Gene editing for autoimmune disorders

Abstract

To provide methods for treating autoimmune disorders.SOLUTION: A provided method for treating a subject having an autoimmune disorder comprises, for instance, decreasing, in one or more cells in the subject, the amount of one or more genetic variants associated with susceptibility to the autoimmune disorder; and / or increasing, in one or more cells in the subject, the amount of one or more genetic variants protective against the autoimmune disorder. Also provided is a method for decreasing, in the subject, the number of cells that have one or more genetic variants associated with susceptibility to the autoimmune disorder; and / or increasing, in the subject, the number of cells that have one or more genetic variants protective against the autoimmune disorder. Also provided are compositions and isolated cells for use in accordance with the methods.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 762,708, filed May 14, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] (Sequence Listing) This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on May 14, 2019, is named M107385_1010WO_Sequence_Listing_ST25.txt and is 136,596 bytes in size. [Background technology]

[0003] Autoimmune and immune-mediated diseases are characterized by the body's abnormal immune response to substances and tissues normally present in the body that cause the death of healthy body tissue. Thus, autoimmune diseases occur when the body's immune system mistakenly attacks healthy body tissue. There are over 80 autoimmune diseases, including multiple sclerosis ("MS"), rheumatoid arthritis ("RA"), type 1 diabetes ("T1D"), ulcerative colitis ("UC"), Crohn's disease ("CD"), eosinophilic esophagitis, celiac disease, psoriasis, and lupus. The exact causes of autoimmune diseases are not fully understood, although many are believed to have both genetic and environmental components. The existing paradigm of drug development for the treatment of autoimmune diseases targets specific signaling pathways. However, the complexity of such diseases makes it difficult to model these pathways, and the resulting treatments are not as effective as desired. Thus, there is a need in the art for new paradigms related to the treatment of autoimmune diseases. Summary of the Invention

[0004] Methods are provided for treating a subject having an autoimmune disease, comprising decreasing the amount of one or more genetic variants associated with a susceptibility to the autoimmune disease in one or more cells of the subject, and / or increasing the amount of one or more genetic variants protective against the autoimmune disease in one or more cells of the subject. In some embodiments, the methods comprise decreasing the amount of a susceptibility genetic variant in one or more immune cells and / or one or more hematopoietic stem cells of the subject, and increasing the amount of a protective genetic variant in one or more immune cells and / or one or more hematopoietic stem cells of the subject.

[0005] Also provided are methods of treating a subject having an autoimmune disease comprising reducing the number of cells in the subject that have one or more genetic variants associated with a susceptibility to the autoimmune disease and / or increasing the number of cells in the subject that have one or more genetic variants that are protective against the autoimmune disease.

[0006] In some embodiments, the cells are immune cells and / or hematopoietic stem cells, hi some embodiments, the immune cells include one or more of leukocytes, phagocytes, macrophages, neutrophils, dendritic cells, innate lymphocytes, eosinophils, basophils, natural killer cells, B cells, and T cells.

[0007] Some embodiments include administering to the subject immune cells and / or hematopoietic stem cells that contain a protective gene variant, and / or immune cells and / or hematopoietic stem cells that contain a protective gene variant and do not contain a susceptibility gene variant. In some embodiments, the ratio of protective protein variants: susceptibility protein variants in the subject (or a cell or cell population of the subject) is increased, i.e., the amount of protective protein variants is increased relative to the amount of susceptibility protein variants.

[0008] Some embodiments include obtaining immune cells and / or hematopoietic stem cells from a first subject, altering the obtained immune cells and / or hematopoietic stem cells to decrease the amount of susceptibility gene variants and / or increase the amount of protective gene variants, and administering the altered immune cells and / or hematopoietic stem cells to a subject in need of treatment. In some embodiments, the immune cells and / or hematopoietic stem cells are obtained from the blood or bone marrow of the subject. In some embodiments, the first subject is a subject in need of treatment. In some embodiments, the altered immune cells and / or hematopoietic stem cells are administered intravenously or by bone marrow transplantation.

[0009] Some embodiments include depleting at least a portion of the subject's hematopoietic stem cells prior to administration of the immune cells and / or hematopoietic stem cells, in some embodiments, depleting includes administering a chemotherapeutic agent or radiation to the subject, administering an anti-c-Kit monoclonal antibody to the subject, and / or administering a CD47 blocking agent to the subject.

[0010] Some embodiments include administering to the subject a genetic modification agent that (a) reduces the amount of a susceptibility genetic variant in one or more cells of the subject, and / or (b) increases the amount of a protective genetic variant in one or more cells of the subject. In some embodiments, the genetic modification agent comprises a nuclease. In some embodiments, the nuclease is (1) a class 2 CRISPR (regularly-interspaced short palindromic repeat)-associated nuclease, (2) a zinc finger nuclease (ZFN), (3) a transcription activator-like effector nuclease (TALEN), or (4) a meganuclease. For example, in some embodiments, the nuclease comprises Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4. In some embodiments, the nuclease comprises Cas9 or Cpf1.

[0011] Also provided are methods of treating a subject having an autoimmune disease, comprising editing DNA of a subject's immune cells and / or hematopoietic stem cells to (a) decrease the amount of one or more genetic variants associated with bacterial distribution in the subject's gut that are associated with (i) resistance to a particular drug for treating the autoimmune disease or (ii) increased susceptibility to the autoimmune disease, and / or (b) increase the amount of one or more genetic variants associated with bacterial distribution in the subject's gut that are associated with (i) increased sensitivity to a particular drug for treating the autoimmune disease or (ii) protective against the autoimmune disease.

[0012] In some embodiments, the genetic variant is an IL23R variant, a CARD9 variant, a NOD1 / 2 variant, a PTPN22 variant, an NADPH oxidase complex gene variant, a TTC7A variant, a XIAP variant, an IL-10 variant, an IL-10RA variant, an IL-10RB variant, an RPL7 variant, a CPAMD8 variant, a PRG2 variant, a PRG3 variant, a HEATR3 variant, an ATG16L1 variant, a TNFsf15 variant, an MHCII variant, an ELF1 variant, an HLA-DB ... * 01:03 variant, HLA-BTNL2 variant, ARPC2 variant, IL12B variant, STAT1 variant, IRGM variant, IRF8 variant, TYK2 variant, STAT3 variant, IFNGR2 variant, IFNGR1 variant, RIPK2 variant, LRRK2 variant, C13orf31 variant, ECM1 variant, NKX2-3 variant, TNF variant, JAK1 variant, JAK2 variant, JAK3 variant, TPMT variant, NUDT15 variant, LOC441108 variant, PRDM1 variant, IRGM variant, MAGI1 variant, CLCA2 variant, 2q24.1 variant or LY75 variant. In some embodiments, the autoimmune disease comprises inflammatory bowel disease, wherein the protective genetic variant encodes one or more of the R381Q mutation, the G149R mutation and the V362I mutation in the IL23R protein. In some embodiments, the protective genetic variant comprises a G to A mutation at rs11209026.

[0013] In some embodiments, the susceptibility and protective genetic variants are determined based on one or more of: (a) a phenotype of one or more family members, sequencing of a gene panel of one or more family members, whole exome sequencing of one or more family members and / or whole genome sequencing of one or more family members; (b) computer simulation of cell signaling and / or immune system response; (c) machine modeling of mutations that affect the phenotype, such as by linear and / or nonlinear regression models, neural networks; (d) data describing gene expression and / or gene signaling; and (e) animal models (e.g., pigs).

[0014] Also provided is an isolated immune cell or hematopoietic stem cell, the DNA of which has been modified by gene editing to (a) decrease the amount of one or more genetic variants associated with susceptibility to autoimmune disease, and / or (b) increase the amount of one or more genetic variants that are protective against autoimmune disease.

[0015] Also provided is a population of immune cells or hematopoietic stem cells, wherein at least about 10% of the cells in the population have been modified by gene editing to (a) decrease the amount of one or more genetic variants associated with susceptibility to autoimmune disease, and (b) increase the amount of one or more genetic variants that are protective against autoimmune disease.

[0016] Also provided is a composition comprising (i) a nucleic acid encoding a CRISPR / Cas nuclease, (ii) a guide RNA or a nucleic acid encoding the guide RNA that hybridizes to a target sequence in the genomic DNA of a cell that encodes a genetic variant that is associated with susceptibility to an autoimmune disease, and (iii) a DNA repair template that encodes a genetic variant that is not associated with susceptibility to an autoimmune disease.

[0017] In some embodiments, the composition comprises (i) a nucleic acid encoding a CRISPR / Cas nuclease, (ii) a guide RNA or a nucleic acid encoding the guide RNA that hybridizes to a target sequence in the genomic DNA of a cell that encodes a non-protective genetic variant for an autoimmune disease, and (iii) a DNA repair template that encodes a protective genetic variant for an autoimmune disease at the location of the target sequence.

[0018] In some embodiments, the compositions comprise: (a) (i) a nucleic acid encoding a CRISPR / Cas nuclease; (ii) a guide RNA or a nucleic acid encoding the guide RNA that hybridizes to a target sequence in the genomic DNA of a cell that encodes a genetic variant associated with susceptibility to an autoimmune disease; and (iii) a DNA repair template that encodes a genetic variant not associated with susceptibility to an autoimmune disease; and (b) (i) a nucleic acid encoding a CRISPR / Cas nuclease; (ii) a guide RNA or a nucleic acid encoding the guide RNA that hybridizes to a target sequence in the genomic DNA of a cell that encodes a non-protective genetic variant for an autoimmune disease; and (iii) a DNA repair template that encodes a protective genetic variant for an autoimmune disease at the location of the target sequence.

[0019] In some embodiments, the composition comprises two or more guide RNAs, which collectively hybridize to two or more target sequences.

[0020] In some embodiments, the composition is an IL23R variant, a CARD9 variant, a NOD1 / 2 variant, a PTPN22 variant, an NADPH oxidase complex gene variant, a TTC7A variant, a XIAP variant, an IL-10 variant, an IL-10RA variant, an IL-10RB variant, an RPL7 variant, a CPAMD8 variant, a PRG2 variant, a PRG3 variant, a HEATR3 variant, an ATG16L1 variant, a TNFsf15 variant, an MHCII variant, an ELF1 variant, an HLA-DB ... *The present invention includes agents for targeting one or more of: 01:03 variant, HLA-BTNL2 variant, ARPC2 variant, IL12B variant, STAT1 variant, IRGM variant, IRF8 variant, TYK2 variant, STAT3 variant, IFNGR2 variant, IFNGR1 variant, RIPK2 variant, LRRK2 variant, C13orf31 variant, ECM1 variant, NKX2-3 variant, TNF variant, JAK1 variant, JAK2 variant, JAK3 variant, TPMT variant, NUDT15 variant, LOC441108 variant, PRDM1 variant, IRGM variant, MAGI1 variant, CLCA2 variant, 2q24.1 variant or LY75 variant, or a combination of the above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] All documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in the documents cited herein, together with manufacturer's instructions, descriptions, product specifications and product sheets for any products mentioned herein or in any documents incorporated herein by reference, are incorporated herein by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated herein by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0022] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th ed. (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (eds. FMA Usubel et al.); Methods in Enzymology (1955) (eds. Colowick); PCR2: A Practical Approach (1995) (eds. MJ MacPherson, BD Hames, and GR Taylor); Antibodies, A Laboratory Manual (1988) (eds. Harlow and Lane); Antibodies, A Laboratory Manual, 2nd ed. (2013) (eds. E.A. Greenfield); Animal Cell Culture (1987) (ed. R.I. Freshney); Benjamin Lewin, Genes IX (2008), The Encyclopedia of Molecular Biology (1994) (eds. Kendew et al.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (1995) (eds. Meyers); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition (1994) (eds. Sainsbury), Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th Edition (1992) (ed. March); and Transgenic Mouse Methods and Protocols, 2nd Edition (2011) (eds. Hofker and Deursen).

[0023] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0024] The term "optional" or "optionally" means that the subsequently described events, circumstances, or substituents may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0025] The recitation of numerical ranges by endpoints includes all numbers and fractions encompassed within each range, as well as the recited endpoints.

[0026] The terms "about" or "approximately" as used herein when referring to measurable values ​​such as parameters, amounts, and temporal durations are intended to encompass variations from the specified value, such as variations of + / - 10% or less, +1-5% or less, + / - 1% or less, and + / - 0.1% or less from the specified value, to the extent that such variations are appropriate for the practice of the disclosed invention. It will be understood that the values ​​to which the modifier "about" or "approximately" refers are themselves specifically and preferably disclosed.

[0027] The term "hematopoietic stem cell" or "HSC" or "hematopoietic bone marrow stem cell" as used herein refers to hematopoietic cells that are pluripotent or multipotent stem cells or lymphoid or myeloid (bone marrow derived) cells that can differentiate into hematopoietic progenitor cells (HPCs) of lymphoid, erythroid or myeloid cell lineages or expand as a stem cell population without initiating further differentiation. HSCs can be obtained, for example, from bone marrow, peripheral blood, umbilical cord blood, amniotic or placental fluid blood or embryonic stem cells. HSCs can self-renew and initiate the pathway to differentiate or become mature blood cells through the hematopoietic process, such as red blood cells, platelets, granulocytes (such as neutrophils, basophils and eosinophils), macrophages, B lymphocytes, T lymphocytes and natural killer cells. The term "hematopoietic stem cell" encompasses "primitive hematopoietic stem cells", i.e. long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC) and multipotent progenitor cells (MPP).

[0028] The term "immune cell" as used herein generally encompasses any cell derived from a hematopoietic stem cell that participates in an immune response. Immune cells include, but are not limited to, lymphocytes such as T cells and B cells, antigen presenting cells (APCs), dendritic cells, monocytes, macrophages, natural killer (NK) cells, mast cells, basophils, eosinophils or neutrophils, and any precursors of such cells. In certain preferred embodiments, the immune cell may be a T cell. The term "T cell" (i.e., T lymphocyte) as used herein is intended to include all cells in the T cell lineage, including thymocytes, immature T cells and mature T cells. The term "T cell" refers to any cell that is a CD4 + and / or CD8 + T cells, T helper (T h ) cells, e.g., T h 1. T h 2 and T h 17 cells and T regulatory (T reg ) cells.

[0029] The term "modified" as used herein broadly means that immune cells or HSCs have been subjected to or manipulated by an artificial process, such as an artificial molecular or cell biology process, resulting in the modification of at least one property of the cell. Such artificial processes can occur, for example, in vitro or in vivo.

[0030] The term "altered expression" means that the expression of the described gene or polypeptide is changed, i.e., altered or regulated, due to the modification of the immune cell or HSC. The term "altered expression" encompasses any direction and any degree of said change. Thus, "altered expression" may reflect a qualitative and / or quantitative change in expression, and specifically encompasses both an increase (e.g., activation or stimulation) or decrease (e.g., inhibition) in expression.

[0031] The term "increased" or "increase" or "upregulated" or "upregulating" as used herein generally refers to an increase by a statistically significant amount. For the avoidance of doubt, "increased" encompasses a statistically significant increase of at least 10% compared to a reference level, such as an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more compared to a reference level, for example, an increase of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more.

[0032] The term "reduced" or "reduced" or "lowering" or "reduced" or "downregulating" or "downregulated" as used herein generally refers to a statistically significant amount of reduction relative to a reference. For the avoidance of doubt, "reduced" encompasses a statistically significant reduction of at least 10% compared to a reference level, such as 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 more reduction, up to and including a 100% reduction (i.e., a level that is not present when compared to a reference sample), or any reduction between 10 and 100% when compared to a reference level. The term "eliminated" or "eliminated" may specifically refer to a 100% reduction, i.e., a level that is not present when compared to a reference sample.

[0033] The terms "quantity", "amount" and "level" are synonymous and generally well understood in the art. These terms may refer in particular to the absolute quantification of a marker in a subject (e.g., in or on a cell, cell population, tissue, organ or organism, e.g., in a biological sample of a subject), or the relative quantification of a marker in a subject, i.e., quantification relative to another value, such as a reference value, or a range of values ​​that represents the baseline of the marker. For example, the baseline or reference value can be obtained based on determining the quantity, level or amount of a genetic variant in a subject (or a cell or cell population from the subject) that has or does not have an autoimmune disease, or in a subject (or a cell or cell population from the subject) that is at risk of developing an autoimmune disease or is not at risk of developing an autoimmune disease. Such values ​​or ranges may be obtained as known in the art. In some cases, the quantity, amount or level is a measured concentration. The amount can be quantified using known techniques, such as PCR, UV absorption, calorimetry, fluorescence-based measurements, diphenylamine reaction methods, and others. See, e.g., Li, Analytical Biochemistry, 451:18-24 (2014); Figueroa-Gonzalez, Oncol. Lett., 13(6):3982-88 (2017); Psifidi, PLOSE ONE, 10(1):e0115960(page 18) (2015); Current Protocols in Protein Science (1996) (eds. Coligan et al.); and Current Protocols in Molecular Biology (2003) (eds. Ausubel et al.).

[0034] Any one or more of several sequential molecular mechanisms involved in the expression of a given gene or polypeptide may be targeted by modifying immune cells or HSCs. These may include, but are not limited to, targeting the gene sequence (e.g., targeting a polypeptide-coding, non-coding or regulatory portion of the gene sequence), transcription of the gene into RNA, polyadenylation and, if applicable, splicing of the RNA and / or other post-transcriptional modifications of the mRNA, localization of the mRNA into the cytoplasm, if applicable, other post-transcriptional modifications of the mRNA, translation of the mRNA into a polypeptide chain, if applicable, post-translational modifications of the polypeptide and / or folding of the polypeptide chain into the mature conformation of the polypeptide. In the case of compartmentalized polypeptides, such as secreted and transmembrane polypeptides, this may further include targeting the transport of the polypeptide, i.e., the cellular machinery that transports the polypeptide to the appropriate subcellular compartment or organelle, membrane, e.g., plasma membrane, or outside the cell.

[0035] Thus, "altered expression" may specifically refer to altered production of a described gene product by an altered immune cell or HSC. As used herein, the term "gene product" includes RNA (e.g., mRNA) transcribed from a gene or a polypeptide encoded by a gene or translated from RNA.

[0036] The term "gene" as used herein refers to a nucleic acid that includes an open reading frame that encodes a polypeptide, including both exon and (optionally) intron sequences. "Gene" refers to the coding sequence of a gene product, including the 5'UTR and 3'UTR regions, introns and promoters of the gene product, as well as the non-coding regions of the gene product. The coding region of a gene may be a nucleotide sequence that encodes an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene may also be an mRNA or cDNA that corresponds to the coding region (e.g., exons and miRNA), optionally including 5' or 3' untranslated sequences associated therewith. Nucleic acids may encompass single-stranded molecules or double-stranded molecules that include one or more complementary strands or "complementary strands" of a particular sequence comprising the molecule. As used herein, a "single-stranded nucleic acid" may be designated by the prefix "ss" and a double-stranded nucleic acid may be designated by the prefix "ds". The term "gene" may refer to a segment of DNA involved in producing a polypeptide chain, including regions before and after the coding region as well as intervening sequences (introns and untranslated sequences, e.g., 5' and 3' untranslated sequences and regulatory sequences) between individual coding segments (exons). A gene may also be an amplified nucleic acid molecule produced in vitro that contains all or a portion of the coding region and / or 5' or 3' untranslated sequences associated therewith. The term "gene variant" is used to refer to a form of a gene, such as a wild type, a mutant form, or a single nucleotide polymorphism. Some gene variants may be associated with increased susceptibility to autoimmune disease. Some gene variants may be protective against autoimmune disease.

[0037] The term "nuclease" as used herein broadly refers to a substance, e.g., a protein or small molecule, capable of cleaving the phosphodiester bonds linking nucleotide residues in a nucleic acid molecule. In some embodiments, a nuclease may be a protein, e.g., an enzyme, capable of binding to a nucleic acid molecule and cleaving the phosphodiester bonds linking nucleotide residues in a nucleic acid molecule. A nuclease may be an endonuclease that cleaves phosphodiester bonds in a polynucleotide chain, or an exonuclease that cleaves phosphodiester bonds at the end of a polynucleotide chain. Preferably, the nuclease is an endonuclease. Preferably, the nuclease is a site-specific nuclease that binds to and / or cleaves specific phosphodiester bonds in a specific nucleotide sequence, which may be referred to as a "recognition sequence," "nuclease target site," or "target site." In some embodiments, a nuclease can recognize a single-stranded target site, while in other embodiments, a nuclease can recognize a double-stranded target site, e.g., a double-stranded DNA target site. Some endonucleases cleave double-stranded nucleic acid target sites symmetrically, i.e., they cut both strands at the same position so that their ends contain base-paired nucleotides (also known as blunt ends). Other endonucleases cleave double-stranded nucleic acid target sites asymmetrically, i.e., they cut each strand at a different position so that their ends contain unpaired nucleotides. Unpaired nucleotides at the ends of a double-stranded DNA molecule are also called "overhangs", e.g., "5' overhangs" or "3' overhangs", depending on whether the unpaired nucleotides form the 5' or 5' end of each DNA strand.

[0038] Target population Provided are methods and compositions for treating or preventing autoimmune disease in a subject. In some embodiments, the subject is diagnosed with an autoimmune disease. In some embodiments, the subject is at risk of developing an autoimmune disease. Some embodiments relate to methods and compositions for treating any disease whose risk or symptoms are reduced by editing the relevant tissue involved in the process that causes the disease to add protective mutations and / or remove susceptibility mutations. Such diseases include, but are not limited to, eosinophilic esophagitis (see Rothenberg, Gastroenterology, 148(6):1143-57 (2015)), celiac disease (see Gutierrez-Achury, Nature Genetics, 47(6):577-78 (2015)), psoriasis (see Tsoi, Nature Communications, 8: paper 15382 (p. 8) (2017)), type 1 diabetes (see Bonifacio, Acta Diabetol. 51(3):403-11 (2014)), rheumatoid arthritis (see Eyrel, Nat. Genet., 44(12):1336-1340 (2012)), and inflammatory bowel disease (see Huang, Nature, 547:173-178 (2017)). The listed references describe a non-limiting set of genes associated with susceptibility or protection for the above phenotypes.

[0039] In some embodiments, the autoimmune disease is inflammatory bowel disease. Inflammatory bowel disease is broadly classified to include ulcerative colitis and Crohn's disease. Ulcerative colitis is a diffuse and non-specific inflammation of unknown etiology that affects the large intestine and mainly affects the mucosa, frequently causing erosions and ulcers. The disease usually presents with bloody diarrhea and various degrees of general symptoms. In general, the disease is classified according to the extent of symptoms (pancolitis, left-sided colitis, proctitis, or right-sided or segmental colitis), disease stage (active or remitted, etc.), severity (mild, moderate, severe) or clinical course (relapsing-remitting, chronic persistent, acute fulminant, or first attack). On the other hand, Crohn's disease is a disease in which granulomatous lesions with ulceration and fibrosis occur discontinuously throughout the digestive tract from the oral cavity to the anus. Symptoms vary according to the site and extent of the lesion and include fever, malnutrition and anemia, and systemic complications such as arthritis, iritis or liver disease may occur. In general, the disease is classified according to, for example, the location of the lesion (small intestine type, small intestine-large intestine type, rectal type or gastroduodenal type) or the stage of the disease (active or inactive, etc.). Furthermore, ulcerative colitis and Crohn's disease have unknown causes, no fundamental treatment exists, and complete cure is difficult to achieve. Thus, relapse and remission occur repeatedly, which significantly impairs the quality of life of the subject.

[0040] In some embodiments, the subject is genetically predisposed to an autoimmune disease, such as inflammatory bowel disease. For example, in some embodiments, the subject has one or more alleles (i.e., susceptibility gene variants) associated with a high risk of having or developing an autoimmune disease. Due to such high susceptibility to an autoimmune disease, the subject may have a higher amount of one or more protein variants (i.e., susceptibility protein variants) associated with an autoimmune disease, when compared with a subject who does not have or does not have a high risk of developing an autoimmune disease.

[0041] Susceptibility gene is not limited to the gene that directly affects the onset of autoimmune disease.For example, in some embodiments, susceptibility gene is related to the subject's response to a certain treatment.In some embodiments, susceptibility gene is related to the subject's microbiota (e.g., gut microbiota), and microbiota is believed to affect the onset and progression of autoimmune disease, without being bound by theory.

[0042] Susceptibility genes can be identified by any means, such as those known in the art. For example, in some embodiments, susceptibility genes are known in the art to be directly or indirectly associated with autoimmune disease. In some embodiments, susceptibility genes are identified based on a family tree that includes relatives who exhibit or are affected by a particular phenotype. In some embodiments, susceptibility genes are identified by whole exome or whole genome sequencing of one or more family members. In some embodiments, susceptibility genes are identified by computer simulation of cell signaling and / or computer simulation of immune system response. In some embodiments, susceptibility genes are identified using machine modeling, such as using neural networks or other linear and nonlinear regression models, and / or gene signaling networks where certain mutations are known to disrupt gene signaling. In some embodiments, susceptibility genes are identified using animal models (e.g., pigs or mice). In some embodiments, various methods of identifying susceptibility genes (e.g., one or more of identifying known susceptibility genes, identifying a particular phenotype with reference to a family tree, and whole exome or whole genome analysis) are combined to identify individuals in need of treatment. That is, in some embodiments, genes common to family members who have an autoimmune disease (eg, a particular autoimmune disease such as ulcerative colitis or Crohn's disease) are determined to be susceptibility genes.

[0043] Non-limiting susceptibility genes include NADPH oxidase complex genes (e.g., NCF2, Annexin A1), TTC7A, XIAP, NOD1 / 2, IL-10, IL-10RA, IL-10RB, Ashkenazi Jewish genes (RPL7, CPAMD8, PRG2, PRG3, HEATR3), ATG16L1 (e.g., T300A is associated with alterations in the microbiota), Asian susceptibility genes (TNFsf15, MHCII), ELF1, HLA-DB1 * The variants may include one or more of 01:03, HLA-BTNL2, ARPC2, IL12B, STAT1, IRGM, IRF8, TYK2, STAT3, IFNGR2, IFNGR1, RIPK2, LRRK2, IL23R, C13orf31, ECM1, NKX2-3, TNF, JAK1, JAK2, JAK3, CARD9, NOD1 / 2, PTPN22, TPMT, NUDT15, LOC441108, PRDM1, IRGM, MAGI1, CLCA2, 2q24.1, and LY75.

[0044] In some embodiments, gene-gene interactions affect the subject's susceptibility to autoimmune disease.Exemplary interactions include HLA-DQA1, RIT1 / UBQLN4, IFNG / IL26 / IL22.As a further example, pediatric CD patients have additive gene-gene interactions including TLR4, PSMG1, TNFRsf6B and IRGM.

[0045] Several susceptibility genes can be identified in terms of environmental influences: for example, SNPs in CYP2A6 may increase the incidence of CD among smokers, whereas SNPs in the GSTP1 gene are associated with a higher risk of UC in former smokers.

[0046] In some embodiments, susceptibility genes affect the subject's response to drugs.For example, leukopenia can be a life-threatening disease for IBD patients taking thiopurines, which is at least partially caused by genetic mutations in TPMT, which encodes thiopurine S-methyltransferase.In patient populations with low frequency TPMT mutations (Asians), SNPs in NUDT15 are associated with thiopurine-related leukopenia.Furthermore, early-onset IBD patients with IL-10R deficiency can benefit from allogeneic stem cell transplantation.

[0047] In some embodiments, susceptibility genes affect the management of IBD. For example, susceptibility loci in the NOD2 gene are associated with ileal location, stricture and fistula behavior, and the need for surgery. Similarly, in CD, disease with fistula is associated with IL23R, LOC441108, PRDM1, NOD2, and the need for surgery is associated with IRGM, TNFSF15, C13ORF31, and NOD2. Stricture phenotype is associated with NOD2, JAK2, and ATG16L1. MAGI1 variants (encoding a protein involved in intestinal epithelial tight junctions) are associated with a complex structured phenotype, and variants at the CLCA2, 2q24.1, and LY75 loci are associated with the presence of ileal involvement, mild disease course, and erythema nodosum. In UC, a SNP-based risk scoring system that includes 46 SNPs can distinguish patients with medically refractory UC from those with medically non-refractory UC, and thus predict the need for surgery. Furthermore, the MHC is also believed to be a genetic determinant for severe UC.

[0048] Treatment method Some embodiments include methods and compositions that change the paradigm for treating autoimmune diseases, including inflammatory bowel diseases such as ulcerative colitis and / or Crohn's disease. Some embodiments include the use of gene editing to edit genes in immune cells that cause inflammatory immune responses or tissues that produce immune cells by hematopoiesis. By changing the causative genes to remove risk alleles and / or create protective alleles, it is not necessary to precisely understand complex gene signaling pathways to eliminate or reduce the severity of the autoimmune phenotype.

[0049] In some embodiments, the subject is administered a therapeutic agent that reduces the amount of one or more protein variants associated with susceptibility to autoimmune disease. Such reduction can be performed in the subject or in one or more cells of the subject. In some embodiments, the number of cells with one or more susceptibility gene variants in the subject is reduced. For example, in some embodiments, the subject is administered a therapeutic agent (e.g., a gene modifying agent) that modifies gene variants in vivo and / or reduces the expression of susceptibility protein variants. In some embodiments, the therapeutic agent targets susceptibility genes, for example, by gene editing or gene silencing techniques. Some embodiments include targeting one or more of susceptibility genes (including but not limited to environmentally mediated susceptibility genes, susceptibility genes that affect drug response, and susceptibility genes that affect autoimmune disease management) and genes that interact with susceptibility genes.

[0050] In some embodiments, the method includes increasing the amount of one or more genetic variants that are protective against autoimmune disease. Such an increase can be performed in a subject or in one or more cells of the subject. In some embodiments, the number of cells in the subject that have one or more protective genetic variants is increased. For example, in some embodiments, the proportion of expression of a protective protein variant is increased in the subject. In some embodiments, a susceptibility gene is mutated to a wild-type variant by a genetic modifying agent. In some embodiments, a susceptibility gene is mutated to a gene that is protective against autoimmune disease (i.e., a protective gene). In some embodiments, a non-protective genetic variant (e.g., a non-protective wild-type genetic variant) is mutated to a gene that is protective against autoimmune disease.

[0051] In some embodiments, the susceptibility gene and the protective gene encode variants of the same protein. In some embodiments, the susceptibility gene and the protective gene encode variants of different proteins.

[0052] Some embodiments include identifying a protective variant in a gene. Protective genetic variants can be identified using the same techniques discussed above for identifying susceptibility genetic variants. In some embodiments, protective genetic variants include a G→A mutation at rs11209026 in the IL23R gene. Without being bound by theory, it is believed that such protective IL23R mutations have an odds ratio of approximately 1 / 3 in CD and UC, and reduce immune response sufficiently to eliminate or improve symptoms of IBD. See, e.g., Duerr, Science, 314(5804): 1461-63 (2006); also see Sivanesan, J. Biol. Chem., 291(16): 8673-8685 (2016). In some embodiments, the protective genetic variant encodes an IL23R variant with one or more of the following mutations relative to wild-type IL23R: R381Q (e.g., rs11209026, corresponding to c.1142G>A), G149R, and V362I. Alternatively or additionally, in some embodiments, the protective mutation (e.g., for IBD) is in one or more of CARD9, NOD2, PTPN22, and SLC11A1. Without being bound by theory, NOD2 and PTPN22 are believed to be protective for UC but susceptibility genes for Crohn's disease. Also without being bound by theory, in PTPN22, the R620W gain-of-function variant is believed to be associated with CD, while the R263Q loss-of-function variant is believed to be associated with UC. In SLC11A1, the -237C / T polymorphism at SNP rs7573065 has an odds ratio of approximately 2 / 3 for inflammatory bowel disease. See, e.g., Archer, Genes and Immunity, 16(4):275-283 (2015). In some embodiments, the protective variants are the alleles P1104A (rs34536443, OR=0.66), A928V (rs35018800, OR=0.53), and I684S (rs12720356, OR=0.86, P=4.6×10 -7) that encodes a TYK2 (tyrosine kinase 2) variant that is protective for rheumatoid arthritis, including E627X. See, e.g., Diogo, PLoS ONE 10(4):e0122271 (page 21) (2015). As another example, in some embodiments, the protective variant encodes an IFIH1 variant that is protective for type 1 diabetes with an odds ratio ranging from 0.51 to 0.74, including an allele such as E627X. See, e.g., Nejentsev, Science, 324(5925):387-89 (2009). There are many more examples of protective variants across several diseases that can be ameliorated by genetic modifications to HSCs. See, e.g., Harper, Nat. Rev. Genetics, 16(12):689-701 (2015).

[0053] In some embodiments, the subject is administered an agent that targets one or more susceptibility gene variants and / or one or more protective gene variants in immune cells and / or hematopoietic bone marrow stem cells. In some embodiments, a genetic modifying agent is administered to the subject to reduce the amount of susceptibility gene variants in the subject and / or increase the amount of protective protein variants in the subject. In some embodiments, modified immune cells and / or HSCs are administered to the subject to reduce the amount of susceptibility gene variants and / or increase the amount of protective gene variants.

[0054] In some embodiments, the ratio of protective:susceptibility gene variants is increased in the subject or in a cell or cell population of the subject, hi some embodiments, the ratio of protective:susceptibility protein variants is increased in the subject or in a cell or cell population of the subject.

[0055] In some embodiments, HSC cells are harvested from blood or bone marrow, and then genetic modification agents are used ex vivo on the harvested cells. In some embodiments, immunomagnetic or immunofluorescence techniques are used to isolate CD34+ cells from blood samples (CD34 protein is a member of the single-pass transmembrane sialomucin protein family expressed on early hematopoietic and vascular-related tissues). It is a cell surface glycoprotein that functions as an intercellular adhesion factor and can mediate the adhesion of hematopoietic stem cells to bone marrow extracellular matrix (or directly to stromal cells). CD34+ is ubiquitously associated with HSCs, but is also present in other cell types. See, for example, Sidney, Stem Cells., 32(6):1380-9 (2014). Clinically, it can be used to select and enrich hematopoietic stem cells for bone marrow transplantation. CD34+ cells can be harvested from blood using antibodies that bind CD34 and are attached to magnetic beads or fluorescent markers to allow for the subsequent isolation and subsequent gene editing of the cells. The cells can also be cultured before or after gene editing. The cells may then be returned to the subject by transfusion or injection. In some embodiments, before returning the edited HSCs or CD34+ cells to the blood, the subject is subjected to chemotherapy or radiation to remove a significant portion of the subject's bone marrow HSCs, so that the bone marrow is recolonized with a significant portion of the edited HSCs. In some embodiments, the application uses lower doses of radiation or lower doses of chemotherapy than would be lethal for bone marrow cancer patients (e.g., to deplete host HSCs but not remove the entire HSC population). In some embodiments, prior to infusion of the edited HSCs, the host is administered an antibody that disrupts HSC function and causes host HSC depletion. For example, HSCs express c-Kit (CD117), a dimeric transmembrane receptor tyrosine kinase involved in HSC function. Anti-c-Kit monoclonal antibodies can be used in conjunction with immunosuppression to deplete HSCs from the bone marrow niche, allowing edited HSC engraftment.In some embodiments, depletion of host HSCs can be achieved without the need for immunosuppression with radiation or chemotherapy, for example by administering antibodies that disrupt HSC function and deplete HSCs together with other antibodies to make the host HSCs more vulnerable. For example, it has been shown that elimination of host HSCs is dependent on Fc-mediated antibody effector function, and that HSCs express CD47, a myeloid-specific immune checkpoint that generates "don't kill" signals and binds SIRPα. See Chhabra, Science Translational Medicine, 8(351):351ra105(p.10) (2016). Thus, enhancing effector activity by blocking CD47 extends anti-c-Kit regulation to fully immunocompetent subjects. Treatment with c-Kit antibodies together with disruption of the CD47-SIRPα axis with CD47 antibodies results in >99% ablation of host HSCs and robust multilineage blood reconstitution following HSC transplantation. Thus, in some embodiments, anti-c-Kit monoclonal antibodies are used in conjunction with CD47 blockade to eliminate the subject's HSCs prior to infusion of the edited HSCs.

[0056] In some embodiments, cells directly involved in the body's immune response, such as lymphocytes (e.g., B cells, T cells, and / or natural killer cells), monocytes, and / or dendritic cells, are harvested from the blood. Once harvested from the blood, these immune cells may be edited in vitro, optionally cultured, and then returned to the subject's bloodstream. Without being bound by theory, it is believed that many such cells have a half-life of approximately one year. Some methods for isolating these cells according to manufacturer's instructions include, for example, cell preparation tubes (CPT) with sodium heparin from Becton Dickinson, FicollPaque Premium (density: 1.077 g / mL) from GE Healthcare, and Lymphoprep with SepMate tubes from STEMCELL Technologies. See, for example, Grievink, Biopreservation and Biobanking, 14(5):410-415 (2016) for the advantages and disadvantages of different approaches with respect to cell isolation efficiency and cell viability.

[0057] Cells encoding a protective gene variant and / or no susceptibility gene variant can be administered to the subject by any known method (eg, intravenous administration or by transplantation).

[0058] Some embodiments include editing bone marrow stem cells that produce blood cells. In some embodiments, gene modifying agents are delivered into the bone marrow. In some embodiments, this delivery includes passive targeting via polymers of neutral charge and suitable size (e.g., about 150 nm), liposomes surface-modified with anionic glutamic acid to increase distribution in the bone marrow by selective uptake by macrophages and greatly reduce distribution in the liver and spleen, and / or molecules such as bisphosphonates that specifically bind to osteogenic surfaces. See, for example, Chao-Feng, Biomaterials, 155:191-202 (2017). These delivery mechanisms, which may include, for example, polymer wrappers, can be used to deliver CRISPR / Cas9 or other gene editing complexes to the bone marrow.

[0059] Some embodiments include direct stem cell transplantation into the bone marrow with stem cells edited with or without radiation to reduce the bone marrow stem cell population containing the original germline allele. Some embodiments include methods of placing bone marrow stem cells into the patient's bloodstream (e.g., allowing the cells to rehome to the bone marrow).

[0060] In some embodiments, cells that have been obtained from the subject and subsequently genetically modified to decrease the amount of a susceptibility gene variant and / or increase the amount of a protective gene variant are administered to the subject in need of treatment. In some embodiments, cells obtained from a separate subject (e.g., cells from a separate subject that have been genetically modified or that naturally encode a desired gene variant) are administered to the subject in need of treatment.

[0061] Genetic Modifiers For example, the present invention provides a method and composition that can be used to perform desired genetic modification using a genetic modification agent.The genetic modification agent can include nucleases such as CRISPR system, zinc finger nuclease system, TALEN, meganuclease or RNAi system.

[0062] CRISPR systems In some embodiments, the genetic modifying agent is a CRISPR-Cas or CRISPR system, which collectively refers to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNAs" (e.g., RNAs for guiding Cas, such as Cas9, e.g., CRISPR RNA and transactivating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from the CRISPR locus. In general, CRISPR systems are characterized by elements (also referred to as protospacers in the context of endogenous CRISPR systems) that promote the formation of CRISPR complexes at the site of the target sequence. See, e.g., Shmakov, Molecular Cell, 60(3):385-97 (2015); Zetsche, Cell, 163(3):P759-771 (2015); WO 2014 / 093622 (PCT / US Patent Application Publication No. 2013 / 074667).

[0063] In the context of forming a CRISPR complex, "target sequence" refers to the sequence to which guide sequence is designed to have complementarity, where hybridization of the target sequence with the guide sequence promotes the formation of a CRISPR complex.Target sequence may comprise DNA polynucleotide or RNA polynucleotide.In some embodiments, the target sequence is located in the nucleus of a cell.In some embodiments, the target sequence is located in the cytoplasm of a cell.

[0064] In certain exemplary embodiments, CRISPR effector protein may be delivered using a nucleic acid molecule that codes for CRISPR effector protein.The nucleic acid molecule that codes for CRISPR effector protein may advantageously be a codon-optimized CRISPR effector protein (e.g., sequence that is optimized for expression in eukaryotes, such as humans (i.e., optimized for expression in humans) or another eukaryote, animal or mammal).See, for example, WO2014 / 093622 (PCT / US2013 / 074667).

[0065] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Cs m2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or their variants. Although Cas9 is the most widely used Cas protein, other complexes can also be used to edit DNA in a manner that is likely improved compared to Cas9. For example, Cpf1 performs staggered cuts in target DNA where there is an overhang on one strand of DNA that allows for more specific DNA reconstruction. Furthermore, Cpf1 requires one CRISPR RNA (crRNA) for targeting, while Cas9 requires both crRNA and transactivating crRNA (tracrRNA). Smaller crRNAs allow for multiplex genome editing since more of them can be packaged in a single vector. Furthermore, Cas9 cleaves the target DNA 3 nucleotide bases upstream, whereas Cpf1 cleaves the target DNA 18-23 bases downstream from the target site, allowing the target region to remain intact and allowing multiple cuts at the target locus to increase the chances of a specific edit. The present invention encompasses all editing methods discussed herein as well as others, and is not dependent on the particular editing method used.

[0066] Some embodiments include vectors for delivering or introducing, for example, Cas and / or RNA (i.e., guide RNA) that can guide Cas to a target locus into a cell, but also for propagating these components. As used herein, a "vector" is a tool that allows or facilitates the movement of an entity from one environment to another. A vector is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. In general, a vector is capable of replication when bound to appropriate regulatory elements. In general, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid that is linked thereto. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends, or no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other variants of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virus-derived DNA or RNA sequence is present in the vector for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0067] A recombinant expression vector can contain a nucleic acid in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more regulatory elements that can be selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. "Operatively linked" in a recombinant expression vector means that the nucleotide sequence of interest is linked to a regulatory element to allow expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell).

[0068] The vector may include regulatory elements, such as a promoter. The vector may include a Cas coding sequence and / or a single guide RNA (e.g., sgRNA) coding sequence (although optionally may include at least 3 or 8 or 16 or 32 or 48 or 50 guide RNAs (e.g., sgRNAs) coding sequences, such as 1-2, 1-3, 1-4, 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNAs (e.g., sgRNAs)). In a single vector, advantageously there may be a promoter for each RNA (e.g., sgRNA) if up to about 16 RNAs are present, and if a single vector provides more than 16 RNAs, one or more promoters may drive expression of two or more RNAs, e.g. if 32 RNAs are present, each promoter may drive expression of two RNAs, and if 48 RNAs are present, each promoter may drive expression of three RNAs.

[0069] Guide molecule The terms "guide sequence" and "guide molecule" encompass any polynucleotide sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with and direct sequence-specific binding of a nucleic acid target complex to the target nucleic acid sequence. The guide sequence prepared using the methods disclosed herein may be a full-length guide sequence, a truncated guide sequence, a full-length sgRNA sequence, a truncated sgRNA sequence, or an E+F sgRNA sequence. In some embodiments, the degree of complementarity of a guide sequence to a given target sequence is about or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% when optimally aligned using a suitable alignment algorithm. In certain exemplary embodiments, the guide molecule comprises a guide sequence that can be designed to have at least one mismatch with the target sequence, so that an RNA duplex is formed between the guide sequence and the target sequence. In some embodiments, the guide sequence is designed to have a stretch of two or more adjacent unmatched nucleotides to further reduce the degree of complementarity to the entire guide sequence. Optimal alignment may be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP and Maq. The ability of the guide sequence (within the nucleic acid target guide RNA) to guide the sequence-specific binding of the nucleic acid target complex to the target nucleic acid sequence may be evaluated by any suitable assay.For example, sufficient components of the nucleic acid target CRISPR system to form a nucleic acid target complex containing the guide sequence to be tested may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with a vector encoding the components of the nucleic acid target complex, and then evaluation of preferential targeting (e.g., cleavage) within the target nucleic acid sequence may be performed, such as by a Surveyor assay, as described herein. Similarly, cleavage of the target nucleic acid sequence (or a sequence nearby) may be evaluated in a test tube by providing components of the nucleic acid target complex, such as the target nucleic acid sequence, the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the rate of binding or cleavage at or near the target sequence between reactions with the test sequence and the control guide sequence. Guide sequences, and thus nucleic acid target guide RNAs, may be selected to target any target nucleic acid sequence.

[0070] In certain embodiments, the length of the guide sequence or spacer of the guide molecule is 15-50 nucleotides (nt). In certain embodiments, the length of the spacer of the guide RNA is at least 15 nucleotides. In certain embodiments, the length of the spacer is 15-17 nt, such as 15, 16 or 17 nt, 17-20 nt, such as 17, 18, 19 or 20 nt, 20-24 nt, such as 20, 21, 22, 23 or 24 nt, 23-25 ​​nt, such as 23, 24 or 25 nt, 24-27 nt, such as 24, 25, 26 or 27 nt, 27-30 nt, such as 27, 28, 29 or 30 nt, 30-35 nt, such as 30, 31, 32, 33, 34 or 35 nt or more than 35 nt. In certain exemplary embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 ... 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nt.

[0071] In certain embodiments, a guide molecule comprises (1) a guide sequence capable of hybridizing to a target locus, and (2) a tracr mate or direct repeat sequence, such that the direct repeat sequence is located upstream (i.e., 5') of the guide sequence. In some embodiments, the seed sequence of the guide sequence (i.e., the sequence involved in recognizing and / or hybridizing to a sequence at the target locus) is within approximately the first 10 nucleotides of the guide sequence.

[0072] In certain embodiments, the guide molecule comprises a guide sequence linked to a direct repeat sequence, where the direct repeat sequence comprises one or more stem loops or optimized secondary structures. In certain embodiments, the direct repeat sequence has a minimum length of 16 nts and a single stem loop. In further embodiments, the direct repeat sequence has a length of more than 16 nts, preferably more than 17 nts, and has two or more stem loops or optimized secondary structures. In certain embodiments, the guide molecule comprises or consists of a guide sequence linked to all or part of a natural direct repeat sequence. A typical type V or type VI CRISPR-cas guide molecule comprises (in the 3'→5' or 5'→3' direction): a guide sequence, a first complementary stretch (the "repeat sequence"), a loop (typically 4 or 5 nucleotides long), a second complementary stretch (the "anti-repeat sequence" is complementary to the repeat sequence), and a polyA (often polyU in RNA) tail (terminator). In certain embodiments, the direct repeat sequence retains its natural structure and forms a single stem loop. In certain embodiments, certain aspects of the guide structure can be modified, for example, by adding, deleting, or substituting features, while certain other aspects of the guide structure are maintained. Preferred locations for engineered guide molecule modifications, such as, but not limited to, insertions, deletions, and substitutions, include the guide ends and regions of the guide molecule that are exposed when complexed with a CRISPR-Cas protein and / or a target, for example, the stem loop of a direct repeat sequence.

[0073] In some embodiments, the CRISPR system effector protein is an RNA targeting effector protein. In certain embodiments, the CRISPR system effector protein is an RNA targeting type VI CRISPR system (e.g., Cas13a, Cas13b, Cas13c or Cas13d). Exemplary RNA targeting effector proteins include Cas13b and C2c2 (also known as Cas13a). The term "Cas13" as used herein refers to any RNA targeting type VI CRISPR system (e.g., Cas13a, Cas13b, Cas13c or Cas13d). When the CRISPR protein is a C2c2 protein, tracrRNA is not required. C2c2 or Cas13 is described in Abudayyeh et al., Science, 353(6299):aaf5573-1-aaf5573-9 (2016); Shmakov, Molecular Cell, 60(3):385-97 (2015); and Smargon, Molecular Cell, 65:618-30 (2017).

[0074] In some embodiments, one or more elements of the nucleic acid targeting system are derived from a particular organism that contains endogenous CRISPR RNA targeting system.In certain exemplary embodiments, the effector protein CRISPR RNA targeting system comprises at least one HEPN domain.In certain exemplary embodiments, the effector protein comprises a single HEPN domain.In certain other exemplary embodiments, the effector protein comprises two HEPN domains.

[0075] In certain other exemplary embodiments, the CRISPR system effector protein is a C2c2 nuclease. The activity of C2c2 may depend on the presence of two HEPN domains. These are known to be ribonuclease domains, i.e. nucleases (particularly endonucleases) that cleave RNA. C2c2 HEPN may also target DNA or potentially DNA and / or RNA. It is preferred that the C2c2 effector protein has ribonuclease function, provided that the HEPN domains of C2c2 in their wild-type form are capable of at least binding and cleaving RNA. See Abudayyeh, Science, 353(6299):aaf5573-1-aaf5573-9(2016).

[0076] TALE System In some embodiments, gene modification is carried out by the transcription activator-like effector nuclease (TALEN) system.Transcription activator-like effector (TALE) can be engineered to bind to virtually any desired DNA sequence.Exemplary methods of genome editing using the TALEN system are described, for example, in Cermak, Nucleic Acids Res., 39(21):e82(2011); Zhang, Nat.Biotechnol., 29:149-153(2011) and U.S. Patent No. 8,450,471, 8,440,431 and 8,440,432.

[0077] Some embodiments include the use of isolated non-naturally occurring recombinant, i.e. engineered, DNA binding proteins that contain TALE monomers as part of their organizational structure, allowing for targeting of nucleic acid sequences with improved efficiency and extended specificity. TALE structure and function are further described, for example, in Moscow, Science, 326:1501 (2009); Boch, Science, 326:1509-1512 (2009); and Zhang, Nat. Biotechnology, 29:149-153 (2011).

[0078] Zinc finger nuclease In some embodiments, the gene modifying agent comprises a zinc finger system. One type of programmable DNA binding domain is provided by artificial zinc finger (ZF) technology, which comprises an array of ZF modules to target new DNA binding sites in genome. Each finger module in the ZF array targets three DNA bases. The customized array of individual zinc finger domains is assembled into ZF protein (ZFP).

[0079] ZFPs can contain functional domains. The first synthetic zinc finger nucleases (ZFNs) were developed by fusing ZF proteins to the catalytic domain of the type IIS restriction enzyme Fokl (Kim, Proc. Natl. Acad. Sci. USA, 91, 883-887 (1994); Kim, Proc. Natl. Acad. Sci. USA, 93, 1156-1160 (1996)). High cleavage specificity can be achieved with reduced off-target activity by using paired ZFN heterodimers, each targeting a different nucleotide sequence separated by a short spacer (Doyon, Nat. Methods, 8:74-79 (2011)). ZFPs can also be designed as transcriptional activators and repressors and have been used to target many genes in a wide variety of organisms. Exemplary methods of genome editing using ZFNs are described, for example, in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626.

[0080] Meganuclease The "editing" disclosed herein can be performed by meganucleases, which are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12-40 base pairs). Exemplary methods for using meganucleases are described in U.S. Patent Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361, 8,119,381, 8,124,369 and 8,129,134.

[0081] RNAi In certain embodiments, gene modifying agent is RNAi (e.g. shRNA).As used herein with reference to the activity of RNAi molecule, e.g. siRNA or miRNA, "gene silencing" or "silenced gene" refers to the reduction of the mRNA level in cells of target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in cells in the absence of miRNA or RNA interference molecule.In a preferred embodiment, the mRNA level is reduced by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[0082] The term "RNAi" as used herein refers to any type of interfering RNA, including, but not limited to, siRNAi, shRNAi, endogenous microRNA and artificial microRNA.For example, the term includes sequences previously identified as siRNA, regardless of the mechanism of downstream processing of the RNA (i.e., siRNA is believed to have a specific method of in vivo processing that results in the cleavage of mRNA, but such sequences can be incorporated into vectors in the context of the flanking sequences described herein).The term "RNAi" can also include both gene silencing RNAi molecules and RNAi effector molecules that activate the expression of genes.

[0083] As used herein, "siRNA" refers to a nucleic acid that forms a double-stranded RNA, which has the ability to reduce or inhibit expression of a gene or a target gene when the siRNA is present in or expressed in the same cell as the target gene. The double-stranded RNA siRNA can be formed by complementary strands. In one embodiment, siRNA refers to a nucleic acid that can form a double-stranded siRNA. The sequence of the siRNA can correspond to the full-length target gene or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is about 15-50 nucleotides in length, and the double-stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length).

[0084] As used herein, "shRNA" or "short hairpin RNA" (also referred to as stem loop) is a type of siRNA. In one embodiment, these shRNAs consist of a short, e.g., about 19 to about 25 nucleotide antisense strand followed by a nucleotide loop of about 5 to about 9 nucleotides and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and be followed by the antisense strand.

[0085] The term "microRNA" or "miRNA" is used herein synonymously with endogenous RNA, some of which are known to regulate the expression of protein-coding genes at the post-transcriptional level. Endogenous microRNAs are short RNAs that naturally occur in genomes and can regulate the productive use of mRNA. The term "artificial microRNA" includes any type of RNA sequence other than endogenous microRNAs that can regulate the productive use of mRNA. MicroRNA sequences are described in publications such as Lim, Genes & Development, 17:991-1008 (2003), Lim, Science, 299, 1540 (2003), Lee and Ambros, Science, 294, 862 (2001), Lau, Science, 294, 858-861 (2001), Lagos-Quintana, Current Biology, 12:735-739 (2002), Lagos Quintana, Science, 294, 853-857 (2001) and Lagos-Quintana, RNA, 9:175-179 (2003). Multiple microRNAs can also be incorporated into the precursor molecule. Furthermore, miRNA-like stem loops can be expressed in cells as vehicles to deliver artificial miRNAs and short interfering RNAs (siRNAs) to regulate the expression of endogenous genes via the miRNA and / or RNAi pathways.

[0086] As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA molecule that is composed of two strands. A double-stranded molecule includes a single RNA molecule that folds back on itself to form a duplex structure. For example, the stem-loop structure of the precursor molecule from which the single-stranded miRNA is derived, called precursor miRNA (Bartel,. Cell, 116(2):281-297 (2004)), includes a dsRNA molecule.

[0087] Some embodiments include a method of producing a eukaryotic cell comprising a modified or edited gene. In some embodiments, the method includes the steps of: (a) introducing into the eukaryotic cell one or more vectors, the one or more vectors driving expression of one or more of a Cas effector module and a guide sequence linked to a direct repeat sequence, the Cas effector module associated with one or more effector domains that mediate base editing; and (b) allowing a CRISPR-Cas effector module complex to bind to a target polynucleotide to effect base editing of the target polynucleotide within the disease gene, the CRISPR-Cas effector module complex comprising a Cas effector module complexed with a guide sequence that is hybridized to a target sequence within the target polynucleotide.

[0088] Further aspects relate to isolated cells obtained or obtainable by the methods described herein, including the compositions described herein or the progeny of the modified cells. In certain aspects, the cells are eukaryotic cells, preferably human or non-human animal cells. In some aspects, the cells are immune cells. In some aspects, the cells are HSCs.

[0089] Some embodiments include isolated immune cells or hematopoietic stem cells whose DNA has been modified by gene editing to (a) reduce the amount of one or more genetic variants associated with susceptibility to autoimmune disease, and / or (b) increase the amount of one or more genetic variants protective against autoimmune disease. Some embodiments include a population of immune cells or hematopoietic stem cells, in which at least about 10% of the cells in the population have been modified by gene editing to (a) reduce the amount of one or more genetic variants associated with susceptibility to autoimmune disease, and / or (b) increase the amount of one or more genetic variants protective against autoimmune disease.

[0090] The present invention further relates to methods for cell therapy comprising administering to a patient in need thereof the modified cells described herein, whereby the presence of the modified cells treats a disease in the patient.

[0091] The following examples are included as illustrative of the compositions and methods described herein. These examples are not intended to limit the scope of the invention in any way. Other embodiments will be apparent to those of skill in the art. EXAMPLES

[0092] Example 1: Addressing immune disorders with gene editing This embodiment uses CRISPR / Cas9 gene editing as understood in the art. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR sequences contain segments of DNA from viruses that have attacked the cell and are used by the cell to produce RNA that can detect DNA from similar viruses and destroy it together with Cas proteins. CAS stands for CRISPR-Associated System, and genes that code for Cas proteins are found in the cell's DNA near CRISPR sequences. Synthetically modified Cas9 proteins combined with guide RNA (gRNA) can bind to specific target DNA corresponding to the gRNA and cleave the DNA in both strands. This double-strand break can either replace the target DNA with a replacement DNA or initiate a homologous repair process in the cell, where the corresponding DNA in the homologous chromosome or template DNA is replicated to repair the cleaved DNA.

[0093] This example further describes experiments performed on a mouse model to demonstrate that these symptoms of IBD can be improved. Colon inflammation in mice is chemically and / or genetically induced. In particular, 2,4,6-trinitrobenzenesulfonic acid (TNBS) is administered intrarectally with ethanol, oxazolone is administered intrarectally, or dextran sulfate sodium (DSS), a sulfated polysaccharide, is administered orally via drinking water. See, for example, Wirtz, Nature Protocols, 12(7):1295-1309 (2017). Alternatively or additionally, mice are genetically modified to induce inflammatory bowel disease. For example, mice are genetically modified to knock out (KO) NOD1 and / or NOD2. See, for example, Natividad, Inflammatory Bowel Diseases, 18(8):1434-46 (2012); Zenewicz, Immunity, 29(6):947-57 (2008). Alternatively or additionally, mice are genetically modified based on models for IL-10 KO, IL-2 KO, TCRa KO, TGFb KO, TAK1 KO, WASP KO or MDR1A KO, etc. See, e.g., Mizoguchi, Progress in Mol. Biol. Transl. Sci., 105:263-320 (2012); Wirtz, Adv. Drug Deliv. Rev., 59(11):1073-83 (2007).

[0094] More specifically, we consider two types of mutations. The first is a susceptibility mutation for IBD, such as a loss-of-function mutation for IL-10, such as 113Gly→Arg, which is believed, without being bound by theory, to prevent IL-10 from inducing STAT3 phosphorylation or inhibiting lipopolysaccharide (LPS)-mediated TNF release in peripheral blood mononuclear cells.See, for example, Glocker, Ann.NYAcad.Sci.,1246:102-07(2011). The second is a protective mutation for IBD for IL23R discussed above (R381Q corresponds to rs11209026, c.1142G>A).

[0095] Four different groups of mice are used by gene editing of standard strain embryos: group A, which has a heterozygous IL-10 sensitivity mutation and no IL23R protective mutation; group B, which has neither IL10 and / or IL23R mutations; group C, which has an IL10 mutation and an IL23R protective mutation; and group D, which has no IL10 mutation and an IL23R protective mutation.

[0096] These mice are generated by CRISPR / Cas9 editing of a common mouse strain, such as C57BL / 6. Alternatively, mice can be generated from strains that are less sensitive to pain, such as Nav1.7KO mice. See, e.g., Shields, J Neurosci., 38(47):10180-10201 (2018). This experiment may be conceptually similar to using humanized mice, i.e., immune-diseased mice transplanted with a functional human immune system. See, e.g., Kenney, Am. J. Transplant., 16(2):389-97 (2016). This experiment may also be performed using Taconic Biosciences C57BL / 6NTac-Il10 mice, which have been edited from a standard C57BL / 6NTac strain using CRISPR / Cas9-mediated gene editing to delete, e.g., the Il10 locus (exons 1-5 including the proximal promoter and UTR).em8Tac This would be conceptually similar to using existing mouse models that do not require specific editing, such as using mouse models.

[0097] In mice in which HSCs have been edited, HSCs are harvested from bone or blood by capturing CD34+ cells (note that CD34 is expressed on nearly all human HSCs, but approximately 20% of mouse HSCs; see Ogawa, Annals of the New York Academy of Sciences, 938:139-45 (2001)). However, to change the proportion of mouse HSCs that express CD34, expression can be stimulated and restored in mice and harvested; see Tajima, Blood, 96(5):1989-93 (2000).

[0098] The harvested cells are then edited ex vivo and returned to the bloodstream of mice that have been substantially depleted of their endogenous HSC population. This can be accomplished using non-radioactive or non-chemotherapy techniques administering anti-c-Kit antibodies such as ACK2 and anti-CD47 antibodies such as CV1mb modified from CV1 to effectively target human CD47 to target mouse CD47, which have been shown to substantially deplete host HSCs, achieving robust engraftment of HSCs delivered by injection from congenic donor mice with over 60% donor-derived HSC chimerism in bone and approximately 60%, 45%, 60% and 30% chimerism in blood for donor-derived myeloid, B, NK and T cells, respectively. See Chhabra, Science Translational Medicine, 8(351):351ra105(p. 10) (2016). Mice can be treated with injections of 500 mg ACK2 on day 1 and 500 mg CV1mb daily for 5 days starting on day 1. Transplantation can begin 6 days after treatment by transferring approximately 1 million edited HSCs by injection daily for 3 days. Approximately 24 weeks after transplantation, the numbers of B cells, T cells, NK cells, granulocytes (neutrophils, eosinophils, basophils and mast cells) with the relevant edits compared to the host's original cells can be counted to determine whether the edited HSCs have successfully engrafted.

[0099] It should be noted that this experiment is conceptually similar to replacing or augmenting with other anti-CD47 antibodies such as CV1, MIAP410, or Hu5F9G4, which are currently undergoing clinical trials in humans for use in solid and hematological cancers (Clinical Trials NCT02216409 and NCT02367196). Additionally, this experiment is conceptually similar to performing more engraftment procedures or augmenting anti-c-Kit and anti-CD47 antibody treatment with radiation or chemotherapy drugs.

[0100] Gene editing can be achieved by CRISPR / Cas9-mediated homologous directed repair (HDR), in which a template DNA that corresponds to a homolog in a region of unedited DNA but with a new sequence is replicated to replace the DNA after the strand is broken. In particular, one-quarter of the mice in group A (group A1) are subjected to gene editing to remove the IL10-sensitive mutation, one-quarter of the mice in group A (group A2) are subjected to gene editing to add a protective IL23R mutation, and one-quarter of the mice in group A (group A3) are subjected to gene editing to add a protective IL23R mutation and remove the IL10 mutation. One-half of the mice in group B (group B2) are subjected to gene editing to add an IL23R mutation. One-half of the mice in group C (group C2) are subjected to gene editing to remove the IL10 mutation. To have a residual number in groups A, B and C similar to the residual numbers in groups A1, A2, A3, B2, C2, D, we can start with a 4:2:2:1 ratio of mice numbers in groups A, B, C and C, respectively.

[0101] After waiting for the edited HSCs to produce immune cells (e.g., after about 6 months) and after considering the engraftment in the edited group to be successful, these mice are chemically induced to develop colitis using one of the methods described above. The phenotypic severity in terms of weight loss, bleeding, and diarrhea is then compared across groups A, B, C, D, A1, A2, A3, B2, and C2. The following table (Table 1) shows one possible classification of the starting genetic state, the theoretically edited genetic state, and the symptom level. Without being bound by theory, if the HSC editing is completely efficient, we would expect the symptoms to be similar between groups A3, B2, C2, and D, between groups A2 and C, and between groups A1 and B. If the protective effect of the IL23R variant is significantly greater than the susceptibility effect of the IL10 variant, we would expect the symptoms to be ordered from worst to worst from A to D. [Table 1]

[0102] CRISPR-Cas9 is used with a guide RNA (gRNA) to introduce a protective mutation into the gene IL23R. More specifically, the gRNA is designed to attach to DNA encoding the IL23R amino acid sequence described below. (SEQ ID NO:1) The corresponding DNA sequence is set forth in SEQ ID NO:2 in the sequence listing.

[0103] The Crispr-CAS9 complex is designed to cleave DNA in the region of R at position 381, and the template DNA is designed to effect a homologous repair, either converting R at position 381 to Q, or converting G at nucleotide position 1142A to A. A template DNA capable of achieving this goal corresponds to the DNA sequence around position 1142 of the reference DNA (SEQ ID NO:2).

[0104] The guide RNA template is designed to target a specific region of the IL23R gene.

[0105] Exemplary mouse IL23R targeting sequences used to construct guide RNA sequences include: ATAGAACAACAGCTCGGATT (SEQ ID NO: 3) ACAACAACTACACGTCCATC (SEQ ID NO: 4) CCCTTAAGCACTGCCGACCA (SEQ ID NO:5) TGTGTCATTTATGAATACTC (SEQ ID NO: 6) ACCATCTGAAGAGCACATAA (SEQ ID NO: 7) ATCTCCACTGACTCACTGCA (SEQ ID NO: 8) Examples include:

[0106] An exemplary guide RNA sequence targeting mouse IL23R is: AUAGAACAACAGCUCGGAUU (SEQ ID NO: 9) ACAACAACUACACGUCCAUC (SEQ ID NO: 10) CCCUUAAGCACUGCCGACCA (SEQ ID NO: 11) UGUGUCAUUUAUGAAUACUC (SEQ ID NO: 12) ACCAUCUGAAGAGCACAUAA (SEQ ID NO: 13) AUCUCCACUGACUCACUGCA (SEQ ID NO: 14) Includes.

[0107] Exemplary human IL23R target sequences used to construct guide RNA sequences include: GTGCAGTACATAGAAGCATG (SEQ ID NO: 15) ACAACAACTACACGTCCATC (SEQ ID NO: 16) CTACATAGACACAAAATACG (SEQ ID NO: 17) ACCAGCTGAAGAGTATGTAA (SEQ ID NO: 18) CCCTTTACATACTCTTCAGC (SEQ ID NO: 19) ACTTCATCAGGAATATCTGG (SEQ ID NO: 20) Examples include:

[0108] An exemplary guide RNA sequence targeting human IL23R is: GUGCAGUACAUAGAAGCAUG (SEQ ID NO: 21) ACAACAACUACACGUCCAUC (SEQ ID NO: 22) CUACAUAGACACAAAAUACG (SEQ ID NO: 23) ACCAGCUGAAGAGUAUGUAA (SEQ ID NO: 24) CCCUUUACAUACUCUUCAGC (SEQ ID NO: 25) ACUUCAUCAGGAAUAUCUGG (SEQ ID NO: 26) Includes.

[0109] Exemplary target sequences used to construct guide RNA sequences targeting G1142A in human IL23R include: TGTCAATTCTTTCTTTGATT (SEQ ID NO: 27) TTTAACAGATCATTCCGAAC (SEQ ID NO: 28) TTAACAGATCATTCCGAACT (SEQ ID NO: 29) CAGATCATTCCGAACTGGGT (SEQ ID NO: 30) CTGCAAAAACCTACCCAGTT (SEQ ID NO: 31) Examples include:

[0110] An exemplary guide RNA sequence targeting G1142A in human IL23R is: UGUCAAUUCUUUCUUUGAUU (SEQ ID NO: 32) UUUAACAGAUCAUUCCGAAC (SEQ ID NO: 33) UUAACAGAUCAUUCCGAACU (SEQ ID NO: 34) CAGAUCAUUCCGAACUGGGU (SEQ ID NO: 35) CUGCAAAAACCUACCCAGUU (SEQ ID NO: 36) Includes. The wild-type human IL-10 sequence is set forth below. MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 37)

[0111] Exemplary target sequences used to construct guide RNA sequences targeting human IL10 include: GAACCAAGACCCAGACATCA (SEQ ID NO: 38) CAAGGCGCATGTGAACTCCC (SEQ ID NO: 39) AAGGCGCATGTGAACTCCCT (SEQ ID NO: 40) AGGCGCATGTGAACTCCCTG (SEQ ID NO: 41) GGCGCATGTGAACTCCCTGG (SEQ ID NO: 42) GGGAGAACCTGAAGACCCTC (SEQ ID NO: 43) GCCTCAGCCTGAGGGTCTTC (SEQ ID NO: 44) GGGTCTTCAGGTTCTCCCCC (SEQ ID NO: 45) GGTCTTCAGGTTCTCCCCCA (SEQ ID NO: 46) Examples include:

[0112] An exemplary guide RNA sequence targeting the G113R amino acid mutation in human IL-10 is: GAACCAAGACCCAGACAUCA (SEQ ID NO: 47) CAAGGCGCAUGUGAACUCCC (SEQ ID NO: 48) AAGGCGCAUGUGAACUCCCU (SEQ ID NO: 49) AGGCGCAUGUGAACUCCCUG (SEQ ID NO: 50) GGCGCAUGUGAACUCCCUGG (SEQ ID NO:51) GGGAGAACCUGAAGACCCUC (SEQ ID NO:52) GCCUCAGCCUGAGGGUCUUC (SEQ ID NO:53) GGGUCUUCAGGUUCUCCCCC (SEQ ID NO:54) GGUCUUCAGGUUCUCCCCCA (SEQ ID NO:55) Includes.

[0113] Example 2: Machine learning approach to select gene mutations for editing To determine which genes or variants should be edited, machine learning techniques are used to account for the probability of developing a phenotype based on genetic mutations. Such techniques include, for example, linear regression models, logistic regression models, nonlinear regression models that include gene or gene variant interactions, principal component analysis or regression models that use constraint functions to increase constraints on regression problems when they are underdetermined or noisy due to too many possible genes or too few patient data. Constraint functions for regression parameters can include L2 regularization used in ridge regression or L1 regularization used in lasso regression. Independent genetic variables can be logically or mathematically combined to create new variables used in linear models to capture nonlinear interactions among the genes while still maintaining model linearity in regression parameters. Nonlinear interactions can also be captured using models that are nonlinear in parameters, such as deep learning neural networks or neural networks including support vector machines. Some of these methods are described, for example, in Rabinowitz, Bioinformatics, 22:541-549 (2006). By looking at the magnitude of regression parameters, which is particularly simple for linear models, or simulating different data and feeding it to nonlinear models, determine the gene or gene mutation that has the greatest effect on disease phenotype or risk of disease phenotype.Other techniques for identifying which variants are associated with disease include tools that use gene function and gene signaling pathway data to identify genes near risk loci that are most likely to cause phenotypes from genome-wide association studies (GWAS).See, for example, Pers, Nature Communications, 6, article 5890 (page 9) (2015).

[0114] In addition to the references already mentioned, the following references are included: Sands, Inflamm. Bowel Dis., 23(1):97-106 (2017); Jinek, Science, 337:816-821 (2012); Salerno, OncoImmunology, 5(12):e1240857-1-e1240857-14 (2016); Sivanesan, J. Biol. Chem., 291:8673-85 (2016); Pidasheva, PLOS ONE, 6(10):e25038 (2011); SNPedia, rs11209026; US National Library of Medicine,dpSNP:rs11209026;Duerr,Science,314(5804):1461-63(2006);Hazlett,Genes&Immunity,13:282-87(2012);Ferguson,Gastroenterology Research and Practice, Paper ID539461 (12 pages) (2010); Mu, Biomaterials, 155:191-202 (2018); Angermann, Nature Methods, 9:283-89 (2012); Gong, JRSoc. Interface, 14:20170320 (13 pages) (2017); Lu, Curr. Drug Targets,15(6):565-72(2014);Bassaganya-Riera,Clin.Nutr.,25(3):454-65(2006) are also well known.

Claims

1. (a) reducing the amount of one or more gene variants associated with susceptibility to autoimmune disease ("susceptibility gene variants") in one or more cells of the subject; and / or (b) increasing the amount of one or more genetic variants that are protective against an autoimmune disease ("protective genetic variants") in one or more cells of the subject.

23. A method of treating a subject having an autoimmune disease comprising:

2. Reducing the amount of the susceptibility gene variant in one or more immune cells and / or one or more hematopoietic stem cells of the subject; and Increasing the amount of said protective gene variant in one or more immune cells and / or one or more hematopoietic stem cells of said subject. The method of claim 1 , comprising:

3. The method of claim 2, wherein the cells are immune cells and / or hematopoietic stem cells.

4. The method of claim 3, wherein the immune cells include one or more of leukocytes, phagocytes, macrophages, neutrophils, dendritic cells, innate lymphocytes, eosinophils, basophils, natural killer cells, B cells and T cells.

5. Immune cells and / or hematopoietic stem cells comprising said protective gene variant, and / or Immune cells and / or hematopoietic stem cells that contain the protective gene variant and do not contain the susceptibility gene variant.

5. The method of claim 4, comprising administering to the subject

6. The method described in claim 5, which increases the ratio of protective protein variants:susceptible protein variants in the subject.

7. Obtaining immune cells and / or hematopoietic stem cells from a first subject. modifying the resulting immune cells and / or hematopoietic stem cells to decrease the amount of the susceptibility gene variant and / or to increase the amount of the protective gene variant; and administering said altered immune cells and / or hematopoietic stem cells to said subject in need of treatment. The method of claim 6 further comprising:

8. The method of claim 7, wherein the immune cells and / or hematopoietic stem cells are obtained from the blood or bone marrow of the first subject.

9. The method described in claim 8, wherein the first subject is a subject in need of treatment.

10. The method of claim 7, further comprising removing at least a portion of the subject's hematopoietic stem cells prior to administration of the immune cells and / or hematopoietic stem cells.

11. The method of claim 10, wherein the removing comprises administering to the subject a chemotherapy agent or radiation, administering to the subject an anti-c-Kit monoclonal antibody, and / or administering to the subject a CD47 blocking agent.

12. A method according to any one of claims 1 to 11, comprising administering to the subject a genetic modifying agent that (a) reduces the amount of the susceptibility gene variant in one or more cells of the subject, and / or (b) increases the amount of the protective gene variant in one or more cells of the subject.

13. The method described in claim 12, wherein the genetic modifying agent comprises a nuclease.

14. The method described in claim 13, wherein the nuclease is (1) a nuclease-related class 2 CRISPR, (2) a zinc finger nuclease (ZFN), (3) a transcription activator-like effector nuclease (TALEN), or (4) a meganuclease.

15. The nuclease is selected from the group consisting of Cas9, Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, 15. The method of claim 14, comprising Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 or Csf4.

16. The method of claim 1, further comprising: (a) reducing the amount of one or more genetic variants associated with bacterial distribution in the gut of said subject that are associated with (i) resistance to certain drugs for treating an autoimmune disease or (ii) increased susceptibility to an autoimmune disease; and / or (b) increasing the amount of one or more genetic variants associated with (i) increased sensitivity to a particular drug for treating an autoimmune disease, or (ii) distribution of bacteria in the gut of said subject that is protective against an autoimmune disease.

23. A method of treating a subject having an autoimmune disease comprising editing DNA of immune cells and / or hematopoietic stem cells of said subject to treat said autoimmune disease.

17. An immune cell or hematopoietic stem cell population, wherein at least about 10% of the cells in the population have been modified by gene editing to (a) decrease the amount of one or more genetic variants associated with susceptibility to autoimmune disease, and (b) increase the amount of one or more genetic variants that are protective against autoimmune disease.

18. The gene variant is an IL23R variant, a CARD9 variant, a NOD1 / 2 variant, a PTPN22 variant, an NADPH oxidase complex gene variant, a TTC7A variant, a XIAP variant, an IL-10 variant, an IL-10RA variant, an IL-10RB variant, an RPL7 variant, a CPAMD8 variant, a PRG2 variant, a PRG3 variant, a HEATR3 variant, an ATG16L1 variant, a TNFsf15 variant, an MHCII variant, an ELF1 variant, or an HLA-DB1* variant.

18. The population of claim 17, which is a 01:03 variant, an HLA-BTNL2 variant, an ARPC2 variant, an IL12B variant, a STAT1 variant, an IRGM variant, an IRF8 variant, a TYK2 variant, a STAT3 variant, an IFNGR2 variant, an IFNGR1 variant, a RIPK2 variant, a LRRK2 variant, a C13orf31 variant, an ECM1 variant, an NKX2-3 variant, a TNF variant, a JAK1 variant, a JAK2 variant, a JAK3 variant, a TPMT variant, a NUDT15 variant, a LOC441108 variant, a PRDM1 variant, an IRGM variant, a MAGI1 variant, a CLCA2 variant, a 2q24.1 variant or a LY75 variant or a combination thereof.