A sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3 is added to said CD34 + 30. The method of any one of embodiments 1 to 29, further comprising the step of introducing into a cell.

[0395] Embodiment 31 The method of embodiment 30, wherein the protein or cytokine is a T cell receptor, a chimeric antigen receptor or IL10.

[0396] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the fourth nucleic acid comprises the sequence shown in SEQ ID NO: 34 or 36.

[0397] Embodiment 33. The CD34 marker is increased by increasing the concentration of the marker. + 33. The method according to any one of embodiments 1 to 32, further comprising a step of selecting the cells.

[0398] Embodiment 34. The CD34 + 34. The method of any one of embodiments 1 to 33, wherein the cell is contacted.

[0399] Embodiment 35: A CD34 antibody for expressing FOXP3, produced by the method according to any one of claims 1 to 34. + cell.

[0400] Embodiment 36. The CD34 of embodiment 35, wherein said FOXP3 is constitutively expressed or is expressed under regulation. + cell.

[0401] Embodiment 37. CD34 for expressing FOXP3, comprising a nucleic acid encoding a gene encoding FOXP3 + cell.

[0402] Embodiment 38. The CD34 according to embodiment 37, wherein said gene encoding FOXP3 is introduced into the FOXP3 gene or into a non-FOXP3 locus. + cell.

[0403] Embodiment 39. The CD34 gene of embodiment 38, wherein the non-FOXP3 locus is the AAVS1 locus or the TRA gene. + cell.

[0404] Embodiment 40. The CD34 + These cells differentiate in the thymus to become T reg 40. The method of claim 39, wherein the CD34 + cell.

[0405] Embodiment 41: The CD34 according to any one of claims 34 to 40. + A composition comprising a cell.

[0406] Embodiment 42. A method of treating, alleviating and / or suppressing a disease and / or condition in a subject, comprising: A subject having a disease and / or condition is administered a CD34 antibody or a combination thereof according to any one of embodiments 33 to 39. + Providing a cell or a composition according to embodiment 41. The method includes:

[0407] Embodiment 43 The method of embodiment 42, wherein the disease...

Claims

1. 1. A system comprising a donor template, the donor template comprising: (a) CD34 + a first homology arm having homology to a first nucleotide sequence within a FOXP3 gene, an AAVS1 locus, or a TRA gene of the cell; (b) a second homology arm that has homology to a second nucleotide sequence within the same gene or locus as the first homology arm has homology; (c) a nucleotide sequence encoding FOXP3 and lacking an intron; and (d) a heterologous constitutive promoter operably linked to the nucleotide sequence encoding FOXP3. Including, The system, characterized in that the heterologous constitutive promoter and the nucleotide sequence encoding FOXP3 are located between the first homologous arm and the second homologous arm.

2. The system of claim 1, further comprising a nuclease or a nucleic acid encoding said nuclease, wherein said nuclease targets said FOXP3 gene, AAVS1 locus or TRA gene.

3. The system of claim 2 , wherein the nuclease is a zinc finger nuclease or a TALEN.

4. 3. The system of claim 2, wherein the nuclease is an RNA-guided DNA endonuclease, and the system further comprises a guide RNA (gRNA) comprising a spacer sequence complementary to a nucleotide sequence within the FOXP3 gene, AAVS1 locus, or TRA gene.

5. The system of claim 4, wherein the RNA-guided DNA endonuclease is a Cas endonuclease.

6. 6. The system of claim 5, wherein the Cas endonuclease is a Cas9 endonuclease.

7. The gRNA is (i) a spacer sequence as set forth in any one of SEQ ID NOs: 1-7, 15-20, and 27-29, or a variant of said spacer sequence having 3 or fewer mismatches compared to any one of SEQ ID NOs: 1-7, 15-20, and 27-29; (ii) a spacer sequence as set forth in any of SEQ ID NOs: 1 to 7, or a variant of said spacer sequence having 3 or fewer mismatches compared to any of SEQ ID NOs: 1 to 7; and / or (iii) a spacer sequence as set forth in any one of SEQ ID NOs: 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any one of SEQ ID NOs: 2, 3, and 5. The system according to any one of claims 4 to 6, comprising:

8. The nucleotide sequence encoding FOXP3 is + The system according to any one of claims 1 to 7, which is codon-optimized for expression in a cell and has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:

110.

9. The system of any one of claims 1 to 8, wherein the first homologous arm has homology to the first nucleotide sequence in the FOXP3 gene, and the second homologous arm has homology to the second nucleotide sequence in the FOXP3 gene.

10. The system according to any one of claims 1 to 9, characterized in that it comprises at least one of the following (a) to (c): (a) the FOXP3 is wild-type human FOXP3; (b) the donor template is contained in an adeno-associated virus (AAV) vector; (c) the heterologous constitutive promoter is an MND promoter, a PGK promoter, an E2F promoter, or an EF-1α promoter.

11. The system according to any one of claims 1 to 10, wherein the heterologous constitutive promoter is an MND promoter.

12. The system of any one of claims 1 to 9, wherein the donor template further comprises a nucleotide sequence encoding a selectable marker.

13. CD34 + The system according to any one of claims 1 to 12, further comprising cells.

14. CD34 + The system of claim 13, wherein the cells comprise a nucleic acid encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).