FOXP3 expression in gene-edited CD34+ cells

Abstract

This provides a novel approach to expressing and regulating FOXP3 in primary human lymphocytes. [Solution] An aspect of the present invention involves incorporating FOXP3 cDNA (for example, the full length of FOXP3 cDNA optimized for human codons) into the FOXP3 gene or a non-FOXP3 locus, thereby enabling the introduction of human primary CD34 + Cells or edited CD34 + This invention relates to the constitutive or controlled expression of FOXP3 in cell-derived cells. In some embodiments, a gene delivery cassette for homologous recombination repair (HDR)-based gene modification is provided, using a guide RNA sequence that is directional to the FOXP3 locus, the AAVS1 locus, or other candidate loci in CRISPR / Cas9-mediated gene regulation.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 663,545, filed on 27 April 2018, 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," which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence listing reference This application was filed together with an electronic sequence listing. This sequence listing was provided as a file of approximately 430kb created on April 24, 2019, with the filename SCRI188WOSEQLISTING. The information contained herein is incorporated herein by reference in its entirety.

[0003] The embodiments of the present invention described herein are CD34 + By incorporating the FOXP3 coding sequence into the FOXP3 gene or non-FOXP3 locus of a cell, gene-edited CD34 + This relates to the constitutive or controlled expression of FOXP3 in cells or cells derived from such cells (such as T cells). [Background technology]

[0004] Lentiviral gene transfer of FOXP3 (also known as forkheadbox protein P3, forkheadbox 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 these publications are explicitly incorporated herein by reference). Furthermore, Passerini et al. (2017) reported that T lymphocytes obtained from patients with FOXP3 mutations exhibited T reg We have previously reported on the development of methods to restore functionality. As reported by Passerini et al. (2017), CD4 + In T cells and effector T cells, lentiviral gene transfer is used, thereby converting these T cells into effector T cells. reg The characteristics of similar cells were demonstrated, and potent in vitro and in vivo inhibitory activity was conferred. Furthermore, Passerini was conferred by the introduction of the FOXP3 gene via lentivirus, resulting in CD4 + T cells reg It has been demonstrated that it can be converted into cells, and this T reg Cells have been shown to be stable in inflammatory states (Passerini et al. (2013)). Chen et al. (2011) also reported adoptive transfer of recombinant T cells infected with a lentiviral vector encoding the FOXP3-IRES-GFP fragment. These cells were shown to prevent GVHD in mouse model recipients. Novel approaches are needed to express and regulate FOXP3 in primary human lymphocytes.

[0005] Regulatory T cells have the potential to induce antigen-specific immune tolerance, so many researchers are interested in treating autoimmune diseases with regulatory T cells. Regulatory T cells (abbreviated as "T reg regs") have various forms. According to the current nomenclature, regulatory T cells that arise in the thymus during T cell development (thymus-derived regulatory T cells or "tT reg regs") and those induced peripherally (peripherally-derived regulatory T cells or "pT reg regs") are classified.

[0006] An important aspect of the regulatory T cell ecosystem is the expression of the transcription factor FOXP3. FOXP3 is thought to be required for the induction of regulatory T cell lineage differentiation. This concept is based on the observation that severe autoimmune diseases develop in neonates in humans lacking FOXP3. Since the expression of FOXP3 is thought to be epigenetically regulated, using tT reg or pT reg in the treatment of autoimmune diseases may not be the best strategy. In tT reg regs, the upstream region known as the "thymus-specific demethylated region" of the FOXP3 gene is demethylated, and thus FOXP3 is thought to be stably expressed. Usually, complete demethylation is not observed in pT reg regs. Also, FOXP3 is thought to be epigenetically silenced in inflammatory pT reg regs, and perhaps also in tT reg regs (however, some researchers think tT reg is completely stable), and as a result, pT reg may change into pro-inflammatory CD4 T cells. Infusing pT reg that revert to an inflammatory phenotype may worsen the symptoms of autoimmune diseases, so the potential lack of stability of pT reg is a serious problem.

Summary of the Invention

Means for Solving the Problems

[0007] In this specification, Deoxyribonucleic acid (DNA) endonuclease, or nucleic acid encoding said DNA endonuclease; CD34 + A guide RNA (gRNA) containing a spacer sequence complementary to the sequence in the cell's FOXP3 gene, AAVS1 locus, or TRA gene, or a nucleic acid encoding said gRNA; and Donor template containing nucleic acid sequences encoding FOXP3 or its functional derivatives This section describes a system that includes this system. In some embodiments, the gRNA is i) A spacer sequence shown in any of sequence numbers 1-7, 15-20, and 27-29, or a variant of said spacer sequence having three or fewer mismatches compared to any of sequence numbers 1-7, 15-20, and 27-29; ii) A spacer sequence shown in any of sequence numbers 1 to 7, or a variant of the spacer sequence having three or fewer mismatches compared to any of sequence numbers 1 to 7; or iii) A spacer sequence shown in any of sequence numbers 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of sequence numbers 2, 3, and 5. Includes. In some embodiments, the FOXP3 or its functional derivative 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 liposomes or lipid nanoparticles.

[0008] Furthermore, in this specification, CD34 + A method for editing the genome of a cell, wherein one of the systems described herein is used with respect to CD34 + A method including the step of providing to cells is described. In some embodiments, the CD34 + These cells are not germ cells.

[0009] Furthermore, this disclosure relates to CD34 by any one of the methods described herein. + Recombinant CD34 cells with edited genomes + Cells, and the recombinant CD34 + A composition containing cells is described. In some embodiments, the recombinant CD34 + These cells are not germ cells.

[0010] Furthermore, this specification provides a method for treating a foxp3-related disease or foxp3-related condition in a subject, wherein one of the systems described herein is used in the subject's CD34 + A method comprising the step of providing to cells is described. The disease or condition may be an inflammatory disease or an autoimmune disease, for example, IPEX syndrome or graft-versus-host disease (GVHD). In some embodiments, the recombinant CD34 + These cells are not germ cells. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram illustrating the design of two different AAV donor templates configured to incorporate a donor cassette into the FOXP3 gene. One design (upper schematic) expresses FOXP3 from heterologous FOXP3 cDNA under the control of an endogenous FOXP3 promoter, while the other design (lower schematic) expresses GFP under a heterologous MND promoter. HA: homologous arm; MND: MND promoter; pA: SV40 polyadenylation signal.

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

[0013] [Figure 3] This figure shows the homologous recombination rate of CD34+ cells edited using Cas9 / gRNA RNP (T3 gRNA or T9 gRNA) and the AAV donor template shown in Figure 1.

[0014] [Figure 4] This bar graph compares the cell viability of CD34+ cells treated with RNP containing Cas9 from two different suppliers (IDT's Alt-R Sp Cas9 nuclease V3 or Aldevron's SpyFi Cas9) and two different FOXP3-targeted gRNAs (T3 or T9).

[0015] [Figure 5] This bar graph compares the cell viability of CD34+ cells edited using two different FOXP3-targeted gRNAs (T3 or T9) with RNP + AAV donor templates containing Cas9 obtained from two different suppliers (IDT's Alt-R Sp Cas9 nuclease V3 or Aldevron's SpyFi Cas9).

[0016] [Figure 6] This is an exemplary result showing the percentage of GFP+ cells among all hCD45+ cells recovered from the spleen of NSGW41 mice transplanted with mock-treated cells or cells edited with SpyFi Cas9 / gRNA RNP (T3) targeting FOXP3. The mean ± SEM is displayed on the graph.

[0017] [Figure 7] This is an exemplary result showing the percentage of GFP+ cells among hCD19+ cells recovered from the spleen of NSGW41 mice transplanted with mock-treated cells or cells edited with SpyFi Cas9 / gRNA RNP(T3) targeting FOXP3. The mean ± SEM is displayed on the graph.

[0018] [Figure 8] This is an exemplary result showing the percentage of GFP+ cells among hCD33+ cells recovered from the spleen of NSGW41 mice transplanted with mock-treated cells or cells edited with SpyFi Cas9 / gRNA RNP(T3) targeting FOXP3. The mean ± SEM is displayed on the graph. [Modes for carrying out the invention]

[0019] This specification describes the expression of FOXP3 from a DNA sequence (e.g., a codon-optimized DNA sequence, e.g., a codon-optimized DNA sequence for expression in human cells) integrated into the FOXP3 gene or a non-FOXP3 locus. Genome editing is performed via CRISPR / Cas by targeting the FOXP3 gene or a non-FOXP3 locus (e.g., in mouse, human, or non-human primates) using a guide RNA. Thus, aspects of the present invention relate to cleaving DNA at the FOXP3 gene or a non-FOXP3 target locus and promoting the integration of the FOXP3 coding sequence by using a novel guide RNA in combination with a Cas protein. In some embodiments, this integration is performed by non-homologous end joining (NHEJ) or homologous recombination repair (HDR) associated with a donor template containing the FOXP3 coding sequence. Some embodiments described herein can be used in combination with a wide range of selection markers such as LNG FR, RQR8, and CISC / DISC / μDISC, and can be multiplexed in combination with editing of another target locus or co-expression of another gene product (such as cytokines).

[0020] As will be explained in more detail later, the applicants have found that by using a novel AAV donor template containing a gene delivery cassette in combination with the Cas9 protein, primary human CD34 + We identified a guide RNA that can frequently induce on-target cleavage in cells and incorporate the gene delivery cassette into the FOXP3 gene. Furthermore, we used edited CD34 in NSG recipient mice. + We were able to sustain cell engraftment and express the GFP reporter construct integrated into the FOXP3 gene over a long period. These findings demonstrate that genome editing systems such as the CRISPR / Cas system described herein can perform efficient editing, thereby enabling the expression of human wild-type FOXP3 in human hematopoietic stem cells and sustaining engraftment at a level that is predicted to provide clinical utility for diseases or disorders with abnormal FOXP3 function, for example, after administration as autologous cell adoptive therapy to subjects with IPEX syndrome. Previous studies have suggested that clinical utility can be observed after allogeneic stem cell transplantation even in subjects with IPEX syndrome with a low donor chimerism rate of 5%. See Seidel, MG et al. (2009). Blood, 113(22):5689-5691.

[0021] The use of a CRISPR / Cas system, including gRNA and donor templates configured to insert the FOXP3 gene cDNA into the endogenous FOXP3 gene, shows promise as a treatment for inflammatory diseases such as IPEX syndrome, an autoimmune disease. Regarding the treatment of IPEX syndrome, since this disease can be caused by various mutations spread throughout the gene, insertion of the entire FOXP3 cDNA (e.g., the entire FOXP3 cDNA optimized for human codons) into the start codon may be desirable. CD34 + By utilizing the endogenous FOXP3 promoter after cell differentiation, it is expected that the transcriptional signals necessary to obtain the optimal FOXP3 expression level can be provided.

[0022] Definition of Terms In this specification, "nucleic acid" and "nucleic acid molecule" include, for example, polynucleotides or oligonucleotides, and are 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 activity, exonuclease activity, and synthesis. Nucleic acid molecules may consist of monomers made from natural nucleotide monomers (DNA, RNA, etc.) or analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications to the sugar moiety and / or the pyrimidine base moiety or purine base moiety. Modifications to the sugar moiety include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be substituted with a structure that is stereochemically similar or electronically similar, such as aza sugars 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. Similar bonds to phosphodiester bonds include phosphorothioate bonds, phosphorodithioate bonds, phosphoroselenoate bonds, phosphorodiselenoate bonds, phosphoranilothioate bonds, phosphoranilidate bonds, and phosphoramidate bonds. "Nucleic acid molecules" also include so-called "peptide nucleic acids," which contain native or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids may be single-stranded or double-stranded.

[0023] In this specification, “coding strand” includes, for example, a DNA strand having the same base sequence as the RNA transcript produced (where thymine is replaced with uracil). The coding strand contains codons, and the non-coding strand contains anticodons.

[0024] In this specification, “regulatory element” includes, for example, segments of nucleic acid molecules capable of increasing or decreasing the expression of specific genes in an organism, and includes, but is not limited to, segments of nucleic acid molecules capable of influencing the transcription and / or translation of operably linked transcriptionable DNA molecules. Regulatory elements such as promoters (e.g., MND promoters), leaders, introns, and transcription termination regions are DNA molecules that have gene regulatory activity and play an essential role in the overall expression of genes in living cells. Therefore, isolated regulatory elements (such as promoters) that function in plants are useful for modifying the phenotype of plants by genetic engineering methods. The regulation of gene expression is a fundamental function of all organisms and viruses. Regulatory elements include, but are not limited to, CAAT boxes, CCAAT boxes, Pribno 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 includes a further segment at either the 5' or 3' end that provides any of the aforementioned features. For example, suitable third segments may include a 5' end cap (e.g., a 7-methylguanylate cap (m7G)); a 3' end polyadenylate tail (e.g., a 3' end poly(A) tail); a riboswitch sequence (e.g., one that stabilizes under control and / or allows access by a protein or protein complex under control); a stability control sequence; a sequence that forms a dsRNA double strand (e.g., a hairpin); a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a traceability modification or sequence (e.g., direct binding to a fluorescent molecule, binding to a region that facilitates fluorescence detection, a sequence that enables fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a DNA-acting protein such as a transcription activator, transcription repressor, DNA methyltransferase, DNA methyl-degrading enzyme, histone acetyltransferase, histone deacetylase, etc.); and combinations thereof.

[0026] Guide RNA and Cas endonucleases (e.g., Cas9 endonuclease) may form a ribonucleoprotein complex (e.g., by binding via non-covalent interactions). The guide RNA provides target specificity to the ribonucleoprotein complex by containing a nucleotide sequence complementary to the target DNA sequence. Site-directed modifying enzymes of the ribonucleoprotein complex provide endonuclease activity. In other words, site-directed modifying enzymes, by association with the protein-binding segment of the guide RNA, are led to the target DNA sequence (e.g., target sequence in chromosomal nucleic acids; target sequence in extrachromosomal nucleic acids (e.g., episomal nucleic acids, minicircles, etc.); target sequence in mitochondrial nucleic acids; target sequence in chloroplast nucleic acids; target sequence in plasmids, etc.).

[0027] In this specification, "FOXP3" includes, but is not limited to, proteins involved in immune system responses. The FOXP3 gene (also known as forkheadbox protein P3, forkheadbox P3, AAID, DIETER, IPEX, JM2, PIDX, XPID, or scurfin) contains 11 coding exons. Foxp3 is involved in endogenous regulatory T cells (nT). reg (T cell line)) and adoptively transferred / inducible regulatory T cells (a / iT reg It is a specific marker for ). In animal studies, induction or administration of FOXP3-positive T cells significantly reduces the severity of disease (autoimmune disease) in diabetes models, multiple sclerosis models, asthma models, inflammatory bowel disease models, thyroiditis models, or kidney disease models. However, previous studies have reported that T cells exhibit plasticity. Therefore, there is a risk in using regulatory T cells for treatment because regulatory T cells transferred to a subject may change into pro-inflammatory helper T17 (Th17) cells instead of regulatory cells. With this in mind, this specification provides a method to avoid the risks caused by the change from regulatory cells to pro-inflammatory cells. For example, iT reg FOXP3, expressed from [source], is used as a master regulator of the immune system, as well as in immune tolerance and immunosuppression. reg Human T1 is thought to play an extremely important 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, such as low-dose IL-2 and expanded culture of autologous T cells. reg A treatment combining adoption and T1 is currently in clinical trials. IL-2 therapy has limited effectiveness due to its multifaceted activity and potential "off-target" effects that can enhance inflammation. reg Therapy also involves large-scale culture of T reg Due to problems with in vivo stability and viability, and a lack of therapeutically useful antigen specificity, its use will likely be limited.

[0028] In this specification, “nuclease” includes, but is not limited to, proteins or enzymes capable of cleaving phosphate diester bonds between nucleotide subunits of nucleic acids. The nucleases described herein are used in “gene editing.” Gene editing is a type of genetic engineering technique that uses one or more types of nucleases to insert, delete, or replace DNA in the genome of an organism. Examples of nucleases include, but are not limited to, nucleases used in the CRISPR / Cas system (e.g., the CRISPR / Cas9 system), zinc finger nucleases, and TALEN nucleases. Nucleases can be used to target gene loci, for example, target gene loci in nucleic acid sequences.

[0029] In this specification, “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 a 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. After introns are removed by RNA splicing, the remaining exons are covalently linked to each other to form a portion of the mature messenger RNA.

[0030] In this specification, “Cas endonuclease” or “Cas nuclease” includes, for example, RNA-induced DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. In this specification, “Cas endonuclease” refers to both natural Cas endonucleases and recombinant Cas endonucleases.

[0031] In this specification, “Cas9” or “CAS9” (also known as Csn1 and Csx12) includes, for example, RNA-induced DNA endonuclease enzymes associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. In this specification, “Cas9” refers to both natural Cas9 and recombinant Cas9.

[0032] In this specification, "zinc finger nuclease" includes, but is not limited to, artificial restriction enzymes created by fusing a zinc finger DNA-binding domain with a DNA-degrading domain. The zinc finger domain can be modified to target a specific DNA sequence of interest, thereby enabling the zinc finger nuclease to target unique sequences within a complex genome.

[0033] In this specification, "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 created by fusing a TAL effector DNA-binding domain to a DNA-degrading domain (a nuclease that cleaves DNA strands). Since transcription activator-like effectors (TALEs) can be modified to bind to virtually any desired DNA sequence, they can be combined with nucleases to cleave DNA at specific locations. Restriction enzymes can be introduced into cells for the purpose of in-situ gene editing or genome editing, a technique known as genome editing using recombinant nucleases. TALENs, along with zinc finger nucleases and CRISPR / Cas, are one of the tools in the field of genome editing.

[0034] In this specification, “knock-in” includes, but is not limited to, genetic engineering methods for, for example, replacing one-to-one DNA sequence information containing a wild-type copy within a gene locus, or inserting sequence information that is not present within a gene locus.

[0035] In this specification, “promoter” includes, but is not limited to, a nucleotide sequence that induces the transcription of a structural gene. In some embodiments, the promoter is located in the non-coding region at the 5' end of the gene and is near the transcription start site of the structural gene. The elements of the promoter sequence that initiate transcription are often characterized by a consensus nucleotide sequence. The promoter is a DNA region that initiates the transcription of a particular gene. The promoter is located near the gene transcription start site upstream (towards the 5' region of the sense strand) within the same DNA strand. 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 approximately 100 base pairs, approximately 200 base pairs, approximately 300 base pairs, approximately 400 base pairs, approximately 500 base pairs, approximately 600 base pairs, approximately 700 base pairs, approximately 800 base pairs, or approximately 1000 base pairs, or within the range defined by any two of these lengths. In this specification, the promoter may be a constitutively active promoter, a repressive promoter, or an inductive promoter. When the promoter is an inductive promoter, the transcription rate increases in response to an inducer. In contrast, when the promoter is a constitutive promoter, the transcription rate is not controlled by an inducer. Repressive promoters are also known. Examples of promoters include, but are not limited to, constitutive promoters, weak heterologous promoters (e.g., endogenous promoters and / or promoters that result in lower expression than constitutive promoters), or inducible promoters. Further examples of promoters include the EF1α promoter, PGK promoter, MND promoter, KI promoter, Ki-67 gene promoter, or promoters that can be inducible 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, which are used in mammalian systems.

[0036] In this specification, “transcriptional enhancer domain” includes, but is not limited to, a short DNA region (50–1500 bp) to which a protein (activator) can bind, which, when an activator binds to the transcriptional enhancer domain, can increase, promote, or enhance the transcription of a particular gene or its transcription level. Such activator proteins are usually called transcription factors. Enhancers are typically cis-acting, located up to 1 Mbp (1,000,000 bp) away from the target gene, and are located upstream or downstream of the transcription start site, and are forward or reverse. Enhancers may be located upstream or downstream of the gene being regulated. In some embodiments, multiple enhancer domains may be used to increase transcription levels; for example, a multimerized activation-binding domain may be used to further enhance or increase transcription levels. Furthermore, since some researchers have found that enhancers are located upstream or downstream, hundreds of thousands of base pairs away from the transcription start site, it is not necessary for enhancers to be located near the transcription start site to influence transcription. Enhancers do not act on the promoter region itself, but bind to activator proteins. Activator proteins interact with the mediator complex, recruiting polymerase II and basal transcription factors to initiate gene transcription. Enhancers can also be located within introns. The direction of the enhancer may be reversed, and this does not affect its function. Furthermore, enhancers may be cleaved or inserted at any location on the chromosome, and such treatments can still affect gene transcription. In some embodiments, enhancers are used to silence repressive mechanisms that inhibit FOXP3 gene transcription. An example of an enhancer-binding domain is the TCRα enhancer. In some embodiments, the enhancer domain is the TCRα 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, the enhancer-binding domain is positioned upstream of the promoter, thereby activating the promoter and increasing the transcription of the FOXP3 gene.

[0037] In this specification, "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 to which the transcription factor can control the rate of transcription of genetic information from DNA to messenger RNA by binding to the transcription activation domain. 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, the activator domain is used to silence repressive mechanisms that block the transcription of the FOXP3 gene.

[0038] In this specification, “Ubiquitous chromatin opening elements (UCOEs)” include, for example, elements located on unmethylated CpG islands derived from housekeeping genes and characterized by bidirectionally transcribed dual promoters. UCOEs are a promising tool for inhibiting the silencing of introduced genes and maintaining their expression in various cell models, including cell lines, pluripotent hematopoietic stem cells, PSCs, and progeny cells differentiated therefrom.

[0039] In this specification, “operably linked” includes, but is not limited to, a functional linkage between a regulatory sequence and a heterogeneous nucleic acid sequence, which results in the expression of the heterogeneous nucleic acid sequence. In some embodiments, a first molecule is linked to a second molecule, and these molecules are arranged such that the first molecule influences the function of the second molecule. These two molecules may be part of a contiguous single molecule or they may be adjacent to each other. For example, if a promoter regulates the transcription of a transcribable DNA molecule of interest within a cell, the promoter is operably linked to this transcribable DNA molecule.

[0040] In descriptions of molecules such as peptide fragments, the term "concentration" refers to the amount of molecules present in a given volume of solution, for example, the number of moles of molecules.

[0041] The terms “individual,” “subject,” and “host” are used interchangeably herein and refer to any subject for which 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 or is suspected of having a FOXP3-related disorder or health condition. In some embodiments, the subject is a human who, at the time of diagnosis or thereafter, has been diagnosed with a risk of a FOXP3-related disorder or health condition. In some cases, the diagnosis of being at risk of a FOXP3-related disorder or health condition may be determined based on the presence of one or more mutations in the endogenous gene encoding FOXP3 or in a nearby genomic sequence that may affect FOXP3 expression. For example, in some embodiments, the subject has or 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 who, at the time of diagnosis or thereafter, has been diagnosed with a risk of an autoimmune disorder. In some cases, a diagnosis of being at risk of autoimmune disorders can be determined based on the presence of one or more mutations in the endogenous FOXP3 gene or in nearby genomic sequences that may affect the expression of the FOXP3 gene.

[0042] When the term “treatment” is used to refer to a disease or condition, it means that the symptoms associated with the condition in which the individual is suffering are at least alleviated, where “alleviation” is used in a broad sense to mean that the degree of a parameter (e.g., symptoms) associated with the condition being treated (e.g., an autoimmune disease) is at least reduced. Thus, treatment also includes a state in which the pathological condition or at least its associated symptoms are completely suppressed, for example, a state in which the onset of the pathological condition or its symptoms is prevented, or a state in which the pathological condition or its symptoms are completely eliminated so that the host no longer suffers from the pathological condition or at least the symptoms that characterize the pathological condition. Thus, treatment includes (i) prevention, i.e., prevention of the onset of clinical symptoms, such as reducing the risk of developing clinical symptoms, for example, prevention of disease progression, and (ii) suppression, i.e., prevention of the onset or further onset of clinical symptoms, for example, reduction or complete suppression of active disease.

[0043] In this specification, “effective dose,” “pharmaceutically effective dose,” or “therapeutic effective dose” means an amount of a composition sufficient to provide the desired utility when administered to a subject having a particular condition. As used in descriptions relating to ex vivo treatment of autoimmune disorders, the term “effective dose” refers to the amount of a population of therapeutic cells or their progeny cells required to prevent or alleviate at least one sign or symptom of an autoimmune disorder, and relates to an amount of a composition containing therapeutic cells or their progeny cells sufficient to provide the desired effect, for example, to treat the symptoms of an autoimmune disorder in a subject. Therefore, the term “therapeutic effective dose” refers to an amount of therapeutic cells or a composition containing 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. Furthermore, “effective dose” may include an amount sufficient to prevent or delay the onset of disease symptoms, an amount sufficient to alter the course of disease symptoms (for example, an amount sufficient to slow the progression of disease symptoms, but not limited to that), or an amount sufficient to improve disease symptoms. In descriptions of in vivo treatment of autoimmune disorders in subjects (e.g., patients) or genome editing in in vitro cultured cells, “effective dose” refers to the amount of components used for genome editing, such as gRNA, donor templates, and / or site-specific polypeptides (e.g., DNA endonucleases), required to edit the genome of cells in subjects or in vitro cultured cells. In any case, an appropriate “effective dose” can be determined by a person skilled in the art simply by performing routine experiments.

[0044] In this specification, “autoimmune disease” includes, but is not limited to, conditions in which the immune system is abnormally underactive or excessively activated. When the immune system is excessively activated, 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 reduced resistance to infection. Examples of autoimmune disorders or autoimmune diseases that can be suppressed, mitigated, 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, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, muscle diseases, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatous disease, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion disorders, NF-κB essential modulator (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX, immune dysregulation, polyglandular endocrine disorders, intestinal diseases, X-linked (IPEX) syndromes (diseases characterized by immune dysregulation, polyglandular endocrine disorders, intestinal diseases, and X-linked inheritance), and ataxia telangiectasia. Immune disorders can be analyzed, for example, by examining the profiles of nerve-specific autoantibodies or other biomarkers if they are detected in the serum or cerebrospinal fluid of a subject. In some of the typical methods provided herein, these methods are for treating, alleviating, or suppressing autoimmune disorders.In some embodiments, autoimmune disorders include systemic lupus erythematosus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, muscle diseases, severe combined immunodeficiency, DiGeorge syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatous disease, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion disorders, NF-κB essential modulator (NEMO) mutations, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferative disorder, IPEX, immune dysregulation, polyglandular endocrine disorders, intestinal diseases, X-linked (IPEX) syndromes (diseases characterized by immune dysregulation, polyglandular endocrine disorders, intestinal diseases and X-linked inheritance), and ataxia telangiectasia or any combination thereof.

[0045] IPEX syndrome refers to a rare syndrome characterized by immune dysregulation, polyglandular endocrine disorders, intestinal diseases, and X-linking, and is associated with dysfunction of the transcription factor FOXP3, which is widely considered to be a master regulator of regulatory T cell lineages. Individuals with IPEX syndrome may exhibit symptoms such as autoimmune intestinal disease, psoriatic or eczematous dermatitis, nail dystrophy, autoimmune endocrine disorders, and autoimmune skin diseases (such as alopecia generalis 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 related to CD4 + CD25 + This leads to a deficiency in regulatory T cells and a deficiency in the expression of the transcription factor FOXP3. The decrease in FOXP3 is thought to be due to the activation of T cells without checks, following the deficiency of regulatory T cells.

[0046] In this specification, “organ transplantation” includes, but is not limited to, replacing a damaged organ in a recipient or transferring an organ that is not present in the recipient’s body, for example, by moving an organ from one body to another, or moving an organ from a donor site within the patient’s own body to another site. The transplantation of organs and / or tissues within the same person’s body is called autologous transplantation. Transplantation between two subjects belonging to the same species, as has been done in recent years, is called allogeneic transplantation. In allogeneic transplantation, organs or tissues obtained from a living or deceased person are transplanted. Some of the embodiments described herein provide methods for treating, suppressing, or mitigating side effects of organ transplantation, such as organ rejection in a subject.

[0047] Transplantable organs include, for example, the heart, kidneys, liver, lungs, pancreas, intestines, and thymus. Transplantable tissues include, for example, bone and tendons (called musculoskeletal grafts), cornea, skin, heart valves, nerves, and veins. The kidneys, liver, and heart are the most commonly transplanted organs. The cornea and musculoskeletal grafts are the most commonly transplanted tissues.

[0048] In some embodiments described herein, methods are provided for treating, suppressing, or mitigating organ transplant side effects, such as organ rejection, in subjects. In some embodiments, the subjects are selected as subjects for administration of an anti-rejection agent. In some embodiments, the anti-rejection agent includes prednisone, Imuran (azathioprine), CellCept (mycophenolate mofetil (MMF)), Myfortic (mycophenolate), Rapamune (sirolimus), Neoral (cyclosporine), or Prograf (tacrolimus).

[0049] In some embodiments, the subject is selected to be subjected to suppression, mitigation, or treatment using the genetically modified cells described in embodiments of the present invention. In some embodiments, the subject is adverse to an anti-inflammatory or anti-rejection drug. Therefore, representative cells or compositions provided herein are provided to the selected subject. Adverse effects of anti-rejection drugs include increased or decreased blood tacrolimus levels due to interaction with other drugs, nephrotoxicity, hypertension, neurotoxicity (tremor, headache, tingling, and insomnia), diabetes (hyperglycemia), diarrhea, nausea, alopecia, or hyperkalemia, or any combination thereof. Therefore, the subject is selected to be subjected to the treatment, suppression, or mitigation methods described herein. Such selection or identification can be performed by clinical or diagnostic evaluation.

[0050] In this specification, “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] In this specification, “graft-versus-host disease (GVHD or GvHD)” includes, but is not limited to, medical complications that occur after tissue transplantation from a genetically different human. While GVHD often occurs in association with stem cell transplantation or bone marrow transplantation, the term GVHD may also be used to refer to complications from other forms of transplanted tissue. Immune cells in donor-provided tissue recognize the recipient as a foreign body rather than “self.” In some embodiments of the present invention, the methods of the present invention can be used to prevent or mitigate complications resulting from GVHD.

[0052] In this specification, “pharmaceutical additives” include, for example, inert substances for adding cells to obtain a composition, but are not limited to these.

[0053] In this specification, “chimeric antigen receptor (CAR)” includes, but is not limited to, artificial T cell receptors or recombinant receptors, also known as chimeric T cell receptors, which can be used to transfer desired specificity to effector immune cells. A CAR may be a synthetically designed receptor comprising a ligand-binding domain of an antibody sequence or other protein sequence that binds to a molecule associated with the disease or disorder, wherein the ligand-binding domain is linked via a spacer domain to one or more intracellular signaling domains (e.g., costimulatory domains) derived from a T cell receptor or other receptor. In some embodiments, cells (such as mammalian cells) containing the chimeric antigen receptor are produced, comprising a nucleic acid encoding a fusion protein. Using the chimeric antigen receptor, for example, the specificity of a monoclonal antibody or its binding portion can be transferred to T cells. In some embodiments of the present invention, the recombinant cell further comprises a sequence encoding the chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is specific to a molecule on tumor cells. Using recombinant cells expressing a T cell receptor or the chimeric antigen receptor, specific tissues requiring FOXP3 expression can be targeted. Some embodiments include methods for providing and delivering FOXP3 by targeting specific tissues. In some embodiments, the tissue is a transplanted tissue. In some embodiments, the chimeric antigen receptor is specific to a target molecule on the transplanted 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 are also called "phenotypic" cells. The aforementioned cells are CD34 + Cells, for example, CD34 + Hematopoietic stem cells are also acceptable.

[0055] In this specification, “protein sequence” includes, for example, a polypeptide sequence of amino acids that constitutes the primary structure of a protein, but is not limited thereto. Furthermore, “upstream” in this specification means the 5' position on a polynucleotide and the position toward the N-terminus on a polypeptide. Similarly, “downstream” in this specification means the 3' position on a nucleotide and the position toward the C-terminus on a polypeptide. Therefore, “N-terminus” refers to the position or specific position of an element on a polynucleotide toward the N-terminus on a polypeptide.

[0056] In this specification, the terms “expression” or “protein expression” refer 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 by quantitative or qualitative indicators. In some embodiments, the protein or the plurality of proteins are expressed in a configuration that allows for dimerization in the presence of a ligand.

[0057] A "functional equivalent" or "fragment of a functional equivalent" relating to a protein description may have one or more conserved amino acid substitutions. A "conserved amino acid substitution" refers to an amino acid substitution to another amino acid that has equivalent properties to the original amino acid. The conserved amino acid group is shown below. [Table 1]

[0058] Conservative substitutions may be introduced at any position in a given peptide or its fragment. However, non-conservative substitutions may be preferable, and in particular, it may be preferable to introduce non-conservative substitutions at any one or more positions, but this is not limited to these cases. Non-conservative substitutions capable of forming a functional equivalent fragment of the peptide may, for example, substantially differ in polarity, charge, and / or steric bulk, while maintaining the functionality of the derivative or variant fragment.

[0059] Sequence identity (%) is determined by comparing two sequences at their optimal alignment on a comparison window. Parts of the polynucleotide or polypeptide sequences on the comparison window may have additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for the optimal alignment of the two sequences. 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 matching positions, dividing the number of matching positions by the total number of positions on the comparison window, and multiplying the result by 100.

[0060] "Identity" or "identity (%)" of two or more nucleic acid sequences or polypeptide sequences refers to two or more sequences or subsequences that are determined to be identical when compared and aligned to obtain the greatest match within a comparison window or specified region, either using one of the sequence comparison algorithms described below or by manual alignment and visual determination, or two or more sequences or subsequences that have a specific percentage of identical amino acid residues or nucleotides (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity in a specific region, e.g., the entire polypeptide sequence or individual domains of the polypeptide). Such sequences are referred to as "substantially identical." This definition also applies to the complementary strand of a test sequence.

[0061] In this specification, the terms “complementary” or “substantially complementary” are used interchangeably and mean that a particular nucleic acid (e.g., DNA or RNA) has a nucleotide sequence capable of linking to another nucleic acid via non-covalent bonds in a sequence-specific and antiparallel manner (i.e., a particular nucleic acid specifically links to a complementary nucleic acid), i.e., forming a Watson-Crick base pair or a G / U base pair. As is known in the art, standard Watson-Crick base pairs include the thymidine (T)-adenine (A) pairing, the uracil (U)-adenine (A) pairing, and the cytosine (C)-guanine (G) pairing.

[0062] A DNA sequence that "codes" a specific RNA is a DNA nucleic acid sequence that can be transcribed into RNA. DNA polynucleotides may code for RNA that is translated into proteins (mRNA), or for RNA that is not translated into proteins (e.g., tRNA, rRNA, or guide RNA; also referred to herein as “non-coding” RNA or “ncRNA”). A “protein-coding sequence” or “sequence that codes for a specific protein or polypeptide” is a nucleic acid sequence that, when controlled by an appropriate regulatory sequence in vitro or in vivo, is transcribed into mRNA (in the case of DNA) and translated into polypeptides (in the case of mRNA).

[0063] In this specification, “codon” refers to a single genetic coding unit in a DNA or RNA molecule, formed by a sequence of three nucleotides. In this specification, “codon degeneracy” refers to a genetic coding property that allows for changes in the nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide.

[0064] "Codon-optimized" or "codon optimization" refers to the modification of codons in the gene or coding region of a nucleic acid molecule to reflect codons typically used in the host organism without altering the polypeptide encoded by the DNA, for the purpose of transforming various hosts. Such optimizations include replacing at least one codon, multiple codons, or a very large number of codons with one or more codons that are frequently used in the organism's genes. Tables showing codon usage frequencies are readily available, for example, from the "Codon Usage Database." Those skilled in the art can utilize their knowledge of codon usage or codon preference in each organism to apply codon frequencies to a given polypeptide sequence, thereby encoding a polypeptide and producing nucleic acid fragments of a codon-optimized coding region that use codons optimal for a given species. Codon-optimized coding regions can be designed by various methods known to those skilled in the art.

[0065] For example, the terms “recombinant” or “engineered” used in reference to cells, nucleic acids, proteins, or vectors indicate that the cell, nucleic acid, protein, or vector has been modified by laboratory methods or is obtained as a result of laboratory methods. Therefore, for example, recombinant or engineered proteins include proteins produced by laboratory methods. Recombinant or engineered proteins may contain amino acid residues not found in natural (non-recombinant or wild-type) proteins, or may contain modified (e.g., labeled) amino acid residues. The terms “recombinant” or “engineered” may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include chemical modifications of a peptide, protein, or nucleic acid sequence (including one or more amino acids, deoxyribonucleotides, or ribonucleotides); the addition, deletion, or substitution of one or more amino acids in a peptide or protein; or the addition, deletion, or substitution of one or more nucleic acids in a nucleic acid sequence.

[0066] "Genome DNA" or "genome sequence" refers to the genomic DNA of an organism, including but not limited to the genomic DNA of bacteria, fungi, archaea, plants, or animals.

[0067] In this specification, "transgene," "exogenous gene," or "exogenous sequence" relating to nucleic acids refers to a nucleic acid sequence or gene that does not exist in the cell's genome but has been artificially introduced into the genome, for example, through genome editing.

[0068] In this specification, “endogenous gene” or “endogenous sequence” with respect to nucleic acids refers to a nucleic acid sequence or gene that is naturally present in the cell genome without being introduced through artificial means.

[0069] A “vector,” “expression vector,” or “construct” is a nucleic acid used to introduce heterologous nucleic acids into cells, and can express heterologous nucleic acids in cells by containing various regulatory elements. Examples of 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 a bacterial system such as Escherichia coli. In this specification, “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 by quantitative or qualitative indicators. In some embodiments, the protein or plurality of proteins are expressed in a configuration that allows for dimerization 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 (for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, etc., and not limited to these).

[0070] In this specification, “fusion protein” or “chimeric protein” includes, but is not limited to, a protein created by ligating two or more genes that originally encoded separate proteins or portions of separate proteins. Fusion proteins can also be created from specific protein domains derived from two or more separate proteins. By translating such a fusion gene, a single polypeptide or a group of polypeptides with functional properties derived from each of the original proteins can be obtained. Recombinant fusion proteins can be artificially created by recombinant DNA technology used in biological research or therapy. Methods for creating such fusion proteins are known to those skilled in the art. Some fusion proteins are combinations of entire peptides and therefore may contain all domains of the original proteins, and in particular all functional domains. However, other fusion proteins, especially those not found in nature, combine only portions of coding sequences and therefore do not retain the original function of the parental genes from which such proteins are derived. In some embodiments, fusion proteins containing interferon or PD-1 protein or both are provided.

[0071] In this specification, a “conditional” promoter or “inducible” promoter refers to a nucleic acid construct that includes a promoter that expresses a gene in the presence of an inducer but substantially does not express a gene in the absence of the inducer.

[0072] In this specification, "constitutive" refers to a nucleic acid construct that expresses a continuously produced polypeptide, as it contains a constitutive promoter.

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

[0074] In some embodiments, when the inducible promoter is expressed, it can induce higher activity compared to when expression is not induced or at the basal level. In some embodiments, the activity level when expression is induced is 2, 4, 6, 8, 9, 10 or more times higher 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 off for a period defined by 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 any two of these periods, in the absence of a transactivator, but is not off for 0 days.

[0075] In some embodiments, the inducible promoter is designed or modified to have low activity at the basal level, induce high levels of expression, and / or be switchable on and off for a short period of time.

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

[0077] In some embodiments, the immunomodulatory imide drugs used in the methods described herein may include thalidomide (including its analogues, derivatives, or pharmaceutically acceptable salts). Examples of thalidomide include Immunoprin, Salomid, Talidex, Talizer, Neurosedyn, α-(N-phthalimido)glutarimide, 2-(2,6-dioxopiperidine-3-yl)-2,3-dihydro-1H-isoindole-1,3-dione); or pomalidomide (including its analogues, derivatives, or pharmaceutically acceptable salts). Examples of pomalidomide include pomalist, Imnovid, (RS)-4-amino-2-(2,6-dioxopiperidine-3-yl)isoindole-1,3-dione; or lenalidomide (including its analogues, derivatives, or pharmaceutically acceptable salts). Examples of lenalidomides include revlimide, (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindole-2-yl)piperidine-2,6-dione; or apremilast (including its analogues, derivatives, or pharmaceutically acceptable salts). Examples of apremilast include otezla, CC-10004, N-{2-[(1S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2,3-dihydro-1H-isoindole-4-yl}acetamide); or any combination thereof.

[0078] In this specification, “extracellular binding domain” refers to one of the domains constituting the complex, configured to bind to a specific atom or molecule, and located outside the cell. In some embodiments, the extracellular binding domain of the CISC is the FKBP domain or a portion thereof. In some embodiments, the extracellular binding domain is the FRB domain or a portion thereof. In some embodiments, the extracellular binding domain is configured to stimulate the dimerization of two CISC components by binding to a ligand or 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 multi-component synthetic protein complex configured to be co-expressed in host cells as two chimeric proteins, as described in international patent application PCT / US2017 / 065746 (this document is expressly incorporated herein by reference). The two chimeric protein components of CISC are one extracellular domain that constitutes half of the rapamycin-binding complex, which is fused to the intracellular signaling complex that constitutes the other half of the rapamycin-binding complex. By delivering the nucleic acid encoding CISC to a host cell, intracellular signaling controlled by the presence of rapamycin or rapamycin-related compounds can be generated in that host cell.

[0080] Although intracellular signaling can be induced by rapamycin-induced dimerization of CISCs, the presence of rapamycin suppresses host cell proliferation and viability, limiting its usefulness for therapeutic and research purposes. Therefore, there is a need for novel compositions and methods that enable the use of rapamycin-mediated intracellular signaling by CISCs while mitigating the adverse effects of rapamycin or rapamycin-related compounds on host cell proliferation and viability.

[0081] In this specification, “dimeric chemical-induced signaling complex,” “dimerized CISC,” or “dimer” refers to two components that constitute a 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 form a single entity, for example, in response to the 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., the binding of two identical entities to dimerize, for example, the binding of two identical CISC components to dimerize. In some embodiments, “dimerization” refers to heterodimerization, i.e., the binding of two different entities to dimerize, for example, the binding of two different separate CISC components to dimerize. In some embodiments, the dimerization of CISC components forms a cellular signaling pathway. In some embodiments, dimerization of CISC components enables selective expansion and proliferation of cells or cell populations. Further CISC systems may include CISC-gibberellin-based CISC dimerization systems or CISC-TMP-based CISC dimerization systems. Other chemically derivable dimerization (CID) systems and their components may also be used.

[0082] In this specification, “chemical-induced signaling complex” or “CISC” refers to a recombinant complex that initiates a signal within a cell and, as a direct result, undergoes dimerization upon ligand induction. A CISC may be a homodimer (a dimer of two identical components) or a heterodimer (a dimer of two different components). Therefore, in this specification, the term “homodimer” refers to a dimer consisting of two protein components described herein that have the same amino acid sequence. The term “heterodimer” refers to a dimer consisting of two protein components described herein that do not have the same amino acid sequence.

[0083] CISCs may be synthetic complexes, as described in more detail herein. "Synthetic" herein refers to complexes, proteins, dimers, or compositions described herein that are not natural and are not found in nature. In some embodiments, "IL2R-CISC" refers to a signaling complex comprising components of the interleukin-2 receptor. In some embodiments, "IL2 / 15-CISC" refers to a signaling complex comprising receptor signaling subunits shared by interleukin-2 and interleukin-15. In some embodiments, "IL7-CISC" refers to a signaling complex comprising components of the interleukin-7 receptor. Thus, CISCs may be named according to the constituent parts that make up a particular CISC. Those skilled in the art will recognize that constituent parts of chemically induced signaling complexes may consist of natural or synthetic components useful for incorporation into CISCs. Therefore, these examples provided herein are not limiting to the invention.

[0084] In this specification, "FKBP" refers to the FK506-binding protein domain. FKBP refers to a family of proteins possessing prolyl isomerase activity, which are functionally related to cyclophyllin but not similar in terms of amino acid sequence. FKBP has been identified in many eukaryotes, from yeast to humans, and functions as a protein folding chaperone for proteins containing proline residues. FKBP belongs to the immunophilin family along with cyclophyllin. 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; including their homologs and functional protein fragments.

[0085] In this specification, “FRB” refers to the FKBP rapamycin-binding domain. The FRB domain is a polypeptide region (protein “domain”) configured to form a ternary complex with the FKBP protein and rapamycin or its rapalog. FRB domains are present in a variety of natural proteins, including mTOR proteins from humans and other species (also referred to herein as FRAP, RAPT1, or RAFT); yeast proteins containing Tor1 or Tor2; and FRAP homologs of the Candida genus. Both FKBP and FRB are major components of mammalian target of rapamycin (mTOR) signaling.

[0086] A "naked FKBP rapamycin-binding domain polypeptide" or "naked FRB domain polypeptide" (also called "FKBP rapamycin-binding domain polypeptide" or "FRB domain polypeptide") refers to a polypeptide containing only the amino acid sequence of the FRB domain, or a protein in which 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the protein's amino acid sequence consists of the FRB domain's amino acid sequence. The FRB domain can be expressed as a 12kDa soluble protein (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 similar to the folding of cytochrome b562 (Choi, J. et al. (1996). Science, 273(5272):239-242). In some embodiments, the naked FRB domain contains the amino acids shown in SEQ ID NO: 37 (MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK; SEQ ID NO: 37) or SEQ ID NO: 38 (MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK; SEQ ID NO: 38).

[0087] In this specification, “activate” means the enhancement of at least one biological activity of the protein of interest. Similarly, “activation” means the state of the protein of interest in which the activity is enhanced. “Activatable” means that the protein of interest is 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 a signal-signaling dimer. In this specification, “signal-signaling” means the ability of a dimer or its configuration to initiate or sustain a downstream signaling pathway.

[0088] In this specification, “signaling domain” refers to a domain of a fusion protein or a component of a CISC involved in a signaling cascade within a cell (such as a mammalian cell). “Signaling domain” refers to a signaling portion 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 primary signal provided by the CD3ζ chain of the TCR / CD3 complex, such as activation, proliferation, differentiation, or cytokine secretion, or any combination thereof. In some embodiments, the signaling domain is located at the N-terminal end of a transmembrane domain, hinge domain, or extracellular domain. In some embodiments, the signaling domain is a synthetic or native domain. In some embodiments, the signaling domain is a linked intracellular 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 γ subunit (IL2Rγ or IL2Rg) domain. In some embodiments, the signaling domain is an interleukin-2 receptor β subunit (IL2Rβ or IL2Rb) domain, or a cleaved IL2Rβ domain (e.g., a cleaved IL2Rβ domain containing the amino acid sequence of SEQ ID NO: 5). In some embodiments, binding of a drug or ligand to the extracellular binding domain causes dimerization of the CISC components, resulting in activation of the signaling pathway and signaling through the signaling domain. In this specification, “signaling” refers to the activation of the signaling pathway by binding of a ligand or drug to the extracellular domain. Binding of the ligand or drug to the extracellular domain causes dimerization of the CISC components and activation of the signal.

[0089] In this specification, “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. The IL-2 receptor has three forms, namely the α chain, β chain, and γ chain, which are also subunits of other cytokine receptors. IL2Rβ and IL2Rγ are members of the type I cytokine receptor family. In this specification, “IL2R” refers to the interleukin-2 receptor, which is involved in T cell-mediated immune responses. IL2R is involved in receptor-dependent endocytosis and the transmission of pro-mitotic signals from interleukin-2.

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

[0091] In some embodiments, the chemically induced signaling complex is a heterodimerization-activated signaling complex comprising two components. In some embodiments, the first component comprises an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracellular signaling domains, which are one of the heterodimerization pairs. In some embodiments, the second component comprises an extracellular binding domain, an optional hinge domain, a transmembrane domain, and one or more linked intracellular signaling domains, which are the other of the heterodimerization pairs. Thus, in some embodiments, two recombination events occur. In some embodiments, these two CISC components are expressed in cells such as mammalian cells. In some embodiments, cells such as mammalian cells, or a population of cells such as a mammalian cell population, are brought into contact with a ligand or factor that induces heterodimerization, thereby initiating signaling. In some embodiments, the homodimerization pair dimerizes, thereby expressing a single CISC component in cells such as mammalian cells, and the homodimerized CISC component initiates signaling.

[0092] In this specification, “selective expansion and proliferation” refers to the ability to expand and proliferate a desired cell, such as mammalian cells, or a desired cell population, such as a mammalian cell population. In some embodiments, selective expansion and proliferation refers to the development or expansion of a population of pure cells (such as mammalian cells) in which two genes have been recombined. One component of a dimerized CISC is involved in one gene recombination, and the other component is involved in the other gene recombination. Thus, each component of a heterodimerized CISC is associated with each gene recombination. By exposing cells to a ligand, it becomes possible to selectively expand and proliferate 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] In this specification, “cytokine receptor modulator” refers to an agent that modulates the phosphorylation of downstream targets of cytokine receptors, the activation of signaling pathways associated with cytokine receptors, and / or the expression of specific proteins such as cytokines. Such agents may directly or indirectly modulate the phosphorylation of downstream targets of cytokine receptors, the activation of signaling pathways associated with cytokine receptors, and / or the expression of specific proteins such as cytokines. Therefore, examples of cytokine receptor modulators include, but are not limited to, cytokines; cytokine fragments; fusion proteins; or antibodies or their binding sites that immune-specifically bind to cytokine receptors or fragments thereof. Furthermore, examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides (e.g., soluble cytokine receptors), fusion proteins, or antibodies or their binding sites that immune-specifically bind to cytokines or fragments thereof.

[0094] In this specification, “hinge domain” refers to a domain that may ligate an extracellular binding domain to a transmembrane domain, thereby conferring flexibility to the extracellular binding domain. In some embodiments, the hinge domain positions the extracellular domain closer to the cell membrane, minimizing the possibility of recognition by antibodies or their binding fragments. In some embodiments, the extracellular binding domain is located at the N-terminus of the hinge domain. In some embodiments, the hinge domain may be naturally occurring or synthetic.

[0095] In this specification, “transmembrane domain” or “TM domain” refers to a domain that is stable within a membrane, such as a cell membrane. The terms “transmembrane span,” “membrane endogenous protein,” and “membrane endogenous domain” are also used herein. In some embodiments, the hinge domain and extracellular domain are located at the N-terminus of the transmembrane domain. In some embodiments, the transmembrane domain is a native or synthetic domain. In some embodiments, the transmembrane domain is the IL-2 transmembrane domain.

[0096] In this specification, “host cell” includes any type of cell (e.g., mammalian cell) that is sensitive to transformation, transfection, or transduction by 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 (abbreviated as "). 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, for example, CD34 + These are hematopoietic stem cells. In this specification, "cell population" refers to a group of cells (such as a group of mammalian cells) that includes two or more types of cells. In some embodiments, cells (such as mammalian cells) containing the protein sequence described herein or an expression vector encoding said protein sequence are produced.

[0097] In this specification, “transformed” or “transfected” means a cell (such as a mammalian cell), tissue, organ, or organism into which a foreign polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule may be incorporated 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 offspring. Furthermore, “transgenic” cells (such as mammalian cells) or “transgenic” organisms, or “transfected” cells (such as mammalian cells) or “transfected” organisms include offspring of transgenic cells or organisms or transfected cells or organisms, including offspring produced by breeding programs that use such transgenic organisms as parents in mating, and which exhibit altered phenotypes resulting from the presence of the foreign polynucleotide molecule. “Transgenic” also means bacteria, fungi, or plants containing one or more heterologous polynucleotide molecules. “Transduction” means the transfer of genes into cells, such as mammalian cells, via viruses.

[0098] In this specification, “mammals” include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates (monkeys, chimpanzees, or apes), and especially humans. In some embodiments, the subject is humans.

[0099] The “marker sequences” described herein encode cells containing the target protein (such as mammalian cells) or proteins used to select or track the target protein. Embodiments described herein provide fusion proteins which may contain marker sequences that can be selected in experiments such as flow cytometry.

[0100] As used herein, “epitope” refers to a portion of an antigen or molecule recognized by an 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 higher-order structural epitope. In some embodiments, cells expressing a fusion protein (such as mammalian cells) are provided, which further include a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor includes an scFv capable of recognizing an epitope on a cancer cell. As used in describing the various polypeptides or nucleic acids disclosed herein, “isolated” or “purified” refers to a polypeptide or nucleic acid that has been identified, separated, and / or recovered from other components in the environment in which it originally resides. In some embodiments, the isolated polypeptide or nucleic acid does not contain any of the other components to which it originally resides. Impurities in the environment in which an isolated polypeptide or nucleic acid originally resides are typically substances that interfere with the diagnostic or therapeutic use of the polypeptide or nucleic acid, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, a method is provided comprising the steps of delivering a nucleic acid according to any one embodiment described herein or an expression vector according to any one embodiment described herein to a bacterial cell, a mammalian cell or an insect cell, growing the cell in a culture medium, inducing the expression of a fusion protein, and purifying and processing the fusion protein.

[0101] The "amino acid sequence identity (%)" for CISC sequences described herein is defined as the percentage of amino acid residues in the extracellular binding domain, hinge domain, transmembrane domain, and / or signaling domain of the candidate sequence that match the amino acid residues in the reference sequence of each domain. This amino acid sequence identity is calculated after aligning the candidate sequence and the reference sequence and inserting gaps as necessary to calculate the maximum sequence identity (%), and conservative substitutions are not considered as part of the sequence identity. Alignment for determining amino acid sequence identity (%) can be performed in various ways that fall within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to maximize alignment over the full length of multiple sequences being compared. For example, calculating amino acid sequence identity (%) using the WU-BLAST-2 computer program (Altschul, SF et al. (1996). Methods Enzymol., 266:460-480) involves using several search parameters, most of which are set to their default values. Parameters not set to default values ​​(e.g., adjustable parameters) are set to overlap span=1, overlap fraction=0.125, word threshold (T)=11, and scoring matrix=BLOSUM62. In some embodiments of CISC, the CISC includes an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, each of which includes a native or synthetic domain, or a mutant or cleaved form of the native domain (e.g., a cleaved interleukin-2 receptor β signaling domain).In some embodiments, a variant or cleavage of a given domain includes an amino acid sequence having sequence identity (%) within a range defined by 100%, 95%, 90%, or 85% sequence identity, or any two of the aforementioned percentages, with respect to the sequence shown in the sequence provided herein.

[0102] In this specification, “T cells” or “T lymphocytes” may be obtained from any mammal, for example, from primates or other species, including monkeys, dogs, and humans. In some embodiments, the T cells are of the same species as the recipient (same species but from a different donor). In some embodiments, the T cells are autologous (the donor and recipient are the same). In some embodiments, the T cells are syngeneic (the donor and recipient are different, but identical twins).

[0103] In this specification, whether in the transitional clause or the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, these terms are to be interpreted as synonymous with the expressions “at least have” or “at least contain.” When the term “comprise(s)” is used in a context relating to a method, it means that the method includes at least the specified steps, and may include other steps. When the term “comprise(s)” is used in a context relating to a compound, composition, or apparatus, it means that the compound, composition, or apparatus includes at least the specified features or components, and may include other features or components.

[0104] genome editing system To regulate the expression, function, or activity of FOXP3, cells (e.g., CD34) may be targeted and incorporated into the cell genome by means of nucleic acids encoding FOXP3 or its functional derivatives. +The present disclosure provides a system for genome editing of cells. Furthermore, the present disclosure provides a system for providing treatment to subjects having or suspected of having a FOXP3-related disorder or health condition using ex vivo and / or in vivo genome editing. In some embodiments, the subjects are those having or suspected of having an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from organ transplantation (e.g., graft-versus-host disease (GVHD)).

[0105] Some embodiments include, (a) DNA endonucleases, or nucleic acids encoding said DNA endonucleases; (b) gRNA (e.g., sgRNA) or nucleic acid encoding such gRNA that can target the FOXP3 gene or a non-FOXP3 locus (e.g., AAVS1 (i.e., an adeno-associated virus integration site in the cell genome)) with the DNA endonuclease; and (c) Donor template including the code sequence of FOXP3 Regarding systems that include this. In some smooth ways, the DNA endonucleases include 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, and 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 gRNA is selected from the group consisting of x10 endonucleases, Csx16 endonucleases, CsaX endonucleases, Csx3 endonucleases, Csx1 endonucleases, Csx15 endonucleases, Csf1 endonucleases, Csf2 endonucleases, Csf3 endonucleases, Csf4 endonucleases, and Cpf1 endonucleases, as well as functional derivatives thereof. In some embodiments, the DNA endonucleases are Cas endonucleases, such as Cas9 endonucleases (e.g., Cas9 endonucleases derived from Streptococcus pyogenes). In some embodiments, the gRNA contains a spacer sequence complementary to the target sequence in the FOXP3 gene. In some embodiments, the gRNA contains a spacer sequence complementary to the target sequence in exon 1 of the FOXP3 gene.In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence complementary to a target sequence in a non-FOXP3 locus (e.g., AAVS1). In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs. 15-20, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs. 15-20. In some embodiments, the FOXP3 coding sequence codes for FOXP3 or a functional derivative thereof. In some embodiments, the coding sequence for FOXP3 is FOXP3 cDNA. A typical FOXP3 cDNA sequence may be contained 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 with the sequence shown in SEQ ID NO: 110 or 111, for example, 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 sequence identity, 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. 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 includes a nucleic acid encoding a gRNA. In some embodiments, the system further includes one or more further gRNAs or nucleic acids encoding the one or more further gRNAs.

[0106] In some embodiments, according to any of the systems described herein, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7, 15-20, and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7, 15-20, and 27-29. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 2, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 2. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 3, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 3. In some embodiments, the gRNA includes the spacer sequence shown in SEQ ID NO: 5, or a variant of the spacer sequence having three 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 derived from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 is a Cas9 derived from Staphylococcus lugdunensis (SluCas9).

[0108] In some embodiments, according to any of the systems described herein, the system comprises a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in a host cell. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in a human 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 mRNA).

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

[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 the MND promoter, the PGK promoter, and the EF1 promoter. In some embodiments, the promoter has the sequence shown 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 with 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 the AAV6 vector.

[0111] In some embodiments, according to any of the systems described herein, the donor template comprises a donor cassette, which comprises 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 cells are CD34 + The donor template is a cell, and the expression of FOXP3 or its functional derivative depends on an endogenous promoter within the cell. 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 containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template being configured to incorporate the donor cassette into a genomic locus targeted by a gRNA included in the system by homologous recombination repair (HDR). In some embodiments, homologous arms corresponding to the sequence of the targeted genomic locus are positioned on both sides of the donor cassette. In some embodiments, the length of the homologous arms is at least 0.2 kb or at least about 0.2 kb (for example, 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. Typical homologous arms include homologous arms contained in a donor template having sequence number 34 or 161. In some embodiments, the donor template is encoded in 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 containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template being configured to be incorporated into a genomic locus targeted by a gRNA included in the system via non-homologous end joining (NHEJ). In some embodiments, gRNA target sites are located adjacent to one or both sides of the donor cassette. In some embodiments, gRNA target sites are located adjacent to both sides of the donor cassette. In some embodiments, the gRNA target sites are target sites of the gRNA included in the system. In some embodiments, the gRNA target sites of the donor template are the reverse complementary strand of the cellular genomic gRNA target site targeted by the 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 containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template further comprising a regulatory element that enhances stable expression. Typical regulatory elements that enhance stable expression include WPRE and UCOE. In some embodiments, the WPRE is the full-length WPRE. In some embodiments, the WPRE is a truncated WPRE. A typical WPRE is a WPRE contained in a donor template having the sequence shown in any of SEQ ID NOs: 33, 34, and 161. In some embodiments, the donor template is encoded in 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 containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, the donor template further comprising a nucleic acid encoding a selection marker. In some embodiments, the selection marker is a surface marker that enables the selection of cells expressing the selection marker. In some embodiments, the selection marker is a low-affinity nerve growth factor receptor (LNGFR) polypeptide, green fluorescent protein (GFP), or a functional derivative thereof. In some embodiments, the LNGFR polypeptide or a functional derivative thereof comprises the 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 with the amino acid sequence of SEQ ID NO: 144. In some embodiments, the nucleic acid encoding GFP or a functional derivative thereof has the nucleic acid sequence of the GFP coding region shown 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 components 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 this order from the 5' end to the 3' end, a nucleic acid encoding the 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 some embodiments, the donor template comprises the nucleic acid shown 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 with SEQ ID NO: 72 or 73. In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector.

[0117] Typical donor templates include those having one of the sequences of SEQ ID NOs: 33-36 and 161. In some embodiments, the donor template includes the sequence of SEQ ID NO: 34 or 161. In some embodiments, the donor template is encoded in 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 the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in liposomes or lipid nanoparticles. In some embodiments, the liposomes or lipid nanoparticles further comprise gRNA. In some embodiments, the liposomes or lipid nanoparticles are lipid nanoparticles. In some embodiments, the system comprises lipid nanoparticles containing a DNA endonuclease and a nucleic acid encoding gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA encoding the DNA endonuclease.

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

[0120] nucleic acid Genome-targeted nucleic acids or guide RNA This disclosure provides genome-targeted nucleic acids that can direct the activity of a relevant polypeptide (e.g., a site-directed polypeptide or DNA endonuclease) to a specific target sequence within a target nucleic acid. In some embodiments, the genome-targeted nucleic acid is RNA. Hereinafter, the genome-targeted RNA is referred to as “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 to form a double helix. In a type V guide RNA (gRNA), the crRNA forms a double helix. In either system, the double helix binds to a site-specific polypeptide to form a guide RNA-site-specific polypeptide complex. This genome-targeted nucleic acid confers target specificity to the complex formed by association with the site-specific polypeptide. Thus, this genome-targeted nucleic acid confers directionality to the activity of the site-specific polypeptide.

[0121] In some embodiments, the genome-targeting nucleic acid is a bimolecule guide RNA. In some embodiments, the genome-targeting nucleic acid is a single-molecule guide RNA. The bimolecule guide RNA has two RNA strands. The first strand has an optional spacer extension sequence, a spacer sequence, and a CRISPR minimal repeat sequence in the direction from the 5' end to the 3' end. 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. The single-molecule guide RNA (sgRNA) of the type II system has 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 in the direction from the 5' end to the 3' end. The optionally provided tracrRNA extension sequence may have elements that confer further functionality (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. An optional tracrRNA elongation 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 in the direction from the 5' end to the 3' end.

[0122] As an example, guide RNAs used in CRISPR / Cas / Cpf1 systems, or other smaller RNAs, can be readily synthesized by chemical means known in the art and described later. Although chemical synthesis procedures are continuously being expanded, the length of the polynucleotides significantly exceeds approximately 100 nucleotides, making the purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC without gels, such as PAGE) difficult. One method used to produce long RNAs involves producing two or more molecules and then ligating them. Very long RNAs, such as those encoding Cas endonucleases (e.g., Cas9 or Cpf1 endonucleases), can be readily produced using enzymes. Various RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic production of RNA, such as modifications that increase stability, modifications that reduce the likelihood or degree of innate immune response, and / or modifications that 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 within the FOXP3 gene of a cell or a genomic sequence in the vicinity of the FOXP3 gene. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7 and 27-29. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer 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 within the AAVS1 gene of a cell or a neighboring genomic sequence within the AAVS1 gene. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 15-20, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 15-20.

[0125] Guide RNA produced by in vitro transcription may contain a mixture of the full-length guide RNA molecule and a portion of the guide RNA molecule. Chemically synthesized guide RNA molecules typically have more than 75% of the full-length guide molecule and may further contain chemically modified bases, such as chemically modified bases to enhance the guide RNA's resistance to cleavage by intracellular nucleases.

[0126] Spacer extension arrangement In some embodiments of genome-targeted nucleic acids, spacer extension sequences can modulate activity, confer stability, or provide sites for modifying genome-targeted nucleic acids. Spacer extension sequences can also modulate on-target or off-target activity or specificity. Several embodiments provide spacer extension sequences. The lengths of the spacer extension sequences are 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, and greater than 200 nucleotides. The length may exceed the creotide length, exceed 220 nucleotides, exceed 240 nucleotides, exceed 260 nucleotides, exceed 280 nucleotides, exceed 300 nucleotides, exceed 320 nucleotides, exceed 340 nucleotides, exceed 360 nucleotides, exceed 380 nucleotides, exceed 400 nucleotides, exceed 1000 nucleotides, exceed 2000 nucleotides, exceed 3000 nucleotides, exceed 4000 nucleotides, exceed 5000 nucleotides, exceed 6000 nucleotides, or exceed 7000 nucleotides, or exceed any of these nucleotide lengths.The length of the spacer extension sequence is 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 approximately 1 nucleotide, approximately 5 nucleotides, or approximately 10 nucleotides. Nucleotide length, approximately 15 nucleotides, approximately 20 nucleotides, approximately 25 nucleotides, approximately 30 nucleotides, approximately 35 nucleotides, approximately 40 nucleotides, approximately 45 nucleotides, approximately 50 nucleotides, approximately 60 nucleotides, approximately 70 nucleotides, approximately 80 nucleotides, approximately 90 nucleotides, approximately 100 nucleotides, approximately 120 nucleotides, approximately 140 nucleotides, approximately 160 nucleotides, approximately 180 nucleotides, approximately 200 nucleotides, approximately 220 nucleotides, approximately 2 The length may be 40 nucleotides, approximately 260 nucleotides, approximately 280 nucleotides, approximately 300 nucleotides, approximately 320 nucleotides, approximately 340 nucleotides, approximately 360 nucleotides, approximately 380 nucleotides, approximately 400 nucleotides, approximately 1000 nucleotides, approximately 2000 nucleotides, approximately 3000 nucleotides, approximately 4000 nucleotides, approximately 5000 nucleotides, approximately 6000 nucleotides, or approximately 7000 nucleotides, or longer than these nucleotide lengths.The length of the spacer extension sequence is 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, and 200 nucleotides. The length may be less than the rheotide length, 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 less 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 portion (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). In some embodiments, the other portion targets a nucleic acid and reduces or increases the stability of the nucleic acid. In some embodiments, the other portion is a transcriptional terminator segment (i.e., a transcription termination sequence). In some embodiments, the other portion functions in eukaryotic cells. In some embodiments, the other portion functions in prokaryotic cells. In some embodiments, the other portion functions in both eukaryotic and prokaryotic cells. Appropriate parts include, but are not limited to, 5' caps (e.g., 7-methylguanylate caps (m7G)); riboswitch sequences (e.g., those that stabilize under control and / or allow access by proteins or protein complexes under control); sequences that form dsRNA double strands (i.e., hairpins); sequences that target RNA to subcellular locations (e.g., the nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that enable tracking (e.g., direct binding to fluorescent molecules, binding to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.); or modifications or sequences that provide binding sites for proteins (e.g., DNA-acting proteins such as transcription activators, transcription repressors, DNA methyltransferases, DNA methyl-degrading enzymes, histone acetyltransferases, histone deacetylases, etc.).

[0128] Spacer array The spacer sequence hybridizes to the sequence within the target nucleic acid. The spacer in the genome-targeted nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer varies depending on the sequence of the target nucleic acid.

[0129] In the CRISPR / Cas system described herein, the spacer sequence is designed to hybridize to a target nucleic acid located at the 5' end of the PAM in the Cas endonuclease used in this system. The spacer may be a perfect match or a mismatch with the target sequence. Each Cas endonuclease has a specific PAM sequence that recognizes the target DNA. For example, Cas9 from Streptococcus pyogenes recognizes a PAM in the target nucleic acid having the sequence 5'-NRG-3' (where 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).

[0130] In some embodiments, the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has 1 or more but 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 compositions and methods of this disclosure as a sequence recognized by Cas9 derived from Streptococcus 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).The spacer arrangement consists of at least 6nt or at least approximately 6nt, 10nt or approximately 10nt, 15nt or approximately 15nt, 18nt or approximately 18nt, 19nt or approximately 19nt, 20nt or approximately 20nt, 25nt or approximately 25nt, 30nt or approximately 30nt, 35nt or approximately 35nt, or 40nt or approximately 40nt, or 6nt to 80nt or approximately 6nt to approximately 80nt, 6nt to 50nt or approximately 6nt to approximately 50nt, 6nt to 45nt or approximately 6nt to approximately 45nt, 6nt~40nt or approximately 6nt~approximately 40nt, 6nt~35nt or approximately 6nt~approximately 35nt, 6nt~30nt or approximately 6nt~approximately 30nt, 6nt~25nt or approximately 6nt~approximately 25nt, 6nt~20nt or approximately 6nt~approximately 20nt, 6nt~19nt or approximately 6nt~approximately 19nt, 10nt~50nt or approximately 10nt~approximately 50nt, 10nt~45nt or approximately 10nt~approximately 45nt, 10nt~40nt or approximately 10nt~approximately 40nt, 10nt~35nt or approximately 10nt~approximately 35nt, 10nt-30nt or approximately 10nt-30nt, 10nt-25nt or approximately 10nt-25nt, 10nt-20nt or approximately 10nt-20nt, 10nt-19nt or approximately 10nt-19nt, 19nt-25nt or approximately 19nt-25nt, 19nt-30nt or approximately 19nt-30nt, 19nt-35nt or approximately 19nt-35nt, 19nt-40nt or approximately 19nt-40nt, 19nt-45nt or approximately 19nt-45nt, 19nt The spacer sequence may be t~50nt or approximately 19nt~50nt, 19nt~60nt or approximately 19nt~60nt, 20nt~25nt or approximately 20nt~25nt, 20nt~30nt or approximately 20nt~30nt, 20nt~35nt or approximately 20nt~35nt, 20nt~40nt or approximately 20nt~40nt, 20nt~45nt or approximately 20nt~45nt, 20nt~50nt or approximately 20nt~50nt, or 20nt~60nt or approximately 20nt~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 long. In some embodiments, the spacer is 17 nucleotides long. In some embodiments, the spacer is 16 nucleotides long. In some embodiments, the spacer is 15 nucleotides long.

[0132] In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at least 30% or at least about 30%, at least 40% or at least about 40%, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 65% or at least about 65%, at least 70% or at least about 70%, at least 75% or at least about 75%, at least 80% or at least about 80%, at least 85% or at least about 85%, at least 90% or at least about 90%, at least 95% or at least about 95%, at least 97% or at least about 97%, at least 98% or at least about 98%, at least 99% or at least about 99%, or at least 100%. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at most 30% or about 30%, at most 40% or about 40%, at most 50% or about 50%, at most 60% or about 60%, at most 65% or about 65%, at most 70% or about 70%, at most 75% or about 75%, at most 80% or about 80%, at most 85% or about 85%, at most 90% or about 90%, at most 95% or about 95%, at most 97% or about 97%, at most 98% or about 98%, at most 99% or about 99%, or at most 100%. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is 100% at six consecutive 5' terminal nucleotides of the target sequence in the complementary strand of the target nucleic acid. In some embodiments, the complementarity (%) between the spacer sequence and the target nucleic acid is at least 60% at 20 or approximately 20 consecutive nucleotides. In some embodiments, the length of the spacer sequence and the length of the target nucleic acid may differ by 1 to 6 nucleotides, and this difference can be considered as one or more bulges.

[0133] In some embodiments, spacer sequences are designed or selected using a computer program. The computer program may use variables, such as, for example, predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC ratio (%), genomic frequency (e.g., changes at one or more locations caused by mismatches, insertions, or deletions in identical or similar sequences), methylation status, and the presence of SNPs.

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

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

[0136] The minimum repeat sequence length for CRISPR is 7 nucleotides to 100 nucleotides or approximately 7 nucleotides to approximately 100 nucleotides. For example, the minimum repeat sequence length for CRISPR is 7 nucleotides (nt) to 50 nt or approximately 7 nt to approximately 50 nt, 7 nt to 40 nt or approximately 7 nt to approximately 40 nt, 7 nt to 30 nt or approximately 7 nt to approximately 30 nt, 7 nt to 25 nt or approximately 7 nt to approximately 25 nt, 7 nt to 20 nt or approximately 7 nt to approximately 20 nt, 7 nt to 15 nt or approximately 7 nt to approximately 15 nt, 8 nt to 40 nt or approximately 8 nt to approximately 40 nt, 8 nt to 30 nt or approximately 8 nt to approximately 30 nt, 8 nt to 2 The minimum repeat length of CRISPR is approximately 5 nucleotides or 8 nucleotides to 25 nucleotides, 8 nucleotides to 20 nucleotides or 8 nucleotides to 20 nucleotides, 8 nucleotides to 15 nucleotides or 8 nucleotides to 15 nucleotides, 15 nucleotides to 100 nucleotides or 15 nucleotides to 100 nucleotides, 15 nucleotides to 80 nucleotides or 15 nucleotides to 80 nucleotides, 15 nucleotides to 50 nucleotides or 15 nucleotides to 50 nucleotides, 15 nucleotides to 40 nucleotides or 15 nucleotides to 40 nucleotides, 15 nucleotides to 30 nucleotides or 15 nucleotides to 30 nucleotides, or 15 nucleotides to 25 nucleotides or 15 nucleotides to 25 nucleotides. In some embodiments, the minimum repeat length of CRISPR is approximately 9 nucleotides. In some embodiments, the minimum repeat length of CRISPR is approximately 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) in a sequence consisting 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 with a reference CRISPR minimal repeat sequence in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides, 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.

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

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

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

[0141] In some embodiments, the minimal tracrRNA sequence has at least 60% or at least about 60% identity with a reference minimal tracrRNA sequence (e.g., wild-type tracrRNA from S. pyogenes) in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides. For example, the minimal tracrRNA sequence has at least 65% or at least about 65% identity with a reference minimal tracrRNA sequence in a sequence consisting of at least 6, 7, or 8 consecutive nucleotides, 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.

[0142] In some embodiments, the double helix formed by the CRISPR RNA minimal sequence and the tracrRNA minimal sequence has a double helix structure. In some embodiments, the double helix 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 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, or at least more nucleotides. In some embodiments, the double strand 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 double helix has mismatches (i.e., the two strands in the double helix do not have 100% complementarity). In some embodiments, the double helix has at least one, at least two, at least three, at least four, or at least five mismatches, or at least about one, at least about two, at least about three, at least about four, or at least about five mismatches. In some embodiments, the double helix has at most one, at most two, at most three, at most four, or at most five, or at most about one, at most about two, at most about three, at most about four, or at most about five mismatches. In some embodiments, the double helix has two or fewer mismatches.

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

[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 (where Y is a nucleotide that can form a fluctuating base pair with a nucleotide on the CRISPR minimal repeat strand).

[0146] In some embodiments, the bulge on the CRISPR minimal repeat chain side forming the double helix has at least one, at least two, at least three, at least four, at least five, or at least more unpaired nucleotides. In some embodiments, the bulge on the CRISPR minimal repeat chain side forming the double helix has at most one, at most two, at most three, at most four, at most five, or at most more unpaired nucleotides. In some embodiments, the bulge on the CRISPR minimal repeat chain side forming the double helix has one unpaired nucleotide.

[0147] In some embodiments, the bulge on the tracrRNA minimal sequence side forming the double helix 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 tracrRNA minimal sequence side forming the double helix 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 second strand forming the double helix (for example, on the tracrRNA minimal sequence side forming the double helix) has 4 unpaired nucleotides.

[0148] In some embodiments, the bulge has at least one fluctuating base pair. In some embodiments, the bulge has one or fewer fluctuating base pairs. 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, one or more hairpins are present at the 3' end of the minimal tracrRNA sequence within the 3' tracrRNA sequence.

[0150] In some embodiments, the hairpin begins with at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more nucleotides from the 3' end of the last paired nucleotide of the double helix consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence, or at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about fifteen, at least about twenty or more nucleotides. In some embodiments, the hairpin may begin with at least one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten or more nucleotides from the 3' end of the double-stranded combo consisting of the CRISPR minimal repeat sequence and the tracrRNA minimal sequence, or at most about one, at most about two, at most about three, at most about four, at most about five, at most about six, at most about seven, at most about eight, at most about nine, at most about ten or more nucleotides.

[0151] In some embodiments, the hairpin has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more consecutive nucleotides, or at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, at least about ten, at least about fifteen, at least about twenty or more consecutive 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 (i.e., two consecutive cytosine nucleotides).

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

[0154] One or more hairpins can interact with the guide RNA interaction region of a site-specific polypeptide.

[0155] In some embodiments, there are two or more hairpins, and in some embodiments, there are three or more hairpins.

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

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

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

[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 one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or at least more nucleotides. In some embodiments, the stem-loop structure of the 3' tracrRNA has at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, at most ten, 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 one, at least two, at least three, at least four, at least five or more functional parts, or at least about one, at least about two, at least about three, at least about four, at least about five or more functional parts. In some embodiments, the stem-loop structure has at most one, at most two, at most three, at most four, at most five or more functional parts, or at most about one, at most about two, at most about three, at most about four, at most about five or more functional parts.

[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 elongation sequence In some embodiments, the tracrRNA elongation sequence is provided when the guide RNA is a single-molecule guide or a bi-molecule guide. In some embodiments, the length of the tracrRNA elongation sequence is 1 nucleotide to 400 nucleotides or approximately 1 nucleotide to approximately 400 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 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 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, or greater than 400 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 20 nucleotides to 5000 nucleotides or more, or approximately 20 nucleotides to approximately 5000 nucleotides or more. In some embodiments, the length of the tracrRNA elongation sequence exceeds 1000 nucleotides.In some embodiments, the length of the tracrRNA elongation sequence is 1 nucleotide or longer, and 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, 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, or less than 400 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence may be 1 nucleotide or more and less than 1000 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 1 nucleotide or more and less than 10 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 10 to 30 nucleotides. In some embodiments, the length of the tracrRNA elongation sequence is 30 to 70 nucleotides.

[0163] In some embodiments, the tracrRNA elongation 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 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 The functional portion is 70nt, 70nt-80nt or approximately 70nt-80nt, 80nt-90nt or approximately 80nt-90nt, 90nt-100nt or approximately 90nt-100nt, 15nt-80nt or approximately 15nt-80nt, 15nt-50nt or approximately 15nt-50nt, 15nt-40nt or approximately 15nt-40nt, 15nt-30nt or approximately 15nt-30nt, or 15nt-25nt or approximately 15nt-25nt. In some embodiments, the functional portion functions in eukaryotic cells. In some embodiments, the functional portion functions in prokaryotic cells. In some embodiments, the functional portion functions in both eukaryotic and prokaryotic cells.

[0164] Examples of suitable functional portions of a tracrRNA elongation sequence include, but are not limited to, a 3' polyadenylated tail; riboswitch sequences (e.g., those that stabilize under control and / or allow access by proteins or protein complexes under control); sequences that form a dsRNA double helix (i.e., hairpins); sequences that target RNA to subcellular locations (e.g., the nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that enable tracking (e.g., direct binding to fluorescent molecules, binding to regions that facilitate fluorescence detection, sequences that enable fluorescence detection, etc.); and modifications or sequences that provide binding sites for proteins (e.g., DNA-acting proteins such as transcription activators, transcription repressors, DNA methyltransferases, DNA methyl-degrading enzymes, histone acetyltransferases, histone deacetylases, etc.). In some embodiments, the tracrRNA elongation sequence has a primer binding site or molecular index (e.g., a barcode sequence). In some embodiments, the tracrRNA elongation sequence has one or more affinity tags.

[0165] Linker sequence of single molecule guide In some embodiments, the linker sequence length of a single-molecule guide nucleic acid is 3 to 100 nucleotides or approximately 3 to 100 nucleotides. For example, Jinek, M. et al. (2012). Science, 337(6096):816-821 used a simple "tetraloop" (-GAAA-) consisting of four nucleotides. The length of the linker is, for example, 3 nucleotides (nt) to 90 nt or approximately 3 nt to 90 nt, 3 nt to 80 nt or approximately 3 nt to 80 nt, 3 nt to 70 nt or approximately 3 nt to 70 nt, 3 nt to 60 nt or approximately 3 nt to 60 nt, 3 nt to 50 nt or approximately 3 nt to 50 nt, 3 nt to 40 nt or approximately 3 nt to 40 nt, 3 nt to 30 nt or approximately 3 nt to 30 nt, 3 nt to 20 nt or approximately 3 nt to 20 nt, or 3 nt to 10 nt or approximately 3 nt to 10 nt. For example, the linker length is 3nt~5nt or approximately 3nt~5nt, 5nt~10nt or approximately 5nt~10nt, 10nt~15nt or approximately 10nt~15nt, 15nt~20nt or approximately 15nt~20nt, 20nt~25nt or approximately 20nt~25nt, 25nt~30nt or approximately 25nt~30nt, 30nt~35nt or approximately 30nt~35nt, 3 The linker length may be 5nt to 40nt or approximately 35nt to 40nt, 40nt to 50nt or approximately 40nt to 50nt, 50nt to 60nt or approximately 50nt to 60nt, 60nt to 70nt or approximately 60nt to 70nt, 70nt to 80nt or approximately 70nt to 80nt, 80nt to 90nt or approximately 80nt to 90nt, or 90nt to 100nt or approximately 90nt to 100nt. In some embodiments, the linker length of the single-molecule guide nucleic acid is 4 to 40 nucleotides long.In some embodiments, the linker length 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. The length is, 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 long, at most 500 nucleotides long, at most 1000 nucleotides long, at most 1500 nucleotides long, at most 2000 nucleotides long, at most 2500 nucleotides long, at most 3000 nucleotides long, at most 3500 nucleotides long, at most 4000 nucleotides long, at most 4500 nucleotides long, at most 5000 nucleotides long, at most 5500 nucleotides long, at most 6000 nucleotides long, at most 6500 nucleotides long, or at most 7000 nucleotides long, 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 various sequences. However, in some embodiments, the linker does not have a sequence with extensive regions of homology to other parts of the guide RNA, because if extensive regions with such homology exist, intramolecular binding that can interfere with other functional regions of the guide RNA may occur. In Jinek, M. et al. (2012). Science, 337(6096):816 - 821, a simple sequence - GAAA - consisting of 4 nucleotides was used, but various other sequences such as longer sequences can also be used similarly.

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

[0168] In some embodiments, the genomic sites targeted by gRNAs according to this disclosure may be located within the FOXP3 gene in the genome (e.g., the human genome), within the FOXP3 gene in the genome, or in the vicinity of 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 the spacer sequence shown in SEQ ID NO: 1 may have spacer sequences including i) the sequence of SEQ ID NO: 1, ii) sequences from positions 2-20 of SEQ ID NO: 1, iii) sequences from positions 3-20 of SEQ ID NO: 1, iv) sequences from positions 4-20 of SEQ ID NO: 1, and so on. As will be understood by those skilled in the art, each guide RNA is designed to contain 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 (together with the corresponding tracrRNA) into a crRNA. 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-strand or single-strand breaks into nucleic acids (e.g., genomic DNA). Double-strand breaks can stimulate intrinsic DNA repair pathways in cells, such as homology-dependent repair (HDR), non-homologous end joining, alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining (MMEJ). NHEJ can repair a cleaved target nucleic acid without requiring a homologous template. This can result in small deletions or insertions (indels) in the target nucleic acid at the cleavage site, potentially leading 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] Homologous donor templates have sequences homologous to the sequences adjacent to the cleavage site of the target nucleic acid. Generally, sister chromatids are used by cells as repair templates. On the other hand, 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. In exogenous donor templates, an additional nucleic acid sequence (such as an introduced gene) or modification (such as a change or deletion of one or more bases) is generally introduced between adjacent homologous regions so that the additional nucleic acid sequence or modified nucleic acid sequence is incorporated into the target gene locus. MMEJ yields genetic results similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ achieves the desired end-join DNA repair result by utilizing homologous sequences consisting of several base pairs adjacent to the cleavage site. In some cases, the expected repair result can be predicted by analyzing the short homologous sequences (microhomology) predicted in the target region of the nuclease.

[0171] Therefore, in some cases, homologous recombination is used to insert an exogenous polynucleotide sequence into the cleavage site of the target nucleic acid. Hereinafter, the exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor, donor sequence, or polynucleotide donor template). In some embodiments, a donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide is inserted into the cleavage site of the target nucleic acid. In some embodiments, the donor polynucleotide is an exogenous polynucleotide sequence, i.e., a sequence that is not naturally present at the cleavage site of the target nucleic acid.

[0172] If a sufficient concentration of exogenous DNA molecules is supplied to the nucleus of a cell where a double-strand break occurs, the exogenous DNA can be inserted into the double-strand break site during the NHEJ repair process, potentially leading to permanent addition to the genome. Such exogenous DNA molecules are referred to as donor templates in some embodiments. If the donor template contains the coding sequence of a target gene, such as the FOXP3 gene (also referred to herein as a “donor cassette”), along with relevant regulatory sequences such as promoters, enhancers, polyA sequences, and / or splice acceptor sequences as needed, the target gene can be expressed from the integrated copy in the genome and may be permanently expressed for the duration of the cell’s life. Furthermore, the integrated copy from the donor DNA template can be transmitted to daughter cells when the cell divides.

[0173] If a donor DNA template containing adjacent DNA sequences homologous to the DNA sequences on both sides of a double-strand break site (called homologous arms) is present in sufficient concentration, this donor DNA template can be incorporated via the HDR pathway. The homologous arms act as substrates for homologous recombination between the donor template and the sequences on both sides of the double-strand break site. This allows the donor template, whose sequences on both sides of the double-strand break site have not been altered from those of the unmodified genome, to be inserted without error.

[0174] Donors provided for HDR editing are highly diverse, but generally, they contain the intended editing sequence and short or long homologous arms on either side, enabling annealing to genomic DNA. The homologous region adjacent to the introduced gene alteration site may be less than 30 bp, or it may be as large as a few kilobase cassette, which may also contain promoters or cDNA. Both single-stranded and double-stranded oligonucleotide donors can be used. The size of these oligonucleotides ranges from less than 100 nt to several kilobases or more, but longer ssDNA can also be generated and used. Double-stranded donors such as PCR amplicons, plasmids, and minicircles are commonly used. Generally, AAV vectors have been shown to be a very effective means of delivering donor templates, but the limit for packaging to individual donors is less than 5 kb. Active transcription of the donor has been shown to triple the HDR rate, indicating that conversion can be increased by including a promoter. Conversely, CpG methylation of the donor may decrease gene expression and HDR rate.

[0175] In some embodiments, donor DNA can be introduced alone or together with a nuclease by various methods, such as transfection, nanoparticles, microinjection, or viral transduction. In some embodiments, the availability of donor in HDR can be enhanced by using various methods for linking donor DNA and nuclease. Examples of such methods include linking the donor to a nuclease, linking it to a DNA-binding protein that binds to the vicinity of the donor and nuclease, or linking it to a protein involved in DNA end joining or DNA repair.

[0176] In addition to genome editing using NHEJ or HDR, site-directed gene insertion can be performed using both the NHEJ pathway and HR. Such combined techniques are applicable in specific situations that may involve intron / exon boundaries. NHEJ has been demonstrated to be effective for ligation in introns, while 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. Examples of functional derivatives of FOXP3 include FOXP3 derivatives having substantially equivalent activity to wild-type FOXP3 (e.g., wild-type human FOXP3), such as FOXP3 derivatives exhibiting at least 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%, 95% or about 95%, or at least 100% or about 100% of the activity of wild-type FOXP3. In some embodiments, functional derivatives of FOXP3 may have at least 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 85% or about 85%, 90% or at least 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% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, those skilled in the art can test the functionality or activity of the compound (e.g., peptides or proteins) using various methods known in the art. Further examples of functional derivatives of FOXP3 include fragments of modified FOXP3 or fragments of wild-type FOXP3 having conservative modifications to one or more amino acid residues of the full length of wild-type FOXP3.Therefore, in some embodiments, the nucleic acid sequence encoding a functional derivative of FOXP3 may have at least 30% 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 with the nucleic acid sequence encoding FOXP3 (e.g., wild-type FOXP3).

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

[0179] In some embodiments of the donor template described herein, which includes a donor cassette, the donor cassette is either adjacent to a gRNA target site at one end or adjacent to gRNA target sites at both ends. For example, such a donor template may include a donor cassette having 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 having a gRNA target site at the 5' end. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 3' end. In some embodiments, the donor template includes a donor cassette having 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 having a gRNA target site at the 5' end and a gRNA target site at the 3' end, wherein these two gRNA target sites contain the same sequence. In some embodiments, the donor template includes at least one gRNA target site, the at least one gRNA target site in the donor template includes the same sequence as the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. In some embodiments, the donor template includes at least one gRNA target site, the at least one gRNA target site in the donor template includes the reverse complementary strand of the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. In some embodiments, the donor template includes a donor cassette having a gRNA target site at the 5' end and a gRNA target site at the 3' end, the two gRNA target sites in the donor template include the same sequence as the gRNA target site in the target locus into which the donor cassette of the donor template is incorporated. 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 complementary strands of the gRNA target sites in the target gene locus into which the donor cassette of the donor template is incorporated.

[0180] In some embodiments, a donor template is provided for targeted incorporation into the FOXP3 gene, comprising a nucleotide sequence encoding FOXP3 or a functional derivative thereof, the donor template comprising, in the direction from the 5' end to the 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 comprises 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 comprises 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 has one end adjacent to a first AAV ITR and / or the other end adjacent to a second AAV ITR. In some embodiments, the first AAV ITR is AAV2 ITR and / or the second AAV ITR is AAV2 ITR. In some embodiments, FOXP3 is human wild-type FOXP3.

[0181] Nucleic acids encoding site-directed polypeptides or DNA endonucleases In some embodiments, based on the foregoing, nucleic acid sequences (or oligonucleotides) encoding site-directed polypeptides or DNA endonucleases can be used in the genome editing method and the composition. The nucleic acid sequence encoding the site-directed polypeptide may be DNA or RNA. If the nucleic acid sequence encoding the site-directed polypeptide is RNA, this RNA can be covalently bound to a gRNA sequence or exist as a separate sequence. In some embodiments, peptide sequences of site-directed polypeptides or DNA endonucleases can be used instead of these nucleic acid sequences.

[0182] vector In another embodiment, the Disclosure provides nucleic acids having a nucleotide sequence encoding the genome-targeted nucleic acid of the Disclosure, site-directed polypeptides of the Disclosure, and / or any nucleic acid or protein molecule necessary to carry out embodiments of the methods of the Disclosure. In some embodiments, such nucleic acids are vectors (e.g., recombinant expression vectors).

[0183] Examples of expression vectors envisioned 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., vectors derived from retroviruses such as mouse leukemia virus; splenic necrosis virus; or Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary cancer virus), or other recombinant vectors. Other vectors envisioned for use in eukaryotic target cells include, but are not limited to, pXT1 vector, pSG5 vector, pSVK3 vector, pBPV vector, pMSG vector, and pSVLSV40 vector (Pharmacia). Further vectors envisioned for use in eukaryotic target cells include, but are not limited to, pCTx-1 vector, pCTx-2 vector, and pCTx-3 vector. Other vectors may also be used as long as they are compatible with the host cell.

[0184] In some embodiments, the vector has one or more transcriptional control elements and / or translational control elements. Depending on the host / vector system utilized, various suitable transcriptional control elements and translational control elements, such as constitutive promoters, inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., can be used in the expression vector. In some embodiments, the vector is a self-inactivating vector that inactivates viral sequences or components of the CRISPR machinery 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 earliest thymidine kinase promoter of herpes simplex virus (HSV), early or late SV40, the long terminal repeat (LTR) derived from retroviruses, the human elongation factor 1 promoter (EF1), a hybrid construct in which the cytomegalovirus (CMV) enhancer is fused to the chicken β-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase 1 locus promoter (PGK), or the mouse metallothionein I.

[0186] For expressing small RNAs such as guide RNAs used with Cas endonucleases, various promoters such as RNA polymerase III promoters such as U6 or H1 may be useful. 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 amplification of expression. Furthermore, since the expression vector is fused to a site-directed polypeptide, it may include nucleotide sequences encoding non-natural tags (e.g., histidine tags, hemagglutinin tags, green fluorescent protein, etc.) that are expressed as part of the fusion protein. In some embodiments, the promoter is an inductive promoter (e.g., a heat shock promoter, a tetracycline-regulating promoter, a steroid-regulating promoter, a metal-regulating promoter, an estrogen receptor-regulating promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., a CMV promoter, or a UBC promoter). In some embodiments, the promoter is a spatially restricted or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). In some embodiments, if at least one gene expressed in a host cell is inserted into the genome and then expressed under the control of an endogenous promoter present in that genome, the vector does not include a promoter for this gene.

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

[0189] Site-directed polypeptides are nucleases used in genome editing to cleave DNA. Site-directed polypeptides can be administered to cells or subjects as one or more polypeptides, or as one or more mRNAs encoding such polypeptides.

[0190] In relation to the CRISPR / Cas system or the CRISPR / Cpf1 system, the site-directed polypeptide binds to a guide RNA, thereby allowing the guide RNA to identify the site on the target DNA to which the site-directed polypeptide is directed. In the embodiments of the CRISPR / Cas system or the 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 is flexible. The length of the linker may be 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, 9 amino acid length, 10 amino acid length, 11 amino acid length, 12 amino acid length, 13 amino acid length, 14 amino acid length, 15 amino acid length, 16 amino acid length, 17 amino acid length, 18 amino acid length, 19 amino acid length, 20 amino acid length, 21 amino acid length, 22 amino acid length, 23 amino acid length, 24 amino acid length, 25 amino acid length, 30 amino acid length, 35 amino acid length, 40 amino acid length, or longer.

[0192] The naturally occurring wild-type Cas9 enzyme has two nuclease domains: an HNH nuclease domain and a RuvC domain. The Cas9 enzyme as envisioned herein has an HNH nuclease domain or an HNH-like nuclease domain, and / or a RuvC nuclease domain or a RuvC-like nuclease domain.

[0193] The HNH domain or HNH-like domain has McrA-like folding. The HNH domain or HNH-like domain has two antiparallel β-chains 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 a single strand of the target nucleic acid (e.g., the complementary strand of the target strand of crRNA).

[0194] RuvC domains or RuvC-like domains possess RNaseH folding or RNaseH-like folding. RuvC domains or RNaseH domains are involved in a diverse range of nucleic acid-based functions, including actions on both RNA and DNA. RNaseH domains have five β-chains surrounded by multiple α-helices. RuvC domains or RNaseH domains or RuvC-like domains or RNaseH-like domains possess metal-binding sites (e.g., divalent cation-binding sites). RuvC domains or RNaseH domains or RuvC-like domains or RNaseH-like domains can cleave a single strand of target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0195] In some embodiments, the site-directed polypeptide has an amino acid sequence having 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 with 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 having 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 with the nuclease domain of a typical wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes (shown above)).

[0197] In some embodiments, the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. In some embodiments, the site-directed polypeptide has at most 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. 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 with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids. In some embodiments, the RuvC nuclease domain of the site-directed polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes) in a sequence of 10 amino acids.

[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 mutations that reduce 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 nucleic acid cleavage activity (but not zero) 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% of the nucleic acid cleavage activity of a typical wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes mentioned above). In addition, the modified form of the site-directed polypeptide may substantially lack nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that substantially lacks nucleic acid cleavage activity, such a modified form is referred to herein as "enzymatically inactive."

[0199] In some embodiments, the modification of the site-directed polypeptide has a mutation that can induce a single-strand break (SSB) on the target nucleic acid (for example, by cleaving only one of the sugar-phosphate backbones of the double-stranded target nucleic acid). In some embodiments, this mutation results in nucleic acid cleavage activity (but not zero) 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% 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 described above). In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave the non-complementary strand of the target nucleic acid while retaining the ability to cleave the complementary strand. In some embodiments, the mutation reduces the ability of one or more of the multiple nucleic acid cleavage domains to cleave the complementary strand of the target nucleic acid while retaining the ability to cleave the non-complementary strand. For example, mutations occur in which one or more nucleic acid cleavage domains (e.g., nuclease domains) are inactivated at residues of a typical wild-type S. pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854, and Asn856. In some embodiments, the residues to which mutations are induced correspond to the Asp10, His840, Asn854, and Asn856 residues of a typical wild-type S. pyogenes Cas9 polypeptide (as determined, for example, by sequence and / or structural alignment). Examples of such mutations include, but are not limited to, D10A, H840A, N854A, or N856A. Those skilled in the art will understand that mutations other than alanine substitutions are appropriate.

[0200] In some embodiments, the D10A mutation, when combined with one or more of the H840A, N854A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the H840A mutation, when combined with one or more of the D10A, N854A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N854A mutation, when combined with one or more of the H840A, D10A, and N856A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. In some embodiments, the N856A mutation, when combined with one or more of the H840A, N854A, and D10A mutations, generates a site-directed polypeptide that substantially lacks DNA cleavage activity. Site-directed polypeptides having a substantially inactive single nuclease domain are called "nickases".

[0201] In some embodiments, variants of RNA-inducible endonucleases (e.g., Cas9) can be used to enhance the specificity of CRISPR-mediated genome editing. Wild-type Cas endonucleases are typically led by a single-strand guide RNA designed to hybridize with a specific sequence of about 20 nucleotides in the target sequence (such as an endogenous genomic locus). However, since a few mismatches can be tolerated between the guide RNA and the target locus, the required homologous sequence length at the target site may be efficiently shortened to, for example, about 13 nt, thereby increasing the likelihood that the CRISPR / Cas complex will bind to another location in the target genome and cleave a double-strand nucleic acid. This is also known as an off-target cleavage. On the other hand, since each Cas endonuclease nickase variant cleaves only one strand, a pair of nickases must bind in close proximity on opposite strands of the target nucleic acid to produce a double-strand break, thereby creating a pair of nicks and resulting in a double-strand break. This requires two separate guide RNAs (one for each nickase) to bind in close proximity on opposite strands of the target nucleic acid. Adhering to this requirement effectively doubles the minimum length of homologous sequence needed to produce a double-strand break, thus reducing the likelihood of the double-strand break occurring elsewhere in the genome, as the two guide RNA sites (if present) are less likely to be close enough to each other to produce a double-strand break. As reported in the art, nickases can also be used to facilitate HDR rather than NHEJ. By performing HDR using a specific donor sequence that effectively mediates the desired modification, the selected modification can be introduced into a target site in the genome. Descriptions of various CRISPR / Cas systems for use in gene editing are found, for example, in WO2013 / 176772 and Sander, JD et al. (2014). Nature Biotechnology, 32(4):347-355, as well as the references cited in these publications.

[0202] In some embodiments, site-directed polypeptides (e.g., variants of site-directed polypeptides, variants of site-directed polypeptides, enzymatically inactive site-directed polypeptides, or site-directed polypeptides enzymatically inactive under specific conditions) target nucleic acids. In some embodiments, site-directed polypeptides (e.g., variants of endoribonucleases, variants of endoribonucleases, enzymatically inactive endoribonucleases, or enzymatically inactive endoribonucleases under specific conditions) target DNA. In some embodiments, site-directed polypeptides (e.g., variants of endoribonucleases, variants of endoribonucleases, enzymatically inactive endoribonucleases, or enzymatically inactive endoribonucleases under specific conditions) target RNA.

[0203] In some embodiments, the site-directed polypeptide has one or more non-natural sequences (for example, 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 with a Cas endonuclease derived from bacteria (e.g., S. pyogenes), a nucleic acid-binding domain, and two nucleic acid-cleaving 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 with a Cas endonuclease derived from bacteria (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 with a Cas endonuclease derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains, one or both of which have at least 50% amino acid identity with the nuclease domain of the Cas endonuclease derived from bacteria (e.g., S. pyogenes).

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

[0208] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity with a Cas endonuclease derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), wherein one or both of the nucleic acid cleavage domains 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 having at least 15% amino acid identity with a Cas endonuclease derived from bacteria (e.g., S. pyogenes), and two nucleic acid cleavage domains (e.g., an HNH domain and a RuvC domain), with a mutation at the 10th aspartic acid position of one of the nuclease domains and / or a mutation at the 840th histidine position of the other nuclease domain, the mutation reducing 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 produce one double-strand break at a specific locus in the genome, or one or more site-directed polypeptides comprise four nickases that cooperate to produce two double-strand breaks at a specific locus in the genome, or one site-directed polypeptide (e.g., DNA endonuclease) acts to produce one double-strand break at a specific locus in the genome.

[0211] In some embodiments, polynucleotides encoding site-directed polypeptides can be used for genome editing. In some such embodiments, the polynucleotide encoding the site-directed polypeptide is codon-optimized for expression in cells containing the target DNA of interest, according to methods known in the art. For example, if the target nucleic acid of interest is present in human cells, it is conceivable to optimize a polynucleotide encoding a Cas endonuclease (e.g., Cas9) for human codons and use this to construct a Cas endonuclease polypeptide.

[0212] The following provides some examples of site-directed polypeptides that can be used in various embodiments of this disclosure.

[0213] CRISPR Endonuclease System CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (such as bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a type of immune system useful in defending prokaryotes from foreign invaders such as viruses and phages. The function of a CRISPR locus consists of three stages: incorporation of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and 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] The CRISPR locus contains numerous short repeat sequences called "repetitive sequences." When expressed, these repeat sequences can form secondary hairpin structures (e.g., hairpins) and / or single-stranded sequences without a fixed structure. Repetitive sequences usually occur as clusters and vary considerably between species. The repeat sequences are spaced at regular intervals by unique intervening sequences called "spacers," forming a locus with a repeat sequence-spacer-repetitive sequence structure. The spacers are either identical to or highly homologous to known invading sequences. The unit consisting of spacers and repeat sequences codes for crisprRNA (crRNA), which is processed to obtain the mature form of the unit consisting of spacers and repeat sequences. crRNA has a "seed" sequence or spacer sequence involved in targeting the target nucleic acid (in its natural form in prokaryotes, the spacer sequence targets the nucleic acid of the invader). The spacer sequence is located at the 5' or 3' end of the crRNA.

[0215] The CRISPR locus further contains polynucleotide sequences encoding CRISPR-related (Cas) genes. Cas genes encode endonucleases involved in the biosynthetic and interference stages of crRNA function in prokaryotes. Some Cas genes have homologous secondary and / or tertiary structures.

[0216] Type II CRISPR System In the biosynthesis of crRNA in the naturally occurring type II CRISPR system, trans-activated CRISPR RNA (tracrRNA) is required. The tracrRNA is modified by endogenous RNase III and then hybridizes to the crRNA repeat sequence in the pre-crRNA array. Endogenous RNase III is recruited to cleave the pre-crRNA. The cleaved crRNA is trimmed by exoribonuclease (e.g., 5' end trimming) to produce the mature form of crRNA. The 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 complex is led by the crRNA to a target nucleic acid that the crRNA can hybridize to. Hybridization of the crRNA to the target nucleic acid activates the Cas endonuclease, which then cleaves the target nucleic acid. In type II CRISPR systems, the target nucleic acid is called a protospacer-adjacent motif (PAM). In fact, PAMs are essential for promoting 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 demonstrates the usefulness 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 The type V CRISPR system has several key differences from the type II system. For example, Cpf1 is a single-strand RNA-inducible endonuclease, but unlike the type II system, it lacks tracrRNA. In fact, a CRISPR array associated with Cpf1 processes into mature crRNA without requiring further transactivated tracrRNA. The type V CRISPR array processes into short mature crRNAs of 42–44 nucleotides in length, each mature crRNA beginning with a 19-nucleotide direct repeat sequence followed by a 23–25 nucleotide spacer sequence. In contrast, mature crRNAs in the type II system begin with a 20–24 nucleotide spacer sequence followed by a direct repeat sequence of approximately 22 nucleotides. Furthermore, Cpf1 utilizes a T-rich protospacer flanking 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 the type II system, where a G-rich PAM following the target DNA is utilized. Therefore, the type V system cleaves the target at a position away from the PAM, while the type II system cleaves the target adjacent to the PAM. Furthermore, in contrast to the type II system, Cpf1 cleaves the DNA double strand at a shifted position, resulting in a 4-nucleotide or 5-nucleotide 5' end overhang. In contrast, double-strand breaks by the type II system result in blunt ends. Cpf1 is predicted to contain a RuvC-like endonuclease domain, similar to the type II system, but unlike the type II system, it lacks a second HNH endonuclease domain.

[0218] Cas gene / polypeptide and protospacer adjacent motif A typical CRISPR / Cas polypeptide is the Cas9 polypeptide, shown in Figure 1 of Fonfara, I. et al. (2014). Nucleic Acids Res., 42(4):2577-2590. The CRISPR / Cas gene naming system has undergone significant revisions since the discovery of the Cas gene. Figure 5 by Fonfara et al. above shows PAM sequences for Cas9 polypeptides derived from various species.

[0219] A complex of genome-targeted nucleic acids and site-directed polypeptides Genome-targeted nucleic acids form complexes by interacting with site-directed polypeptides (e.g., nucleic acid-inducible nucleases such as Cas9). The genome-targeted nucleic acid (e.g., gRNA) leads the site-directed polypeptide to the target nucleic acid.

[0220] As described above, in some embodiments, site-directed polypeptides and genome-targeted nucleic acids can be administered to cells or subjects separately. On the other hand, in some other embodiments, site-directed polypeptides can be pre-complexed with one or more guide RNAs, or site-directed polypeptides can be pre-complexed with tracrRNA and one or more crRNAs. The pre-complexed material can be administered to cells or subjects. Such pre-complexed material is known as ribonucleoprotein particles (RNPs).

[0221] Genome editing methods In organisms requiring the expression of the FOXP3 protein or its functional derivatives, one method for expressing the FOXP3 protein or its functional derivatives involves targeting an endogenous FOXP3 gene or a non-FOXP3 gene that is sufficiently expressed in a related cell type (e.g., T cells) using genome editing, such that a nucleic acid containing a coding sequence encoding the FOXP3 protein is incorporated into the non-FOXP3 gene, thereby inducing the expression of the incorporated coding sequence by the endogenous promoter of the endogenous FOXP3 gene or the non-FOXP3 gene. In some embodiments where the non-FOXP3 gene is targeted, the expression of the non-FOXP3 gene is controlled to prevent expression in unrelated cell types (e.g., CD34). + CD34 cells such as hematopoietic stem cells + It is desirable that the assay be specific to the cell or cells derived from it (e.g., T cells).

[0222] In some embodiments, the knock-in method involves knocking in a sequence encoding FOXP3 or a functional derivative of FOXP3 into a genomic sequence, such as a wild-type FOXP3 gene (e.g., a wild-type human FOXP3 gene), FOXP3 cDNA, or a FOXP3 minigene (having 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). In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located in, within, or near the FOXP3 gene. In some embodiments, the genomic sequence into which the FOXP3-encoding sequence is inserted is located in, within, or near exon 1 of the FOXP3 gene.

[0223] In some embodiments provided herein, a method for knocking in a sequence encoding FOXP3 or a functional derivative thereof into a genome is provided. In one embodiment, the disclosure provides insertion of a nucleic acid comprising a sequence encoding FOXP3 or a functional derivative thereof into a cellular genome. In some embodiments, the sequence encoding FOXP3 encodes wild-type FOXP3. Functional derivatives of FOXP3 include FOXP3 derivatives having substantially equivalent activity to wild-type FOXP3 (e.g., wild-type human FOXP3), such as FOXP3 derivatives exhibiting at least 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%, 95% or about 95%, or at least 100% or about 100% of the activity of wild-type FOXP3. In some embodiments, functional derivatives of FOXP3 have at least 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 85% or about 85%, 90% or about 90%, 95% or about 95%, 96% or about 96%, 97% or about 97%, 98% or about 98%, or 99% or about 99% amino acid sequence identity with FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 is encoded by an intron-deleting nucleotide sequence (e.g., FOXP3 cDNA). Those skilled in the art can test the functionality or activity of FOXP3 derivatives using methods known in the art. Examples of functional derivatives of FOXP3 include fragments of wild-type FOXP3 that have conservation modifications to one or more amino acid residues of the full length of wild-type FOXP3.Therefore, in some embodiments, the nucleic acid sequence encoding a functional derivative of FOXP3 may have at least 30% or 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 with the nucleic acid sequence encoding FOXP3 (e.g., wild-type FOXP3). In some embodiments, FOXP3 or its functional variant 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) sequences encoding FOXP3 or its functional derivatives. In some manner, the DNA endonucleases include 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, and 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 The DNA endonucleases are CsaX endonucleases, Csx3 endonucleases, Csx1 endonucleases, Csx15 endonucleases, Csf1 endonucleases, Csf2 endonucleases, Csf3 endonucleases, Csf4 endonucleases, or Cpf1 endonucleases, their homologs, recombinants of natural molecules, codon-optimized or modified versions thereof, or any combination thereof. In some embodiments, the DNA endonucleases are Cas9. In some embodiments, the Cas9 are Cas9 derived from Streptococcus pyogenes (spCas9).In some embodiments, the Cas9 is Cas9 derived from Staphylococcus lugdunensis (SluCas9).

[0225] In some embodiments, the cells targeted for genome editing have one or more mutations in their genome that reduce the expression of the endogenous FOXP3 gene compared to normal cells without the mutation. The normal cells may be healthy cells or control cells derived from (or isolated from) another subject that does not have the FOXP3 gene abnormality. In some embodiments, the cells targeted for genome editing may be cells derived from (or isolated from) a subject that requires treatment for a FOXP3 gene-related condition or FOXP3 gene-related disorder (e.g., a subject suffering from an autoimmune disorder (e.g., IPEX syndrome)). Therefore, in some embodiments, the expression of the endogenous FOXP3 gene in such cells is reduced by 10% 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 the expression of the endogenous FOXP3 gene in normal cells.

[0226] In some embodiments, CD34 + A method for editing the genome of a cell, (a) Cas DNA endonucleases (e.g., Cas9 endonucleases) or nucleic acids encoding said Cas DNA endonucleases; (b) a gRNA (e.g., sgRNA) or nucleic acid encoding the gRNA that can target the Cas DNA endonuclease to the FOXP3 gene or a non-FOXP3 locus (e.g., AAVS1) in the genome of the cell; and (c) Donor template including the code sequence of FOXP3 CD34 + The present invention provides a method that includes a step of supplying cells. In some embodiments, the Cas DNA endonuclease is a Cas9 endonuclease (for example, a Cas9 endonuclease derived from Streptococcus pyogenes). In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence in the FOXP3 gene. In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence in exon 1 of the FOXP3 gene. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7 and 27-29. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence complementary to the target sequence in a non-FOXP3 locus (e.g., AAVS1). In some embodiments, the gRNA includes the spacer sequence shown in any of SEQ ID NOs 15-20, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 15-20. In some embodiments, the FOXP3 coding sequence encodes FOXP3 or a functional derivative thereof. In some embodiments, the FOXP3 coding sequence is FOXP3 cDNA. A typical FOXP3 cDNA sequence may be contained in an AAV donor template having the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the method is described in CD34 + The method includes the step of providing the Cas DNA endonucleases to cells. In some embodiments, the method provides the nucleic acid encoding the Cas DNA endonucleases to the CD34 +The method includes the step of providing the gRNA to cells. In some embodiments, the method provides the gRNA to the CD34 + The method includes the step of providing the gRNA to cells. In some embodiments, the gRNA is sgRNA. In some embodiments, the method provides the nucleic acid encoding the gRNA to the CD34 + The method includes the step of providing to cells. In some embodiments, the method provides one or more further gRNAs or nucleic acids encoding the one or more further gRNAs to the CD34 + This further includes the process of supplying the product to cells.

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

[0228] In some embodiments, according to any of the cell genome editing methods described herein, 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 a human cell.

[0229] In some embodiments, according to any of the methods for editing the cell genome described herein, the method uses a nucleic acid encoding a DNA endonuclease. In some embodiments, the nucleic acid encoding the DNA endonuclease has codons optimized for expression in the cell. In some embodiments, the cell is a human cell, for example, a human CD34 cell. +It 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 mRNA).

[0230] In some embodiments, according to any of the methods for editing the cell genome described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor template is configured such that the donor cassette is incorporated by homologous recombination repair (HDR) into the genomic locus targeted by the gRNA of (b) above. In some embodiments, homologous arms corresponding to the sequence of the targeted genomic locus are positioned on either side of the donor cassette. In some embodiments, the length of the homologous arm is at least 0.2kb or at least about 0.2kb (for example, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb or at least more, or at least about 0.3kb, at least about 0.4kb, at least about 0.5kb, at least about 0.6kb, at least about 0.7kb, at least about 0.8kb, at least about 0.9kb, at least about 1kb or at least more). In some embodiments, the length of the homologous arm is at least 0.8kb or at least about 0.8kb. Typical homologous arms include homologous arms contained in a donor mold having sequence number 34 or 161. Typical donor molds include donor molds having sequence number 34 or 161. In some embodiments, the donor template is encoded in 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 cell genome described herein, the donor template comprises a donor cassette containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof, wherein the donor template is configured to be incorporated by non-homologous end joining (NHEJ) into the genomic locus targeted by the gRNA of (b) above. In some embodiments, gRNA target sites are located adjacent to one or both sides of the donor cassette. In some embodiments, gRNA target sites are located adjacent to both sides of the donor cassette. In some embodiments, the gRNA target sites are target sites of the gRNA included in the system. In some embodiments, the gRNA target sites of the donor template are the reverse complementary strand of the cellular genome gRNA target site targeted by the 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 for editing the cell genome described herein, the DNA endonuclease or the nucleic acid encoding the DNA endonuclease is formulated by encapsulation in liposomes or lipid nanoparticles. In some embodiments, the liposomes or lipid nanoparticles further comprise gRNA. In some embodiments, the liposomes or lipid nanoparticles are lipid nanoparticles. In some embodiments, the method uses lipid nanoparticles containing a DNA endonuclease and a nucleic acid encoding gRNA. In some embodiments, the nucleic acid encoding the DNA endonuclease is mRNA encoding the DNA endonuclease.

[0233] In some embodiments, according to any of the methods for editing the cell genome described herein, the DNA endonuclease is pre-complexed with gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex is provided to the cells by electroporation. In some embodiments, the donor template is an AAV donor template encoded in an AAV vector (e.g., an AAV6 vector). In some embodiments, the AAV donor template is provided to the cells at the same time as, or approximately at the same time as, the RNP complex. For example, in some embodiments, the RNP complex is electroporated into the cells, and the AAV donor template is transduced on the same day. In some embodiments, the RNP complex is electroporated into the cells, and the AAV donor template is transduced, with a time difference of 12 hours or less or approximately 12 hours (for example, 11 hours or less or approximately 11 hours, 10 hours or approximately 10 hours, 9 hours or approximately 9 hours, 8 hours or approximately 8 hours, 7 hours or approximately 7 hours, 6 hours or approximately 6 hours, 5 hours or approximately 5 hours, 4 hours or approximately 4 hours, 3 hours or approximately 3 hours, 2 hours or approximately 2 hours, 1 hour or approximately 1 hour, or less). In some embodiments, after electroporating the RNP complex into the cells, the cells are seeded, and the cells are transduced using 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 (e.g., UM171 or StemRegenin(SR1)) that can promote the proliferation or self-renewal of HSCs before providing RNP and AAV donor templates.In some embodiments, pre-stimulation is carried out over a period of at least 12 hours or at least about 12 hours (for example, over a period of 16 hours or about 16 hours, 20 hours or about 20 hours, 24 hours or about 24 hours, 36 hours or about 36 hours, 48 ​​hours or about 48 hours, or longer). In some embodiments, pre-stimulation is carried out over a period of at least 48 hours or at least about 48 hours. In some embodiments, pre-stimulation is carried out in a cell composition containing the cells, and the concentration of the cells in the cell composition and / or culture medium is at least 10% or at least about 10% of the cells in the cell composition (for example, at least 20% or at least about 20%, at least 30% or at least about 30%, at least 40% or at least about 40%, or at least 50% or at least about 50%), but at a concentration that allows the cells to remain in a quiescent state after the completion of pre-stimulation. In some embodiments, 10% to 60% or about 10% to about 60% (for example, 10% to 50% or about 10% to about 50%, 10% to 40% or about 10% to about 40%, or 10% to 30% or about 10% to about 30%) of the cells in the cell composition remain at rest after the completion of pre-stimulation. In some embodiments, the concentration of the cells in the cell composition is 5 × 10⁻¹⁰. 5 Less than 5 pieces / ml or approximately 5 x 10 5 less than or equal to 4 x 10 5 Less than 10 pieces / ml or approximately 4 x 10 5 pcs or less, 3×10 5 Less than 10 cells / ml or approximately 3 x 10 5 pcs or less, 2.5×10 5 Less than 10 pieces / ml or approximately 2.5 x 10 5 pcs or less, 2×10 5 Less than 10 cells / ml or approximately 2 x 10 5 pcs or less, 1×10 5 Less than 1 / ml or approximately 1 x 10 5 pcs or less, 0.5×10 5 Less than 10 cells / ml or approximately 0.5 x 10 5The concentration is less than or equal to 10¹⁶ cells. In some embodiments, the concentration of the cells in the cell composition is 2.5 × 10¹⁶ cells. 5 Less than or equal to 10 cells / ml or approximately 2.5 x 10 5 The number of particles per ml is less than or equal to 1 / ml.

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

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

[0236] In a first embodiment (but not limited to) of such target sequence selection, many endonuclease systems have rules or criteria for initially selecting a cleavable target site, for example, CRISPR type II or V endonucleases require specific requirements for PAM sequence motifs at particular positions adjacent to the DNA cleavage site.

[0237] In another embodiment (but not limited to) relating to the selection or optimization of target sequences, the frequency of “off-target” activity (e.g., the frequency of double-strand breaks occurring at sites other than the selected target sequence) with a particular combination of target sequence and gene-editing endonuclease is evaluated relative to the frequency of on-target activity. In some cases, cells precisely edited at a desired locus may have a selective advantage compared to other cells. Specific examples of selective advantages include, but are not limited to, the acquisition of various attributes such as improved replication rate, persistence, tolerance to specific conditions, improved engraftment success or persistence after in vivo introduction into a target, and other attributes related to maintaining or improving the number or viability of such cells. In another case, cells precisely edited at a desired locus may be selected as positive cells by one or more screening methods used for the identification, sorting, or other selection purposes of precisely edited cells. Selective advantages and targeted selection methods can utilize phenotypes associated with the modification. In some embodiments, cells may be edited two or more times to produce a second modification for creating a novel phenotype used for the selection or purification of an intended cell population. Such a second modification can be made by adding a second gRNA for a selection marker or screening marker. In some cases, a DNA fragment containing cDNA and a selection marker can be used to precisely edit cells at the desired locus.

[0238] In embodiments, regardless of whether selective advantages or targeted selection are applicable in particular cases, target sequences are selected considering off-target frequency to improve the effectiveness of applying selective advantages or targeted selection and / or reduce the possibility of undesirable modifications at sites other than the desired target. As further described and illustrated herein and in the Art, off-target activity arises under the influence of various factors, such as the similarities and differences between the target site and various off-target sites, and the specific endonuclease used. Bioinformatics tools are available to assist in predicting off-target activity, and in many cases, such tools can be used to identify the sites most likely to occur, and such predictions and identifications can be experimentally evaluated to estimate the relative frequency of off-target activity to on-target activity, thereby enabling 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 relates to homologous recombination events. Sequences sharing homologous regions can play a central role in homologous recombination events that delete intervening sequences. Such recombination events occur during the normal replication process of chromosomes and other DNA sequences, as well as at other points in DNA sequence synthesis, such as during double-strand break repair, which occurs regularly in the normal cell replication cycle. However, they can also be enhanced by various events (such as UV light and other DNA break inducers) or the presence of certain drugs (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 even in normal cells. During double-strand break repair, the original sequence can be reconstructed with complete fidelity, but in some cases, short insertions or deletions (called "indels") may be introduced at the DSB site.

[0240] Furthermore, as can be seen with the endonuclease systems described herein, double-strand breaks (DSBs) can be specifically induced at particular locations, which can then be used to trigger directed or selective recombination events at selected chromosomal locations. The tendency for homologous sequences to be recombined in DNA repair (and replication) can be utilized in a variety of situations and forms one basis for the application of gene editing systems such as CRISPR, in which the target sequence provided by a "donor" polynucleotide is inserted into a specific location on the desired chromosome by homologous recombination repair.

[0241] Desired deletions can be created using homologous regions between specific sequences, and these homologous regions can be short "microhomology" regions, sometimes as short as 10 base pairs or less. For example, a single double-strand break (DSB) is introduced at a site that exhibits microhomology with a neighboring sequence. In the normal repair process of such DSBs, deletion of the intervening sequence occurs frequently, which results from recombination facilitated by the DSB and its associated cellular repair processes.

[0242] However, in some situations, selecting a target sequence within a homologous region may result in much larger deletions, such as gene fusion (deletion within the coding region), and such results may or may not be desirable in certain circumstances.

[0243] The examples provided herein further illustrate the selection of various target regions for constructing double-stroke bonds (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, target loci are selected from the FOXP3 gene, the AAVS1 locus, and the TRA gene.

[0244] Nucleic acid modification In some embodiments, as described in detail herein and as known in the art, the polynucleotides introduced into cells have one or more modifications that can be used individually or in combination for, for example, enhancing activity, stability or specificity, altering delivery, reducing or otherwise enhancing the innate immune response of the host cell.

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

[0246] When using the CRISPR / Cas system for such an example (but not limited to) applications, modifying the guide RNA can improve the formation or stability of the CRISPR / Cas genome editing complex containing the guide RNA, which may be formed from a single-molecule guide or a bi-molecule guide and a Cas endonuclease. Alternatively, modifying the guide RNA can improve the initiation, stability, or dynamics 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, modifying the guide RNA can improve specificity, for example, by improving the relative genome editing rate at the on-target site compared to the effect at other sites (off-target).

[0247] Alternatively, or in addition to the above, the stability of guide RNA can be improved by modifying its resistance to degradation by ribonucleases (RNases) present in cells, thereby extending the half-life of the guide RNA in cells. Modifications that extend the half-life of guide RNA may be particularly useful in embodiments in which Cas endonucleases are introduced into cells and edited using RNA that needs to be translated to produce endonucleases, because the extended half-life of the guide RNA introduced simultaneously with the endonuclease-encoding RNA allows for an extension of the time that the guide RNA and the encoded Cas or Cpf1 endonuclease coexist in the cell.

[0248] Alternatively, or in addition to the foregoing, modifications can be utilized to reduce the likelihood or extent to which RNA introduced into cells induces an innate immune response. As will be discussed later herein and as is known in the art, such immune responses have been well evaluated with respect to RNA interference (RNAi), such as small interfering RNAs (siRNAs), and tend to be associated with shortening of the RNA half-life and / or induction of cytokines or other factors related to the immune response.

[0249] It is also possible to modify the RNA encoding the endonuclease introduced into the cell with one or more modifications, including, but not limited to, modifications that improve the stability of the RNA (such as modifications that increase degradation by RNAse present in the cell), modifications that enhance the translation of the product (e.g., endonuclease), and / or modifications that reduce the likelihood or degree to which the RNA introduced into the cell induces an innate immune response.

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

[0251] delivery In some embodiments, nucleic acid molecules used in the methods provided herein, for example, nucleic acids encoding the genome-targeted nucleic acids or site-directed polypeptides of this disclosure, are packaged inside or on the surface of a delivery carrier for delivery to cells. Possible delivery carriers include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, or micelles. Various targeting moieties can be used to enhance the selective interaction between such carriers and desired cell types or locations, as reported in the Art.

[0252] The introduction of the complexes, polypeptides, or nucleic acids of this disclosure into cells can be carried out by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, or nucleic acid delivery via nanoparticles.

[0253] In some embodiments, guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) can be delivered by a delivery carrier using a virus known in the art or a non-virus delivery carrier. Alternatively, one or more endonuclease polypeptides can be delivered by a delivery carrier using a virus known in the art or a non-virus delivery carrier, such as electroporation or lipid nanoparticles. In some embodiments, DNA endonucleases 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, polynucleotides can be delivered by non-viral delivery carriers, including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. Several specific examples of non-viral delivery carriers are described in Peer, D. et al. (2011). Gene Therapy, 18: 1127-1133 (this document focuses on non-viral delivery carriers for siRNA, which are also useful for the delivery of other polynucleotides).

[0255] In embodiments, polynucleotides such as guide RNA, sgRNA, or mRNA encoding an endonuclease can be delivered to cells or targets by lipid nanoparticles (LNPs).

[0256] While non-viral nucleic acid delivery methods have been tested in both animal and human models, the most well-developed system is lipid nanoparticles. Lipid nanoparticles (LNPs) generally consist of an ionizable cationic lipid and three or more additional components, which are typically cholesterol, DOPE, and lipid-containing polyethylene glycol (PEG) (see, for example, Example 2). The cationic lipid can bind to positively charged nucleic acids to form a dense complex that protects the nucleic acid from degradation. As they pass through a microfluidic system, each component self-assembles to form particles 50–150 nM in size, with the nucleic acid encapsulated in a core complexed with the cationic lipid and surrounded by a lipid bilayer-like structure. After injection into the target circulatory system, these particles can bind to apolipoprotein E (apoE), a ligand for the LDL receptor, which mediates uptake into hepatocytes of the liver via receptor-mediated endocytosis. This type of LNP has been shown to efficiently deliver mRNA and siRNA to hepatocytes of the liver in rodents, primates, or humans. After endocytosis, LNPs reside in endosomes. The encapsulated nucleic acid escapes the endosome due to the ionizable properties of cationic lipids. This allows the nucleic acid to be delivered to the cytoplasm, where the mRNA is translated into the encoded protein. After escaping from the endosome, Cas mRNA (e.g., Cas9 mRNA) can be translated into Cas protein and form a complex with gRNA. In some embodiments, nuclear translocation of the Cas protein / gRNA complex is facilitated by including a nuclear localization signal in the Cas protein sequence. Alternatively, a small gRNA can pass through the nuclear pore complex and form a complex with Cas protein in the nucleus. Once in the nucleus, the gRNA / Cas complex scans the genome for homologous target sites and selectively generates double-strand breaks at the desired target sites in the genome. The half-life of RNA molecules in vivo is generally short, ranging from a few hours to a few days. Similarly, the half-life of proteins also tends to be short, ranging from a few hours to a few days.Therefore, in some embodiments, delivery of gRNA and Cas mRNA using LNPs can result in only transient expression and activity of the gRNA / Cas complex. This can offer the advantage of reducing the frequency of off-target cleavage and, therefore, minimizing the risk of genotoxicity in some embodiments. LNPs are generally less immunogenic than viral particles. While many humans already have immunity to AAV, they do not have pre-existing immunity to LNPs. Furthermore, the likelihood of an adaptive immune response to LNPs is low, allowing for 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, and MD1. In one type of LNP, the GalNac moiety is attached to the outside of the LNP and functions as a ligand for liver uptake via the asialoglycoprotein receptor. LNPs are formulated using one of these cationic lipids to deliver gRNA and Cas mRNA to the liver.

[0258] In some embodiments, LNPs refer 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 be in the range of 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 prepared from cationic lipids, anionic lipids, or neutral lipids. Neutral lipids such as the fusion phospholipid DOPE and the membrane component cholesterol can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to low stability and rapid clearance, and the possibility of inducing inflammatory or anti-inflammatory reactions. LNPs can also contain hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0260] Any lipid or combination known in the art can be used to prepare LNPs. Examples of lipids used to prepare 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 this embodiment, LNPs can be prepared by combining multiple types of lipids in any molar ratio. Furthermore, LNPs can also be prepared by combining single or multiple polynucleotides with single or multiple lipids in various molar ratios.

[0262] In embodiments, site-directed polypeptides and genome-targeted nucleic acids can be administered separately to cells or targets. Alternatively, site-directed polypeptides can be pre-complexed with one or more guide RNAs, or with tracrRNA and one or more crRNAs. The pre-complexed material can then be administered to cells or targets. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs).

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

[0264] In some embodiments, the endonuclease contained in the RNP may or may not be modified. Similarly, the gRNA, crRNA, tracrRNA, or sgRNA may or may not be modified. Various modifications are known in the art and can be used.

[0265] Endonucleases and sgRNAs can typically be combined in a 1:1 molar ratio. Alternatively, endonucleases, crRNAs, and tracrRNAs can typically be combined in a 1:1:1 molar ratio. However, RNPs can be prepared using various molar ratios.

[0266] In some embodiments, delivery can be carried out using recombinant adeno-associated virus (AAV) vectors. Techniques for producing rAAV particles to provide cells with the functions of an AAV genome and a helper virus, packaged to contain the polynucleotides, rep gene, and cap gene to be delivered, are known in the art. The production of rAAV requires the presence of the functions of the rAAV genome, the AAV rep gene and cap gene isolated from this rAAV genome (e.g., not contained internally), and the helper virus within a single cell (referred to herein as a packaging cell). The AAV rep and cap genes may be derived from a serotype of AAV that allows for the creation of recombinant viruses, or from a serotype of AAV different from the ITR in the rAAV genome. Examples of AAV serotypes include, but are 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 creation of pseudotyped rAAVs is disclosed, for example, in international patent application WO01 / 83692. Table 1 shows the AAV serotypes and Genbank accession numbers of several selected AAVs. [Table 2]

[0267] In some embodiments, the method for producing packaging cells involves creating a cell line that stably expresses all the components necessary for producing AAV particles. For example, a single plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, and AAV rep and cap genes separate from this rAAV genome, along with a selection marker such as a neomycin resistance gene, is incorporated into the cell genome. The AAV genome is introduced into a bacterial plasmid by methods such as GC tailing (Samulski, RJ et al. (1982). Proc. Natl. Acad. Sci. USA, 79(6):2077-2081), addition of a synthetic linker 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 cell selection and the suitability of the cells for mass production of rAAV. Another preferred method involves introducing the rAAV genome, as well as the rep and cap genes, into packaging cells using adenovirus or baculovirus instead of plasmids.

[0268] General principles for the production of rAAV are reviewed, for example, in 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; US Patent No. 5,173,414; WO 95 / 13365 and its corresponding US Patent 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; as described in U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595.

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

[0270] In some embodiments, the AAV vector includes 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 with any one of sequence numbers 33-36 and 161. In some embodiments, the AAV vector includes 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 with sequence number 33. In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 34 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 35 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 36 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more). In some embodiments, the AAV vector includes a nucleic acid sequence having at least 90% or at least about 90% sequence identity with sequence number 161 (for example, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more).

[0271] In addition to adeno-associated virus vectors, other viral vectors can also be used. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpesviruses, Epstein-Barr virus, papovaviruses, poxviruses, vaccinia viruses, and herpes simplex viruses.

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

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

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

[0275] In some embodiments relating to the delivery of genome editing components for therapeutic procedures, at least two components, namely a sequence-specific nuclease and a DNA donor template, are used to transform cells (e.g., CD34). + It is delivered into the nucleus of the cell. In some embodiments, the AAV is selected from the serotypes AAV2 or AAV6. In some embodiments, the DNA donor template packaged in the AAV is first administered to the subject (e.g., patient) by peripheral intravenous injection, followed by the administration of a sequence-specific nuclease. The advantage of first delivering the donor DNA template packaged in the AAV is that the delivered donor DNA template is transduced CD34 +The sequence-specific nuclease is stably maintained within the cell nucleus, enabling the subsequent administration of the sequence-specific nuclease, which in turn creates double-strand breaks in the genome, leading to the integration of the DNA donor via HDR or NHEJ. In some embodiments, it is desirable that the sequence-specific nuclease maintains its activity in the target cell for the duration required to promote 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 prolonged period, this increases the frequency of off-target double-strand breaks. Specifically, the frequency of off-target breaks is a function of the off-target break efficiency multiplied by the duration of nuclease activity. Because mRNA and the proteins translated from it are short-lived in cells, delivery of the sequence-specific nuclease in mRNA form results in a short duration of nuclease activity, ranging from a few hours to several days. Therefore, it is expected that delivery of the sequence-specific nuclease to cells already containing the donor template will 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 consisting of a sgRNA and Cas endonuclease directed to the FOXP3 gene. In some embodiments, the Cas endonuclease is delivered as 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 in lipid nanoparticles to CD34 + Cells (for example, CD34) + It is delivered to hematopoietic stem cells.

[0277] In some embodiments, to promote the nuclear localization of the donor template, a 366 bp region consisting of the origin of replication and initial promoter of Simian virus 40 (SV40), which can promote plasmid nuclear localization, can be added to the donor template. Other DNA sequences that bind to cellular proteins can also be used to improve DNA nuclear translocation.

[0278] Genetically modified cells and genetically modified cell populations In one embodiment, the disclosure herein provides a method for producing genetically modified cells by editing the genome of cells. In several embodiments, a population of genetically modified cells is provided. Thus, genetically modified cells refer to cells having at least one genetic modification introduced by genome editing (e.g., genome editing using the CRISPR / Cas system). In some embodiments, genetically modified cells are genetically modified hematopoietic stem cells, for example, genetically modified CD34 + Recombinant CD34 cells in hematopoietic stem cells, etc. + These are cells. Genetically modified cells having an incorporated FOXP3 coding sequence are assumed herein. In some embodiments, the genetically modified cells are not germ cells.

[0279] In the embodiments described herein, FOXP3 is stably expressed by modifying the regulatory elements of the FOXP3 gene using a gene-editing nuclease, thereby creating therapeutic cells that stably express FOXP3. In the representative data provided herein, FOXP3 expression was induced by placing a promoter (examples of constitutive promoters include the EF1α promoter, PGK promoter, or MND promoter) upstream of the coding exon of FOXP3. However, it is assumed that FOXP3 can also be stably expressed by modifying the regulatory elements using various other approaches. By modifying the endogenous regulatory elements using several approaches, the endogenous FOXP3 gene can be constitutively expressed in the therapeutic cells of the present invention, resulting in a loss of sensitivity to regulation that causes silencing of the FOXP3 gene or conversion to a non-repressive cell phenotype. Therefore, the typical method described herein solves the problem of deletion of FOXP3 expression due to epigenetic effects on endogenous regulatory sequences and endogenous promoters.

[0280] CD34 + Methods for enhancing FOXP3 expression in bulk cell populations are also anticipated. The endogenous TCR repertoire of inflammatory T cell populations in subjects with autoimmune diseases or organ graft rejection exhibits TCRs with specificity to recognize and precisely bind to inflamed or foreign tissues within organs. Such T cells are thought to mediate autoinflammatory responses or organ rejection. By converting a portion of the bulk T cell population to a regulatory phenotype, the TCR specificity of the pro-inflammatory population can be utilized in a therapeutically effective cell population. In this respect, the method of the present invention is superior to other therapies utilizing thymus-derived regulatory T cells (which are thought to have a different TCR repertoire that does not overlap with inflammatory T cells). Furthermore, in subjects with autoimmune diseases or organ rejection, it is likely that the in vivo tT regThe group is thought to fail in inducing the immune tolerance necessary for avoiding inflammation. The methods described herein can be used for the treatment of autoimmune diseases and for inducing immune tolerance to transplanted organs.

[0281] As a significant drawback, it is necessary to use a gene editing tool that can efficiently perform recombination in the FOXP3 gene. In the methods provided herein, it has been shown that this reaction can be efficiently carried out using TALEN nuclease. In principle, however, it is considered that any nuclease platform can perform recombination similarly and well.

[0282] Regulatory T cell therapy can be used to induce immune tolerance in transplantation and autoimmune diseases. Currently, T reg infusions are expanded ex vivo. In the phase 1 trial, only limited efficacy has been observed against type 1 diabetes (T1D), but there have also been some cases where a benefit has been observed against GvHD after transplantation. In some embodiments, the next generation of genetically engineered regulatory T cells are endogenous T reg that are directed by a chimeric antigen receptor (CAR). By expressing FOXP3, effector T cells can also be converted into T reg .

[0283] However, there are thought to be some differences between the endogenous T reg used in the treatment method and the genetically engineered T reg . The endogenous T reg therapy is considered safe, but because the number of endogenous T reg is too small, it causes autoimmunity. Also, T reg plays an important role in various autoimmune diseases (IPEX, T1D, SLE, RA, EAE, etc.). Various approaches to enhancing the number or function of human T reg cells are currently in clinical trials. For example, low-dose IL-2 and autologous T regA treatment method combined with adoptive transfer is in clinical trials. IL-2 therapy has limited effectiveness because of its multifaceted activities and potential "off-target" effects that can enhance inflammation. Also, adoptive transfer of T reg therapy also has problems with the stability and viability of expanded T reg in vivo and lacks the antigen specificity useful for treatment, so it can probably be used only limitedly.

[0284] Endogenous T reg (nT reg ) also has potential drawbacks in its use. For example, subjects with autoimmune diseases may have a genetic predisposition to unstable T reg . For example, conversion from CAR-expressing nT reg to CAR-expressing effector T cells can occur. Furthermore, nT reg may be epigenetically regulated by FOXP3, so the induction of the desired FOXP3 may be downregulated. Furthermore, endogenous T reg may not have the exact specificity for TCR (T cell receptor). The function of T reg is also related to selection markers, and it is considered that inflammatory cells are always mixed in the expanded population of endogenous T reg cells. Therefore, the method provided in this specification uses recombinant cells, so it can induce the function of regulatory T cells by the expression of CAR and can avoid the possibility that engrafted CAR T reg is converted into inflammation-promoting CAR T cells. In this regard, it is an improved method compared to the method of transplanting natural T reg cells.

[0285] In some embodiments, the genome of a cell can be edited by inserting a nucleic acid sequence encoding FOXP3 or a functional derivative thereof into the cell's genome sequence. In some embodiments, the cells targeted for genome editing have one or more mutations in their genome that reduce the expression of the endogenous FOXP3 gene compared to its expression in normal cells that do not have the mutation. Normal cells may be healthy cells or control cells derived from (or isolated from) another subject that does not have the FOXP3 gene abnormality. In some embodiments, the cells targeted for genome editing may be cells derived from (or isolated from) a subject that requires treatment for a condition or disorder related to the FOXP3 gene. Therefore, in some embodiments, the expression of the endogenous FOXP3 gene in such cells is increased by 10% 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 the expression of the endogenous FOXP3 gene in normal cells.

[0286] For example, if the insertion of a transgene such as a nucleic acid encoding FOXP3 or a functional derivative is successful, the expression of the transgenerated nucleic acid encoding FOXP3 or a functional derivative in cells will be at least 10% or at least about 10%, at least 20% or at least about 20%, at least 30% or at least about 30%, at least 40% or at least about 40%, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 70% or at least about 70%, at least 80% or at least about 80%, at least 90% or at least about 90%, at least 100% or at least about 100%, and at least 200% of the expression of the endogenous FOXP3 gene in cells. Or it may be at least approximately 200%, at least 300% or at least approximately 300%, at least 400% or at least approximately 400%, at least 500% or at least approximately 500%, at least 600% or at least approximately 600%, at least 700% or at least approximately 700%, at least 800% or at least approximately 800%, at least 900% or at least approximately 900%, at least 1,000% or at least approximately 1,000%, at least 2,000% or at least approximately 2,000%, at least 3,000% or at least approximately 3,000%, at least 5,000% or at least approximately 5,000%, at least 10,000% or at least approximately 10,000%, or more.In some embodiments, the activity of the introduced FOXP3 coding sequence product (including functional derivatives of FOXP3) in genome-edited cells is at least 10% or at least about 10%, at least 20% or at least about 20%, at least 30% or at least about 30%, at least 40% or at least about 40%, at least 50% or at least about 50%, at least 60% or at least about 60%, at least 70% or at least about 70%, at least 80% or at least about 80%, at least 90% or at least about 90%, at least 100% or at least about 100%, at least 200% or at least about 200%, at least 300% or at least about 300%, at least 400% or at least about 400%, at least 500% or at least about 500%, at least 600% or at least about 600%, at least 700% or at least about 700%, at least 800% or at least about 800%, at least 900% or at least about 900%, at least 1,000% or at least about 1,000%, at least 2,000% or at least about 2,000%, at least 3,000% or at least about 3,000%, at least 5,000% or at least about 5,000%, at least 10,000% or at least about 10,000%, or even more.In some embodiments, the expression of the introduced FOXP3 coding sequence in cells is at least twice or about twice, at least three times or about three times, at least four times or about four times, at least five times or about five times, at least six times or about six times, at least seven times or about seven times, at least eight times or at least about eight times, at least nine times or at least about nine times, at least ten times or at least about ten times, at least fifteen times or at least about fifteen times, at least twenty times or at least about twenty times, at least thirty times or at least about thirty times, at least fifty times or at least about fifty times, at least 100 times or at least about 100 times, at least 1000 times or at least about 1000 times, or more. Furthermore, in some embodiments, the activity of the introduced FOXP3 coding sequence product (including functional derivatives of FOXP3) in genome-edited cells may be comparable to, or even exceed, the activity of the endogenous FOXP3 gene product in normal, healthy cells.

[0287] In one embodiment, CD34 in Exvivo + By genetically modifying cells and then reintroducing them into a target organism, genetically modified T cells expressing the inserted FOXP3 gene are produced in the target organism.

[0288] Manufacturing method In some embodiments, a method for producing genetically modified cells, CD34 containing the first nucleic acid which includes at least one gene locus + The process of supplying cells; A step of providing a Cas endonuclease (e.g., Cas9 endonuclease) or a second nucleic acid encoding the Cas endonuclease; The Cas endonuclease or the second nucleic acid is the CD34 + The process of introducing the substance into cells; A step of introducing a third nucleic acid encoding at least one gRNA configured to hybridize to at least one gene locus, or a set of nucleic acids encoding said at least one gRNA; and The fourth nucleic acid, which includes a gene delivery cassette, is the CD34. + The process of introducing the cells This provides a method that includes this.

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

[0290] In some embodiments, according to the method for producing genetically modified cells provided herein, the at least one gene locus is the FOXP3 gene, the AAVS1 gene locus, or the TRA gene.

[0291] In some embodiments, a second nucleic acid, a third nucleic acid, the set of nucleic acids and / or a fourth nucleic acid are provided incorporated into 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 mRNA. In some embodiments, the at least one gRNA includes a spacer sequence containing the sequence shown in SEQ ID NOs: 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 includes a sequence encoding a FOXP3 cDNA sequence optimized for human codons. In some embodiments, the fourth nucleic acid further includes 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 includes a sequence encoding a low-affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB, or LNGFR (a sequence encoding the LNGFR epitope). In some embodiments, the fourth nucleic acid further comprises a sequence encoding a low-affinity nerve growth factor receptor (LNGFR) or LNGFRe (a sequence encoding an LNGFR epitope).

[0293] In some embodiments, the method includes a second gene delivery cassette containing a fifth nucleic acid on the CD34 +The process further includes the step of introducing the substance into cells. In some embodiments, the fifth nucleic acid is contained in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid includes sequences encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid includes sequences encoding a marker protein. In some embodiments, the fourth sequence and / or the fifth sequence further include sequences encoding a P2A self-cleaving peptide. In some embodiments, the fourth sequence and / or the fifth sequence further include sequences encoding a poly-A sequence. In some embodiments, the poly-A sequence includes the 3'UTR of SV40 poly-A or FOXP3. In some embodiments, the fourth sequence includes the sequence shown in any of SEQ ID NOs. 37-42.

[0294] In some embodiments, the fourth and fifth arrays are the CD34 + The fourth and fifth sequences are introduced into cells and include sequences encoding an expression cassette configured to express FOXP3cDNA-LNGFR and DISC, FOXP3cDNA-LNGFR and μDISC, LNGFR-FOXP3cDNA and DISC, LNGFR-FOXP3cDNA and μDISC, CISCβ-DN and CISCγ-FOXP3cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3cDNA.

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

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

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

[0298] In some embodiments, the method involves a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3, which is then used in the CD34 + This further includes the process of introducing the cells into the cell.

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

[0300] In some embodiments, the culture medium containing hTPO, hFlt3, hSCF, or hIL6 contains the CD34 + Bring the cells into contact.

[0301] In some embodiments, CD34 for FOXP3 expression is produced by the method described in any one of the embodiments described herein. + The cells are provided. In some embodiments, the FOXP3 is expressed constitutively or under controlled conditions.

[0302] In some embodiments, CD34 for FOXP3 expression contains nucleic acid encoding the gene encoding FOXP3. + Cells are provided. In some embodiments, the gene encoding FOXP3 is introduced into the FOXP3 gene locus or a non-FOXP3 gene locus. In some embodiments, the non-FOXP3 gene locus is the AAVS1 gene 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. In some embodiments, the CD34 + Cells are, T regIncludes phenotype.

[0304] In some embodiments, the CD34 described in any one of the embodiments above + The present invention provides a composition containing cells.

[0305] In some embodiments, a method for treating, alleviating and / or suppressing a disease and / or condition in a subject, wherein the subject having the disease and / or condition is subjected to any one of the embodiments described herein by CD34 + A method is provided comprising the step of providing cells or a composition. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is an (IPEX) syndrome characterized by immune dysregulation, polyglandular endocrine disorders, intestinal diseases and X-linking. In some embodiments, the condition is graft-versus-host disease (GVHD).

[0306] In some embodiments described herein, a method for producing genetically modified cells, CD34 containing the first nucleic acid which includes at least one gene locus + The process of supplying cells; A step of providing a Cas endonuclease (e.g., Cas9 endonuclease) or a second nucleic acid encoding the Cas endonuclease; The Cas endonuclease or the second nucleic acid is the CD34 + The process of introducing the substance into cells; The steps of introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one gene locus, or a set of nucleic acids encoding said at least one CRISPR spacer sequence; and The fourth nucleic acid, which includes a gene delivery cassette, is the CD34. + The process of introducing the cells This provides a method that includes this. In some embodiments, the fourth nucleic acid further comprises a promoter. In some embodiments, the promoter is a MND promoter, a PGK promoter, or an E2F promoter. In some embodiments, the promoter is a MND promoter. As described in the embodiments described herein, the MND promoter is provided incorporated into vector #3008 (pAAV_FoxP3.0.6kb.MND.GFP.WPRE3.pA) (SEQ ID NO: 33).

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

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

[0309] Therapeutic approach One embodiment provided herein is a gene therapy approach for providing treatment to a subject having or suspected of having a disorder or health condition related to the FOXP3 protein by editing the genome of said subject. For example, in some embodiments, said disorder or health condition is an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from organ transplantation (e.g., GVHD). In some embodiments, said gene therapy approach incorporates a nucleic acid containing a sequence encoding a functional FOXP3 gene into the genome of a relevant cell type of the subject, thereby curing said disorder or health condition. In some embodiments, the cell type targeted by the gene therapy approach incorporating the FOXP3 encoding sequence is CD34 + Cells, for example, CD34 + These are hematopoietic stem cells, and the reason for this is that these cells can efficiently differentiate into T cells in the target environment.

[0310] In another embodiment, an ex vivo and in vivo intracellular method is provided for causing a permanent change in a cell's genome by using genome engineering tools to knock in a coding sequence encoding FOXP3 or a functional derivative thereof into a locus in the cell's genome, thereby restoring FOXP3 activity. Such a method uses an endonuclease, such as a CRISPR-related nuclease (CRISPR / Cas9, Cpf1, etc.), to permanently delete, insert, edit, modify, or replace any sequence from the cell's genome, or to insert an exogenous sequence (e.g., a sequence encoding FOXP3) into a cellular genomic locus. Thus, as illustrated in the examples described herein, FOXP3 activity is restored in a single therapeutic step (without requiring the delivery of alternative therapies throughout the subject's lifetime).

[0311] In some embodiments, the ex vivo cell-based therapy involves CD34 isolated from the subject. + Cells, for example, CD34 derived from umbilical cord blood +The procedure is carried out using cells. Next, the chromosomal DNA of the cells is edited using the systems, compositions, and methods described herein. Finally, the edited cells are transplanted into the subject.

[0312] One advantage of the ex vivo cell therapy approach is that a comprehensive analysis of the therapeutic agent can be performed before administration. Nuclease-based therapies always come with some degree of off-target effects. By performing gene modification ex vivo, the characteristics of the modified cell population can be comprehensively confirmed before transplantation. Aspects of this disclosure include sequencing the entire genome of the modified cells and, if off-target cuts are present, confirming that these cuts are located in genomic locations that minimize risk to the target. Furthermore, specific cell populations, such as clonal populations, can be isolated before transplantation.

[0313] Another embodiment of such a method is an in vivo treatment. In this method, 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 the ease of manufacturing and administering the drug. The same therapeutic approach and method can be used to treat one or more subjects, for example, multiple subjects with the same or similar genotype or allele. In contrast, ex vivo cell therapy typically uses the subject's own cells, isolating them, genetically modifying them, and then returning them to the same subject.

[0315] In some embodiments, subjects requiring the treatment described herein are subjects having symptoms of a foxp3-related disease or foxp3-related condition. For example, in some embodiments, the subjects have symptoms of an autoimmune disease (e.g., IPEX syndrome) or a disorder resulting from organ transplantation (e.g., GVHD). In some embodiments, the subjects may be humans suspected of having the disease or condition. Alternatively, the subjects may be humans diagnosed as being at risk of the disease or condition. In some embodiments, subjects requiring the treatment are subjects having one or more genetic abnormalities (e.g., deletions, insertions, and / or mutations) in the endogenous foxp3 gene or its regulatory sequence, resulting in significantly reduced foxp3 expression or functionality compared to normal healthy subjects.

[0316] In some embodiments, a method for treating or suppressing a foxp3-related disease or foxp3-related condition (e.g., autoimmune disease) in a subject, (a) Guide RNA (gRNA) that targets the FOXP3 gene in the cell genome; (b) DNA endonucleases, or nucleic acids encoding said DNA endonucleases; and (c) Donor template containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof. The present invention provides a method that includes the step of providing a substance to target cells. 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 of SEQ ID NOs: 1-7, 15-20, and 27-29.

[0317] In some embodiments, a method for treating or suppressing a foxp3-related disease or foxp3-related condition (e.g., autoimmune disease) in a subject, (a) gRNA containing a spacer sequence complementary to the genomic sequence within the endogenous FOXP3 gene of a cell or to the genomic sequence adjacent to the endogenous FOXP3 gene; (b) DNA endonucleases, or nucleic acids encoding said DNA endonucleases; and (c) Donor template containing a nucleic acid sequence encoding FOXP3 or a functional derivative thereof. The present invention provides a method that includes the step of providing a substance to target cells. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7 and 27-29, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7 and 27-29. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 1-7, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 1-7. In some embodiments, the gRNA includes a spacer sequence shown in any of SEQ ID NOs 2, 3, and 5, or a variant of the spacer sequence having three or fewer mismatches compared to any of SEQ ID NOs 2, 3, and 5. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 2, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 2. In some embodiments, the gRNA includes a spacer sequence shown in SEQ ID NOs 5, or a variant of the spacer sequence having three or fewer mismatches compared to SEQ ID NOs 5. In some embodiments, the cells are human cells, such as human stem cells, such as human CD34 + These are hematopoietic stem cells. In some embodiments, the subjects are patients who have or are suspected of having an autoimmune disease (e.g., IPEX syndrome) or graft-versus-host disease (GVHD). In some embodiments, the subjects are subjects diagnosed as being at risk of an autoimmune disease (e.g., IPEX syndrome) or graft-versus-host disease (GVHD).

[0318] In some embodiments, the present invention provides a method for treating or suppressing a foxp3-related disease or foxp3-related condition (e.g., an autoimmune disease) in a subject, comprising the step of providing a genetically modified cell prepared by one of the cell genome editing methods described herein. In some embodiments, the nucleic acid sequence encoding foxp3 or a functional derivative is expressed under the control of an endogenous foxp3 promoter. In some embodiments, the nucleic acid sequence encoding foxp3 or a functional derivative is codon-optimized for expression in the cell. In some embodiments, the cell is CD34 + The cells are autologous cells obtained from a subject. In some embodiments, the genetically modified cells are autologous cells obtained from a subject. In some embodiments, the method further comprises the step of obtaining a biological sample from the subject, the biological sample comprising input cells, and genetically modified cells being prepared from these input cells. In some embodiments, the input cells are CD34 + It is a cell.

[0319] Some embodiments include pharmaceuticals for use in the treatment or suppression of FOXP3-related diseases or FOXP3-related conditions (e.g., autoimmune diseases) in a subject. Another embodiment includes recombinant CD34 genome edited by any one of the methods described herein for use in the suppression or treatment of FOXP3-related diseases or FOXP3-related conditions such as inflammatory diseases or autoimmune diseases. + The invention relates to cells. Further embodiments include recombinant CD34 genome edited by any one of the methods described herein. + Regarding the use of cells as pharmaceuticals.

[0320] Cell transplantation to the target In some embodiments, the ex vivo methods of the present disclosure include transplanting genome-edited cells into a subject requiring such a method. This transplantation step can be carried out using any transplantation method known in the art. For example, the genetically modified cells can be injected directly into the subject's blood or administered to the subject by other means.

[0321] In some embodiments, the methods disclosed herein include “administration,” but this term can be used interchangeably with “introduction” and “transplantation,” 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 be localized to a desired site in order to obtain a desired single or multiple effect. The therapeutic cells or progeny cells differentiated therefrom can be administered via a preferred route that allows delivery to a desired site in the subject’s body in which at least a portion of the transplanted cells or at least a portion of their components can remain viable. The viability of the cells after administration to the subject may range from a short period of time (e.g., several hours to several days) to several years or even the lifetime of the subject (e.g., long-term engraftment).

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

[0323] In some embodiments, when recombinant stem cells are provided for therapeutic purposes, the recombinant stem cells are provided at (or after) the onset of symptoms or signs of a foxp3-related disease or foxp3-related condition (e.g., an autoimmune disease such as IPEX syndrome), for example, at the onset of the disease or condition.

[0324] The effective amount of therapeutic cells (e.g., genome-edited stem cells) used in the various embodiments described herein is at least 10 2 each, at least 5 × 10 2 pieces, at least 10 3 each, at least 5 × 10 3 pieces, at least 10 4 each, at least 5 × 10 4 pieces, at least 10 5 each, at least 2 × 10 5 each, at least 3 × 10 5 each, at least 4 × 10 5 each, at least 5 × 10 5 each, at least 6 × 10 5 each, at least 7 × 10 5 each, at least 8 × 10 5 pieces, at least 9 × 10 5 each, at least 1 × 10 6 each, at least 2 × 10 6 each, at least 3 × 10 6 each, at least 4 × 10 6 each, at least 5 × 10 6 each, at least 6 × 10 6 each, at least 7 × 10 6 each, at least 8 × 10 6 pieces, at least 9 × 10 6 The number of cells may be one or multiples thereof. The therapeutic cells may be derived from one or more donors or may be autologous. In some embodiments described herein, the therapeutic cells are cultured and grown before being administered to a subject requiring the administration of the therapeutic cells.

[0325] In some embodiments, a moderate and progressive increase in the expression of functional foxp3 in cells of subjects having a foxp3-related disease or foxp3-related condition (e.g., IPEX syndrome) may be beneficial in alleviating one or more symptoms of the disease or condition, improving long-term survival rates, and / or reducing side effects associated with other treatments. Administering such cells to human subjects is beneficial because it provides therapeutic cells that increase the production of functional foxp3. In some embodiments, effective treatment of a subject results in functional foxp3 accounting for at least 1%, 3%, 5%, or 7% or at least about 1%, about 3%, about 5%, or about 7% of the total foxp3 in the treated subject. In some embodiments, functional foxp3 accounts for at least 10% or at least about 10% of the total foxp3. In some embodiments, functional FOXP3 accounts for 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 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, even introducing a relatively small number of cells with significantly elevated levels of functional FOXP3 may be beneficial in a variety of subjects, because, in some situations, normalized cells may have a selective advantage over diseased cells. However, even a moderate number of therapeutic cells with increased levels of functional foxp3 may be beneficial in alleviating one or more aspects of the aforementioned disease or condition in the subject.In some embodiments, 10% 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 subjects to which such cells were administered showed increased production of functional FOXP3.

[0326] In embodiments, at least a portion of a therapeutic cell composition (e.g., a composition comprising multiple cells of any of the cells described herein) can be localized to a desired site by delivering the composition to a target by a specific method or route. The cell composition can be administered by any suitable route that provides effective treatment to the target, for example, by administration of the cell composition, at least a portion of the composition (e.g., at least 1 × 10⁶ cells) can be localized to a desired site. 4 Cells can be delivered to a desired site within the target body over a specified period of time. Methods of administration include injection, intravenous infusion, intra-infusion, and oral ingestion. "Injection" includes, but is not limited to, intravenous injection, intramuscular injection, intra-arterial injection, intracerebroventricular injection, intra-articular injection, intraorbital injection, intracardiac injection, intradermal injection, intraperitoneal injection, transtracheal injection, subcutaneous injection, subepidermal injection, intra-articular injection, subcapsular injection, subarachnoid injection, intraspinal injection, intracerebrospinal injection, and intrasternal injection, as well as intravenous infusion. In some embodiments, the route of administration is intravenous. Cell delivery can be performed by injection or intravenous infusion.

[0327] In one embodiment, the cells are administered systemically; in other words, the therapeutic cell population enters the target circulatory system and undergoes metabolism and other similar processes, rather than being directly administered to a target site, target tissue, or target organ.

[0328] The effectiveness of treatments using compositions for the treatment or suppression of FOXP3-related diseases or conditions (e.g., IPEX syndrome) can be determined by a skilled clinician. However, a treatment is considered effective if one or all of the signs or symptoms of the disease (e.g., the amount of functional FOXP3) are changed in a beneficial manner (e.g., by at least a 10% increase), or if other clinically recognized symptoms or markers are improved or alleviated. Effectiveness can also be measured by determining that the individual's deterioration has stopped (e.g., the progression of the disease has stopped or at least slowed) based on an assessment of the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatments include any treatment or suppression of a disease in an individual or animal (e.g., humans or mammals), including (1) suppression of the disease, e.g., cessation or delay of the progression of symptoms, or (2) alleviation of the disease, e.g., induction of symptom regression, and (3) prevention of the onset of symptoms or a reduction in the likelihood of symptom onset.

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

[0330] In some embodiments, the composition comprises a nucleotide sequence encoding a genome-targeted nucleic acid (e.g., 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 a site-directed polypeptide.

[0332] In some embodiments, the composition comprises a polynucleotide (e.g., 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., 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 comprises (i) a nucleotide sequence encoding a nucleic acid (e.g., gRNA) that targets the 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 (e.g., a donor template) to be inserted into the genome.

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

[0337] In some embodiments of the composition, the composition comprises a genome-targeted nucleic acid using a single-molecule guide. In some embodiments of the composition, the composition comprises a genome-targeted nucleic acid using a bimolecule guide. In some embodiments of the composition, the composition comprises two or more bimolecule guides or single-molecule guides. In some embodiments, the composition comprises a vector encoding a nucleic acid that targets the nucleic acid. In some embodiments, the genome-targeted nucleic acid is configured to be used in combination with a DNA endonuclease, particularly a Cas endonuclease (e.g., Cas9 endonuclease).

[0338] In some embodiments, the composition may include one or more gRNAs that can be used for genome editing, particularly for the insertion of sequences encoding FOXP3 or derivatives into the cellular genome. The one or more gRNAs may target a genomic site on the endogenous FOXP3 gene, a genomic site within the endogenous FOXP3 gene, or a genomic site near the endogenous FOXP3 gene. Thus, in some embodiments, the one or more gRNAs may have a spacer sequence complementary to the genomic sequence on the FOXP3 gene, a spacer sequence complementary to the genomic sequence within the FOXP3 gene, or a spacer sequence complementary to the genomic sequence near the FOXP3 gene.

[0339] In some embodiments, the gRNA for the composition comprises spacer sequences shown in SEQ ID NOs: 1-7, 15-20, and 27-29, and a spacer sequence selected from any one of the variants having at least 50% or about 50%, at least 55% or about 55%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, or at least 95% or about 95% identity or homology with any one of SEQ ID NOs: 1-7, 15-20, and 27-29. In some embodiments, the gRNA variant for the kit includes 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% sequence identity with one of sequence numbers 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 to 20 base pairs long. 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 nucleic acids for the kit can be encoded in an adeno-associated virus (AAV) vector. Therefore, in some embodiments, gRNA can be encoded in an AAV vector. In some embodiments, nucleic acids encoding DNA endonucleases can be encoded in an AAV vector. In some embodiments, donor templates can be encoded in an AAV vector. In some embodiments, two or more nucleic acids can be encoded in a single AAV vector. Therefore, in some embodiments, nucleic acids encoding DNA endonucleases and gRNA sequences can be encoded in a single AAV vector.

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

[0344] In some embodiments, the composition further comprises one or more additional reagents, such additional reagents selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for producing polypeptides from DNA in vitro, sequencing adapters, etc. The buffer may be a stabilizing buffer, a reconstitution buffer, a dilution buffer, etc. In some embodiments, the composition may further comprise one or more components used to promote or enhance on-target binding, cleave DNA by endonucleases, or improve targeting specificity.

[0345] In some embodiments, any of the components of the composition are formulated with pharmaceutically acceptable additives such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the specific method of administration and dosage form. In embodiments, the guide RNA composition is typically formulated to achieve a physiologically compatible pH, which is in the range of 3 to 11 or about 3 to about 11, or 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 about 5.0 to pH about 8. In some embodiments, the composition comprises a therapeutically effective amount of at least one compound described herein and one or more pharmaceutically acceptable additives. Optionally, the composition may have a combination of compounds described herein, or may contain a second active ingredient useful for treating or preventing bacterial growth (e.g., antimicrobial agents or antimicrobial agents), or may contain a combination of reagents of the present disclosure. In some embodiments, the gRNA is formulated with one or more other 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 may be formulated separately or in combination with another nucleic acid in the manner described above for the gRNA formulation.

[0346] Suitable additives include carrier molecules containing macromolecules with slow metabolic progression, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and inactive virus particles. Other typical additives include antioxidants (e.g., ascorbic acid), chelating agents (e.g., EDTA), carbohydrates (e.g., dextrin, hydroxyalkylcellulose, or hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., oil, water, saline solution, glycerol, or ethanol), wetting agents or emulsifiers, or pH buffering agents.

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

[0348] In some embodiments, “composition” refers to a therapeutic composition comprising therapeutic cells used in ex vivo therapeutic treatments.

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

[0350] Typically, the recombinant therapeutic cells described herein are administered as a suspension containing a pharmaceutically acceptable carrier. Those skilled in the art will understand that the pharmaceutically acceptable carrier used in the cell composition does not contain amounts of buffers, compounds, cryopreservants, preservatives, or other agents that substantially inhibit the viability of the cells delivered to the target. The cell-containing formulation may, for example, contain an osmotic buffer to maintain the integrity of the cell membrane, and may optionally contain nutrients to maintain cell viability at administration or to enhance cell engraftment. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with progenitor cells according to the description herein using routine experiments.

[0351] In some embodiments, the cell composition can be emulsified, or provided as a liposome composition, provided that the emulsification process does not adversely affect the viability of the cells. The cells and other active ingredients can be mixed with one or more pharmaceutically acceptable and compatible additives in amounts suitable for use in the therapeutic methods described herein.

[0352] Further agents included in the cell composition include pharmaceutically acceptable salts of the components in the cell composition. Examples of 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. Salts formed with free carboxyl groups can be derived from inorganic bases such as sodium, potassium, ammonium, calcium, and iron hydroxide, or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0353] Physiologically acceptable carriers are well known in the art. Typical liquid carriers are sterile aqueous solutions containing no materials other than the active ingredient and water, or sterile aqueous solutions containing a buffer such as sodium phosphate or physiological saline with a physiological pH value, or both (such as phosphate-buffered saline). Furthermore, aqueous carriers may contain one or more buffer salts, salts such as sodium chloride or potassium chloride, dextrose, or polyethylene glycol or other solutes. Liquid compositions may also contain another liquid phase in addition to water, or may contain another liquid phase after removing 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 effective for treating a particular disorder or condition varies depending on the characteristics of the disorder or condition and can be determined by known clinical techniques.

[0354] kit Some embodiments provide a kit comprising one or more of the above compositions, for example, a composition for genome editing or a cell composition (for example, a therapeutic cell composition), and one or more further components.

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

[0356] In some embodiments, the kit may further include instructions for carrying out the method using each component of the kit. 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 also be included in the kit as an accompanying document printed on a label of the container containing the kit or its components (i.e., a label affixed to the packaging or sub-packaging). Alternatively, the instructions may be included as an electronic data file on a suitable computer-readable storage medium (e.g., a CD-ROM, diskette, flash drive). In some cases, the actual instructions may not be included in the kit, and a means for obtaining the instructions from a remote source (such as via the internet) may be provided. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Similar to the instructions, the means for obtaining the instructions may be recorded on a suitable substrate.

[0357] Further embodiments In some embodiments, a method for producing genetically modified cells, CD34 containing the first nucleic acid which includes at least one gene locus + The process of supplying cells; A step of providing a CAS9 protein, or a second nucleic acid sequence encoding the CAS9 protein; The CAS9 protein or the second nucleic acid is the CD34 + The process of introducing the substance into cells; The steps of introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one gene locus, or a set of nucleic acids encoding said at least one CRISPR spacer sequence; and The fourth nucleic acid, which includes a gene delivery cassette, is the CD34. + The process of introducing the cells This provides a method that includes this.

[0358] In some embodiments, the method is described as the CD34 + The process further includes a step of activating the cells, wherein the activation is performed by the CD34 + This is performed before introducing a second nucleic acid into the cells. In some embodiments, activation is performed on a cytokine selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), FLT3L, and IL-6, and CD34 + This is done by bringing cells into contact. In some embodiments, the at least one gene locus is the FOXP3 gene, the AAVS1 gene locus, or the 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 incorporated into one or more vectors. In some embodiments, the one or more vectors are viral vectors. In some embodiments, the viral vectors are adeno-associated virus (AAV) vectors.

[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 mRNA. In some embodiments, the at least one spacer sequence includes the sequence shown in SEQ ID NOs: 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 includes a sequence encoding a FOXP3 cDNA sequence optimized for human codons. In some embodiments, the fourth nucleic acid further includes 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 sequences encoding the coding sequence for low affinity nerve growth factor receptor (LNGFR), μCISC, CISCγ, FRB, and / or LNGFRe (the coding sequence for the LNGFR epitope).

[0360] In some embodiments, the method includes a second gene delivery cassette containing a fifth nucleic acid on the CD34 +The process further includes the step of introducing the nucleic acid into cells. In some embodiments, the fifth nucleic acid is contained in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid contains sequences encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid contains sequences encoding a marker protein. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further contain sequences encoding a P2A self-cleaving peptide. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further contain sequences encoding a polyA sequence. In some embodiments, the polyA sequence contains the 3'UTR of SV40 polyA or FOXP3. In some embodiments, the fourth nucleic acid contains a WPRE3 element. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid is the CD34 + The fourth and / or fifth nucleic acid is introduced into cells and contains a sequence encoding an expression cassette that expresses FOXP3cDNA-LNGFR and DISC, FOXP3cDNA-LNGFR and μDISC, LNGFR-FOXP3cDNA and DISC, LNGFR-FOXP3cDNA and μDISC, CISCβ-DN and CISCγ-FOXP3cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3cDNA. In some embodiments, the fourth and / or fifth nucleic acid is the CD34 +The fourth nucleic acid and / or fifth nucleic acid are introduced into cells and contain sequences encoding an expression cassette. In some embodiments, the fourth nucleic acid includes at least one homologous arm having a locus-specific sequence, the length of which is configured to achieve efficient packaging into an AAV vector. In some embodiments, the length of the at least one homologous arm is within the range defined by 0.25kb, 0.3kb, 0.45kb, 0.6kb, 0.8kb, or 1kb, or any two of these values. In some embodiments, the marker is LNGF, RQR8, or EGFRt. In some embodiments, the method includes a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3, which is located on the CD34. + The method further includes the step of introducing the marker into cells. 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 the sequence shown in SEQ ID NO: 34 or 36. In some embodiments, the method increases the concentration of the marker by CD34 + The process further includes the step of selecting cells. In some embodiments, the CD34 is added to a culture medium containing hTPO, hFlt3, hSCF and / or hIL6. + Bring the cells into contact.

[0361] In some embodiments, CD34 for FOXP3 expression is prepared by the method according to any one of the embodiments described herein. + The cells are provided. In some embodiments, the FOXP3 is constitutively expressed or under controlled expression. The method is CD34 containing the first nucleic acid which includes at least one gene locus + The process of supplying cells; A step of providing a CAS9 protein, or a second nucleic acid sequence encoding the CAS9 protein; The CAS9 protein or the second nucleic acid is the CD34 + The process of introducing the substance into cells; The steps of introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one gene locus, or a set of nucleic acids encoding said at least one CRISPR spacer sequence; and The fourth nucleic acid, which includes a gene delivery cassette, is the CD34. + The process of introducing the cells Includes. In some embodiments, the method is described as the CD34 + The process further includes a step of activating the cells, wherein the activation is performed by the CD34 + This is performed before introducing a second nucleic acid into the cells. In some embodiments, this activation involves activating a cytokine selected from the group consisting of thrombopoietin (TPO), stem cell factor (SCF), FLT3L, and IL-6 with CD34 +This is done by contacting cells. In some embodiments, the at least one gene locus is the FOXP3 gene, the AAVS1 gene locus, or the TRA gene. In some embodiments, the second nucleic acid, the third nucleic acid, the pair of nucleic acids, and / or the fourth nucleic acid are provided incorporated into 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 mRNA. In some embodiments, the at least one spacer sequence includes the sequence shown in SEQ ID NOs: 2, 3, or 5. In some embodiments, the second nucleic acid, the third nucleic acid, the pair 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 FOXP3 cDNA sequence optimized for human codons. 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) coding sequence, μCISC, CISCγ, FRB, and / or LNGFRe (a coding sequence for the LNGFR epitope). In some embodiments, the fourth nucleic acid further comprises a sequence encoding a low affinity nerve growth factor receptor (LNGFR) coding sequence and / or LNGFRe (a coding sequence for the LNGFR epitope). In some embodiments, the method comprises a fifth nucleic acid comprising a second gene delivery cassette on the CD34 +The process further includes the step of introducing the nucleic acid into cells. In some embodiments, the fifth nucleic acid is contained in a vector. In some embodiments, the vector is an AAV vector. In some embodiments, the fifth nucleic acid contains sequences encoding CISC, FRB, a marker protein, μCISC, and / or βCISC. In some embodiments, the fifth nucleic acid contains sequences encoding a marker protein. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further contain sequences encoding a P2A self-cleaving peptide. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid further contain sequences encoding a polyA sequence. In some embodiments, the polyA sequence contains the 3'UTR of SV40 polyA or FOXP3. In some embodiments, the fourth nucleic acid contains a WPRE3 element. In some embodiments, the fourth nucleic acid and / or the fifth nucleic acid is the CD34 + The fourth and / or fifth nucleic acid is introduced into cells and contains a sequence encoding an expression cassette that expresses FOXP3cDNA-LNGFR and DISC, FOXP3cDNA-LNGFR and μDISC, LNGFR-FOXP3cDNA and DISC, LNGFR-FOXP3cDNA and μDISC, CISCβ-DN and CISCγ-FOXP3cDNA-LNGFR, or CISCβ-DN and CISCγ-LNGFR-FOXP3cDNA. In some embodiments, the fourth and / or fifth nucleic acid is the CD34 +The fourth nucleic acid and / or fifth nucleic acid are introduced into cells and contain sequences encoding an expression cassette. In some embodiments, the fourth nucleic acid includes at least one homologous arm having a locus-specific sequence, the length of which is configured to achieve efficient packaging into an AAV vector. In some embodiments, the length of the at least one homologous arm is within the range defined by 0.25kb, 0.3kb, 0.45kb, 0.6kb, 0.8kb, or 1kb, or any two of these values. In some embodiments, the marker is LNGF, RQR8, or EGFRt. In some embodiments, the method includes a sixth nucleic acid encoding a protein or cytokine for co-expression with FOXP3, which is located on the CD34. + The method further includes the step of introducing the marker into cells. 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 the sequence shown in SEQ ID NO: 34 or 36. In some embodiments, the method increases the concentration of the marker by CD34 + The process further includes the step of selecting cells. In some embodiments, the CD34 is added to a culture medium containing hTPO, hFlt3, hSCF and / or hIL6. + Bring the cells into contact.

[0362] In some embodiments, CD34 for FOXP3 expression contains nucleic acid encoding the gene encoding FOXP3. + Cells are provided. In some embodiments, the gene encoding FOXP3 is introduced into the FOXP3 gene locus or a non-FOXP3 gene locus. In some embodiments, the non-FOXP3 gene locus is the AAVS1 gene 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. In some embodiments, the CD34+ From the cells, T differentiates within the thymus. reg Progenitor cells that produce T cells with the phenotype are generated.

[0363] In some embodiments, CD34 according to any one of the embodiments described herein + The present invention provides a composition containing cells.

[0364] In some embodiments, a method for treating, alleviating and / or suppressing a disease and / or condition in a subject, wherein the subject having the disease and / or condition is subjected to any one of the embodiments described herein by CD34 + A method is provided comprising the step of providing cells or a 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] Typical Embodiments Embodiment 1. A method for producing genetically modified cells, CD34 containing a first nucleic acid that includes at least one target gene locus + The process of supplying cells; A step of providing a CAS9 protein, or a second nucleic acid sequence encoding the CAS9 protein; The CAS9 protein or the second nucleic acid is the CD34 + The process of introducing the substance into cells; The steps include introducing a third nucleic acid encoding at least one CRISPR spacer sequence configured to hybridize to at least one target gene locus, or a set of nucleic acids encoding said at least one CRISPR spacer sequence; and The fourth nucleic acid, which includes a gene delivery cassette, is the CD34. + The process of introducing the cells A method that includes this.

[0366] Embodiment 2. The CD34 +The process further includes a step of activating the cells, wherein the activation is performed by the CD34 + The method according to Embodiment 1, performed before introducing a second nucleic acid into the cells.

[0367] Embodiment 3. The activation is the CD34 + The method according to Embodiment 2, which is carried out by bringing cells into contact with CD3 and / or CD28.

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

[0369] Embodiment 5. The method according to 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 incorporated into one or more vectors.

[0370] Embodiment 6. The method according to Embodiment 5, wherein one or more vectors are viral vectors.

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

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

[0373] Embodiment 9. The method according to Embodimen...

Claims

[Claim 1] The inventions described herein.

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