CN112384230A - Gene editing for autoimmune disorders - Google Patents

Abstract

Methods of treating a subject having an autoimmune disorder are provided, the methods including, for example, reducing the amount of one or more genetic variants associated with susceptibility to the autoimmune disorder in one or more cells of the subject; and/or increasing the amount of one or more genetic variants that prevent the autoimmune disorder in one or more cells of the subject. Also provided are methods of reducing the number of cells in the subject having one or more genetic variants associated with susceptibility to the autoimmune disorder; and/or increasing the number of cells in the subject having one or more genetic variants that prevent the autoimmune disorder. Also provided are compositions and isolated cells for use according to the methods.

Description

Gene editing for autoimmune disorders

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/762,708 filed on 5, 14, 2018, which is incorporated herein by reference in its entirety.

Sequence listing

This application contains a sequence listing filed in ASCII format via EFS-Web and hereby incorporated by reference in its entirety. The ASCII copy created on day 5, month 14, 2019 was named M107385_1010WO _ Sequence _ Listing _ st25.txt and was 136,596 bytes in size.

Background

Autoimmune and immune-mediated disorders are characterized by abnormal immune responses of the body to substances and tissues normally present in the body, resulting in the killing of healthy body tissues. Thus, when the body's immune system mistakenly attacks healthy body tissue, an autoimmune disorder occurs. There are more than 80 types of autoimmune disorders, 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 cause of autoimmune disorders is not fully understood, but many are thought to be associated with both genetic and environmental factors. Existing paradigms of drug development for the treatment of autoimmune disorders target specific signaling pathways. However, the complexity of such barriers makes modeling these pathways difficult, and the resulting treatments are not as effective as expected. Therefore, there is a need in the art for new paradigms associated with the treatment of autoimmune disorders.

Disclosure of Invention

Methods of treating a subject having an autoimmune disorder are provided, the method comprising reducing the amount of one or more genetic variants associated with susceptibility to the autoimmune disorder in one or more cells of the subject; and/or increasing the amount of one or more genetic variants that prevent the autoimmune disorder in one or more cells of the subject. In some aspects, the method comprises reducing the amount of the susceptibility genetic variant in one or more immune cells and/or one or more hematopoietic stem cells in the subject and increasing the amount of the protective genetic variant in one or more immune cells and/or one or more hematopoietic stem cells in the subject.

Also provided are methods of treating a subject having an autoimmune disorder, the method comprising reducing the number of cells in the subject that have one or more genetic variants associated with susceptibility to the autoimmune disorder; and/or increasing the number of cells in the subject having one or more genetic variants that prevent the autoimmune disorder.

In some aspects, the cell is an immune cell and/or a hematopoietic stem cell. In some aspects, the immune cell comprises one or more of a leukocyte, phagocyte, macrophage, neutrophil, dendritic cell, innate lymphoid cell, eosinophil, basophil, natural killer cell, B cell, and T cell.

Some aspects include administering to the subject immune cells and/or hematopoietic stem cells containing the protective genetic variant; and/or immune cells and/or hematopoietic stem cells containing the protective genetic variant and not containing the susceptibility genetic variant. In some aspects, the proportion of protective protein variant to susceptible protein variant in the subject (or in a cell or population of cells in the subject) is increased. That is, the amount of the protective protein variant is increased relative to the amount of the susceptible protein variant.

Some aspects 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 the susceptibility genetic variant and/or increase the amount of the protective genetic variant, and administering the altered immune cells and/or hematopoietic stem cells to a subject in need of treatment. In some aspects, the immune cells and/or hematopoietic stem cells are obtained from the blood or bone marrow of the subject. In some aspects, the first subject is a subject in need of treatment. In some aspects, the altered immune cells and/or hematopoietic stem cells are administered intravenously or via bone marrow transplantation.

Some aspects include depleting at least a portion of the hematopoietic stem cells in the subject prior to administering the immune cells and/or hematopoietic stem cells. In some aspects, the ablating comprises administering chemotherapy or radiation to the subject; administering an anti-c-Kit monoclonal antibody to the subject; and/or administering a CD47 blocker to the subject.

Some aspects include administering a genetic modifier to the subject, wherein the genetic modifier (a) reduces the amount of the susceptibility genetic variant in one or more cells in the subject, and/or (b) increases the amount of the protective genetic variant in one or more cells in the subject. In some aspects, the genetic modifier comprises a nuclease. In some aspects, the nuclease is (1) a class 2 regularly interspaced clustered short palindromic repeats (CRISPR) -associated nuclease, (2) a Zinc Finger Nuclease (ZFN), (3) a transcription activator-like effector nuclease (TALEN), or (4) a meganuclease. For example, in some aspects, the nuclease comprises Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cpf1, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, cs 3, Csxl5, Csfl 2, Csf3, or Csf 4. In some aspects, the nuclease comprises Cas9 or Cpf 1.

Also provided is a method of treating a subject having an autoimmune disorder, the method comprising editing DNA in immune cells and/or hematopoietic stem cells in the subject to: (a) reducing the amount of one or more genetic variants associated with: (i) resistance to a particular drug used to treat the autoimmune disorder or (ii) a bacterial distribution in the gut of the subject that is associated with increased susceptibility to the autoimmune disorder; and/or (b) increasing the amount of one or more genetic variants associated with: (i) increased sensitivity to a particular drug used to treat the autoimmune disorder or (ii) bacterial distribution in the gut of a subject that prevents the autoimmune disorder.

In some aspects, the genetic variant is an IL23 variant, CARD variant, NOD/2 variant, PTPN variant, NADPH oxidase complex gene variant, TTC7 variant, XIAP variant, IL-10RA variant, IL-10RB variant, RPL variant, CPAMD variant, PRG variant, hetr variant, ATG16L variant, TNFsf variant, MHCII variant, ELF variant, HLA-DB 01:03 variant, HLA-BTNL variant, ARPC variant, IL12 variant, STAT variant, IRGM variant, IRF variant, TYK variant, STAT variant, IFNGR variant, RIPK variant, LRRK variant, C13orf variant, ECM variant, NKX-3 variant, TNF variant, JAK variant, TPMT variant, NUDT variant, PRDM variant, IRGM variant, mag variant, clq variant, 24. In some aspects, the autoimmune disorder comprises inflammatory bowel disease, wherein the protective genetic variant encodes one or more of a R381Q mutation, a G149R mutation, and a V362I mutation in IL23R protein. In some aspects, the protective genetic variant comprises a G to a mutation at rs 11209026.

In some aspects, the susceptibility genetic variant and the protective genetic variant are determined based on one or more of: (a) a phenotype of one or more family members, sequencing of a set of genes in one or more family members, sequencing of a full exome in one or more family members, and/or sequencing of a full genome of one or more family members; (b) computer modeling of cellular signaling and/or immune system responses; (c) machine modeling of phenotype-affecting mutations, such as using linear and/or non-linear regression models, neural networks; (d) data describing gene expression and/or gene signaling; and (e) animal models (e.g., swine).

Also provided are isolated immune cells or hematopoietic stem cells, wherein the cellular DNA has been modified by gene editing to (a) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and/or (b) increasing the amount of one or more genetic variants that prevent the autoimmune disorder.

Also provided are populations 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) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and (b) increasing the amount of one or more genetic variants that prevent the autoimmune disorder.

Also provided are compositions 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 within the genomic DNA of the cell that encodes a genetic variant associated with susceptibility to an autoimmune disorder, and (iii) a DNA repair template that encodes a genetic variant not associated with susceptibility to an autoimmune disorder.

In some aspects, the compositions comprise (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 within the genomic DNA of the cell that encodes a genetic variant that does not prevent an autoimmune disorder, and (iii) a DNA repair template that encodes the genetic variant that prevents an autoimmune disorder at the location of the target sequence.

In some aspects, 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 within the genomic DNA of the cell that encodes a genetic variant associated with susceptibility to an autoimmune disorder, and (iii) a DNA repair template that encodes a genetic variant that is not associated with susceptibility to an autoimmune disorder; 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 within the genomic DNA of the cell that encodes a genetic variant that does not prevent an autoimmune disorder, and (iii) a DNA repair template that encodes the genetic variant that prevents an autoimmune disorder at the location of the target sequence.

In some aspects, the composition comprises two or more guide RNAs, wherein the guide RNAs collectively hybridize to more than one target sequence.

In some aspects, the composition comprises an agent for targeting one or more of: IL23 variants, CARD variants, NOD/2 variants, PTPN variants, NADPH oxidase complex gene variants, TTC7 variants, XIAP variants, IL-10RA variants, IL-10RB variants, RPL variants, CPAMD variants, PRG variants, HEATR variants, ATG16L variants, TNFSf variants, MHCII variants, ELF variants, HLA-DB 01:03 variants, HLA-BTNL variants, ARPC variants, IL12 variants, STAT variants, IRGM variants, IRF variants, TYK variants, STAT variants, IFNGR variants, RIPK variants, LRRK variants, C13orf variants, ECM variants, NKX-3 variants, TNF variants, JAK variants, TPDT variants, LOCDT variants, LOC441108 variants, PRDM variants, IRGM variants, MACA variants, CLCA variants, 2q24.1 variants, or a combination thereof.

Detailed Description

Any manufacturer's specifications, descriptions, product descriptions, and product tables for any products mentioned in all documents cited or referenced herein ("herein cited documents") and in all documents cited or referenced in herein cited documents, as well as in any document herein or incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the present invention. More specifically, all cited documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Unless defined otherwise, 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 may be found in one or more of the following documents: molecular Cloning A Laboratory Manual, 2 nd edition (1989) (Sambrook, Fritsch, and Maniatis); molecular Cloning A Laboratory Manual, 4 th edition (2012) (Green and Sambrook); current Protocols in Molecular Biology (1987) (edited by F.M. Ausubel et al); methods in Enzymology (1955) (edited by Colowick); PCR 2A Practical Approach (1995) (edited by M.J.MacPherson, B.D.Hames, and G.R.Taylor) Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.); antibodies, a Laboratory Manual, 2 nd edition (2013) (edited by e.a. greenfield); animal Cell Culture (1987) (r.i. freshney, eds.); benjamin Lewis, Genes IX (2008), The Encyclopedia of Molecular Biology (1994) (edited by Kendew et al), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (1995) (edited by Meyers); singleton et al, Dictionary of Microbiology and Molecular Biology, 2 nd edition (1994) (Sainsbury, eds.), Advanced Organic Chemistry Reactions, mechanics and Structure, 4 th edition (1992) (March, eds.); and, Transgenic Mouse Methods and Protocols, 2 nd edition (2011) (edited by Hofker and Deursen).

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

The term "optional" or "optionally" means that the subsequently described event, circumstance, or alternative may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range and the recited endpoint.

The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, amount, time distance, and the like, are intended to encompass variations in the specified value and from the specified value, such as +/-10% or less, + 1-5% or less, +/-1% or less, and +/-0.1% or less of the specified value and from the specified value, provided such variations are suitable for carrying out the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed per se.

The term "hematopoietic stem cell" or "HSC" or "hematopoietic bone marrow stem cell" as used herein refers to a hematopoietic cell which is a pluripotent or multipotent stem cell or lymphoid or myeloid (bone marrow-derived) cell capable of differentiating into a Hematopoietic Progenitor Cell (HPC) of lymphoid, erythroid or myeloid lineage or proliferating as a stem cell population without starting further differentiation. HSCs can be obtained, for example, from bone marrow, peripheral blood, umbilical cord blood, amniotic fluid or placental blood, or embryonic stem cells. HSCs are capable of self-renewal and differentiation into mature blood cells, such as erythrocytes (erythrocytes), platelets, granulocytes (e.g., neutrophils, basophils, and eosinophils), macrophages, B lymphocytes, T lymphocytes, and natural killer cells, or to open a pathway to become mature blood cells through the process of hematopoiesis. The term "hematopoietic stem cells" encompasses "primitive hematopoietic stem cells," i.e., long term hematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs), and pluripotent progenitor cells (MPPs).

The term "immune cell" as used herein generally encompasses any cell derived from a hematopoietic stem cell that plays a role in an immune response. Immune cells include, without limitation, lymphocytes (e.g., 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 progenitor cell of such cells. In certain preferred aspects, the immune cell may be a T cell. As used herein, the term "T cell" (i.e., T lymphocyte) is intended to include all cells within the T cell lineage, including thymocytes, immature T cells, mature T cells, and the like. The term "T cell" may include CD4⁺And/or CD8⁺T cell, T helper (T)_h) Cells (e.g. T)_h1、T_h2 and T_h17 cells) and T regulation (T)_reg) A cell.

The term "modified" as used herein broadly refers to an immune cell or HSC that has been subjected to or manipulated by an artificial process (e.g., an artificial molecular or cellular biological process) resulting in a modification of at least one characteristic of the cell. Such artificial processes may be performed, for example, in vitro or in vivo.

The term "altered expression" means that the modification of the immune cell or HSC alters or modulates the expression of one or more of the genes or one or more polypeptides. The term "altered expression" encompasses any direction and any degree of the alteration. Thus, "altered expression" may reflect one or more qualitative and/or quantitative changes in expression, and specifically encompasses an increase (e.g., activation or stimulation) or a decrease (e.g., inhibition) in expression.

The term "increased" or "increase" or "upregulated" as used herein generally means an increase in a statistically significant amount. For the avoidance of doubt, "increased" encompasses a statistically significant increase of at least 10% as compared to a reference level, including 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, including at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more as compared to a reference level.

The term "reduced" or "decrease" or "reduction" or "reduced" or "downregulated" as used herein generally means 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, for example a reduction 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% or more up to and including a 100% reduction (i.e. a level that is not present compared to a reference sample), or any reduction between 10% and 100%. The term "abolish" or "abolished" may particularly denote a reduction by 100%, i.e. an absence level, compared to a reference sample.

The terms "amount", "quantity", and "level" are synonymous and are generally well understood in the art. The term may particularly refer to an absolute quantification of a marker in a test subject (e.g. in or on a cell, cell population, tissue, organ or organism, e.g. in a biological sample of a subject), or to a relative quantification of a marker in a test subject, i.e. relative to another value, such as relative to a reference value or relative to a range of values indicative of a baseline for the marker. For example, the baseline or reference value may be obtained based on a determination of the number, level or amount of genetic variants in a subject (or in a cell or population of cells from the subject) with or without an autoimmune disorder, or in a subject (or in a cell or population of cells from the subject) at risk of developing or not at risk of developing an autoimmune disorder. Such values or ranges may be obtained as conventionally known. In some cases, the amount, quantity, or level is a measured concentration. Quantification may be performed using known techniques such as PCR, UV absorption, calorimetry, fluorescence-based measurements, diphenylamine reaction methods, and the like. 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) (Coligan et al, eds.); and Current Protocols in Molecular Biology (2003) (Ausubel et al, eds.).

Any one or more of several sequential molecular mechanisms involved in the expression of a given gene or polypeptide may be targeted by the immune cell or HSC modification. Without limitation, these may include targeting: a gene sequence (e.g., a polypeptide coding, non-coding and/or regulatory portion that targets a gene sequence), transcription of a gene into RNA, polyadenylation, and where applicable splicing of the RNA into and/or other post-transcriptional modification to mRNA, localization of the mRNA to the cytoplasm, where applicable other post-transcriptional modification of the mRNA, translation of the mRNA into a polypeptide chain, where applicable post-translational modification of the polypeptide, and/or folding of the polypeptide chain into the mature conformation of the polypeptide. For compartmentalized polypeptides (e.g., secreted polypeptides and transmembrane polypeptides), this may also include targeting the transport of the polypeptide, i.e., the cellular mechanism by which the polypeptide is transported to the appropriate subcellular compartment or organelle, membrane (e.g., plasma membrane), or extracellularly.

Thus, "altered expression" may particularly denote an alteration of the production of the gene product by a modified immune cell or HSC. As used herein, the term "one or more gene products" includes RNA (e.g., mRNA) transcribed from a gene, or a polypeptide encoded by a gene or translated from an RNA.

As used herein, the term "gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including exon and (optionally) intron sequences. "Gene" refers to the coding sequence of a gene product as well as to the non-coding regions of the gene product (including the 5 'UTR and 3' UTR regions, introns, and promoters 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 (e.g., tRNA, rRNA, catalytic RNA, siRNA, miRNA, and antisense RNA). The gene may also be an mRNA or cDNA corresponding to the coding region (e.g., exons and miRNA), optionally comprising 5 'or 3' untranslated sequences attached thereto. Nucleic acids may encompass single-stranded molecules or double-stranded molecules comprising one or more complementary strands or "complements" comprising the particular sequence of the molecule. As used herein, a single-stranded nucleic acid may be denoted by the prefix "ss", and a double-stranded nucleic acid may be denoted by the prefix "ds". The term "gene" may refer to a segment of DNA involved in the production of polypeptide chains and which comprises regions preceding and following the coding region as well as intervening sequences (introns and untranslated sequences, e.g., 5 'and 3' untranslated sequences and regulatory sequences) between the individual coding segments (exons). A gene may also be an in vitro generated amplified nucleic acid molecule comprising all or part of the coding region and/or 5 'or 3' untranslated sequence linked thereto. The term "genetic variant" is used to refer to a form of a gene, such as a wild-type form, a mutant form, or a single nucleotide polymorphic form. Some gene variants may be associated with increased susceptibility to autoimmune disorders. Some gene variants may prevent autoimmune disorders.

The term "nuclease" as used herein broadly refers to an agent, such as a protein or small molecule, that is capable of cleaving phosphodiester bonds that join nucleotide residues in a nucleic acid molecule. In some aspects, the nuclease may be a protein, such as an enzyme that can bind to a nucleic acid molecule and cleave phosphodiester bonds that link nucleotide residues within the nucleic acid molecule. The nuclease may be an endonuclease that cleaves a phosphodiester bond within a polynucleotide strand, or an exonuclease that cleaves a phosphodiester bond at the end of the polynucleotide strand. Preferably, the nuclease is an endonuclease. Preferably, the nuclease is a site-specific nuclease that binds to and/or cleaves a specific phosphodiester bond within a specific nucleotide sequence, which may be referred to as a "recognition sequence", "nuclease target site" or "target site". In some aspects, the nuclease can recognize a single-stranded target site, in other aspects, the 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., cleave both strands at the same position such that the ends comprise base-paired nucleotides, also referred to as blunt ends. Other endonucleases cleave double-stranded nucleic acid target sites asymmetrically, i.e., each strand is cleaved at a different position such that the ends comprise unpaired nucleotides. Unpaired nucleotides at the ends of double-stranded DNA molecules are also referred to as "overhangs", e.g., "5 'overhangs" or "3' overhangs", depending on whether one or more unpaired nucleotides form the 5 'or 5' end of the respective DNA strand.

Target population

Methods and compositions for treating or preventing an autoimmune disorder in a subject are provided. In some aspects, the subject has been diagnosed with an autoimmune disorder. In some aspects, the subject is at risk for developing an autoimmune disorder. Some aspects relate to methods and compositions for treating any disease in which risk or symptoms are reduced by editing the relevant tissues involved in the pathogenic process to increase 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: article 15382(8pages) (2017)); type I 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 of the above phenotypes.

In some aspects, the autoimmune disorder is inflammatory bowel disease. The inflammatory bowel diseases are broadly classified as including ulcerative colitis and crohn's disease. Ulcerative colitis is a diffuse, non-specific inflammation of unknown etiology that affects the colon and mainly attacks the mucous membranes, and often causes erosions and ulcers. Typically, it manifests as bloody diarrhea and various degrees of general symptoms. Generally, symptoms are classified according to their spread (pan colitis, left-side colitis, proctitis or right-side or segmental colitis), stage of disease (e.g. active or remission), severity (mild, moderate, severe) or clinical course (relapsing remission, chronic persistence, acute outbreak or first onset). On the other hand, crohn's disease is a disease in which granulomatous lesions accompanied with ulcers and fibrosis discontinuously occur in the entire digestive tract from the oral cavity to the anus. Although varied depending on the location and extent of the lesion, symptoms include fever, nutritional disorders, and anemia, and systemic complications such as arthritis, iritis, or liver disorders may also occur. Generally, the disease is classified according to, for example, the location of the lesion (small intestine type, small intestine-large intestine type, rectum type, or gastroduodenal type) or the period of the disease (e.g., active or inactive period). Furthermore, the etiology of ulcerative colitis and crohn's disease is unknown, there is no fundamental therapy, and complete cure is difficult to achieve. Thus, recurrence and remission occur repeatedly, thereby greatly impairing the quality of life of the subject.

In some aspects, the subject is genetically predisposed to an autoimmune disorder, such as inflammatory bowel disease. For example, in some aspects, the subject has one or more alleles associated with increased risk of having or suffering from an autoimmune disorder (i.e., a susceptible genetic variant). Such increased susceptibility to the autoimmune disorder can result in a subject having an increased amount of one or more protein variants associated with the autoimmune disorder (i.e., susceptibility protein variants) as compared to a subject that does not have the autoimmune disorder or has an increased risk of developing the autoimmune disorder.

Susceptibility genes are not limited to genes that directly affect the development of the autoimmune disorder. For example, in some aspects, the susceptibility gene is associated with a subject's response to a particular therapy. In some aspects, the susceptibility gene is associated with a microbiome (e.g., gut microbiome) of the subject that is believed, without being bound by theory, to affect the development and progression of autoimmune disorders.

Susceptibility genes can be identified by any means, including those known in the art. For example, in some aspects, the susceptibility gene is known in the art to be directly or indirectly associated with an autoimmune disorder. In some aspects, the susceptibility gene is identified based on a family tree comprising relatives exhibiting or affected by a particular phenotype. In some aspects, the susceptibility gene is identified via whole exome or whole genome sequencing of one or more family members. In some aspects, the susceptibility gene is identified via a computer simulation of cell signaling and/or a computer simulation of immune system response. In some aspects, the susceptibility gene is identified by machine modeling, such as using neural networks or other linear and non-linear regression models and/or using gene signaling networks, where particular mutations are observed to disrupt gene signaling. In some aspects, the susceptibility gene is identified using an animal model (e.g., pig or mouse). In some aspects, the various methods of identifying susceptibility genes (e.g., identifying known susceptibility genes, identifying specific phenotypes for family trees, and one or more of whole exome or whole genome analysis) are combined to identify individuals in need of treatment. That is, in some aspects, genes shared by family members having an autoimmune disorder (e.g., a particular autoimmune disorder such as ulcerative colitis or crohn's disease) are identified as susceptibility genes.

Non-limiting susceptibility genes may include one or more variants of: NADPH oxidase complex genes (e.g. NCF2, annexin A1), TTC7A, XIAP, NOD1/2, IL-10RA, IL-10RB, DE-Jersey genes (RPL7, CPAMD8, PRG2, PRG3, HEATR3), ATG16L1 (e.g. T300A is associated with changes in microbiome), Asian susceptibility genes (TNFSf15, MHCII), ELF1, HLA-DB 1:03, HLA-BTNL2, ARPC2, IL12B, STAT1, IRGM, IRF8, TYK2, IFNGR2, RIPK2, LRRK2, IL23 2, C13orf 2, ECM 2, NKX 2-NKMT, ATPR 2, CLNGR 4472, CLNGR 2, NL2, LOCGI 2, LOC 2, JAK2, TRK 2, CLGI 2, LOC 2, TROP 72, TRK 2, and TROP 72.

In some aspects, the interpgenic interaction affects the susceptibility of the subject to an autoimmune disorder. Exemplary interactions include HLA-DQA1, RIT1/UBQLN4, IFNG/IL26/IL 22. As another example, pediatric CD patients have additive intergenic interactions involving TLR4, PSMG1, TNFRsf6B, and IRGM.

Some susceptibility genes may be identified in the context of environmental influences. For example, SNPs for CYP2a6 may increase the incidence of CD in smokers. On the other hand, SNPs in the GSTP1 gene were associated with increased UC risk in former smokers.

In some aspects, the susceptibility gene affects the subject's response to the drug. For example, in IBD patients receiving mercaptopurine, leukopenia may be a life-threatening condition that is caused, at least in part, by genetic variation in TPMT encoding mercaptopurine S-methyltransferase. In a population of patients with low frequency of TPMT mutations (asians), the SNP in NUDT15 was associated with mercaptopurine-associated leukopenia. Still further, early-onset IBD patients deficient in IL-10R may benefit from allogeneic stem cell transplantation.

In some aspects, the susceptibility gene affects the management of IBD. For example, a susceptible locus in the NOD2 gene is associated with ileal location, stenotic and penetrating behavior, and surgical needs. Also in CD, fistulous disease is associated with IL23R, LOC441108, PRDM1, NOD2, while surgical requirements are associated with IRGM, TNFSF15, C13ORF31 and NOD 2. The stenotic phenotype is associated with NOD2, JAK2 and ATG16L 1. The MAGI1 variant (which encodes a protein involved in the tight junctions of the intestinal epithelium) is associated with a complex structural phenotype, whereas the variants in the CLCA2, 2q24.1 and LY75 loci are associated with ileal involvement, a mild course and the presence of erythema nodosum. In UC, a SNP-based risk scoring system comprising 46 SNPs is able to distinguish patients with medically refractory UC from non-medically refractory patients, thereby predicting the need for surgery. In addition, MHC is also considered to be a genetic determinant of severe UC.

Method of treatment

Some aspects include methods and compositions that alter paradigms for treating autoimmune disorders, including inflammatory bowel disease, such as ulcerative colitis and/or crohn's disease. Some aspects relate to the use of gene editing to edit genes of immune cells or genes of tissues that produce immune cells by hematopoiesis, the immune cells or the tissues eliciting an inflammatory immune response. By altering potential genes to eliminate risk alleles and/or generate protective alleles, there is no need to precisely understand the complex gene signaling pathways to eliminate or reduce the severity of the autoimmune phenotype.

In some aspects, the subject is administered a therapy that reduces the amount of one or more protein variants associated with susceptibility to an autoimmune disorder. This reduction may occur in the subject or in one or more cells of the subject. In some aspects, the number of cells having one or more susceptibility genetic variants is reduced in the subject. For example, in some aspects, a subject is administered a therapy (e.g., a genetic modifier) that modifies a genetic variant and/or reduces expression of a susceptibility protein variant in vivo. In some aspects, the therapy targets the susceptibility gene, e.g., using gene editing or gene silencing techniques. Some aspects relate to targeting one or more of the susceptibility genes (including, without limitation, environmentally mediated susceptibility genes, susceptibility genes affecting drug response, and susceptibility genes affecting management of the autoimmune disorder) and genes that interact with the susceptibility genes.

In some aspects, the method comprises increasing the amount of one or more genetic variants that prevent the autoimmune disorder. This increase may occur in the subject or in one or more cells in the subject. In some aspects, the number of cells having one or more protective genetic variants is increased in the subject. For example, in some aspects, the expression ratio of a protective protein variant is increased in the subject. In some aspects, the susceptibility gene is mutated to a wild-type variant via a genetic modifier. In some aspects, the susceptibility gene is mutated to a gene that prevents the autoimmune disorder (i.e., a protective gene). In some aspects, a non-protective genetic variant (e.g., a non-protective wild-type genetic variant) is mutated to a gene that prevents the autoimmune disorder.

In some aspects, the susceptibility gene and the protective gene encode variants of the same protein. In some aspects, the susceptibility gene and the protective gene encode variants of different proteins.

Some aspects include identifying protective variants in a gene. The same techniques discussed above with respect to identifying susceptibility genetic variants can be used to identify protective genetic variants. In some aspects, the protective genetic variant comprises a G → a mutation at rs11209026 in the IL23R gene. Without being bound by theory, it is believed that this protective IL23R mutation has a odds ratio of about 1/3 for CD and UC, and will suppress the immune response sufficiently to eliminate or reduce the symptoms of IBD. See, e.g., Duerr, Science,314(5804):1461-63 (2006); see also Sivanesan, J.biol.chem.,291(16):8673-8685 (2016). In some aspects, the protective genetic variant encodes an IL23R variant having one or more of the following mutations to wild-type IL 23R: R381Q (e.g., corresponding to rs11209026, c.1142G>A) G149R and V362I. Alternatively or additionally, in some aspects, the protective mutation (e.g., for IBD) occurs in one or more of: CARD9, NOD2, PTPN22 and SLC11A 1. Without being bound by theory, NOD2 and PTPN22 are believed to be protective against UC and susceptible genes to crohn's disease. Again without being bound by theory, it is believed that for PTPN22, R620W of the functional variant was associated with CD whereas R263Q loss of the functional variant was associated with UC. In SLC11A1, the odds ratio of the-237C/T polymorphism at SNP rs7573065 to inflammatory bowel disease is about 2/3. See, e.g., Archer, Genes and Immunity,16(4): 275-. In some aspects, the protective variant encodes a polypeptide protective against rheumatoid arthritisTYK2 (tyrosine kinase 2) variants, the protective variants including alleles P1104A (rs34536443, OR ═ 0.66), A928V (rs35018800, OR ═ 0.53) and I684S (rs12720356, OR ═ 0.86, P ═ 4.6x 10)^-7). See, e.g., Diogo, PLoS ONE 10(4): e0122271 (page 21) (2015). As another example, in some aspects, the protective variant encodes an IFIH1 variant protective against type I diabetes with a odds ratio ranging from 0.51 to 0.74, the protective variant including an allele such as E627X. See, e.g., Nejentsev, Science,324(5925):387-89 (2009). There are more examples of protective variants in several diseases that can be alleviated by genetic modification of HSCs. See, e.g., Harper, nat. Rev. genetics,16(12):689-701 (2015).

In some aspects, the subject is administered an agent that targets one or more susceptibility genetic variants and/or one or more protective genetic variants in immune cells and/or hematopoietic bone marrow stem cells. In some aspects, a genetic modifier is administered to the subject to reduce the amount of a susceptibility genetic variant in the subject and/or to increase the amount of a protective protein variant in the subject. In some aspects, immune cells and/or HSCs that have been modified to reduce the amount of susceptibility genetic variants and/or increase the amount of protective genetic variants are administered to the subject.

In some aspects, the ratio of protective genetic variant to susceptible genetic variant is increased in the subject or in a cell or population of cells of the subject. In some aspects, the ratio of protective protein variant to susceptible protein variant is increased in the subject or in a cell or population of cells of the subject.

In some aspects, HSC cells are harvested from blood or bone marrow and then the genetic modifier is applied to the harvested cells ex vivo. In some aspects, CD34+ cells are isolated from blood samples using immunomagnetic or immunofluorescence methods (CD34 protein is a member of the single-pass transmembrane sialoprotein protein family expressed on early hematopoietic and vascular-related tissues. Although CD34+ is commonly associated with HSCs, CD34+ is also found on other cell types. See, e.g., Sidney, Stem cells, 32(6):1380-9 (2014). Clinically, it can be used for selection and enrichment of hematopoietic stem cells for bone marrow transplantation. CD34+ cells can be collected from blood using antibodies that bind to CD34 and are attached to magnetic beads or fluorescent labels to enable subsequent isolation and subsequent gene editing of these cells. These cells can also be cultured before or after gene editing. The cells can then be returned to the subject by transfusion or injection. In some aspects, the subject is subjected to chemotherapy or radiation to eliminate most of their bone marrow HSCs, and then the edited HSCs or CD34+ cells are returned to the blood such that the bone marrow is re-colonized by the majority of the edited HSCs. In some aspects, the dose of radiation or chemotherapy used herein is lower than the lethal dose for patients with bone marrow cancer (e.g., to deplete host HSCs but not eliminate the entire HSC population). In some aspects, an antibody that disrupts HSC function and causes depletion of host HSCs is administered to the host prior to infusion of the edited HSCs. For example, HSCs express c-Kit (CD117), a dimeric transmembrane receptor tyrosine kinase, which is involved in HSC function. anti-c-Kit monoclonal antibodies can be used with immunosuppression to deplete HSCs from the bone marrow niche, allowing for edited HSC engraftment. In some aspects, depletion of host HSCs can be achieved without immunosuppression by radiation or chemotherapy, for example, by administering antibodies that disrupt HSC function and deplete HSCs and other antibodies that make host HSCs more vulnerable. For example, it has been shown that host HSC clearance is dependent on Fc-mediated antibody effector function, and HSCs express CD47 (a myeloid-specific immune checkpoint), which CD47 generates a "no-kill" signal and binds to sirpa. See Chhabra, Science relative Medicine,8(351):351ra105 (page 10) (2016). Thus, enhancement of effector activity by blocking CD47 may extend anti-c-Kit opsonization to fully immunocompetent subjects. Treatment with the c-Kit antibody together with interference of the CD 47-sirpa axis with the CD47 antibody resulted in elimination of > 99% of host HSCs and achieved robust multilineage blood reconstitution following HSC transplantation. Thus, in some aspects, an anti-c-Kit monoclonal antibody is used to clear HSCs from a subject along with a blockade of CD47 prior to infusion of edited HSCs.

In some aspects, cells directly involved in the body's immune response are harvested from blood, including, for example, lymphocytes (e.g., B cells, T cells, and/or natural killer cells), monocytes, and/or dendritic cells. Once harvested from the blood, these immune cells can be edited in vitro, optionally cultured, and then returned to the subject's bloodstream. Without being bound by theory, it is believed that the half-life of many such cells is about one year. Some methods for isolating these cells include, for example, cell preparation tubes containing heparin sodium (CPT) manufactured by Becton Dickinson, FicollPaque Premium (density 1.077g/mL) manufactured by GE Healthcare, and Lymphoprep using SepMate tubes manufactured by stem cell Technologies, according to the manufacturer's instructions. For advantages and disadvantages of different methods with respect to cell separation efficiency and cell viability, see for example Grievink, Biopreservation and Biobanking,14(5): 410-.

Cells that encode the protective genetic variant and/or do not encode the susceptibility genetic variant can be administered to the subject via any known method (e.g., via intravenous administration or transplantation).

Some aspects relate to editing bone marrow stem cells that make blood cells. In some aspects, the genetic modifier is delivered into the bone marrow. In some aspects, the delivering comprises passive targeting via: polymers of neutral charge and suitable size (e.g., about 150 nm); liposomes surface-modified with anionic glutamate to increase distribution in the bone marrow via selective uptake by macrophages and greatly reduce distribution in the liver and spleen; and/or a molecule that specifically binds to a bone-forming surface (e.g., a diphosphate). See, e.g., Chao-Feng, Biomaterials,155:191-202 (2017). These delivery mechanisms, which may, for example, include polymeric encapsulation, can be used to deliver CRISPR/Cas9 or other gene editing complexes to the bone marrow.

Some aspects relate to direct stem cell transplantation in bone marrow with edited stem cells with or without the use of radiation to reduce a population of bone marrow stem cells with primordial germline alleles. Some aspects include methods of placing bone marrow stem cells into a patient's bloodstream (e.g., returning the cells to the bone marrow).

In some aspects, cells obtained from a subject in need of treatment are administered to the subject, followed by genetic modification to reduce the amount of susceptibility genetic variants and/or increase the amount of protective genetic variants. In some aspects, cells obtained from a different subject (e.g., cells from a different subject that have been genetically modified or naturally encode a desired genetic variant) are administered to a subject in need of treatment.

Genetic modifier

Methods and compositions are provided that can be used, for example, to perform a desired genetic modification using a genetic modifier. The genetic modifier can comprise a nuclease, such as a CRISPR system, a zinc finger nuclease system, a TALEN, a meganuclease, or an RNAi system.

CRISPR system

In some aspects, the genetic modifier is a CRISPR-Cas or CRISPR system, collectively referring to the transcript and other elements involved in expression of or directing the activity of a CRISPR-associated ("Cas") gene, including sequences encoding the Cas gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (encompassing "direct repeats" and partial direct repeats of tracrRNA processing in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or "one or more RNAs" (e.g., one or more RNAs for guiding Cas (such as Cas9), e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgrna chimeric)) or other sequences and transcripts from the CRISPR locus. Generally, CRISPR systems are characterized by elements (also referred to as protospacer in the context of endogenous CRISPR systems) that promote the formation of CRISPR complexes at target sequence sites. See, e.g., Shmakov, Molecular Cell,60(3):385-97 (2015); zetsche, Cell,163(3) P759-771 (2015); WO 2014/093622(PCT/US 2013/074667).

In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of the CRISPR complex. The target sequence may comprise a DNA polynucleotide or an RNA polynucleotide. In some aspects, the target sequence is located in the nucleus. In some aspects, the target sequence is located in the cytoplasm of the cell.

In certain exemplary aspects, a CRISPR effector protein can be delivered using a nucleic acid molecule encoding the CRISPR effector protein. The nucleic acid molecule encoding a CRISPR effector protein may advantageously be a codon optimized CRISPR effector protein (e.g., a sequence optimized for expression in a eukaryote such as a human (i.e., optimized for expression in a human) or optimized for expression in another eukaryote, animal, or mammal). See, for example, WO 2014/093622(PCT/US 2013/074667).

Non-limiting examples of Cas proteins include cassl, CaslB, Cas (also known as Csnl and Csxl), CaslO, Cpf, Csyl, Csy, Csel, Cse, Cscl, Csa, Csn, Csm, Cmrl, Cmr, Csbl, Csb, Csxl, CsaX, Csx, Csxl, Csfl, Csf, or homologs or modified forms thereof. Although Cas9 is the most widely used Cas protein, other complexes can also be used to edit DNA in potentially improved ways compared to Cas 9. For example, Cpf1 enables staggered cleavage of target DNA, where an overhang is present on one strand of the DNA, thereby enabling more specific DNA reassembly. In addition, Cpf1 requires one CRISPR RNA (crRNA) for targeting, while Cas9 requires both crRNA and transactivating crRNA (tracrrna). Smaller crrnas enable multiplexed genome editing because more crrnas can be packaged in a single vector. Furthermore, Cas9 cleaves the target DNA at3 nucleotide bases upstream of the target site, while Cpf1 cleaves the target DNA at 18-23 bases downstream of the target site, allowing the target region to remain intact and multiple rounds of cleavage at the target locus to increase the likelihood of specific editing. The present invention encompasses all editing methods discussed herein, as well as others, and is not dependent on the particular editing method used.

Some aspects relate to vectors, e.g., for delivering or introducing Cas and/or RNA capable of directing Cas to a target locus (i.e., guide RNA) in a cell, and also for propagating these components. As used herein, a "carrier" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon (e.g., plasmid, phage or cosmid) into which another DNA segment may be inserted to cause replication of the inserted segment. In general, a vector is capable of replication when associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other various 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 (e.g., by standard molecular cloning techniques). Another type of vector is a viral vector, wherein viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). The viral vector also comprises a polynucleotide carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell 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 a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Common expression vectors for use in recombinant DNA technology are typically in the form of plasmids.

A recombinant expression vector may comprise a nucleic acid in a form suitable for expression of said nucleic acid in a host cell, which means that said recombinant expression vector comprises one or more regulatory elements, which may be selected on the basis of the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows for 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).

The one or more vectors may comprise one or more regulatory elements, such as one or more promoters. The one or more vectors can comprise a Cas coding sequence and/or a single guide RNA, but can also comprise at least 3or 8 or 16 or 32 or 48 or 50 guide RNA (e.g., sgRNA) 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 when there are up to about 16 RNAs, there may be one promoter for each RNA (e.g., sgRNA); and when a single vector provides more than 16 RNAs, one or more promoters may drive expression of more than one RNA, for example when there are 32 RNAs, each promoter may drive expression of two RNAs, and when there are 48 RNAs, each promoter may drive expression of three RNAs.

Guide molecules

The terms "guide sequence" and "guide molecule" encompass any polynucleotide sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. The guide sequence prepared using the methods disclosed herein can 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 aspects, the degree of complementarity of the guide sequence to a given target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. In certain exemplary aspects, the guide molecule comprises a guide sequence that can be designed to have at least one mismatch with a target sequence such that an RNA duplex is formed between the guide sequence and the target sequence. In some aspects, the guide sequence is designed as a stretch with two or more adjacent mismatched nucleotides such that the degree of complementarity across the guide sequence is further reduced. Optimal alignments can be determined 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 targeting guide RNA) to direct sequence-specific binding of the nucleic acid targeting complex to the target nucleic acid sequence can be assessed by any suitable assay. For example, components of the nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex can be provided to a host cell having a corresponding target nucleic acid sequence, e.g., by transfection with a vector encoding the components of the nucleic acid-targeting complex (including the guide sequence to be tested), followed by assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, e.g., by a surfyor assay described herein. Similarly, cleavage of a target nucleic acid sequence (or a sequence adjacent thereto) can be assessed in vitro by providing the target nucleic acid sequence, a component of the nucleic acid targeting complex (including the guide sequence to be tested and a control guide sequence different from the test guide sequence) and comparing the rate of binding or cleavage at or adjacent to the target sequence between the test and control guide sequence reactions. The guide sequence, and thus the nucleic acid targeting guide RNA, can be selected to target any target nucleic acid sequence.

In certain aspects, the guide sequence or spacer of the guide molecule is 15 to 50 nucleotides (nt) in length. In certain aspects, the spacer of the guide RNA is at least 15 nucleotides in length. In certain aspects, the spacer length is from 15 to 17nt (e.g., 15, 16, or 17nt), from 17 to 20nt (e.g., 17, 18, 19, or 20nt), from 20 to 24nt (e.g., 20, 21, 22, 23, or 24nt), from 23 to 25nt (e.g., 23, 24, or 25nt), from 24 to 27nt (e.g., 24, 25, 26, or 27nt), from 27 to 30nt (e.g., 27, 28, 29, or 30nt), from 30 to 35nt (e.g., 30, 31, 32, 33, 34, or 35nt), or 35nt or more. In certain exemplary aspects, 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, or 100 nt.

In certain aspects, the guide molecule comprises (1) a guide sequence capable of hybridizing to a target locus and (2) a tracr mate or direct repeat sequence, wherein the direct repeat sequence is located upstream (i.e., 5') of the guide sequence. In some aspects, the seed sequence of the guide sequence (i.e., the sequence associated with the sequence that recognizes and/or hybridizes to the target locus) is within about the first 10 nucleotides of the guide sequence.

In particular aspects, the guide molecule comprises a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem loops or optimized secondary structures. In a particular aspect, the direct repeat sequence has a minimum length of 16nt and a single stem loop. In a further aspect, the direct repeat sequence has a length greater than 16nt, preferably greater than 17nt, and has more than one stem loop or optimized secondary structure. In particular aspects, the guide molecule comprises or consists of a guide sequence linked to all or part of a naturally occurring direct repeat sequence. Typical type V or type VI CRISPR-cas guide molecules comprise (in the 3 'to 5' direction or in the 5 'to 3' direction): a guide sequence, a first segment of complementary sequence ("repeat"), a loop (which is typically 4 or 5 nucleotides in length), a second segment of complementary sequence ("anti-repeat sequence" complementary to the repeat), and a poly a (typically a poly U in RNA) tail (terminator). In certain aspects, the direct repeat sequence retains its native architecture and forms a single stem loop. In certain aspects, certain aspects of the guide architecture may be modified, for example, by the addition, removal, or substitution of features, while certain other aspects of the guide architecture are maintained. Preferred positions for engineered guide molecule modifications (including but not limited to insertions, deletions and substitutions) include guide ends and regions of the guide molecule that are exposed when complexed with the CRISPR-Cas protein and/or target, such as the stem loops of the direct repeats.

In some aspects, the CRISPR system effector protein is an RNA-targeted effector protein. In certain aspects, the CRISPR system effector protein is a type VI CRISPR system targeting RNA (e.g., case 3a, case 3b, case 3c, or case 3 d). Exemplary RNA-targeted effector proteins include Casl3b and C2C2 (also known as Casl3 a). As used herein, the term "case 3" refers to any type VI CRISPR system targeting RNA (e.g., case 3a, case 3b, case 3c, or case 3 d). When the CRISPR protein is a C2C2 protein, no tracrRNA is required. C2C2 or Cas13 have been described in the following documents: 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).

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

In certain other exemplary aspects, the CRISPR system effector protein is C2C2 nuclease. The activity of C2C2 may depend on the presence of two HEPN domains. These have been shown to be rnase domains, i.e. nucleases (particularly endonucleases) that cleave RNA. C2C2 HEPN may also target DNA, or potentially target DNA and/or RNA. The HEPN domain of C2C2 is at least capable of binding to RNA and cleaving RNA in its wild type form, based on which it is preferred that the C2C2 effector protein has rnase function. See Abudayyeh, Science,353(6299) aaf5573-1-aaf5573-9 (2016).

Tale system

In some aspects, the genetic modification is by a transcription activator-like effector nuclease (TALEN) system. Transcription activator-like effectors (TALEs) can be engineered to bind virtually any desired DNA sequence. Exemplary methods of genome editing using TALEN systems can be found in the following documents: for example, Cerak, Nucleic Acids Res.,39(21): e82 (2011); zhang, nat.Biotechnol.,29: 149-.

Some aspects include the use of an isolated, non-naturally occurring, recombinant or engineered DNA binding protein comprising a TALE monomer as part of its tissue structure, enabling targeting of nucleic acid sequences with improved efficiency and increased specificity. The structure and function of TALEs are also described in the following documents: for example, Moscou, Science,326:1501 (2009); boch, Science,326: 1509-; and Zhang, nat. Biotechnology,29: 149-.

Zinc finger nucleases

In some aspects, the genetic modifier comprises a zinc finger system. Artificial Zinc Finger (ZF) technology, which involves arrays of ZF modules to target new DNA binding sites in the genome, provides one type of programmable DNA binding domain. Each finger module in the ZF array targets three DNA bases. Custom arrays of individual zinc finger domains are assembled into ZF proteins (ZFPs).

The ZFPs may comprise functional domains. The first synthetic Zinc Finger Nuclease (ZFN) was developed by fusing the ZF protein to the catalytic domain of the type IIS restriction enzyme Fokl. (Kim, Proc. Natl. Acad. Sci. U.S.A.,91, 883-containing 887 (1994); Kim, Proc. Natl. Acad. Sci. U.S.A.,93, 1156-containing 1160 (1996)). Increased cleavage specificity and reduced off-target activity can be obtained by using paired ZFN heterodimers, thereby targeting each ZFN heterodimer to 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 variety of organisms. Exemplary methods of genome editing using ZFNs can be found in the following documents: for example, U.S. Pat. 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.

Meganucleases

As disclosed herein, editing can be performed by meganucleases, which are deoxyriboendonucleases characterized by large recognition sites (double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methods of using meganucleases can be found in the following references: 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.

RNAi

In certain aspects, the genetic modifier is an RNAi (e.g., shRNA). As used herein, "gene silencing" or "gene silenced" with respect to the activity of an RNAi molecule (e.g., siRNA or miRNA) refers to a reduction in the mRNA level of a target gene in a cell 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% relative to the mRNA level found in a cell in the absence of the miRNA or RNA interfering molecule. In a preferred aspect, the mRNA level is reduced by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

As used herein, the term "RNAi" refers to any type of interfering RNA, including but not limited to siRNAi, shRNAi, endogenous microrna, and artificial microrna. For example, it includes sequences previously identified as sirnas regardless of the mechanism of downstream processing of the RNA (i.e., although sirnas are believed to have a specific method of in vivo processing leading to mRNA cleavage, such sequences can be incorporated into vectors in the context of flanking sequences described herein). The term "RNAi" may include both gene-silencing RNAi molecules and RNAi effector molecules that activate gene expression.

As used herein, "siRNA" refers to a nucleic acid that forms a double-stranded RNA that has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene. The double-stranded RNA siRNA can be formed from a complementary strand. In one aspect, siRNA refers to a nucleic acid that can form a double stranded siRNA. The sequence of the siRNA may 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 a 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 in length, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

As used herein, "shRNA" or "small hairpin RNA" (also referred to as stem-loop) is one type of siRNA. In one aspect, these shrnas consist of: short (e.g., about 19 to about 25 nucleotides) antisense strands, followed by nucleotide loops of about 5 to about 9 nucleotides, and similar sense strands. Alternatively, the sense strand may precede the nucleotide loop structure, and the antisense strand may follow the nucleotide loop structure.

The terms "microrna" or "miRNA" are used interchangeably herein and are endogenous RNAs, some of which are known to regulate expression of protein-encoding genes at the post-transcriptional level. Endogenous micrornas are small RNAs that occur naturally in the genome that are capable of modulating the productive utilization of mRNA. The term artificial microRNA includes any type of RNA sequence, except endogenous microRNAs that are capable of modulating the productive utilization of an mRNA. Microrna sequences have been described in the following publications: such as Lim, Genes and Development,17: 991-. Multiple micrornas can also be incorporated into a precursor molecule. In addition, miRNA-like stem loops can be expressed in cells as vehicles for delivering artificial mirnas and short interfering rnas (sirnas) with the aim of modulating the expression of endogenous genes via miRNA and/or RNAi pathways.

As used herein, "double-stranded RNA" or "dsRNA" refers to an RNA molecule consisting of two strands. Double-stranded molecules include double-stranded molecules composed of a single RNA molecule that is folded upon itself to form a double-stranded structure. For example, the stem-loop structure of an ancestral molecule from which a single-stranded miRNA is derived (known as a pre-miRNA) (Bartel,. Cell,116(2):281-297(2004)) comprises a dsRNA molecule.

Some aspects relate to methods of generating eukaryotic cells comprising modified or edited genes. In some aspects, the method comprises (a) introducing one or more vectors into the eukaryotic cell, wherein the one or more vectors drive expression of one or more of: a Cas effector module and a guide sequence linked to a direct repeat, wherein the Cas effector module associates with one or more effector domains that mediate base editing, and (b) allows binding of a CRISPR-Cas effector module complex to a target polynucleotide to effect base editing of the target polynucleotide within the diseased gene, wherein the CRISPR-Cas effector module complex comprises the Cas effector module complexed with a guide sequence that hybridizes to a target sequence within the target polynucleotide.

Another aspect relates to an isolated cell obtained or obtainable from a method described herein, comprising a composition described herein or a progeny of said modified cell. In a particular aspect, the cell is a eukaryotic cell, preferably a human or non-human animal cell. In some aspects, the cell is an immune cell. In some aspects, the cell is a HSC.

Some aspects include an isolated immune cell or hematopoietic stem cell, wherein the cellular DNA has been modified by gene editing to (a) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and/or (b) increasing the amount of one or more genetic variants that prevent the autoimmune disorder. Some aspects include 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) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and/or (b) increasing the amount of one or more genetic variants that prevent the autoimmune disorder.

The invention also relates to a method for cell therapy, the method comprising administering to a patient in need thereof a modified cell described herein, wherein the presence of the modified cell treats a disease in the patient.

The following examples are included to illustrate the compositions and methods described herein. The examples are in no way intended to limit the scope of the invention. Other aspects will be apparent to those skilled in the art.

Examples

Example 1: addressing immune disorders using gene editing

This example uses CRISPR/Cas9 gene editing as known in the art. CRISPR stands for regularly interspaced clustered short palindromic repeats. CRISPR sequences contain DNA segments from viruses that have attacked a cell and are used by the cell to produce RNA that can be detected and combined with Cas proteins to destroy DNA from similar viruses. CAS stands for CRISPR-associated system and the gene encoding CAS protein is found in the cell DNA in the vicinity of the CRISPR sequence. A Cas9 protein that is synthetically modified and integrated with a guide rna (gRNA) is capable of binding to a specific target DNA of a matching gRNA and cleaving the DNA on both strands. This double-stranded cleavage results in the replacement of the target DNA by the replacement DNA, or initiates a homologous repair process in the cell, wherein the corresponding DNA in the homologous chromosomal or template DNA is copied to repair the cleaved DNA.

In addition, this example will describe experiments performed on murine models to demonstrate that their IBD symptoms can be reduced. Chemically and/or genetically induces colonic inflammation in mice. In particular, 2,4, 6-trinitrobenzenesulfonic acid (TNBS) is administered intrarectally with ethanol; administering oxazolone intrarectally; or the polysaccharide sulfate dextran sulfate sodium sulfate (DSS) is administered orally via drinking water. See, e.g., Wirtz, Nature Protocols,12(7): 1295-. Alternatively or additionally, the mice are genetically modified to induce inflammatory bowel disease. For example, mice are genetically modified to Knock Out (KO) NOD1 and/or NOD 2. See, e.g., Natividad, Inflummatory Bowel Diseases,18(8):1434-46 (2012); zenewicz, Immunity,29(6):947-57 (2008). Alternatively or additionally, the mouse is genetically modified based on models involving IL-10KO, IL-2KO, TCRa KO, TGFb KO, TAK1 KO, WASP KO, or MDR1A KO, and the like. See, e.g., Mizoguchi, Progress in mol. biol. Transl. Sci.,105:263-320 (2012); wirtz, adv. drug deliv. rev.,59(11):1073-83 (2007).

More specifically, we consider two mutations. The first is a susceptibility mutation to IBD, such as a loss of function mutation on IL-10, such as, for example, 113Gly → Arg, which, without being bound by theory, is believed to result in the inability of IL-10 to induce STAT3 phosphorylation or to inhibit Lipopolysaccharide (LPS) -mediated TNF release in peripheral blood mononuclear cells. See, e.g., Glocker, ann.n.y.acad.sci.,1246:102-07 (2011). The second is a protective mutation on IL23R for IBD as discussed above: R381Q, corresponding to rs11209026, c.1142g > a.

Four different groups of mice were used, depending on the gene editing performed on embryos of standard strain: group A, having heterozygous IL-10 susceptibility mutations and no IL23R protective mutations; group B, no IL10 and or IL23R mutations; group C, with IL10 mutation and with IL23R protective mutation; and group D, no IL10 mutation and with an IL23R protective mutation.

The mice were generated by CRISPR/Cas9 editing of common mouse strains (e.g., C57 BL/6). Alternatively, mice can also be generated from strains that are less sensitive to pain, such as, for example, Nav1.7 KO mice. See, e.g., Shields, J neurosci, 38(47) 10180-10201 (2018). The experiments can also be conceptually similar using humanized mice, i.e., immunodeficient mice implanted with a functional human immune system. See, e.g., Kenney, am.j.transplant, 16(2):389-97 (2016). Furthermore, the experiments will be conceptually similar using existing mouse models that do not require specific editing, such as, for example, using C57BL/6NTac-Il10 from Tastic Biosciences^em8TacA mouse model edited using CRISPR/Cas 9-mediated gene editing to delete the Il10 locus (exons 1 to 5, including the proximal promoter and UTR) from a standard C57BL/6NTac strain.

In HSC-edited mice, HSCs are harvested from bone or Blood by capturing CD34+ cells (note that although CD34 is expressed in almost all human HSCs, it is expressed on approximately 20% of murine HSCs see Ogawa, Annals of the New York Academy of Sciences,938:139-45 (2001); however, murine expression can be stimulated and reversed to alter the fraction of murine HSCs that express CD34 and can be harvested see Tajima, Blood,96(5):1989-93 (2000)).

The harvested cells are then edited ex vivo and returned to the bloodstream of mice whose native HSC population has been largely depleted. This can be achieved by using non-radioactive non-chemotherapy techniques that administer anti-c-Kit antibodies (such as ACK2) and anti-CD 47 antibodies (such as CV1mb modified from CV1 that effectively targets human CD47 to target mouse CD47) that have been shown to deplete host HSCs in large numbers, enabling robust engraftment of HSCs delivered from syngeneic donor mice by injection, with greater than 60% chimerism of donor-derived HSCs 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 relative Medicine,8(351):351ra105 (page 10) (2016). Mice can be treated as follows: 500mg of ACK2 was injected on day 1 and 500mg of CV1mb were daily for 5 days from day 1. Transplantation can be initiated 6 days after treatment by: approximately 1 million edited HSCs were transferred daily by injection for 3 days. Approximately 24 weeks after transplantation, the number of B cells, T cells, NK cells, granulocytes (neutrophils, eosinophils, basophils, and mast cells) carrying the relevant edits compared to the host primitive cells can be counted to determine whether the edited HSCs were successfully engrafted.

It should be noted that the experiments will be conceptually similar to be replaced or enhanced with other anti-CD 47 antibodies (such as CV1, MIAP410, or Hu5F9G4), which are currently undergoing clinical trials in humans for solid and hematologic cancers (clinical trials NCT02216409 and NCT 02367196). Furthermore, the experiments will be conceptually similar if more implantation procedures are performed or anti-c-Kit antibody and anti-CD 47 antibody treatments are enhanced using radiation or chemotherapy.

Gene editing can be achieved by Homology Directed Repair (HDR) via CRISPR/Cas9, where template DNA matching the homologue in the unedited DNA region but carrying the new sequence is copied to replace the DNA after cutting the strand. In particular, one quarter of the mice in group a (group a1) were genetically edited to remove IL10 susceptibility mutations; gene editing of one quarter of the mice in group a (group a 2) to add a protective IL23R mutation; and one quarter of the mice in group a (group a 3) were gene edited to add a protective IL23R mutation and to remove the IL10 mutation. Half of the mice in group B (group B2) were gene edited to add the IL23R mutation. Half of the mice in group C (group C2) were gene edited to remove the IL10 mutation. To make the number remaining in A, B and group C similar to the number in groups a1, a2, A3, B2, C2, D, we can start with the number of mice in the 4:2:2:1 ratio in A, B, C and group C, respectively.

After waiting for the edited HSCs to generate immune cells (e.g., after about 6 months), and assuming successful engraftment in the edited group, one of the above methods was used to chemically induce colitis in mice. The phenotypic severity was then compared between A, B, C, D, A1, a2, A3, B2 and C2 groups in terms of weight loss, bleeding and diarrhea. The following table (table 1) shows the initial genetic state, the theoretically edited genetic state and one possible grouping of symptom levels. Without being bound by theory, if HSC editing is perfectly efficient, we 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 far counteracted the susceptibility effect of the IL10 variant, we expect symptoms to rank from worst to lightest as a to D.

Table 1: genetic status and symptom level of each group

CRISPR-Cas9 with guide rna (grna) was used to confer a protective mutation to gene IL 23R. More specifically, grnas were designed to attach to DNA encoding IL23R amino acid sequence as shown below:

MNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNTGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK(SEQ ID NO:1)。

the corresponding DNA sequence is shown in SEQ ID NO 2 of the sequence Listing.

The criprpr-CAS 9 complex is designed to cleave DNA in the region of R at position 381, and the template DNA is designed to effect homologous repair such that R at position 381 is converted to Q, or alternatively such that G at nucleotide position 1142A is converted to a. Template DNA that can accomplish this will match the DNA sequencer around position 1142 on the reference DNA (SEQ ID NO: 2).

The guide RNA template was designed to target a specific region of the IL23R gene.

Exemplary murine IL23R target sequences for constructing 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)

an exemplary guide RNA sequence targeting murine IL23R comprises the following sequence:

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)

exemplary human IL23R target sequences for constructing 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)

an exemplary guide RNA sequence targeting human IL23R comprises the following sequence:

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)

exemplary target sequences for constructing a guide RNA sequence 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)

an exemplary guide RNA sequence targeting G1142A in human IL23R comprises the following sequence:

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)

the wild-type human IL-10 sequence is shown below:

MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:37)

exemplary target sequences for constructing 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)

an exemplary guide RNA sequence targeting the G113R amino acid mutation in human IL-10 comprises the following sequence:

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)

example 2: machine learning method for selecting gene mutations to be edited

Machine learning techniques are used to describe the likelihood of developing a phenotype based on gene mutations to determine which genes or variants should be edited. Such techniquesIncluding, for example, linear regression models, logistic regression models, nonlinear regression models that contain interactions of genes or gene variants, regression models that use principal component analysis or limiting functions to add constraints to the regression problem in cases of underdetermined or noisy due to too many possible genes or too little patient data. The limiting function of the regression parameters may include L used in ridge regression₂L used in norm or LASSO regression₁And (4) norm. Non-linear interactions between genes can be captured while still keeping the model linear in regression parameters by logically or mathematically combining independent genetic variables to create new variables to be used in the linear model. Nonlinear interactions can also be captured using models that are nonlinear in terms of parameters, such as neural networks including deep learning neural networks, or support vector machines. Several of these methods are described, for example, in Rabinowitz, Bioinformatics,22:541-549 (2006). The genes or genetic mutations that most affect the disease phenotype or disease phenotype risk are determined by looking at the size of the regression parameters, which is particularly simple for linear models, or by modeling different data and presenting them to a non-linear model. Other techniques to identify which variants are associated with disease include tools to identify genes most likely to cause a phenotype near the risk locus from Genome Wide Association Studies (GWAS) using gene function and gene signaling pathway data. See, e.g., Pers, Nature Communications,6, article 5890 (page 9) (2015).

In addition to the already mentioned references, the following should be mentioned:

sands, inflam. bowel dis.,23(1):97-106 (2017); jinek, Science,337: 816-; 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, rs 11209026; US National Library of Medicine, dpSNP, rs 11209026; duerr, Science,314(5804):1461-63 (2006); hazlett, Genes & Immunity,13:282-87 (2012); ferguson, Gastroenterology Research and Practice, article ID 539461 (page 12) (2010); mu, Biomaterials,155:191-202 (2018); angermann, Nature Methods,9:283-89 (2012); gong, j.r.soc.interface,14:20170320 (page 13) (2017); drug Targets,15(6) 565-72 (2014); Bassaganya-Riera, Clin. Nutr, 25(3) 454-65 (2006).

Sequence listing

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Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr

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Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn Ser Gly Leu

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acaagggtgg cagcctggct ctgaagtgga attatgtgct tcaaacaggt tgaaaggtaa 60

ataggcagcg atcatccaaa tacttaaaga cagctatatt tccttttgga aatacatggg 120

ccaagttgtt ttctcttgca agttctcatg atattccatg cagtggttac tgatgaaaag 180

ggaatctata atattatatg gcatcacatt actttaaata ataattattg catatttcca 240

tcagttaact tctaactagg aacattttta gggcttctgt tgcttcttat gaagagaaga 300

attactattg ctaacaaata atgaaaggga tccctatatt tctgagccaa ggggacccca 360

gtttttttaa atgttactag atattttatg cgatatgatg agccatgcat tgcgttctct 420

tgtagtttta tagaaaaagc tcaggtttta ttaaacagat aaaataactg aataatgttg 480

atcattgagt aaatgagtta attcatgtaa ctgattaatc aaatagtgta actctagttg 540

taaactaaca acggtattat taggaatttc tataacgtgt tcctttcgcg ttctctacag 600

atgaaaggct ttttcctctt ttttaggggg tgcgggggac aaggtctcac tctgtcgccc 660

aggctggagt gcaatggtgt gatcatggtt cactgcaacc ttcacctccc gggtagctgg 720

gatgaccgtc acatgccacc acactgggct aatttttgta tttttagtag ggacagggtt 780

tcaccctgtt gcccagcctg atctcgaact cctgggctca agcaatccac cggcctctgc 840

ctcccaaagt gctgggatta caggcatgag ccaccacgcc tccccagatg aaacgctttt 900

tagagtctca ctggtgagag cacatattgc ttaatctcac ttcacttttc ttaattttga 960

atccattttc tctgggagtt ctgaaatttt tggctcttcc caagtttagc tcatcaaaga 1020

ctagtatttc tggagtgtga taagtatgta actttggcag ggagaagaat tcatcaccag 1080

aagttagctc agcccaggca ccatggttca tgcctgtaat cctagcactt tgggaggttg 1140

agttgggagg accgcttgag cccaggagtt tgaggttaca gtgagctatg ataatgccgc 1200

tgcactccag cctgggctac agagcctggc ccttgtctct aaaaaaattt aaaacataaa 1260

ataaaaaaag aagttaactc agaggaggag gtttctgatg agaaaggcta gattaggagg 1320

cactgtaatg gatcaaatgc cctactgagt taattcatag tgacttttga agagtataaa 1380

attccattta tttgctttac ttctccatct cttgttatat ctcctaaata taccagtaac 1440

atttctttgc tctgtttcct tccttccttt cttccttcct tctttccttc ttcctcccta 1500

atcaaaggtt cccatcaaat acaataattc ttagggaaaa atgttatgct ttttattatt 1560

ttactaaaat actacaattt aaacattttt catatttttt ttccagaggg aaacagtctt 1620

ttcctgcttc cagacatgaa tcaggtcact attcaatggg atgcagtaat agccctttac 1680

atactcttca gctggtgtca tggaggtatg gtgttttatg tatctattgc tatctttcat 1740

tcaacaaatg gaatattgag tgattatcat tttcatggtt ttatgttaga atctctgaag 1800

aatacaaata ttattagact agaaaaaatg tgtgcataaa aattacatag aatgaaataa 1860

aaatggccca atctccttcc aaatgcagga tttctttctc aaatttccct tctagacagc 1920

cctcttactc acatgtggat atttccacaa aggagcttat tagtttgagt tggcatgttc 1980

tattgtcaaa ctattataac tgttatataa ttacctgtca aacatttgga tgcaattcta 2040

tttcacaaac cacttgttga tagttacttt gtgccgggca catagtaact atgtgggcca 2100

ttggcaagag tatgatttta ttcattatat tcaaacttta ctgaaaaaag taggctttat 2160

ctaaaaaaga acaaaagatt ggttgagtag ctagagaatg gggttcagat tctacagtgc 2220

ctagtgttat ttctaatctt acctttgaaa aatactgtag ctttataatc tctcggttct 2280

ctactttaaa gtaggagagc taattatatt tgctctttag acacttagag aagcttccag 2340

gctgactcta gtcttgtgtc atggtgctga acatcaataa aagcataaca gaggccagac 2400

gaggtggcta atgcctgtaa tcccagcact ttgggaggcc gaggcgggtg gatcacctga 2460

ggtcaggagt tcaagatcag cctggccaac atggtgaaac gccatattag ccgggcgtgg 2520

tggtgctcac ctgtaatccc agctactcag gaggctaagg caggagaatc gcttgaacct 2580

ggaaggcgga tgttgcagtg agccgagatc gcaacactgc actgcagcct gggtgacaga 2640

atgagactgt ctcaaaaaaa aaaaaaaaaa aaaaagccta acagaaacac actgtaatag 2700

gctagtttat gaaaagatac atctagtcct aaaactgttt tcaatggaat catttagtta 2760

atagaaatga taaatttaaa atgcaatagc atattcttct gaatctcttg atttaatgtt 2820

ttacgtgtaa tttattattt ttccatttat ttctaggaat tacaaatata aactgctctg 2880

gccacatctg ggtagaacca gccacaattt ttaagatggg tatgaatatc tctatatatt 2940

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aagaaagatt tcaaatcaca aggattaata aaacaacagc tcggctttgg tataaaaact 3060

ttctggaacc acatgcttct atgtactgca ctgctgaatg tcccaaacat tttcaagaga 3120

cactgatatg tggaaaagac atttcttctg gatgtaagtg ttggggcaca tttgaaatgc 3180

aaacaaaagc tagtttaaca attaactggt cattaccaca ctagtctaaa aatagggaca 3240

aaataatata gttcagtttt tagattagtt tcatacagga gctgcaaact caaatgtctc 3300

cagggccagg ttggtgaact atatgaactg tagccactgc ccagatccag taatcacagg 3360

tgcccggtat tcctacatct tttggctttt caaaggaaga cagaaacata aaattttaat 3420

gtgtactctc taaattttaa aatgttgcca atacattcaa aaaacaattt ttcacctcta 3480

ccctgaacga acactgcata tgtgagggcc agccccgtta gatttaaaat gttgctcttt 3540

taatcttctt gtttattttt ctaaactaat ataatataat taaccttgaa gcagatccca 3600

ttgagaaatt ttgaggccaa ggtttaggga gagttgcaaa actagttttg tgctcccact 3660

gtactgctag gtttgtgacc ttggacaagt tgcttaagcc tcaattcctc gcctgtaaaa 3720

ggaggctaat tatacaggct gtgttatgca cttaatttca ataatttatt tggaagcata 3780

tgataaagtt taaagtacat cagtacccaa atccttaaaa atgcatcctt ttgataaaac 3840

atagggagag tctcctacag tcctctgttc ctaatctcac tattccaatt tagaatcact 3900

gtgtttgtta ttgtaactat taattttttt ctttttctta aaacccacct tgaagggaat 3960

aattattaat ttatttaaaa gcttagtcaa gtcaatctta aaaatatacg ctaaattcaa 4020

tgtgtgctct gagtgtcctg gaggtgttta aatcactata gtgatatggg ctggctcgta 4080

taattatata cctagtttgg gttctactat tatgaagggt gtaaatcaat aagttcagca 4140

ctgttttaac tcagatattc attctctata gcacttgaac ccattttcag taaaaaaata 4200

aaggaatttg tgtctttgcc atgttcccgt cctccacatt tttgagaatc aagcctcagt 4260

tcttgcttct tcttcacctt tacaacttag ctggggggca gacccatatc accactacca 4320

cctgacccca ttgtggctgc ttggaaagta agaggcttcc ttgctctata atgaggccat 4380

tgtcctactt aactctgagc agctgatctt gcaagctttt caagagaagg aactatagaa 4440

agttgtagag gtgagttgag gaaggaaagt cataggtttt ggagtcaaag aataaatcta 4500

ccttaaactg atgccttaaa gctgctaaac ttgccctaaa ttgtggaaat taaaccgcag 4560

gcagacatct tatctgatgt aagaaatctt cttagcttgt ccacatcctc aggaaacata 4620

accaaacctc aatgcccaaa tactttacta tttatccttt gggggtggga gttgggtaga 4680

gagactatac aatttctttg gagggcaaat aagcaacttt cacagttaca gaaaaagctt 4740

agttgttaat ttaagccaga ggaaaaaaaa ggaagcaaaa taaacattgt ctctgttaaa 4800

aaacaaaatt tcagcaaatt tagtttttag atctgatagg tttttatgtg tgattcatga 4860

atcggcagca tctctttcta aaaaactgat atgtgctctg agtgtcctgg aggtgtttaa 4920

gttactatag tgatatgggc tggcttgtat aattatatac ttagttttgg ctctactatt 4980

ctaacagaac agttggtttt tgtaaggtag gaacaagaaa tagaaatttt tataaaaact 5040

accagattgg ttaacattgt tacttcaggt tattttcctt gtaatggtta aagcagaggg 5100

gatttcctta tcatccctgc tcaggttgac tgggctcttt ctacgtggtt gctgtgaatt 5160

ttctgttttc aggaaaaact agtctgtggg agttttacct gcttccttaa agtttcagct 5220

tggttatatg tcacttaaca ggagtgactt cattttggtt tggtatgtgg gggcctagtg 5280

caggagctca gtccaaaaca atggtctcct atacatttta tttaacacca ccttccgtgt 5340

gttgcagttt gtataactga ttactcccaa aagccctccc aagtgttggc tggggggcct 5400

ctgtccccaa acccaacacc aggcaatgta gaaggtaaga gaatggactt tgttcctctt 5460

ccaggacagc ctaatagaaa tacattctct gccgatggct gcttagaaag aacatcactc 5520

tttccactta tttaactttt gagtttgtga attgtaactc cttacatagg cctctggtta 5580

tgagtgtaaa aatgtaactg ccaggtgaga atttactcaa acccctggat tcaagctgaa 5640

tgtcttcttg agctctacat tcttagcaaa caaaacttcc tatttaagta ggtcaaaaaa 5700

gggtttttga gggttttgtg gcattctaga aatcacattg ctttcactgg accactttga 5760

tcacaggata tttgtcatgt gagatttatt atgtcaactt atcatcactg ctttgaacaa 5820

aatgattata tgtatttttt tagactagag acaaattgta aaagacaaga taaatgagtc 5880

attgctttcc ttgaaaagct atccaaatat attttaaagt gctgtgcttt aatgtataat 5940

ttgtgaaata taaaccatat tatcacaatt tccattttta gccacaatcc tagacacttc 6000

ttagattctt atctaagact aaatgcacac atattatggg aaatttgttc taatcaggcc 6060

acaccaatca tgggataaaa tacaagatgt ctttattcat ttaaaaatac caaattaaac 6120

catgcacagt catgtttcca cttaatcatt tgtttaaaat tttaaatatc tttgactttt 6180

taaattttaa cttttggact ttatttacaa tgatgaaagc acatttgtta ttcaataaaa 6240

gcctctgaat atttgtttct aacattgcta ttaagtatga ttacggaaca attaggctgg 6300

cctgtgccct gcttgagtaa gaaattatag agtgatcttt gccagaagca ttgcatctct 6360

ctccatttta atccttcatg ttattatctt aatcaagcca atcaattcat ggaataaact 6420

ttattgagtg gttactatgc accaaaaatg tgttaaatgc tagaagtaca aagaagagga 6480

aatcacggac tgttttcaag aagttctcag tagaaaggga tccttggcta gactctgaat 6540

taacaagaaa cctggcctgt gattcctgag tgtcccaata ataggggagg ggtgggattg 6600

gcaagaaaga gcaaaaacat aagccccctt ttgtatacct cagagtattc tctagtatct 6660

agttgtatag ccaggttttg ttcttggata ttgaataacc tggcaatgag gcctaacaac 6720

aacttgatgt tattatcata agcatactca caggcccatc tttacattgc ttcatgttaa 6780

caaacactta tttgcatgtg tgtgtatagt catctttttg atcttttgca aagttaattg 6840

accaccagac atttgtaacc ccaagaattc acttcatgta gatgccgtgg agtaacacag 6900

cccaaacaat ctgatagcca aatcaatggc actctgtatc tgaaaccaga ggaaaaaata 6960

acttcagcac aaatcattgt aatcaatgag ggaccacaac tcaaactcca aaatgattta 7020

aatttatgtt tccacaccta cttcatttaa ggctcaatta aaacaaagat ttattttacc 7080

ctacttggag cccagtttct ctccattgta actcaatgct acaagtcgcc aaaatgttgt 7140

aaagtacttt ttagtgtgtt cttactgttt gaatataggt ttttttaaaa ggatattaga 7200

ccttcgatat atgaaaatag ttttttaatt ataaaatcaa attgcattga atatagagag 7260

tttttaaaat gtagaccagc aaaaaagaaa tacctatcat tcccataaaa ccataacttt 7320

actagtaacc ataatccctc tttaataatt tggaatatgt ctttgaaatt tcagaccctc 7380

tctttctcag tctctcactt aaatgaaacc actaattgaa ccattttgca acctgctttt 7440

ttcactttgc catatgatca tgacttcact acatatactt ttaattcctt ccaaaggcca 7500

agtacttaaa ctgtcttctt caacattgct tttggatgtg tttaggactg tattatagtt 7560

tatttaacct tattttggaa aattaagttt gtttacaatt ttttcattgt tatgaacaat 7620

aagggaacat cctttaagcc aaatatttta tatatctatg aacatttcaa taaggatgca 7680

ttcttagatg tataattgct gagttgcaaa atattacacc tgcttttaca gattgtgata 7740

tgtaaagtgc tctccataaa ggctgtacca atttgtactt ccaccagaag tgtgttagag 7800

gatttccatg agtccactcc aacactgtgt actatcattt ttcaaaatcc ttgctaattt 7860

gataggcttg aatgacatct agtattgcct tgatttgcag tgagaataaa atttttaata 7920

tatttgttta ttgttctaca agcaatattt ttggtaagca atttgcaggt gaagtgaaag 7980

catatatgta cttaagataa atctttggat gtaacttcag acgcataaat gtctgtattt 8040

tcattcttta tgagccccta tccttccatt tgtcttgtca ttctctgagc agtcaagtgt 8100

cccagcagta tttcacacgt agggatgaac taagagagcc acacctgaaa agtgaatatt 8160

taacaaacac tcaatatttc tatacttaaa aaaatcttaa tcttattcac tattgaatga 8220

tataactgaa aattctaggt gtcattctat agttgttggg gtttcatagg ctttagaggc 8280

ccccaaaact caatgtcatc agaactctct catcttttcc agtctgctag gtattgtttt 8340

cccctgtgtt ggcttcattc ttaaatagac taaagactgt gaaaaagcct gtttcacaat 8400

gacagcaaga tggccaccag caacttacag ctcacatgct actcagagat agcagtttca 8460

ggaggaaaga gtgtgcttct tttacagtgg tttagaaaaa gtgccttgga gaattctgat 8520

tggttcatct tgagtcatat gttcattccc aaaggcacac tcctattagt cggcctacct 8580

cagaggcaca cttctgtgga gagcctcttg attgacaggt cttggggttg gggtgggggt 8640

gagattggag tgaggggctg gaggggatgt cagttctcaa gagggaaaga cgctagccaa 8700

tgaaaaataa aaatgtttgc cataacatgg ccacctggag aaagagctgg gcaggaatgg 8760

gggtgggatg agttgagtgg agtgacagga aaagagcctt gaaatagagt tgcatgaata 8820

ccaaacttta aaaaaacctt gttgtgttgg tggtaaaata cataacataa aatgtaccat 8880

ttttaccatg gttaagtgtg cagttcagtg gcactaagta cacttacact gttgtacaac 8940

catcaccacc atttatctcc agaaccctct tattgcaaat ggaaagtctg cagcctttaa 9000

acaaatatcc acttccctct cctccaagcc ccagcaactg ccattctact ttctacaagt 9060

ttgactcttc cagatacctt gtgtaagggg tcatgcaatg tttttccttt agggtctgtc 9120

ttacttcgct taatataatg tcttcaagtt tcatccacat tgtagcatgt gttaaaattt 9180

ccttcctttt taaggctgag taatatctta ttgcatgtat gaacacccaa cttttaaaat 9240

gagtagtgga aaatgtgcaa gagaagaaga tagtctgcca gagtagtaga agaaaactac 9300

aaagaaccca ttcgcaagag ccaaaggcaa agaggtttct tttcttgtct cgtttttgag 9360

acaaggtctc actgtgtcac ccaggctgga gtgcagtgtc ttggtcatag ctcactgcag 9420

cctcaaactc ctaggctcca ggaatccttc cagcctcagc ctcccaagta gctgagacta 9480

cagatatgag ccaccacacc cagctttaaa acagagagct ttcaaggagg gagtgagcaa 9540

aatatcaaat gctgctcaat gacatcaaac tcaaactcaa tgacaaatga ccagagatgg 9600

ggtctgttga agagaaagtc atcaatgacc ttagcaacaa tggttttggt ggaattgtgg 9660

aggctgaatc ctgattgttt tgggtttaaa tgcatatggg aaatgaagaa taataggctc 9720

tttaaaagtg tttagctgtt aagaaaaaaa gaaagataca ggagtagctg ggggagactg 9780

aatttaggat aatatagtca agtataagaa gaagaggaat ggatatttga tggtaaagtg 9840

ggttgcaagg taaggtagta gggatggaat gaggagcagg gaggtgctga gtactgattg 9900

gcctgacttc tcaaataaaa ttgtcagttt ctcaacggac tatagagaac ttcatatttc 9960

tttttttttt attcttccat tacaaattta ttatcttttc agacaagttg agacatacta 10020

atttaaaata aattcccttt tcagtgaaaa gttgataagt ttaatcaggt aaagctaatg 10080

attaggtaag caatcaatgt ttataattgc atcacaactt ttctggccag tgacttttta 10140

tttatctatt taattttttt aaattatact tgaagtttta gggtacatgt gcacaacgtg 10200

caggcttgtt tcatacatat acatgtgcca tgttggtgtg ctgcacccat taactgatca 10260

tttaacatta ggtatatctc ctaatgctat ccctcccccc tcctcccacc acacaactgg 10320

ccccggtgtg tgatgttccc cttcctgtgt ccatgtgttc tcattgttca gttcccacct 10380

atgagtgaga acatgtggtg tgatagtttg ctgagaatga tggtttccag cttcatccat 10440

gtccctacaa aggacatgaa ctcatcattt tttatggctg catagtattc catgctatat 10500

atgtgccaca tttttttaat ccactctatc attgttggac atttgggttg gttccaagtc 10560

tttgctattg tgaatagtgc cacaataaac atacgtgtgc atgtgtcttt acagcagcat 10620

gatttataat cctttgggta tatactcagt agtgggatgg ctcagtcaaa tggtatttct 10680

agttctagat ccctgaggaa tcgccacact gacttccaca atggttgaac tagtttacag 10740

tcccaccaac agtgtaaaag tgttcctatt tctccacatc ctctccagca cctgttgttt 10800

cctgactttt taatgatcac cattctacct ggtgtgagat ggtatctcat tgtggttttg 10860

atttgcattt ctctgatggc cagtgatgat gagcattttt tcatgtgtct gttggctgca 10920

taaatgtctt cttttgagaa gtgtctgttc atatctgttg cccacttttt gatggggttg 10980

tttgtttttt tcttgtaaat ttgtttgagt tctttgtaga ttctggatat tagccctttg 11040

tcagatgagt agattgcaaa aattttctcc cattctgtag gttgcctgtt cactctgatg 11100

gtagtttctt ttgctgtgca gaagctcttt agtttaatta gatcccattt gtcaattttg 11160

gcttttgttg ccattgtttt tagtgttttc atcatgaagt ctttgcccat gcctatgtcc 11220

tgaatggtat tatctaggta ttcttctagg gtttttatgg ttttaggtct tatgtttaag 11280

tattgaatcc atcttgagtt aatttttgta taaggtgcaa ggaagggatc cagtttcagc 11340

cttatgcata tggctagcca gtttcccaac acaatttatt aaatagggaa tcctttccct 11400

attgctcgtt tttgtcaggt ttgtcaaaga tcagatggtt gtagatgtgt ggtgttattt 11460

ctgaggtctc tgttctgttc cattggtcta tatatctgtt ttggtaccag tatcctgctg 11520

tttttgttac tgtagccttg tagtatagtt tgaagtcagg tagcatgatg cctccggctt 11580

tgtccttttg gcttaggatt gacttggcga tgcaggcttg tgtttggttc catatgacct 11640

ttaaactagt tttttccaat tctgtgaaga aagtcattgg tagcttgatg gggatggcat 11700

tgaatctata aattaccttg ggcagtatgg ccattttcac gatattgatt cttcctcccc 11760

atgagcatgg aatgttcttc catttgtttg tgtcctcttt tatttcattg tacagtggtt 11820

tgtagttctc tttgaagagg tccttcacat cccttgtaag ttggattcct aggtatttta 11880

ttctctttga agcaattgtg aatgggagtt cacttttgat ttggctctcc atttgtctgt 11940

tattggtgta taagaatgct tgtgattttt gcacattgat tttgtatcct gagactttgc 12000

taaagttgcc tatcagctta aggagatttt gggctgagac gatggggttt tctagatata 12060

caatcatgtc atctgcaaac agggacaatt tgacttcctc ttttcctaat tgaataccct 12120

ttatttcctt cacctgcctg attgccctgg ccagaacttc caacactatg ttgaatagga 12180

gtggtgagag agggcatccc catcttgtgc cagttttcaa agggaatgct tccagttttt 12240

gcccattcag atgatattgg ctgtgggttt gtcatagata gctcttatta ttttgagata 12300

cgtcccacta atacctaatt tattgagagt ttttagcatg aagcttgttg aattttgtca 12360

aaggcctttt ctgcagctat tgagacaatc atgtggtttt tgtcattggt tctgttgata 12420

tgctggatta cgtttattga tttgcatatg ttgaaccagc cttgcctccc agggaggaag 12480

cccacttgat catggtggat aagctttttg atgtgctgtt ggattcggtt tgccagtatt 12540

ttattgagga tttttgcatc gatgttcatc agagatattg gtctaaaatt ctcttttttg 12600

gttgtgtctc tgccaggctt tggtatcagg aagatgctgg cctcataaaa tgagttaggg 12660

aggattccct ctttttctat tgattggaat agtttcagaa ggaatggtac cagcttcttc 12720

ttgtacctct ggtagaattt gtctgtgaat ccatctggtc ctggactttt tttggttggt 12780

aggctattaa ttattgcctc aatttcagag cctattattg gcctattcag agattcaact 12840

tcttcctggt ttagtcttgg gagggtgtat gcgtccagga atttatccat ttcttctaga 12900

ctttctagtt tatttgcatc aaggtgttta tagtattctc tgatggtagt ttgtatttct 12960

gtgggatcgg tggtgatatc ccctgtatca ttttttattg catctgtttg atgcttctct 13020

cttttcttct ttattagtct tgctagtggt ctatcaattt tgttgatttt ttcaaaaaaa 13080

acagctcctg gattcattga ttttttgaag ggttttttgt gtctctattt ccttcagttc 13140

tgctctgatc ttagttattt cttgccttct gctagctttt gaatgtgttt gatcttgctt 13200

ctccagttct tttaattgtg atgttaggtt gtcaatttta gatctttcct gctttctctt 13260

gtgggcattt agtgctataa atttccctct acacactgct ttaaatgtgt cccagagatt 13320

cgggtatgtt gtgtctttgt tctcgttggt ttcaaagaac atctttattt ctgccttcat 13380

ttcgttatgt acctagtagt cattcaggag caggttgttc agtttccatg tagttgagca 13440

gttttgagtg agtttcttaa tcctgagtcc tagtttgatt gcactgtggt ctgagagaca 13500

gtttgttaga atttctgttt ttttacattt gctgaggagt actttacttc cgactatgtg 13560

gtcaatttgg aagaggtgtg gtgtggtgct gagaagaata tatattctgt ggatttaggg 13620

tggagagttc tgtagatgtc tattaggtct gcttggtgca gagctgagtt caattcctgg 13680

atatcattgt taactttctg tctcgttgat ctgtctaatg ttgacagtgg ggtgttaaag 13740

tctcccatta ttattgtgtg gggagtctaa gtctctttgt aggtctctaa ggacttgctt 13800

tatgaatctg ggtgctcctg tattgggtgc atatatattt aggatagtta gctcttcttg 13860

ttgaattgat ccctttacca ttatgtaatg gccttttttg tctcttttga tctttgttgg 13920

tttaaagtct gttttatcag agactaggat tgcaacccct gccttttttt gttttccatt 13980

tgcttggtag atcttcctcc atccctttat tttgagccta tgtgtgtctc tgcacgtgag 14040

atgggtttcc tgaatacagc acgctgatgg gtcttgactc tttatccaat ttgccagtct 14100

gtgtctttca attggagcat ttagcccatt tacatttaag gttaatattg ttatgtgtga 14160

atttgatcct gtcattatga tgttagctgg ttattttgct cattagttga tacagtttct 14220

tcctagcctc gatggtcttt acaatttggc acgtttttgc agtggctggt actggttttt 14280

cctttccatg tttattgctt ccttcaggag ctcttttagg gcaggcctgg tggtgacaaa 14340

atctctcagc atttgcttgt ctgtaaagta ttttatttct ccttcactta tgaagcttag 14400

tttggctgga tatgaaaatc tgagttgaaa attcttttct ttaagaatgc tgaatattga 14460

cctccactct cttctggctt ttagagtttc tgctgagaga tccgctgtta gtctgatggg 14520

cttccctttg tgggtaaccc gacctttctc tctggctgtg cttaacattt tttccttcat 14580

ttcaactttg gtgaatctga caatcatgtg tcttggagtt ggtcttctcg aggagtatct 14640

ttgtggcgtt ctctgtattt cctgaatctg aatgttggcc tgccttgcta gatttgggaa 14700

gttctcctgg atcctactct ggggaagtat cctgcagagt gttttccagc ttggttccat 14760

tctccctgtc actttcaggt acaccaatca gacatagatt tggtcttttc acatagtccc 14820

gtatttcttg gaggctttgt tcatttcttt ttattctttt tcctctaaac ttctcttctc 14880

acttcatttc attcatttga tattccatca ctgataccct gtcttccagt tgatcgaatt 14940

ggctgctgag gcttgtgcat tcatcatgta gttctcgtgc catggttttc agctccatca 15000

ggtcctttaa ggacttctct gcattggtta ttctagttag ccatccgtct aatctttttt 15060

caaggttttt aacttctttg ccatgggttc aaacttcctc ctttagctcg gagaagtttg 15120

atcttctgaa gccttcttct ctcaacttgt caaagtcatt ctccatccag ctttgttcca 15180

ttgctggtga ggagctgcat tcctttggag gaggagaggc gctctgattt ttagaatttt 15240

cagtttttct gctctgttct ttccccatct ttgtggtttt atctatcttt ggtctttgat 15300

gatggtgatg tacagatggg gttttggtgt ggatgtcctt tctgtttgtt agttttcctt 15360

ctaacagtca ggaacctcag ctgcaggtct gttggagttt gctggaggtc cattccagcc 15420

ctgtttgcct gggtatcagc agtggaggct gcagaacagc gaatattggt gaacagcaaa 15480

tgttgctgcc tgattgttcc tctggaagtt ttgtctcaga ggagtacctg gccatgtgac 15540

gtgtcagtct gcccctactg gggggtgcct cccagttagg ctactcagag gtcagggacc 15600

cacttgagga ggcagtctgt ccattctcag atctcaagct gtgtgctggg agaaccacta 15660

ctctcttcaa agctgtcaga cggggacatt taagtctgca gaggtttctg ctgccttttg 15720

tttagctatg ccctgccccc ggaggtggag tctacagagg caggcagtca ggcctccttg 15780

agctgcagtg ggctccaccc agttcgagct tcccagccac tttgtttacc tactcaagcc 15840

tcagcaatgg cgggcacccc tctcccagcc tcgctgccac cttgcagttt gatctcagac 15900

tgctgtgcta gcaatgagtg aggcttgatg ggcataggac cctctgagcc aggcatggga 15960

tataagctcc tggtgtgcca tttgctgaga ctgtcggaaa agtgcagtat tagggtggga 16020

gtgaccctat tttccaggtg ccatctgtca cccctttcct tggctaggaa agggaattcc 16080

ctgacccctt gcacttcccg ggtgaggcaa tgcctcgccc tgcttcggct cacactctgt 16140

gcactgcacc cactgtcccg cacccactgt ctgataaacc ccagtgagat gaacccggta 16200

cctcagttgg aaatgaagaa atcgttcgtc ttctgcatca ttcacgctgg gagctgtaga 16260

ctggagctgt tcctattcag ccatcttggc ttgggaccag agaacttcgt atttcttaca 16320

gcacctccta agtgttatgt tttgttgcag atccgccaga tattcctgat gaagtaacct 16380

gtgtcattta tgaatattca ggcaacatga cttgcacctg gaatgctggg aagctcacct 16440

acatagacac aaaatacgtg gtacatgtga agaggtaggt cacttcctca cggcttcata 16500

taagcagttc caccccagtt cagccagagc tctgcctcca gcagagatcc aagaaatcag 16560

cctcaaacat taaatatata ccctgattta tcccttattt cctacttatg atcagtgaaa 16620

ctaccaaagc ccttttcaag ccattaatat tttcactctg gcaggcaaag tgctctatga 16680

tctttctgtc ctttttattc attagtaggt aactggtagc aggctcctaa tggtggtaca 16740

gcctagcaca gttttaagcc attgcttagg acgggcacag tggctcacac ctgtaatccc 16800

acactttggg aggccaaggt gggtgaataa cctgaggtca ggagtttgag accagcctgg 16860

ccaacatggt gaaacctggt ttctactgaa aatacaaaaa ttagccaggc attgtggcag 16920

gcatctgtaa ttccagctgc tcaggaggct gaggcaggag aattgcttga acccaggagg 16980

cagaggttgc agtgagccga gactgtgcca ctgcactcta gcctgggcga cagaacaaga 17040

ctccatctca aaaaataaaa taaaataata aattattgct taaatccctg ctctattctt 17100

cttctagggt ttcaacaact taatttcccc gaattgctat tatttttaat ctttaaatgg 17160

gcctaaaaat aaaacataac acacaggttt gttctaatta attatgtgga atttggcttt 17220

taaagtgtat ataattatgc catattacca ctaaattcat ggaagctatt gttaataatg 17280

accctcagat cttaaattta gtgtctacat agctgatcaa ctttcctgtt tgccttatac 17340

taagggggtt ttcaggacaa aggattttca gttttaaaac ctggaaagtt gtgggcaaac 17400

taggacaagt tgcttacctt atccgtgttt cttgttttgt ttagtttatt aaaaaataaa 17460

attccaggcc aggcatagtg gctcatgcct gtaatcccag cactttggga gactgaggct 17520

ggtggatcac ccgaggtcag gagttcaaga tcagcctggc caacatggtg aaaccccatc 17580

tctactaaaa atacaaaaat tagctgggcg tggtggtggg cgcctgtaat cccagctact 17640

caggaggctg aagcagggag aattgcttga acccaggagg cagaggttgc agtgagctga 17700

gatcacgcca ctgcactcca gcctgggcga aagagcgaga ctccatctca aaaataataa 17760

taaataaaat aagatgccgg ctgggcgcgg tggctcccgc ctgtaatccc agcactttgg 17820

gaggctgagg tgggtgcatc acctgagatc aggaatttga aatgagcctg gccaacatgg 17880

tgaaaccccg tctctactaa aaatgcaaaa attagccagg tgtggtggca cacacttgta 17940

atcccagcta ctggggaggc cgagacagga gaattgcttg aactcaggag acaggagctt 18000

gcagtgagcc aagatcgtgc cattgcactc cagcctgggc aacaagaggg aaactctgtc 18060

tcaaaaaata aataaaataa aataaataaa ataaaataaa ataaaataaa ataaaataaa 18120

ataaaataaa ataagccttc tgtggtgttg ttgaagtatg cttttttttt tctgtcacta 18180

attcttaaaa atttcacaaa ccattttgta aagcatttat tggaccctta ctgtttgcca 18240

agcactgggc caagagcttt ataaatgctg cctcatttaa taccctatac aaccatgtga 18300

agccctggga tcatacaaat gcttatatcc tcctaaaatt catatgttga aacaaacccc 18360

caatgcaata gtaacaagag ttggggcctt tgaagtgaga ttaggtcatg agggtaaagc 18420

cctcatggat ggaattattg cccttccata agaggcctga aagagagctt gtttgccctt 18480

ctaccctgtg aggacacaac cagaaggtgc tgtctgtaaa gcagagagtg agccctcacc 18540

agacactgaa tctgctgatg ccttgatctt ggacttccaa gcctccagaa ctgtaaacaa 18600

taaatttctg ttgtttataa attgcctagt ctaaggtatt tgtatggcag ctcaaagaga 18660

ctaagacagc tgcatacaat tatttttccc atttgtatag acaagggaac tgagacttag 18720

tgaggtcagg taatgaatgt atccaggacc acacagctgc agagaaggag aggtggaatt 18780

tgaacatagg ttgcctgact gtcttctcaa cttattttct gctgaaggtg tgtggaggac 18840

cagatcgcag ggcatattta agatattcta ctcagttctc atcattatgg aagggaataa 18900

ccttcttttt aaaaaaaatc tttttattta tttatagggt ttttgttttg ttttgttttc 18960

gtttttgttt ttgtttttga gccacggtct ctgtcgccca ggctggagtg cagtggcaca 19020

atctcagctc actgcaacct ctgcctcctg ggtttcaagc aattctccca gccaccaagc 19080

ctgatctcta tggaagagaa taacctaatc ttgctctctc tctccacttt ttggatactc 19140

agaataattt tgattaagtg aaacctaaaa agaaacacct catcagtcaa gaaagtccac 19200

gtagaaacta gacagatttt atgaacacat tgccaggcct agagttacca gctacatgcc 19260

attggcaaga ggcgtacacc tttcattctg aacaggagtt aaacagaggc atattctggt 19320

accaaagagg tgatgaaaac tctgatatca gggatgggtg gaattgattg aagagatgca 19380

atatgctaag tactgtgctc tgtacttcat gtgcatgaca tcttcaattc tcaacttaac 19440

cttgtgaggg aactatcgtt attgttccca ttttacagat ggcgtgggga gctcacatta 19500

cctctccaaa gtcacttgac taagaagtaa aggagcaggg attctaacag ggatttctct 19560

ggctacacca ctggagacta aacctctacc ccttgcctat agggtaaagt agaaaagcca 19620

tgagagcaag acaactagag tctcattgct aagtcaactt taggaatgtg aaggtgaggg 19680

cagtatggca gcaaactttg cacttcaagc acacagaaaa accaggcagt gccagattga 19740

cagagatagc ctagtgtctt gaggatcttc actatctccc agaacctgga aggatctcct 19800

gaactgctta tcctggttgt ttggttaatt cattctttct tttattttta atataaccct 19860

ttattaagta taattcatct accatacaat tcaccttatt taaagtatat cattccttga 19920

gttttagcac ccatcactac aatcaatatt gaacattttc atcatgccaa aaaaaaaatc 19980

acatccatga gcagtcactt cctatttccc ctcaatcttc ccagtctatg gcagccacta 20040

tttgctttcc gtttctgttg atttgcctac actggacact tcatgtaaaa ggaatcatat 20100

aatatgtggt cttttgtgac tggcttcttt catttagtat aatgtcttca aggttatcta 20160

tggtgtaaca tgtatcaggc tggagtgcaa tggtgtgatc tcggctcact acaacctctg 20220

cctcctgggt tcaagtgatt ctcctgtctc agcctcctga gtagctggga ttacaggtgc 20280

acgccaccac gcccagctga ttttttttgt attttagtag agactgggtt tcaccatgtt 20340

gcccaggctg gtatggaact cctgagctca ggcaaccctc ctgccttggc ctcccaaagt 20400

gctaggatta caggcataag ccaacgcgcc tggcccttta ttcctttttt taataatgtt 20460

ccattatttg gaacacattt tatttattta ttccttggtt gatgaacttt ttggttgttt 20520

ccacttttca gctattatga attattcttc tattaatatt tgtgtacacg ttttgtgtag 20580

acatattttt atttctcttg ggcatatatt taggaataga attcctgggt ctcttggtaa 20640

ctctatgttt aatcttttgt tcaactgcca taatgttttc caaagcagcc acaccatttt 20700

acattcccaa caacagtgta taagggttcc agtatctcca cattcttgcc aacctttgtt 20760

tttatcttgg ttattgccat cctagtggat gcaggttaat tctttcaagc ctgacttacc 20820

tgccttctga ggttcctgat agtaaaactg cattgcaaat tccagacaga tgaaccacaa 20880

tttgaaaaaa tatatttgtt ggatacaatt ttaaacttgc taacccccac ctaatttcct 20940

atttagtatc ataaaaatat tttctgaata ttttatatga ctatgtatta tatatttctt 21000

ttaaaatcct ttaatgtgtt caaaattatg tatgtttaca tttatttaca catgactttt 21060

gaggaacaca ttcactttat aaagtaagga atatttgtag tagttatgag tactaagatt 21120

gttctcagta ctcaaaaagt cactctgaaa gtattaagaa tgagtttact aaagagaatg 21180

ctttcagaaa tattcattgg gaagagcatg ggctttagag ttttagattt aatttccagt 21240

tctgaaactt accagtcagg tgtcttttat gatacaaata tcagaaaact caagtgctgg 21300

ccaggcgcag tggcacacgc ttgtaatccc agcactttgg gaggctgagg cagatggatc 21360

acttgaggtc cggagttcaa gaccagcctg gccaatatgg tgaaacccca tctctactaa 21420

gaatacaaaa attagcaggg catgtaatcc cagctacttg ggaggctgag gcaggagaat 21480

cgcttgaacc tgggaggtga aggttgcagt gagctgagtt catgccattg cactccagcc 21540

tgggctacag agtgagactc catctcaaaa aacaaaaaaa taaaagaaaa ataaaaaaac 21600

tcaagtgcag tcacttaaat agacatggac ttatttatat caggtcataa ttatatgtag 21660

taacagatgg catgataatt cagtaatacc ccagggatct aggctctttt cctctttcta 21720

ctctgccatc ttcagtgtgt tgaccttaat cttcagatgc tgtatctcca ggcttcttat 21780

ccaagttcca ggcaggaaaa gtggtaaaga gcaaaggagt tcctcctgtg gacttcgacc 21840

tacactgagt tggccacaac agtgtcacat gtctactctg tctggatggg aacctggcag 21900

aagggcattg taaatggagt tggataccgt catccaacag tatctgccat agttgctagc 21960

tgtgtaatct tagaagagtt atttagcctt gctgagcctc agcctcctgt gaaatgagga 22020

taattaatgc cacaaagctt ataaaaaagc aaattatagg aagtgacttg catataggaa 22080

atgcttagaa ttaagtacaa cacaaaatga tgtcttagtc ccttcaggtg actatgacaa 22140

agcacctaga ctgcgtaatt tgtaaaaaac agaaattcgt tgctcacagt tctggaggct 22200

aggaagtccc agatcaagtg tcaacagatt ttgaaggtct gttcttcata gatggtgact 22260

tctatatgtt ctcacatagt agaaggggcc aggcagctcc cttcaacctc ttttataggg 22320

gcactaaatt gattcatgag ggaaaagccc tcatgactta accactttct aaaggcccca 22380

cctcttaata ctattacatt ggggtttaag tttcaacatg taaattttga gggtacacta 22440

atattcagac tatagcaaag gatattataa tattatggcc taagggaaac ctgaccgatt 22500

tgattgaggg aaatattaat gtaaagccat aaaaattatt atttatagga tattatagat 22560

aattttattc atatattttt aagtaataca ataattttgg aaaaatctag aaagagatgt 22620

tgcatgtgct ctaatagaaa gacttatttt ttgtatgtca atagtacatt tattcattca 22680

aactagaatg taggcttaat gtgggcaaga atttttgttt gattttttca ttgctatgtt 22740

gattgaatct agaatggggc ctgacacata gtaaatgctc aataaatatt tgttgagtaa 22800

ttattctagt tgataactag aaatagacat ttcctgaatc tccccttcac tgtctagtta 22860

agtaataata taaggtcata tatattttat aaattatttt tcccatcttc tttcttaaaa 22920

tccaacaata aactataacc aaactctgat aaacctaacc aagttctgaa ttgatggagg 22980

ccagattaat gggattttat tacattctaa acaggacata aaagatataa aaggttagaa 23040

taaaagtgaa tgttctcctc tagaaaagtc aaaggactaa cttataaata tagacaggtc 23100

agaaaaggaa acaaacacct atttatctaa tgattagtta caaacaaact aatgagttga 23160

aaaaaattag agaagagggc tgggcgcagt ggctcatgcc tgtaatccca gcactttggg 23220

aggctgaggc aggtggattg cttgagctca ggagtttgag accaacctgg gcaacatggc 23280

gaaaaataca aaaaatataa aaatttctac aaaaaataca aaaactagcc aggcgtgttg 23340

gcgggtgcct ggagtccaag ctacttggga agatggcttg aacccaggag gcagaggttg 23400

cagtgagcca ctgtactcca gcctgggcga cttagccaga ccctgtctca aaaaaaaaga 23460

aagaaaagag caagtcaatt tccctaacta ccagaaaaaa tctttccctc aaggagaaag 23520

ctgacttata atgaatgttg tggtcgaggt agtattatgt ttaaaggagc tcttctaaac 23580

gttcagttat tagggtattt tccacattgg taactaggca gagatcagca tgtgcaaggt 23640

ttcacttcta actcatgtgg ttcagttttc cagtaaaatc acatgtagag agaagctttt 23700

tacctgagac cacctactca tttctccagt tgcagaaagg cagtgtttcc cctatggcaa 23760

cataggtgcc atcacaggcc tcctcatgtt ggggtgaagt tgtaaagtag aattttgtct 23820

gttttgcttc aagagctttc tttcttccca gctcctttgc ggtggcctca ccagacagag 23880

cctcctggct gttcacatac cttcttttac ctcctgtatt ttgctgtttc aaagggtaat 23940

gatagggaaa aaaaaaaacc aaaaaaaaaa caaaaccagt gctaataacc atctccctac 24000

tgtagtccca ctatcttcag caatttcatg taaatatatt ttttatttat cttggaaaca 24060

ctggtaatgt ctggctcagt gtattctact ccataaatat ttgttgaata aaaatgcatg 24120

aatgatagcc ctgcattctg ttccattcaa gtcatcttag caaatattta tttagcattt 24180

gatggtttcc tggaattgta ttaaactgta gatatttaaa aaaaagagat catctctgtg 24240

catgacaagc tcataaacag tttagaataa catcaatgtt gggattttca cgcaccgtag 24300

aagaccaata aggcaaatgt tttcagatgc agtatcatgt tagagccctt aaattcagcg 24360

gcctactctg ctaattcata aggcctgttt gtttttgcca cagcttctct acatgcatat 24420

ttttaataac cagaaagagt tcttaggtgt gattccaaat actattttta aggctacgtt 24480

aggtttcagt aattccttta ggactcaaaa tgtagtttat attttagaat ccatttcctc 24540

actgattatg agcccagaga gctaattgtg gctggagaca aacagaacct tgctgactga 24600

tactgcttta tattcatgac cactaacctc aactgggccc ggagagccac acagtaatcc 24660

tactacattt cactagccaa gtcactgtct tgctaagcca ggtcattgct tcattacttt 24720

ttctgtcatc tctcctctct agtatcttcc cttcctcact ctctattgaa gacctgcttc 24780

ctactttcct gagaaaacag aagcaatgag aaataccaca gcctccctca ttgactaatg 24840

cattgacatc tgtacccata tagttgatct ttcctgctat tgctatggat gaattaccat 24900

tgccctaagg tcaactctcc acttgggtac tagatcttat cctgcctgcc tactggagga 24960

cactgctcca aaactttcct ctcttccttc tgcattgtca ttgtctctgt ctttactgga 25020

tcattcccat tggcttttaa gcatgctgta gcctttgccc ttttgaaaaa tataaccctc 25080

cctttgttcc tgacctctac cacccaattt ctctgtttct ttttatgaaa aaaaatcaaa 25140

agagttgtct gcactgtctg tctctatttt ctctcttctc ttttatctag tttagtcatg 25200

ttttatgtcc tcaccactcc actgaaacag ctcctgtcaa agtcactgat gatttcccca 25260

ttgccagatt gaatggctta ttctcagtct ttatcttact tatgaaacat ttactatccg 25320

agcatttaat gtagctcttc acttcctcca tttggcttcc aggacaggac tcccttttgc 25380

tgctcctccc accttcctgg aaactttatc tcagtctctt ttgctgattc atcttcattt 25440

cagataaatt catagtgacc cagagctcta tattttgtca gcctaaaaaa agacactagg 25500

gaagattatc ttcaaatttg ttgggtttac ttgagaatat aaataaggag tataatacag 25560

aatgcatgac atggcaagcc agcagtgcat ttggtgaggg aaaggataaa gggaagcttt 25620

tattaccaaa aaggaatata tgtgagctat ttaggaacag agttcattag ttccagaggc 25680

tcaaagccag agtgattgtc aattaataag tggagatgta ttactgggta agcggtcttc 25740

caaaaacatc ttatctggat tactgcagtc ctaaaggatg tctagtgata aaccttatca 25800

aagcagggga tgactgaaag gtttttagaa agtccttggg aacagttctt atctcagaca 25860

tgtaagcatg ggcctcttct ccttcaggcc tttcttgccc tattttgtct gggtctgaca 25920

aaagtgattt catcctggta tgtgcaactt tcacacctag gaccttttat tatctctatt 25980

tacagtcatt acctaaagga tagccttcaa atttgtggtt taaaattcct caatgtatat 26040

ctgtgtctaa atatctccac caaatacagc tgccatgttt ctaacctgtc attcaaggta 26100

tcttgaagtt aacattttca aaaccaaaca cccagtttcc tctcccaaga cttgcatttt 26160

ccatttcagt aaatggcaac tgtatccttt gcattattca ggtcaaaatc aggtagattc 26220

tcctggattc ctctctatta tggccttcat ctaattgatc agcaaaacct tccggctcta 26280

tcttttaaaa tgacctcacc acctacactg caatccaagc cagatcatct ctcacttggg 26340

agtataagaa cttctaactg gtccactcgc ttcctccaga gtcccttatt catgagtttt 26400

cacagtaatc cttttcacat gcacctcagg ccatgtcacc cttctgctca tgaacctccc 26460

atttcattca gagatcaaag ccagagtccc tcaaggactc tatgtgacct tgcaagtctt 26520

ctaaactttc ccctcttcca aaactctctg attttgtctt taatcccttt cctagtaaaa 26580

cctcactgac ctccttgctg ctctttgaac aggccaagca aggtcctgcc tcagggcctt 26640

tgcatgttac ctagaatgct cttcctccag agagccacag ggtttcctta ttgccctaat 26700

gtcaccctct cagataagac tttcctcatc tctctaaaaa acaaaaacct taaaacccac 26760

ccctactctc agaactgcct aaccttttta ctccgcttta gttttcttct gaacatttct 26820

caccatacat agatcagatt ctatatttat ttgtttccac caaaatgtgt gcttcatgac 26880

agttgggact ttatctgttt tgttcattgt agccccaata tctaaaaata tacttggaat 26940

atagtcagtg ttaaataaat atttattgaa tgagtgatta aatgaatgaa agacactatt 27000

taccattatc aacataaatc tgcaatgttt tttgaaaatc tataaattac aactatatct 27060

aggtgctcag ggaacatcag aaaaatatga attatactaa ctcccttcac aaatcatctg 27120

tcatgtatta gcccatgaaa aaaagaaata gaaataagat attcaaaaac tttgtaatat 27180

acagttatgt aaaagataga ggaaaaacca aagtgttgtt ctcctttcta ctctcaacag 27240

cactcctgac acgaaatgtg ggaagatttt ccccacacac caatcaattc tcctgcagac 27300

atcagctaga tgtcctctaa tttaattcaa ttctgactct gtctacctgg aggtagcatc 27360

agatcccaca gctaaaggct cagtcccaca agactcaacc ctgttttaaa tgtcaatcac 27420

aagccccagg tcatgacctg tgcttttgac caaccggcta aaaattgtgg tttccacgat 27480

acactcctca ggtttgatta acttgctaga gtgtctcata gaactcagga agacacttta 27540

cttatattta tccatttatt ataaagaata taacaaaggc tacagatgaa cagccagatg 27600

gaagagatgc atggggcaaa gcatgcggga acaccaccct gcaggcatcc tcatatgttc 27660

agatatctgg aagctccctg aacccagtcc ttttgggttc ttatggaggc ttcattacat 27720

aagcacggtt catcatatca ttggccattg gggatcattc agcccttctc tcttcccagg 27780

agatgggggt gggtaaaggg caagggggtt ggggggactg aaagtttcaa tcttctcatc 27840

acattgttgg ttcccctggc aaccagcccc taccctaagc ctatccagga gcccccagcc 27900

atcagccatc tcattagcac tttggagatt ccaagggttt taggagctgt gtgctgggaa 27960

ccagacagaa accaaatata tatttcttat tatgtcacaa tatcacaagt tctatataaa 28020

aataacccta aggtgtaatt agtcattatt atcagattct tcaattaaaa cagtgctttg 28080

tggtctaggt gctaatatgg ggtagacttt aaaagcagaa ttaaattact tagtaactat 28140

ggttactgtc agtttgaaaa tacatttcca ttttgacttg tggtctcagt atgtgtggta 28200

aaagaattag ccacaaacca atgaagaaca tataatgtgg ttttctctaa ctggaaaata 28260

ttcaaagatg tcctacttac catttacaca tagttgccat tttcttgttt tcacatttga 28320

attcttactt agcatgaggt taacatactt taacaagttc tctgttaagg tatttacgtt 28380

tcatcttatt tcagaaagaa tataaggtaa ctatgtaaac atctcaattt aacggcataa 28440

tttaacttaa gatacagtga tataagagaa attttactgg taagaaatat agcaatttta 28500

aaagtttcat ttgtattctt tagaatgtgt caaagtcaca tatcataaat atcttctttt 28560

tttatttaaa tggacacata gtctcactct gtcatccagg ctggagtgca gtggtgcaat 28620

gttggctcac tgcagcctcc atctcctggg ctcaggcaat cctcagcttc ctgagtagct 28680

gggaggacag gtgcctgcca ccacacctgg ctaatttttg tatttttagt agagacaggg 28740

tttcaccacg ttggccaggc tagtctcaaa ctcctgacct caagtgatct gcctgcctca 28800

gcctcccaaa gtgctgggat tacaggtgtg agccaccatg cctggcctca gccagtatct 28860

cttaattcag aaaaattgtt gtttaatgag tccttcatta ggtgagaagc catctactct 28920

gtttcttatt taatttactc atgataacaa caacgttatc attcctatac tgaggatgag 28980

taaactgagc tgtatagagc ttaagtaact tgctccaagt cttacaactt atcaaggggc 29040

agagctggga tttgaaggta ggtctgtctc taaaatccaa gccctgtgat tttagtcacc 29100

atgacatacc ctcctcgtcc tctcatggtg ttcatagtga tctcacgctg gtatgaggaa 29160

agaggtaaca cagaaaaaat gcactggaac cgaaaaacct gagctgaata ggaggagttg 29220

ctcctcaccc ctccaatgac tatcagcctt agagagattc cttgaccact tgctgaggtt 29280

gtcttgttct gatagtagca ataagtttat ataaattata taaaatatag aatttctata 29340

gtacttagat gcacacatct gcttattgct tggtattaat agcataaggc tggttccttt 29400

tctctgcgtc tcagcaagca cgtttcttgg attccaaaag atgtaagtgg ctagcaagtt 29460

taatcctggt gggatatttt ggggctagaa aaatttagga gttgctgaaa agatatatag 29520

tagtaacagg acttggtgga gccttggtgg agggacagtg atttttaagc tttatcttct 29580

ccacaaatcc aatcaaggca gtcccctgag agagagggca gttgatggta ttagatttaa 29640

aaaatcaggt tagagtctgc ctcaacagca caaacagtgt aattggaaca agacaaagaa 29700

gatggcatga cccctgatca aggacggcat gcaaatttgt gaagtatttc catttgatac 29760

aaataagaat gtatatggct gagcacggtg gctcatgctt gtaatcctca cgtgggagga 29820

tcccttgagg ccaagaattt gagaacagcc tgggcaacat agcgaggctg catctcttaa 29880

aaaaaaatta ttttaaataa aaaaattagt taggcctggt ggcacatgcc tgcgatccca 29940

gctactcagg aggctgaggt gggaggatca cttgagccca gaaagtcaag gctgcagtga 30000

gttatgattt gtgccactgc actcaaacct gggtgacaga gtgagaccct gtctctaaaa 30060

aatagaagaa aataagaatg taggctactt ggctttccag cattgttcag atttcaaggt 30120

tactttcctt ctaaagtagt atatctatat caagttaagt ttcatgatgt aatgcaatta 30180

acttaggagt ctgaaagtca taaaccaaac aggtaacaaa gacctagacc attgccatag 30240

ctatcaaaac agggaaaaaa gataacatag gaaaataata ttttaatgct taaaaaaatg 30300

cttaaccttc tttagtaatt ggttggctct taaaggtgac gaagagaaaa ttgttaaaaa 30360

tggcaaactg agaaaggtgg accgcatgcc tttgacaaca atgaagacac tggcaagaag 30420

tgctgatttg catggaaaga taatttagtt gtcagttttc tatagttctg gttccagtca 30480

gacatccaaa tgaagctccc agtgagggca caggtatgcg tgagacacac atgtgaaata 30540

gcaggtgaag ctgagaaggg agtgagccca gaaaaagggg tcagggaaaa ggcaaacacc 30600

agggctgacc ttcaatgtga gtgaactcag agagtaggga aagaaaccca ggtggaaggc 30660

tggaggggga catgccacta agtaggtgtg agagaggaag gccctctaac ccagaagaca 30720

ggctttgggt agcaaggaag tgagaattat aaccaatcaa gagggataaa tgcttaaatg 30780

aggattcaga ggacttcacc ttgtaaagga caccatttta aaagagcagt ttcaaggaaa 30840

agtaaaggca aagatcacaa taaaaggact aaagaccaaa tgggaataag ttggatgtag 30900

ttactaagga ggtagaaaat ctgagaagct taagaacatg aaataatgga gtaggtagtt 30960

caaggggctc aagaaatggg aactgaggaa attctagtga aaatacttcc atgttacaaa 31020

tgaatctgat tacagattat gttgtgtttt ggagtctgag gggggcccta ttcatctctg 31080

cagaagaatg agtgaacaag gggtgggaca agaggctagg agatttgagg acactcctgt 31140

gttagtccat tcttgccttg ctataaagaa atatctagac caggtgtggt ggctcacacc 31200

tgtaatccct gaactttggg aggccaaggc aagaggatca cttgagctca gagctcgagg 31260

ctggcctggg caatgtggca aaaccctgtc tctacaaaaa attagctgag agtggtggtg 31320

catgcctgta gtcccagcta ctcaggaggc tgagatggga ggacctcctg agcctgggag 31380

gtcaagactg cagtgagctg tggccatacc actgcactcc agcctgggtg acagagtgag 31440

accctgtcat taaaaaagaa aagaaaagaa aagaaaagaa aagaaatacc tgaggctcgg 31500

taatttatga agaaaagagt tttatttcag ctcacagttc tgcaggctgt ccaggaagat 31560

tgtgaacaag atagagggag gggggatatg ccacactctt taaaatgacc agatctgcct 31620

gaactcagag ggagaagtca ctcattactg ccaggagggc agcaagccat tcatgaaggg 31680

tctgccccac aagtcctcac ctccaacact ggtgatgact ttgcaacatg agatttggag 31740

aggatgaggg tggaaacaat atcaaatctc ttcacagaaa tcagaggagc aaagaagcat 31800

tctccagcac tcctttcgcc accgcctcca accctcgtcc actaaaaaca tcatgtgatt 31860

atttaggggt agagatttaa aaacagaggc tcaaacattc tgtccaaagc cagtgaaaac 31920

aatttgaaac tctggcatta acagatatca ttcggaattc taccaagttt attgggctac 31980

ttggatattc tcattcaatt ataaatactt agataaatat gctttgaaat gatacttaat 32040

agccctctct attcccctgt tctaaactag atctttcctc tttatctttt ccatcctctt 32100

ccacagtgca tgactccctt tcccctcatc aaacctgtca ttaggtccag tcagttctac 32160

actcaaaatc cttcatgggt tgggcgaggt ggctcatgcc tataatccca gcactttggg 32220

aggccaagac gggcagagtg cttgagttca agagttggag actagcctgg gtatcatggg 32280

aaaaccccat gtctacacaa aatacaaaaa ttaattgggc atggtagtac gtgcctgtgg 32340

tcccggctac tcaggaggct ggggtgggag gatcactcga gcccaggagg tcaaggttgc 32400

agtgagccaa gatggcccca ctgcacgcca gcctgggtga cagagccaga ccctgtctca 32460

aaatccttca tatccacccc cattggcttc cacctatttc agactaataa tccaattctg 32520

gattattgta tagccttttg atttttccac taccaccact ttcatttccc acaaatcctc 32580

cgtaaaggtg ctagacagtc ccagagaccc tcaaaatagt gtgaatctca ctatatgcac 32640

tcactttcta tcacttacat gatacagtcc tgatccatca gtagctcatg gaaggtcttc 32700

tgacaggcct gctccactac aagtttcttc ttgcattttc aggatccagg ctattgcact 32760

tgttgaacct tctcagaaat gtcatgttat ttcacgtatt tgtttcagaa catgctggtt 32820

gtctctgtcc aggaggttgg ccttccccct accccattgc gatctctcac cccatcctga 32880

ctcctcccca ccactccatc ctcaccctat gtcctgatag aaatgtctga cttcacagtc 32940

cttgcactga accaaaatgc agtgctcttt tccaaaggca gctattctgg ctattccaag 33000

ctatgtccag tgccgacttc cctaacaggc acagtaggca cagtggctag ggcccacaat 33060

aattttagga gtccatgaaa atgtttaatt ttacttaaaa tcagaagaga aaaataactg 33120

ttatgttcat gtatatgcat gtatatggca tgcatatgta tacatctatg tatatgtgtg 33180

tatatttttg tgtattttat atatttatat aatatatacg ctcattgttt ttttaatgga 33240

ggaaagggtc catgaaggca gaagtgttca gggcccattg aaatcatact gtggcccttg 33300

tcagatttga atttattttt taaaattgtg aaaataaaat caattttgaa atatttttca 33360

gtaaaaggtt gcatatttga aagcaggctt aaaaagttag cgtttttgtc caggcacagt 33420

ggctcatgcc tgtaatccta gcactttaga aggctgaaat gcacggattg cttgagtcca 33480

ggagtttgag accagcctga gcaacacagc aagaccctgt ctctactgtt tagaaaatta 33540

aattttaatt ttttgttttt gtttttgttt ttgagacgga gtctcgctct gtcgcccctg 33600

ctggagtgca gtggtgtgat ctcggctcac tgcaagctcc tcctcctggg ttcacgccat 33660

tctcctgcct cagcctcccc agtagctggg actacaggca cctgccacca cgcccggcta 33720

atttcttttt gtatttttgg tagagacagg gtttcaccat gttagccagg atagtctcga 33780

tctcctgacc ttgtgatccg cccgcctcgg cctcccaaag tgctgggatt acaagcgtga 33840

gccataaatt ctaatttttt ttaaaaaggt aacattttaa aaatctggaa taattaattc 33900

aaactttttt tctttttttt ttttttgaga cagagtctca ctctgttgcc caggctggag 33960

tgaatggcgt gaccttggct cactgcaggc tctgcctccc gggttccagc aattctcctt 34020

tcgcagcctc ccaagtaact gggattacag gcacatgcca ccaattattg tattttttta 34080

atagagacag ggtttcacca tgttggccag gctggtctcg aactcctgac ctcaggtgat 34140

ccaacccgct tggtctccca aaatgctagg attacaggtg tgagccacca tgcctggcca 34200

attaattcaa actaaataca atttatgatc atcttttttt tttgttttaa gtttagagac 34260

agaagaagag caacagtatc tcacctcaag ctatattaac atctccactg attcattaca 34320

aggtggcaag aagtacttgg tttgggtcca agcagcaaac gcactaggca tggaagagtc 34380

aaaacaactg caaattcacc tggatgatat aggtaaagaa taagaaattc tgtaagtctt 34440

taataataac cagtttgtgc tgaccttgtc aaatgaggtc tagatctgaa agaagttccc 34500

ctaaagttcc tgcagagcat gataaaagaa acagaaatta cccataattc tacctgccca 34560

agacaactta gagatgagta gtgctgctgc cattttgttg tatatccttc cagtcttatt 34620

tctaaattgt tgtatgtggc tgggccggtg gctcacgcct gtaatcccag cattttggga 34680

ggtcgaggtg ggcgatcact tgaggtcagg atttcgagac cagcctggtt aacatggtga 34740

aaccctgtct ctactaaaag tacaaaaaaa aaattagcca ggcatggtgg cgggcacctg 34800

tagtcccagc tactcaggag gctgaggcag gagaattgct tgaatctggg aggcagaggt 34860

tgcagtgagc caagattgtg ccactgcact ccagcctggg tgacacagca aaactcttgt 34920

ctaaaaaaaa aaaaaaaaat ccatatatgt gtttttacaa atatttattg gaaacttctt 34980

attttcaagc attgttctaa aggctagagt gaataagata gacaaggtgc ctgccctgaa 35040

ataacttaca atctactagg gaaggcaaaa aaaaaaaaaa caaaaaaaca ctatgatcat 35100

gataaagttt gttgagtctt acgttgtgaa aagtacagca tgctaaattc ttatactcta 35160

tacactactt ggggacttat gtaaggtttt tttgtttgtt tgtttttatt ttgaaaagtt 35220

ttacaaaata catagaaaat tgcagagaat tatgtagacc tctctatgtt catcagccag 35280

atttaacaaa cattaatagt ttttgtttca gatattttat accctgcctt tttatactta 35340

tatattagta ataaacattt ctattaattt aatctatagt tttattttta atgttgtctg 35400

aatatatagc agtttaatcc ctattgttgg agattaacac tgttttcagt ttttaaccat 35460

ataaactata ttgccatgaa caactttgtg gctgaatttt agcatagttt ttgattgttt 35520

ccttaagata aattccaaaa agtaattacc cttgagcctt ttacaactac atccaagtcc 35580

taaaaccaca attcagccaa atgacaccaa gtctgaatca tcctgtcttt gcagatccca 35640

gtaccaatga ttcctgttgt tataattaaa gatatttcaa taagctaaca tttattgagc 35700

actttcttat gtgctaacta ctttttcttt ttttgaggca gagtctcact ctgtggccca 35760

ggctggagtg cagttgcaca atctcggctc actgcatcct ctgcctcctg gcttcaagca 35820

attctcatgc ctcagcctcc ctagtaactg ggactacagg tgtgtgccac catgcccagc 35880

tgatctatat tgccacaaag ttgttcatgg ctatatagat ttttgtattt ttagtagaga 35940

tgggattttg ctatgttggc caggctggtc tcaaactcct gagttcaggc aatcctccca 36000

cctcagcctc ccaaagtgtt aggattacag gcgtgagcca ctgccagcta acagcttttc 36060

acatacgtta tttaaccctc atatcaaccc tattttgtag atgagattat aatttatata 36120

ttagaaaact gaggtttgat ggcattaaaa aatttgagat cacagaggta gggaacattt 36180

aattcagggg atcctcctgc cttagcctct cgagtagctg ggacgacagg ccacaggcca 36240

cagtgcctgg ctaatttttt taaatctctt ttagagacag agtctcattt tgttgcccaa 36300

acttgtctcg aactctgtgg ctcaagcgat cctcctgcct tggcctcaca aaatgctggt 36360

atttcaggcg tgagccacca tgaccggcct gaatattaga tttgaaaaca cactctgttg 36420

aatatgatga tggacaatta ttccatttta ttgtatatcc ttggagttac agttctcttg 36480

actatgctct atacattttt gtgaatttac attttgaggt agaatgcagt aatataccct 36540

tttcttacga tagataaatt aatgatgttt aagagctatc atgaccagta tcattttagc 36600

agtgcaattt ttaggaacta aataataata ataatacttg tattattatt accattctga 36660

tcttaatatc attgcatcaa tttatttagc accttctgtg ttccagacac ctctcatgca 36720

catcacgttt cattctggaa acaaccatac atgatactac tgtccccatt ttacagatga 36780

agaaagtgat aaaatgattt gcgcaaagtc acttagctag taatgaggaa agccagattt 36840

catcccagac atgctgactt gctatagttt accaccatcc cacctcccac aaaggaaaaa 36900

atcccatctg gcctctactt ccagactgac ttctcacact gggaactagg agtttggagt 36960

tgtggttcct tttccttctt ctccttcttt ctctttccct cctcctcttc ctccacctcc 37020

tcctcctcca cctcttcctc cccttttgtc tctattcctt tccttgcctt ttcattatcc 37080

cagaatgtac tcagaaactc aggttgaaaa atatggatta attgtatagg aggccccagc 37140

caacaaaacc accagattat taacagatga gccgtcttca tgggtaacac tttaagaatg 37200

agacaacgca aattagataa atgaactgtt ctatgccact gagaacagaa gactgtcctg 37260

ggaacaattt ccacatagca gctacgggct gggctttaat gacattaggg aggttatgct 37320

gaatggcagc gcagagttag tagcacatta acacttgcca aaaaagcata tgagaggaat 37380

gcttttaact tctcttgtag ggaaataaat tcgggtacag aaattgctgt gctttctgcc 37440

tgccattttg aatattactt ctccccatta tataaatatg gataaatatg tgttggaaag 37500

atgctaaaag gctataatct cgcttttatt tcaatacgag tagaatttaa aaatatgaca 37560

tctataataa aattgatact ttttttttct ttttttgaga cggagtctcg ctctgtcgcc 37620

caggctggag tgcagtggcg cgatctcggc tcactgcaag ctccgccttc cgggttcacg 37680

ccattctcct gcctcagcct cccgagtagc tgggactaca ggcgccagcc acctcgcccc 37740

gctaattttt tgtattttta gtagagatgg ggtttcacca tgttagccag gatggtctcg 37800

atctcctgac ctcatgatcg gcccgcctcg acctcccgat acttttttaa aatgtaagat 37860

ctggtggaaa tatgtgaaac ctaatcagta agaataaaaa ttcaacatct gagtcttgtg 37920

taacaaacta aatattcaag tatgtattac ataataaatg ccagtttgca aaaatatgaa 37980

ctcattcaaa cttaaagact taaatctgga ttgcactgac ctgctttatg ctgtgattct 38040

tactgtgcta tctgcatttc tcataagact aaacagattt ttactttctc ctcagaataa 38100

ttcttttgcc aaaagggtcc tagaatgctt gtgctgctat aacagtggcc aagccgaact 38160

cagaggtgga ggtgggtgac aggaggaggt gagaaattta tggccttgag ggtgcttaaa 38220

atggtcctaa gcaatttcag agccccaggg atccatgccc ttgactctag aatctcaata 38280

ctgatttttt ttttttttcc agggttgggg ttgggcagag tcttgctctg ttacccaggc 38340

tggagtgcag tggcatgatc tcagctcgct gcaacctcta cttcttggat tcaagtgatt 38400

cttgtgcttc agcctcccaa gtagctggga ttacaggcgt ctgccacaac acctggctaa 38460

gttttgtatt tttagtagag acaggtgttt cgctatgttg gccaggctgg tcttgaactc 38520

ctgacctcaa atgatctgcc cacctaggcc tccccaaagt gttgggatta caggtgtgag 38580

ccactgcgtc cagcctgaat actgattctt attacaagaa gcttgcacta gcaaaaggaa 38640

atcagctatg gaattcagat gataaaggtt gagtacagta aacttgttaa ctaaattaaa 38700

gctactgcta cttctacgcc aggtgtagcc cccaaaattt tattagagga ctgtgttact 38760

agacagcaaa tccttaattt ttgtttttct ctgtaaatca tctacctttg cacataattt 38820

tagataataa tagtataata gtcattgtag taaaacaata attttcttaa agaaagtttt 38880

ataaacctag aataaatgaa aataaactaa ttcatagaga atattaacct gtaatagtgg 38940

tctctaaaac cacgcctaca ggcccttgag ttctttgtta aatagtacag ctatcaaagt 39000

cataggtttt cattgttcct taggggagaa atggaaagtg ttaatgattc tattctgttc 39060

agacaaaaca aacttacaag tatttacaca gtttagaaac atagcgattc caactatata 39120

cctcctacta taacaaaaca actaataaaa taaccaatca gaatttcagt tccttctgtg 39180

aaggttatta ttatagaaca tgccaacaat ttacaaagcc ttagagtctt atgagatctg 39240

ctttcttcaa ataaaatttc aaggaaggac agatttattt taaaggagaa caaaacaagt 39300

tagcaataga tataaatatt catatgtttc cctaatacac aagagaagga gaagattatt 39360

gtgataaaag atctgaattg aacatccatc tttctttctt tctttctttc tttctttctt 39420

tctttctttc tttctttctt tttctttctt tctttctctt tttttttctt tctttctttc 39480

ttgctctttc tgtctcttcc tttctttctc ttttccttcc tcctttccct tcctttcttt 39540

cttttttgac agagtctcgc tccgccaccc aggctggagt gcagtggcat gatctcagct 39600

cactgtaacc tctgcctccc gggttcaagc gatcctcctg cctcagcctc ctgagtagct 39660

gggattggca cgcaccacca ccacgccggg ctaatttttt ctgtttttag tagagacagg 39720

gttttgccac gacgaccagg ctggtctcaa actcctgacc ttaagtgatc tgcccacctc 39780

ggcctcccaa atgctgggaa tacagacatg agccaccatg cccagccttt ctttcttttt 39840

tttgagacaa gctctcattc tgtcgtccag ggtggagtac agttgcacca tcatggctca 39900

ctgcaacctt gaactcctgg gctcaagtga ccctcccatc tcagcctcct gagtagttgg 39960

aatttcaggc acacatcacc acacctggct caatttaaca tcttttcaaa tctgatcatc 40020

tgaactaaat ttgggtgcat atcatctctc ctcgaacaga tggagggaag aggggatagc 40080

tcctttatct catttttata ctcttttgtt aacaaggcta ggctctggaa ttttattagt 40140

taggattgta agggagtgaa aggtggggga aagacaggac tattagagtt cagttttttt 40200

ttttttaatg gggaatgggc tgaatagaat tttccttaac agcagttttc ttctatgtaa 40260

caaattagca aataagaaaa tgtccataat tgttcattag accatgaatt ttattttttt 40320

ttactttgag ctttctcata tctagatatt gacgaaaaga tgccagtttc tccctaggca 40380

agttttaaac agccaggtct tttttttttt tttttttttt ctagtgatac cttctgcagc 40440

cgtcatttcc agggctgaga ctataaatgc tacagtgccc aagaccataa tttattggga 40500

tagtcaaaca acaattgaaa aggtttcctg tgaaatgaga tacaaggcta caacaaacca 40560

aacttggaat gtaagctcaa ctttcattat gctttagcat gtgaatgaat gatttaaaag 40620

ccaaccatca gtggctacag tggacttatt atgtctattt tacatgtttt taatctgatt 40680

gtttgcatga tattcaagcc actttagttt ttgttttata atttcaactt ttattttaga 40740

ttcgggggca catgtgcagg tttgttacct ggatatattg catggtattg aggtttgggg 40800

tatgattgat cctgtcaccc aggtgctaag catagttacc aataatttgc ttttcaaccc 40860

ttgccttcct accttcctcc acactctagt gtggtcccca gtgtctatta ttgccatctt 40920

tatgtccatg agtacctgat atgatttggc tgcatcccca ccaaatcgca acttgaattg 40980

tgtctcccag aattcccagg tgttgtggga gggacccagg gggaggtaat tgaatcatgg 41040

aggccagtct ttcccacact agtctcgtga tagtgaataa gtctcacgag atctgatgcg 41100

tttatcagag gtttccgctt ttgcttcttc ctcattttct cttgccacca ctaagtaaga 41160

agagcctttt gcctcccacc atgattctga ggccttacca gccatgtgga actgtaagtc 41220

caattaaacc tctttttctt cctagtcttg ggtatgtctt tatcattagc atgaaaatgg 41280

attaatacag taaattggta ccagtagagt ggggcattgc ttaaaagata cccgaaaatg 41340

tggaagcaac tttggaactg ggtaataggc agaaattgga acagtttgaa gggctcagaa 41400

gaagacagga aaatgtggga aatttggaac ttcctggaga cttgttgaat ggctttgccc 41460

aaaatgctga tagcaatatg gacaataaaa tctaggctgg ggtggtctca gatggagatg 41520

aggaacttgt taggaactgg aggaaaggtg actcttgtta tgttttagca aagagactgg 41580

cagcattttg cccctgccct agagatctgt ggaactttga actcgaaaga tgatttaggg 41640

tgtctggtag aagaaatttc tacgcagcaa agcattcaag tggtgatttg ggtactatta 41700

aaggcattca gttttaaaag ggaaacagaa cataaaagtt cagaaaattt gtggcctgac 41760

tatgcaatag aaaagaaaaa cccattctgg gggggagaaa ttcaagccag ctgcagaagt 41820

ttgcacaagt agcaaggagc ctaatgttaa ttccctagac catggggaaa atgtctccag 41880

gccacatcag agacctttac agcagcccct cccatcacgg gcttggaggc ccaggagaaa 41940

aaagtggttt catgggctgg gaccagggtc actgtgctgt gtgcagctta gggacttggt 42000

gccctgtgtc ccagctgctc caaccatggc tcaaagggcc aacatccagc ttgggctgtg 42060

gcttcagaag gtggaagccc caagctttga cagcttccac atggtgttga gcctgcgggt 42120

acacagaagt cagaattgag gtttgggaac ctccacctag atttcagaag atgtatggaa 42180

atgcctggat ggccaggtga aagtttgctg cagggacggg gccctcatgg agaacctctg 42240

gtagggcagt gtggaagcaa aatgtggggt ctgagcctcc acacagagtc cctagtgggg 42300

cattgcctag tggagctgtg agaagagggc caccatcctc cagattccag aatggtagat 42360

ccaccaacag cttgaaccgt gcacctggaa aagttgcaga cactcaatac cagcccatga 42420

aagcagctgg gagggagact gtaccctgca aagtcacagg gttggagctg cccaagacca 42480

tgggaaccca cctcttttat cagcataacc tggatgagag acatggaata aaagaagatc 42540

atttgggagc tttaaagttg ccctgctgga ttttggactt gcatgggcgc tgtaacccct 42600

ttgttttggc caatttctcc catttggaat ggctgtatta cctaatacct gtaccctcat 42660

tgtatctagg aagtaactaa cttgcttttg attttatagg ctcataggtg gaagggactt 42720

gccttgtctc agatgagact ttggactgtg gacttttggg ttaatgctga aatgagttat 42780

gactttgggg gacaattggg aaggcatagt tggttttgaa atgtgaggac acgaatttgg 42840

aggggccagg gatggaatga catggttttg ctgtgccccc atcaaatctc aacttgaatt 42900

gtatctccca gaattcccac atgttgtggg agggagccag gggaggtaat tgaatcctgg 42960

gagcccgtct ttcccatact attctcatga tagtgaataa gtctcatgag atctgatgca 43020

tttatcaggg gtttccgctt ttgctttttc ctcattttct cttgctgctg ccatgttaag 43080

tagtgccttt caccacccat catcattctg aggcctctcc agccatatgg agctataagt 43140

ccaattaaac ctctttttct tcccagtctc cagtgtgtct ttatcaacag catgaaaaca 43200

gactaatgca gtacccagtg tttagctccc actcataagt gagaacatgt ggtatttgat 43260

tttctgttcc tgtgttaatt cacttaggaa aaccactcta gctttaataa tgattattgt 43320

atgcttgcaa acagagaact gtttcctcaa acgatccact tgccttttat tagttgctaa 43380

atagacaaag ctccgactaa gaggaatcta attagctatt tgtaattcag tgtctcctag 43440

ggtgaaattt atatcagtga tcatggataa aaaatttaag tattctgtgc tgaaatttga 43500

caggcatcca gggaaacaaa aattgttcgg aaaggagaac taagatgtat gaatgtttct 43560

ttttaagtga aaagtgtata gttcagagtg taatatttat taccagtata ggcctgagtc 43620

ttaggtgagc ttaaagatga tgataatccg agtaaaaatg agtttaaaaa agtgaacgtc 43680

tagagaaaca aactaaccat ccttaaatgc caaggttaaa ttatctagtg tcttaagaaa 43740

ggcatagctg caaggttcat taataaagtc acatcagata acagcacctg ggaacaacgt 43800

aataaaactc agtaatttca gctgtggagg aacactagcc tgatttagga agaagttcca 43860

attttgatat attttaaaag aaatgctatt tgattatttt taagcgtaac aaactgccat 43920

ttagtccaaa taattaagaa atagtttctg gccttttttt ttcccttccg atactttttt 43980

aaggtttttt ttttagagat agactctcac ttcatcaccc aggctgaagt tcagtggtgc 44040

aatcatagct cactgcagcc acaacctctt gggctcaagt gatcctcctg ccttagcctc 44100

ccaagtagct gggactacag tcactcatca ctatgcctgg ctaatttatt tttattttta 44160

tttttttgta gattcttgcc atgttgccca ggccggtctt gaactagcct caaataatcc 44220

tccccctggc tttctcgtca gcctcccaaa gtgctaggat tacaagcatg agccacagca 44280

cctggcctgg cttttcttaa ttaacagtta tgtatcagct gtgaaattac cagttttcag 44340

cgcgttaaat aaagtgatat gtatttaaca ctaaaataca ttaaacataa cttaattttt 44400

ctttggtgct aagcatgatt ccaaatcctt ttgctgttta agactgattc tgagtatcgg 44460

ttttgctata gagtaatata tcttaaaagt atcaagaaga tgggggcaaa actatactaa 44520

ttttattata taccctaaaa attactcatt aaaagtaaat tccttacgtc aacttgttta 44580

cctttgttca ctcaaatcat aaatgtgaac tttatggttg tttgcataca cttaaatggg 44640

atccacgttc tgcatcattt gattgataat caagtgaaga tcctgctgaa ttccttttgc 44700

atatgcagaa tttagattaa atttcaaaac aacacaaata caattctcaa gtcctagatt 44760

ctgaattaat ggggttttat cctaataaga cacctggggt ccttgtatag tatcacagtc 44820

atagaatgat attaaagaat actgagtttc ttaggctggg tgcagtggct catgcctgta 44880

atcccagcac tttgggaggc caaggcaggc ggatcacctg agctcaggga ttgaagacca 44940

gactggccat catggcaaaa ccccgtctct actgaaaata caaaaaattt agccaagcct 45000

ggtggtgtgt gcctgtaatc ccagctactc agaaggctga ggcaagagaa tcgcttgaat 45060

ctgggaggtg gaggttgcaa tgagccaaga tggagccact gcactccagc ctgggtgaca 45120

gagtgactct gtctccagag gaaaaaaaaa aaaaggatac caaatcctct tacttcatgc 45180

aaataggagt atgtaataga ctagaaaaag tgtttagaaa atagaaagga attatattat 45240

cagtgtctct gaataagttt tcagaagcca actgttttct ggttgaaact cttattctct 45300

gctccccctg gtggtgctac ataggccatc ttggtaacag gtacatttga gctcactttt 45360

caaaaccttc tctttatcag aagtggcaaa tagagtagaa agaatatttg ttatctcata 45420

tgctcttcat aacaatcctt tgtgataggt agtattagct ccattatata aatagggaaa 45480

tagagtttga aagaagtcaa gccagatttt tttgaactta taccgtcagt aactaagtgc 45540

ctctcacaga ctctacatca ctttaaagac caaataaata tttagaaaat gaaaagacag 45600

gtttcaatcc aaagccactt ctgtctcctc caccattatt tttttcagaa agttttttta 45660

aactcatgac tccctcagtc aatctaccac gtttccttta aacacaacca ctaacacaga 45720

aaaagtgaat taccatttct atccagatca ctcaagccaa gtcactgtag ccagaatgaa 45780

gctttgttta catttgctac tgtcaatttc atctgggtca tgcatgaagt gttgtctctg 45840

catgtctgta ggaagacagg aaagtcagag tcaagaggag tgggaggata ccaaagatta 45900

caggtctctt ctaccacttt agctccgtgg tggcattgcc tccattagat attgctggag 45960

gtggggggtt tacttcaaag cgacaggaaa gccttgtggc caagagcaca ggctctagag 46020

ttcaaagcct ggctctgcta ctttctagtc atgcaatctt tttttttttt ttccttttga 46080

gacagggtct tgctctactg cccaggctgg agcgcagtgg cgcaatctca gctcactgca 46140

gccttgacct cctggactca agcaatcctc ctgcctcagt ctcccaagta gctgaccaca 46200

ggagagtgcc accatgccca gctaaatttt aaaaattttt ctgtagagac agggttgtgt 46260

catgttgccc aggctagtgt caaggtctct aactgctgag ctcaagcaac catcccgcct 46320

cagcctccca aagtgctggt attacaggca cgagccactg ctcctggcta agtcatgcaa 46380

tctttctttt tctttttttt tctttttaga cagagtctca ctgtgttgcc caggctggag 46440

tgcagtggcg tgatcttggc tcactgcaac ctctgcttcc cgggttcaag cgattctcct 46500

gactcagcct ccctagtagc tgggattaca ggcatcgggc accacgtctg gcttcttttt 46560

gtatttttag tagagatggg gtttcaccac gttggccagg ctgatcccaa gtgatctgcc 46620

catctcggcc tcccaaaatg ctgagattac aggtgcgagc cactgcgccc ggcctagtca 46680

tgcaatcttg agcatgtttc ttatcttctc tgtgcctcgt tattcccata cataacatgg 46740

ggataagata agcccttata tcatgtgttt gttgtgggaa aggggatcca atgatgtaag 46800

aagttagcta caagcctgga atagtgtaat tgttcactaa atgctagcta gtaatgttac 46860

tctctagcct ctaggggagc catgcagcag ggatagtaga gcaaaggaat ccaactgaaa 46920

gtcacagtat tgagggtgct tttcagcccc tttagctaag gtacatacag actgtgaagt 46980

atctaaggga attaggctga cgaggcaagg agaatatgtg ccacgcagca gtccatgttt 47040

caggcaatga tggatggcat atatgatggt ggtcccataa acttatcatg cagctgaaaa 47100

atttctgtca cctagtgatt tatattacaa ttgcctgcag gattcagtac agtaacatgc 47160

tttataggtt tgtagcctag aagcaacagg ctataccata tagcctaggt gtgtagtagg 47220

ctataccacc caggtttgtg taattacaat ctatgatgtt cgtacaatga tgaaatcgct 47280

taatgatgca tttctcagaa tgtatccctg tcgttaagtg acacatgact ctagttatct 47340

cttgaaagat ggttccaggt caaagcttca gattttgtct ctggagacac agctgcctct 47400

gggagaacat ttacaaacat cttgctccca cctcatttac ctctctccct tctgaattcc 47460

ctagaagtct agcttagacc aggttggaat atatacagat tttagtaaag gctcagagat 47520

agcaagaaga aaataaatct cagctgggag gggtgataca agcctatatt cagctacctg 47580

agagactgag gcaggaagat cacttgagcc agggttttga ggctgtaatg cactatgatc 47640

ccaccaggga tagccactgc actgcagccg gggcaataca gtgagacccc catctctata 47700

aaaagaaaga aaataaacat ctcagagaaa aaccacctac tatgtaatct tgattacaaa 47760

atataactag cttttcaaag ttggtttgag acctgtcttt tcattttcta aatatttatg 47820

tggtctttaa agtgatgtag agtctgtgag aggcactcag ttactgatgg tataagttca 47880

aaaaaatctg gcttgacctc tttcaaactc tgtttcccta tttatataat ggagctaata 47940

ctacctgtca caaaggattg ttatgaagac catatgagat aacaaatatt ctttcagttc 48000

catttgccac ttctgacaaa gagaaggttt tgaaaagtga gctcaaatgt acctgttacc 48060

aagatggcct atgtagcacc accaggggga gcagagaatg agagtttcaa ccagaaaaca 48120

gttggtttct gaaaacttat tcagagacac tggtaatata attcctttct attttctaaa 48180

catttttcta gtctattaca tactcctatt tacatgaaat aagagggatt ggtatatcgg 48240

ctatctactg ctacgattga gggaagagag agaccctctc atattgtttt atattttttt 48300

atactcagta cctgttttaa gaaaaaacaa caaggaagta aaagcaaaga caggcaaccc 48360

agcaccaggc ccgaaaccag gactgggcct gcctggccaa aacccagtag ttaaaaatca 48420

actcataact tagaaagcga tgttattcat agattccaga cattgtatag aagaacattg 48480

tgaaactccc tgccctgttt tgtttctctc tgaccactgg tgcatgcagc ctctgtcacg 48540

taccgcctgc ttgctcaaat caatcacgac cctttcatgt gaaatctgta gtgttgtgag 48600

ccctttaaaa ggacagaaat tgtgcattcg gggagctcgg attttaaggc ggtagattgc 48660

cgatgctccc agctgaataa agcccttcct tctataactc ggtgtctgag aggttttgtc 48720

tgcggctcgt cctgctacac aataatgtgt aacaaactgc cacaaaagct gaagggacat 48780

ataacaataa acatttattt ctcaatgtca gtggggtcca gctggtctaa ggtgagcttt 48840

gctggaattg ggcatgtgtt tgcagctcag ctggctcact cctatgtctt tgcatgagct 48900

ctcctctaca tgtctcctgg tcctctttct gccatcaaca ggctagcctg ggcatgtcct 48960

tatggtgatg gcagagggca agagtaccta aggcccaatc tttccagtgt ctttcaatcc 49020

tctgcttata tcaggcttac caatgtctgt tggctcaaag caagctacat ggacaaacca 49080

gagccagaat gggagaacgt tcaaagctac atggtaaagg gccaggatat ggggctggga 49140

aattacctgg gcattattgc aatccacctc aaaatgtcat caaggctctc cacctgctgc 49200

tggaaccagt tcgtgtgagg agaggaagat ggtagcatct ttccaaacaa taaagaccaa 49260

gaaccttggc agtcttcatc ctactctagc atttgacact gttgaccacc ttgtcctgaa 49320

aatgtctctc ctcttggttt tcccatttca agaatgtgtt agtcttctat caatatttct 49380

ggatgccctt tcctttgtaa attatttttt ctgccaattc cctaaacatt gacattccct 49440

tcatacctac catctcactg tctcctctct ttcttactcc tttcacctat tcccaaggcc 49500

tcagctacac ctgtatgtag gctagaaact ggagccctcc caactctcat gaaagtcaga 49560

cctatatttc taactatttg tttgtatctt tatgggctgt ctcctaggcc cctcaaattc 49620

atgtcaaaaa ttgagctccc agttgcgaac ggcaatccac acctacttcc gtatactctg 49680

tttcagggca tttgccacat tgtatatact tgcctgttta cacttctatt tctctactct 49740

ttaactgtga actcatggaa ggcagagatg tgccatgatc attttttgca tctccattga 49800

tttttataat gtcaagttca tgtggataat attttgaaac attagctatt actattatta 49860

ttattatttg tctaatggtg tccaacttta gtgacagagg cagggaaatc tcagctatgt 49920

gttcaagtaa gtggattatt ggtgactcat ttctctaatt aagctacacc agggtctcag 49980

caaacttttt attcaagggc caaatggtaa acattttagg ctttgcagaa cataagattt 50040

ctgtctcaac tactcaactc tgtgtttgta gcgtgaaagc agtcacagac aattatgtaa 50100

atgaatgggc atagtctgtt ccaattaaaa tttcattttt ggacactgaa atttgaattt 50160

cacataattt ccatacatca tgaaatagtc tttttttaaa aaaaatttct ttcaaccatt 50220

taaaagtgag aaaaccattc ttaacttagg ctgcacaaaa ccaagtattt attcttttct 50280

tgaaagggct gatagaacag catacagata gcaaagacaa atccttggct tcggcatttc 50340

caagtctgaa ggttggtata agacagaaaa tgtggaaaaa tgtatgatta gtcacaaata 50400

agtttcccca tcaccacccc atttccctcc tctaccaaac actagataaa aattacttgg 50460

gcagaaaggg gaggaggttg tagggtaaag ggagaaacca agggtttaga cactggtggg 50520

ttaccaagga ccctctcatc cagtgccatg ccccatactg atagttgggg aaggtaggaa 50580

ttatagaact ctaactaggt tggagatttt ttcttccctc cacccaagga tgttcccagg 50640

aacttatatt taacataaga aaagagagat ctcaaactga agaatattac gtttcaatga 50700

cccctaacct tcagagttga ggctctaagt agaaatggac ttacttcaaa atttcttgca 50760

ctcttgaaat tatactattt acagaggcac tcaattaagt ccagctcttt caatgttcat 50820

tttagcctcc aaagatatca tcctgaggta tcatgttgta cttgcagggc cctaagcagc 50880

cacctggttt gcctcatggt ttaaacaact gataacagtt ccataacgtt cttcttacta 50940

ttttggaatt ctggctgtac tttacggtat ttctttcctt taagaggact tatctataaa 51000

tgtaatggca gcaaacatat tggagactaa caacttcctt acaagagaca ctaatatgaa 51060

gcagagtgca tttgagagtc aaatctttgg cacagtaagt aaaatttctc tagatcccag 51120

gagacctcag acactctcca gttatattaa cattgcagaa aatttccagg gccatgaata 51180

gagacatggc ttatggaaac tctgaaagat atctttcatg cagagtttga aaaatatcat 51240

tcagagtttc cataagccat gtctcaatag acaaaactat tctagggcag atgacatccc 51300

ccaaacaaat attttcatga attttacatt ttcagtgcat tgcaagtttt aaggattaat 51360

gttaaggttg ggttttaaat ggaagcatcc ctgaatgagg ttgatacctc attctttttt 51420

ttttttaatc tcaatcgttg taactttctc tttcatgagt atctgataac tgctttcacc 51480

tagttgttgc ataaatgcat ttctagacat attgacatat tttatgtctg cctttcaatc 51540

atgcttcact ttttccagta aatcagttac tagaacatac tgaagatgct gtacaaatgc 51600

cttggtttct ttctgccaac tgaaacagct tgtttggggc tcatttttca ttacgtttta 51660

tagcagggtg tagtgctcca gggaaaaaag gatttttgtg tttatccctc agctattttc 51720

tttatcctcc attggatttc tcagttcaac tttcttaata ttttaatttt tctctgacac 51780

atacacatat atatatatat acatacatat acatatatac atatatacac atatatgcat 51840

atgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtatatat atatatatgt atgtattatt 51900

gggccttgta gaatctgcaa taacctgcaa tgaggaccaa cttacagctt agcttatagg 51960

tatttttttc taaaagtcac cattagcgag taaaattatt tcatcattca ggaagaatta 52020

ttgatctaaa tatccagctg caggggagag gggaattcag acaattgcta tcagctacca 52080

tttctccacc ccattaaaag agtatattcc aaaattaaga atatattcca aaattaagaa 52140

tatattccaa aattaaggct gggtatggtg gctcactcct gtaatctcaa cactttggga 52200

ggccaaggca gagagatgac ttgtgcccag gagaccagcc tgggcaatat aatgagaact 52260

tatctctaca gaaaaattta aaaattatcc aatcatggta gtgcatgcct gtagtcccag 52320

ctacttggga ggctgaggca ggaggatcac ttcagcccag gaggaggtgg aggttgcagt 52380

gagctgtgat cgagccactg cactccacag tccagcctgg gcaacagagt gggaccctat 52440

ctagaaaaaa aataaaataa aaaatatata tatacacaca cacacatata aataaataaa 52500

tatatataca cacataaata aatatatata cacatatata taatatcaca tttggacttt 52560

ctggagattt gagacagttg tcaaacataa agcagtatgg gctgggcacg gtggctcaca 52620

cctgtaatcc cagcactttg ggaggccaag gtgggcggat cacttgaggt caaaaattca 52680

agaccagcct ggccaacatg atgatacccc atctttacta aaaatacaaa aaagtagcca 52740

ggtgttgtag tgcatgactg taatcccagt tacttgggag gctgaggcag aagaatcgct 52800

tgaacccggg aggcggaggt tgcagtgaac tgagatcgag ccaccgcact ccagcctggg 52860

caatagagcg agactccatc tcaaaaaaag cagtgtgtgt ttcagtttta atgtatttca 52920

gagacagtat ttgattatgt acggccacgt tttatataaa gaacactttg ttttcctaga 52980

gtctagaaga cagcttggaa cataataggt gttccataca tttctgctaa ataaaatagt 53040

tgttttaaaa gcacaccaca ttttattatt gttacccatc cattttaggt taaagaattt 53100

gacaccaatt ttacatatgt gcaacagtca gaattctact tggagccaaa cattaagtac 53160

gtatttcaag tgagatgtca agaaacaggc aaaaggtact ggcagccttg gagttcactg 53220

ttttttcata aaacacctga aacaggtgag tgtacttata tattttattc tgttgggctt 53280

ttctttatat atcttttctg ctgagcacag tggctcacac ctataattcc agcactttga 53340

gaggccaagg caggaagatt gcttgagcct aggagtttga gactggcctg ggcaacatag 53400

tgagacccta gtctgtacag aaaaataata attattatta gcctgggtgg tagaatgcat 53460

ttgtagtcgc agctacttgg gaagctgagg tagtaggatt gcgtgagccc gggagtttga 53520

tgctgcagtg agctatgatc atcccactgc tctctagcct ggaggaaaga ccaagaccct 53580

gtttcctaaa aagtttaaaa cagccaggtg cagtggctta tgtctgtaat cccagcactt 53640

tgggaggcca aggtgggtgg attaccttag gtcaggactt caagacctcc tcggccgaca 53700

tggtgaaacc ctgtctctac taaaaatacg aaaattagct gggcatggtg gcaggtgcct 53760

gtaatctcag ctactcggaa ggctgaggca ggaaaattgc ttgaacccaa gaagtggagg 53820

ttgcagtgaa ctgagattgt accaccgcac tccagcctgg ccaagagaga gagacttggt 53880

ctcaaaaaaa aataaaaata aaaataataa taataaataa gttaaaaaca aaataaagct 53940

acaagatatt ttttttctct ttacctttga ccaaaattga caaaactatt ctagggcaga 54000

tgataacatt taaattgtca ttttgctttg attttcttaa ggcttactac aatttatata 54060

atggttctca aacattcctg ggtataaaaa ccatctggga ggctgggtac gctggtttac 54120

acctgtaatc ccagcacttt gggagaccaa ggcagaaaga ttgcttgagc ccaggagttc 54180

aagaccagac tggacaagat agggagaccc catctctacc aaaaatacaa ataaaattac 54240

ccaggcatgg tggcccacgc ccatagtccc agctacttgg ggactacagg aagaattatt 54300

gatctaaaca ggcttcaatc atagctcact gaagcctctg cctcctgggc tcaagcaatc 54360

ctcctgcctc agcctcccaa gtggctcaga ctacagatgc ggtccaccat gctagctgag 54420

gcaggaggat cacttgaacc tgggaggtcg agaatgcagt gagccacgtt catgccactg 54480

aactctagcc tgggtgacat cctgggcaat tgtgagccat acaataagct aagaacttgt 54540

tttgaaaaat attatctggg tgtggtggct caagcctgta atcccagcac tttcagaggc 54600

ggaggcagtt ggatcacttg agcccaggag cttgagacca gcctggccaa cgtggcaaaa 54660

tcccatctct actgaaaata caaaaattag ctgggcatgg cggtacatgc ctgtaatccc 54720

agctattcag gagtctgagg caggaaaatg gcttgaaccc aggagacaga ggttggagtg 54780

agccgagatc gcgccactgc actccagcct gggtgacaga gtgagactcc atctcagaaa 54840

attttattaa gaacatattc tcctaaaaga gacacaaaac aatcatatca ataaacgtta 54900

tctactatat gtaaagcaat cagaaagtta aaactcctaa acacagaatt ctcttctcct 54960

caacaggatt tagctgattt attgtttatc tttagatatt ttcctgcttc taatattatt 55020

agtttaattt tatattcatt tattcatgcc attcaataac atcttagcca aatagacttc 55080

agttgaagca cataataaaa tgaatctatt gtaattgtaa taatactcct aatggcccca 55140

ttaacctaaa gatgtctccc tactgtaggt accttataca ctagatttaa cattttcttt 55200

tcagaactga aggactttat tataatccca gtatgaatag catcattatt ataacattct 55260

ggaattgtgt tttatgtttt caaggcactt tgacatacat tataaatatt gatggctgcc 55320

aaattagtag agggagcaaa gagcatgact tgctgcaaag ctcttctggt atctcctact 55380

gcccaaatct tagaaatact ttggccaggt gcagtggctc atgcctgtaa tcccagcaca 55440

ttgggaaatg gaggcaggtg gattgcttga gcctaggagt tcaaacccag cctgggcaat 55500

atagtgagac tctgcttcta tttaaaaaaa ataaaaataa atagaaatta aatttaaaaa 55560

tgttaaaatg aagaaacaac ttattaggga atccttttct ctttctttct ttctttcttt 55620

tttttttttt tttttttttt tttttgacag tcttgctctc ttgctctttt gcccagcctg 55680

aggtacagtg gtgtgatctc ggctcactgc aacctccacc tcctgggttc aagagattct 55740

tgtgcctcag cctaccgagt agctgggatt acaggcaccc accactatgc ctggctaatt 55800

tttgtatttt taatagaggc ggggtttcac catggtgggc aggctggtct cgaactcacg 55860

acctcaggtg atccgcccgc tttggcctcc caaagtgctg ggattacagg cgtgagccat 55920

ggcacccagc cagggaatta ttttcagtat cagttctctt ttttaaaagt tagtttttaa 55980

ttgaaggtat tctgtcaagt tgatactgtt aacaattcgt ggactttaga tgccatttta 56040

taatagtcag aatttagttg ataacccttt tttttaatga actctataac tgcctagcaa 56100

gattatgcaa attgataact accatttatc atttacgaag tactcctgtg tataagcttg 56160

tttgattatg atgtcagcca tatttggtag tgtaattagc gctactttac aaaagcggaa 56220

actgggcatg acttactaaa tagtacattg ctggtgggta atgacaccta aactataaca 56280

aaacttttct tattcaaaat attgaactgc ttggctaggt cagttggtag agtatgagac 56340

ttttaatccc agtgtgaaaa tactgtgcat tttccccacc atccctcagc aatttcattc 56400

tttaatttca gggaagcaga ggagcaactt acttaagtat tctaagtata ggactacaaa 56460

tgttcttctt taaacataaa agtcttggcg aggtgtggtg gctcatgcct gtaaccccag 56520

cactttaaga ggccaaggcg agtggatcac ctaaggtcag gagtttaaga ccaccctggc 56580

caacatggtg aaaccccgtc tctactaaaa atacaaaaat taactgggtg tggtggcagg 56640

tgcctgtaat cccagctact agggaggctg aggcaggaga atctcttgaa cttgagaggc 56700

ggaggttgca gtgagccaag atcctgccac tgcactccag cctgggtgac agagcgagac 56760

tctgtctcta aataaataaa taaataaagt aaaataaaga taaaagtctt aagcttcagg 56820

tagaaggaaa taggaacacc acagtttaaa tttaaggtct gtttcctgag gagaaaaatc 56880

acttaagaga caaaaatacc aattaaaatt aagtatccct gaaaacttgg atttattaaa 56940

gtttaacatg ttagctaaga gaaaccatag actgttctct tggtacaaat tcccttctaa 57000

gacacattac atgagaaaca gtaaaagtgt gttagggaaa gtgctcatgt taaatctctt 57060

tgaaaatgta cctttttttc tgtgtgtaaa agcaatgtaa gtttactgta gtatgcaacc 57120

taaaaacatc cactatttac atttatttaa tttaatacta gtttttccta tgcatttttt 57180

taatgtttgg ggctatgatg tatatactat tttatatcct gattttctta cttaatctac 57240

cctgttaagt ttttaaaaac tgatttcatg gctgcacggc attctcgttt atgtatgtac 57300

tataatttat ttaggcattt ccctacctaa ataacatcta ggtcatttcc attttatcca 57360

ttatcaataa acttctttgc atagctttgt atataaatgg tctttattcc tttagttcta 57420

aagaagaatt attgcatcaa gagttaagca ccttttaaga tgctgatgta tgttgtcgaa 57480

ctgcttttta ccgaatcttt aatattgatt gctttttaaa aagggaccta tgaaaagaca 57540

gtcattcaca aaatactcat ttagcctcta ccatgtgcca ggcattttta gatccttgca 57600

gaacctcagt gagcaaagga gacaaaattc cctgccttgg acaagtttcc ttctggagaa 57660

gacacacagt aaaaatcaag cataataatt acataaatta cattgcatca tacataaaga 57720

tgtaaataaa agcagtaaga atcagacttc acagctgaag tatgaattgc ctcacaaatg 57780

aaaaaataaa atgatatcat ttctgtaatt atttatttaa ccaacttgtt tgataatgag 57840

gttttttggc cagccaaact tttccagtgc ttaaaggttt atttatgaag aaacacagcc 57900

caggcagtaa tgcctcttcc aagcaggact tttctgaatt agtctctaag attctgacat 57960

gtgtattact gaattgattc atctcaatac aatgtgtttg caaaattctg cccagctaag 58020

accagaggac aaatcctgaa gtgggtatgg cttgtcctgc agcctcggca ttcttattct 58080

tttctgttgc tttccctctg aatagtgcct ggcttggcca ttaaaaacct ggtctcaaaa 58140

atgaaccaaa ccaactcact tataatcaac tttaatgact tattctcttt ttcttactta 58200

gagtatttag actttaatag gacaaacaaa gccttatact gaaaaagaaa atcaatcctc 58260

cagaataccc atttattcta tctgatataa tagcaagagg ccaattaaca gactgaatcc 58320

cagggtgact tcaggataat aaataataat agctaacatt tattggatgc ttactaagtg 58380

ttgggcattt tgataagcat tttacatatg aattgttctt tatttatcac tgcagctcta 58440

taaaaggagt gtaattatta tcccagttgc acagatgaaa ccatatggcc caagcagttt 58500

tgtcaaacat tacaaggctg gtaaacagta gagccagggt ctaactcagg ctatgggatt 58560

ccagcaccca tgttatttat agctctgcta gtctccttcc taagagacct aggagggtcc 58620

tgcatccgcc tgtgaaacaa aatcattctc tacacattag ctagtaataa aactatgcca 58680

acagagcttc atttctaatg cagtgtttgt tatggaaatg caaacgcatg agaaaagaac 58740

cagaagatac tactgactac tgctactttg gcttgaatac aagatgtgga agcagatttg 58800

cagtagatga tgagaatgaa accatagtac tataggaggt ttctgatagg ataggtaagg 58860

aaggggcagt ctgagatgag gataaaggat aggtaaaaaa ggcagcctta gccaagacag 58920

gaccatcatg ggcaattcat catgtgcttt accagctaat tatgggctat ctagaaagca 58980

ctgtttctca tagtagggtc tgtggacaat ctacattaag aatgcttggg cacttttttt 59040

ttttttgata cagtctcgct ctgtcaccca ggctagagtg tagtgcacca tctcagatca 59100

ctgcaacctc cgcctcctgg ttcaaacaat tctcctgcct cagcctctca agtagctggg 59160

actacagaca tccaccacac ctggctattt ttttttgcat ttttagtaga gacagggttt 59220

cgtcatgttg gccaggctgg tctcgaactc ctgacctcaa atgatccccc tgccttggcc 59280

tcccagagtg ctgggattac aagcacgagc cactgtgcct ggcctgaatg cttgggcact 59340

tcttaaagta cagattcctg agccctttgc caagcgatac ttctgcagtg aagcataaaa 59400

tccattgctg tagaggtcag acacactctt taagagaagg aagtgtcatc ataaaagaca 59460

acatagggaa tggacagaaa atgtggacag aaaggcagag tggatatgat tgcccaagcc 59520

attgaaacgg gagagttccc tgactcctgt cgcatatcat gtggctcatc tattctgcca 59580

aggcacatgc tcaaacccgt tacaggaggg ggagcacaca gatggacagg tgcgcaggag 59640

ctggggtgag caccttgggg ctccagcccc acagtagcat ctaggggtgg atgcctgtga 59700

ctcccaaagc ccaagtggga atgtgataca gttcactatt ttagctttgc tgtctgcaga 59760

cagcttaact gttaaccagc tcagtgccct cttggtaccc aggtccttgt ccagtgtcca 59820

ggaagaatca ggtcacacac agatttgaag gatgaatgtg ggggttttat tgagtggtgg 59880

aggtggctct tagcgggata gatagggagc tggaaggggg atagagtggg aggatgatct 59940

tcccctggag ttggctgtcc agcggccgat ctcctctctg atcgtcccca ggggaacttc 60000

tctcagcatt cagatgctcc ttttcttctg tccttctctg ccgagccatt ctgccattct 60060

tctgctcttc tatttatctc tctgtctgct tctggaacct ggggtctgga gtttatgagg 60120

gtacaggata gcggggcata gcaggccaaa aggcaacttt tgagcacgaa aacaagaatg 60180

cctgcttcta tttagggcta tgggtttcca agcttgaggg tgggacctct gccagggaac 60240

caccctcatt tattcagtat tttcctgttt gctgtttgta tcaccatcgc cttagtagta 60300

gtggtttggg tgacagaaaa gggtgggcat gtgagaatct taggccatta tctctagtac 60360

tatctggtca cctctctgtt tcctatttct cctgagtcac tgaccacccc agcccacctg 60420

ggcattacag gaacccccca aagcacagga tataggactg ttgtcgtaat taaccatcac 60480

atcacacatg accctcttct gcctagaact caccctgtct ctggatgtga ggtttcagct 60540

gagattatga gaaccgctga gagctgctga agttagagct tctgatccac caaccttgat 60600

aggcactgtt atcctggggt ctttcagcag tgcggaaaga gccccaaagg ggaaaagaca 60660

atttgtcatt gtagaaaacc aacactgaaa atgtataagt cagtagtcta aatttgctac 60720

tactgactta aaaaagctga aacaacaatg gctagccagg aatctttttc aattctgcat 60780

tgtaatatag atcccttttt aacgaagaaa agacacaggt tctataattc ctttaataac 60840

aggttaaagg cttaagggaa gggcagtcac atttcatgat gactaattca cttgcttgaa 60900

ttgctgcttg ccatagaaaa gtttcccaga gaactcatat tactcttttt ttttttttga 60960

cagttttcca gaggaaggca agtatgagca agatattaaa caacattttg ctttcctctc 61020

acaaaacagt attggttgtt gcactaccag tgactccttg aagatgctca ttaatttttc 61080

aggtttttaa gtggctaaaa gggaaaaagc aaatattatg ttaacggaac taaatatcag 61140

tgctattaaa tataatgcaa attagatgat ggcacacaga atgtattttt caaacatttt 61200

attatgtgta attagagatt tgtgaattgt ccacatagag atgggttctg aacttgtaga 61260

ctctattggt tactaaccca tgttaaacag acacggtgtt tcttgaaatt ctaatcactt 61320

tagacttttc atttgggttt aactttcttc aggtcctgcc tctgataaac caccaagttt 61380

ctgcagaata ggaaacaatt cagaagagtt ccataatctc cctgaagttt gcaacctatt 61440

actacagaac aaagatcttt ctttctttct ttctttcttt ctttctttct ttctttcttt 61500

ctttctttct ttctttcttt ctttctttct ttcttccttt ctttcttttc tttctttctc 61560

tttctttctt tctttctctg tctctctctt tctttctttt ctttctttct ttctttcttt 61620

ctctctcttt ctttctttct ttccttcttt ctttctgtct ttctttttcc ttccttcctt 61680

ccttccttcc ttccttcctt ccttccttcc ttcctccctt cttttttaga cagggtctct 61740

ctctgtcacc caggctggag tgcagtggtg tgatctcagc tcataagatc tgggctcaaa 61800

caatcctctc accttggcct ttcaagtacc tggaactaca ggcatgcacc accataccca 61860

gctaattttt tttcttcctt tttttttttt tttgtagaga cagggtttca ccatgtttgc 61920

caagttggtc ttgaactcct ggactcaaga actctgccca ccttggcctc ccaaagtgct 61980

gggcttacag gcaggagcca ccatgcctgg cctatgatta tgctttttct tgaagtcatc 62040

atcttctata ttagtttcct attactactg tcacaaatca tcacaaactt gaaagcttaa 62100

aacaacatga atttattatc ttatagttct ggaggtgaga aatccaaaat cacaaccact 62160

gggctgaaat cagggtgtca atagggttat gttccttctg gaggctacag gacagaatca 62220

gtttcattgc tttttctata ttctagaggc tgcctacatt cctcagctca tggctccatc 62280

cttcattttc aaagccagaa gcatagcatc ttccaatctc tttctctgac tctcaacctt 62340

ctcccttctg cttataagaa ctcttgtgat tttaccagat ccacccatgt aatctaggat 62400

agccacttcc tctcaattcc cttttgcctt ataaggtaac tgagtagaaa gaacatactc 62460

aatttctctt gctatatagt actttcacaa cacaggtcac ttctgtgacc tctgattacc 62520

aacatgtgta tggggatttc tccccctcag caacaaatta tctggcagat tctttgaggg 62580

acaccagctg tgtgtcctcc aagtcaattc attctggcac tatctgccta gacatagtgt 62640

cagatcctac aactcccacc tcagatgcca gtctcaagta ataggtggtt acctatactt 62700

ccatctgatt ggctataaat tagagtttgt ttcctgatct ttttttcaga tttgatcaat 62760

ttgctaggat gccccacaga acttgggaaa acacttatga ttactggttt atgataaaca 62820

gcattacaaa ggatacagtc gaacagccag atggaagaga tgcagtgggc aaggcatgtg 62880

gggaggggca tggagcttcc ataagctttc caggtgcatc acctgccagg aacctccatg 62940

tgttcagcta tcctgaagct cctagaactt aggcctttgg ggttttttgt aggcttcatt 63000

acataagcat gattgattaa accattggcc attggtgatc aacttaatct tcatcccctc 63060

tcccctctgg gaggttgggg atgggttgaa aaatcccaac cttctaatcg tgccttgatc 63120

tttcctctga acaaccccca tcctgaaact acataggggc ggccagccac tagtcagctc 63180

gttagcatac aaaatgaccg ttaccacttt gggcattcta aggattttag gacttgtatg 63240

ccagaaaacc aggatgaaga cacaatatgt acttcgtaat attatagtaa cctgttcaca 63300

gattccaggg attaggacac ggacatcttt atgatgccat tatttgtcct actacatctc 63360

cctacctccc caaccactac caccaattgc agtttttact agtcaggatg attaggtgta 63420

agcaaaataa atcaaggctg gttatcttaa gcagaaagag aattattaga aaggtagctg 63480

aaaatcagta gaaagcctgg caaaatgggc atgaacagga tccgagtgag agcaggaccc 63540

agaaccacag ccaaaatcac agcctaggaa aagtctggcg aggaccctgc agctgacgct 63600

gatcactgcc actgcctgcc ctggtgccac tgtcactgta ccctggatac agctatggtg 63660

cccacattgg attctaagtg gtttctggaa gccctatgtc aacccttcaa cattcacagc 63720

ctgggtgtta ggggaggggg catctggttg gtgaggttta ggccatttgc ttgtagccct 63780

actgcctctc agctttttta ggatgcccac aaagaagtat atcctagtgc tgggcaaata 63840

aaacatggca aatgtctgct agactataca gattggaaga tgctatgcct ggagagcaag 63900

ggtgtacttc tgaggaatct gttggccact gtgtagactt ggaagctgtc cattctttca 63960

tatagagttt tgctacactt tgacatcaga ccttctctgt atggtccttg tcatttagga 64020

aaatttcctg ggtggctact gctttttgcc ttttcctcct cttattcttt ctttttaaca 64080

atgtaactgc catatgtaga tttcttacct tctccctaca taccaagtac ctaaagccca 64140

ggaatataag aaggaagata aacccttgaa tatgtctgtg tgtaccctgc tacccttata 64200

tacaccgctg atactataat ccactaaatg attcattgct tgacttgttt ttcaaggtgt 64260

ggtaatgagt ttgttctgac acaccgtttt ccctgccctg ggcttctggt gcctatttga 64320

tatgttggca ttagttttgc cttgctaatt gacatactca actctttacc cttggcccga 64380

taatgttgaa ttctgaccac agagctaaac tggcctggct tggagaaatg gcacttaagt 64440

gctgtgaaac agctgaatta ttaaaaggag atgcgaaaga gagaaaccaa aaggaggtat 64500

ttgtgttttg agaaaaatag gagggagggt gggggcgaat ctccaatgat ttaaaaaaaa 64560

acacatcctt tgtctccttt aatatgactc ctctttcttg aatatctaga acccctgaaa 64620

ggtccagaaa agtccatgtg tctgctctaa tcaaagcctt tatgatgcag gccttggtta 64680

ctgtggcttt gacatcaaga ttggcaagcg caaaagtaaa taaagaaacc ttagagatgt 64740

ctataccctt tggctcagta attcctcttg tagagatctg gccttaagaa ataatcagtg 64800

gaaggaaatt tatgtacaat gatatacatc caaaacctaa ttataatagt aagaatttgg 64860

aaccagtcta aaagttcaac aatagcaata attagtgctt attaaggttt tatttgtgta 64920

aaacatggta ctaagtgctt gatatgaact gtctcatcaa acctcatgac ctcactgctt 64980

ttatctaatt ttacaaatga agaaactgag tcactgaggg gttaagcaat ttgcccaggg 65040

ccatggaact ccatctagta gagccaggac ttggactgag tttcgtttga gtctgcagtt 65100

ggtgtttgaa accacctagc tatacagttt ctcaataggg gtaagattga ataaatacag 65160

taaaacatga tgaaacatta actagccata gcatgatgtt ttgaacaatt tttaataaaa 65220

tgaggaaatt tttattaagt aatattgaat aaggctgggt gtggtggctc acacctgtaa 65280

ttctagcact ttgggaggct gaggcaggca gatcacctga ggtcaggagt ttcagaccag 65340

cctagccaac acagcaagac ctcatctcta ctaaaagtac aaaaattagc tgggcgtggt 65400

agtgtgcacc tgtaattcca gctactcagg aggctgaggc tggaggatag cttgagccca 65460

gggggcagag gttacagtga gccaagatca caccactgca ctctagcttg ggtgatggtg 65520

tgagactctg tctcaaaaga aaaaaattga ataaaaaagg caagacaaaa catttataaa 65580

gagtatgatc tccactaggc taccaacata cacaaaagac tgttaacatt ttttttaaat 65640

aatagtttct agtttgcaaa caaatataaa attgtattat cttctccata cccttctata 65700

cttttcaagc atcctgtaat tagcatgtat tacttgtgta ataaaggaaa gtagtcggtt 65760

ttaagaagaa aataactcta cgctagaaga tttaaagcat cagtgctaaa tgggttagca 65820

cagtagggct tgtggggtcc agagccccac aaggccaggc agccccaaca caccatggcg 65880

agtcctcagt ggagcaatgc taagtctcat cctgggagag gctgcttgca gggctgactc 65940

tgcctcccac cctgagccat cctgctgtgg aagtgaggtt attaatttaa tgagatcagt 66000

gaccagcaca aactacatgt cagaactgga tgggaatttc cactcttcac cctctaccta 66060

ggtgactttg agcaagttat ttatcctttc tactcttttg tttccttatc cgtataatga 66120

ggataataat attgtctgtg tgattgggtt gttgtgagga ttaaatgaga gaaaacatgt 66180

aaagtttagc atgttacctg tgacataatt aatactcatt aaatggtcac tttaaaagga 66240

tgacaaaaca cactttgccc catggttgat gatatggttt ggctgtgtct ccacccaaat 66300

ctcatcttga attgtagttc ccataatccc catgagtcgc gggaggtaat tgaatggtgg 66360

gggcagttgc agtcatgctg ttcttgtgat agtaagttct catgagatat gatggtttta 66420

taaggggctt ttcccccttt gcttggcact tatctggcct gttgtcatgt aagacatgcc 66480

tgtttccccg atcaccatga ttataagttt cctgaggcct ccccagacat gtggaactgt 66540

gagtcaatta aaattctctt ctttataaat ttcccagttt tgtccaggtg cggtgcctca 66600

cacctgtaat cccagcattt tgggaggctg aagcaggtgg atcacctgag gtcaggagtt 66660

cgagaccagt ctgaccaata tggtgaaatc ctgtctctac taaaaattcc aaaaaaaaaa 66720

aaaaaaaaaa aaaagccacg cgtggtggca tgctcctgta atcccagcta cttgggaggt 66780

tgagacagga gaattgctag aacccaggag gcagaagttg cagtgagcca ggatcatgcc 66840

actgcactcc agcctgggca acagagggag attctgtctt aaaaaaaaaa atccggtttt 66900

gattatgtct tcatagcagt gtgaaaacag actagtacgg ttgatgtaga aagaagagct 66960

gaggtgatga tttggcatca tccttaaaat acagatggaa tacgttattg ctaaaaccag 67020

gtccttttga gtggatttga ttaaactagc ctggtgtttt ggtaggccaa aaaatatagt 67080

tgttatgctt taaattttgt ccaacaataa gaaaccatat ttctcgtttg agatcactct 67140

aaattcccac aggcacattg tcttcttgta agactaaagt ttggtgccag tgtgtacaag 67200

ttatataaaa attcttccca aattaaagat aatttggatt ttttttagta tattcaagta 67260

tgtcctgtga gattaatagg cataagttaa tattctgttg caaggatgga actgtctcat 67320

tatggacgta acaccacaaa caccaaatac agtaagatat catgaggttt tttttttttt 67380

tagcaaatta ggcaatgcac tgatgacaga actgatataa aaaacagttt ccagtcataa 67440

aggctgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgag attctgtgtt 67500

tgctaatgct tttgtatgtt aaaatacttg caaataccat aaagcactaa tagcaggtgg 67560

attaaaagga tgggattagg gatggttctt tttttctcct tttaatttct cctgttgaaa 67620

atttttctgt actttaatta tcgcttttac agtcaaagat gttaagtata atttaaaaag 67680

aaatgacgta gccacgtggt ggcagtattc catgaagaat gtaatctaca tagctttgtt 67740

tcagtcctat gtctaacatt ggcttgattt cttctctgac aattttgttt ataaattgtc 67800

ttattttaga taactgattc cttgtgaaat ttccctatga ggattaagca cctcttataa 67860

gtgactttaa tttaatgcac ctgtttgatt tggaactatg ataacaaata gactctaaag 67920

gaagattgta tttattccct gttagggcta ataaaaccct agtcagatat gaaaattgtt 67980

tttatatgat gcaaattaat aactgaatga agtttcattg ccaataacta cctgtagttt 68040

atacaacaga attacattaa cctgtattaa aagccaacaa caccatatat tacttttaaa 68100

ttaaaaatgc taattttaaa ataataaaag tgaaaacaaa aatactacag cagcatatgc 68160

tataagccca tgattttgct aaattgaaaa ttgtgttacc ttttaatgta gtatttttac 68220

tctttttttt tttttttttt gagatggagt cttgttctgt tgcccaggct ggagtgcagt 68280

ggtgtgatct cagctcactg cagcctctgc ctcctgggtt caagtgattc tcctgcctca 68340

gcctcccgag tagctgggac tataggcacc tgccaccatg cctggctaat ttttgtattt 68400

ttagtagaga tggggtttca ccatattggc caggctgatc ttgaactcct gacaccctcc 68460

tcagcatccc aaagtgctgg gattacaggc atgagccact gcaactggcc agtattttta 68520

ttcttaaaat ttcaaaataa tggagattca gactcctgcc tgagctccct gcaaatgaac 68580

attggcctgt aaagatagca ctttagatga ataaacctga ctgttggcca tcagggcata 68640

accttgttcc ctcagagttt ggtggaacca tatacccatg gagcaaaaga gacttagcga 68700

ggcattgaga agccaaaaac aatttctgat ttgtcacaat tccgggtgaa acattttcct 68760

cttgctgtag agaagccagg acaaaaaaca cgggaaataa cttttggctt ttctgatgtt 68820

ttcatcttct aaaatccctc acccactgtt ctcagaaaac taccctcctc acgcttatgc 68880

ctgcacccag agcagccttt tttctttttt cttttttttt ttttgtgaaa tggagtctcc 68940

ctctgtcacc caggccggag tgctatggca cgatctagac tcactgcagc ctccacctcc 69000

cgggttcaag tgattctcct gtctcagcct cccaagtagc tggtattaca ggcacgcacc 69060

accatacctg gctaattttt gtatttttag tagaaacagg gtttcaccat gttgcccagg 69120

ctggtctcaa ctcctggcct caagtgatcc acctgccttg gcttcccaaa gtgctgagat 69180

tacaggcatg agccactgtg tctggccaga ccagcctttt tcaaaaattt ctgaagcccc 69240

gtagaaacct tttcattgga aatgtaggac acttttctca aagacagtca catgggccac 69300

actactcctt atacaagcct gtgatatctc agcctcaagt tgtgatttct ccctgccccc 69360

aaaagtaatt tttttaaaag aaaaatttga gcactgaaat cagagtgacg gaatacacag 69420

ggcttgaagc aatctcaggg atccaaggct tagagccatc ttcagaaatg tcttctgcca 69480

gttgggagaa ttgtgtcctt gaagtcactt ctgtcagctt ttaattatca ggaaggagga 69540

gactggcaag gctgcaccag gacccctttg agttcagact gaaagttagg taccagggtt 69600

gctcacccca ccctggtcag aatcattcat tagcagtttc ctgacagcct ttataactag 69660

accaggctgc caggaaaaga aaagagcaga gagaagtcat tggtgacatc ccacccaggt 69720

gttacaacac caaagtgcct gctaaccaaa attaggtttc ttatgaggag ccaggagaag 69780

gtgaacagca tcccaaagat tgtgcaaagg caaaagtgac acatcttggg gaccctcaag 69840

gaaatctgaa ccttattgca ccatcaattg caaggaatca aatagagatt agttatcatg 69900

cgttttttct ggctcagtaa aataaagctc tttggtattt gctgcctaga agcctcatga 69960

aattagtgga cacaccttga agttttacaa cctaggtaga ttatagttgc ccttgcagtg 70020

tttcctcctt tgagatttac ccaattaaat aaaaagaaga agaaaagaca catactcgaa 70080

gactattgct tttattccac ccttcaagcc cattaaagtt attggtttac tttagtcatc 70140

tctaaacaag ggaagaaact ccgttgggaa atttggcaag cagagtgcaa aatgtaagaa 70200

actgacactc tttccttttg gtttaagttc cccaggtcac atcaaaagca ttccaacatg 70260

acacatggaa ttctgggcta acagttgctt ccatctctac agggcacctt acttctggta 70320

agaaaataca acttaggctt tttgagtagt cttttagtaa ttgcccattt taacccatca 70380

tactgaaaaa atcacatcag gtgttaagtt tctggacaat aagatatgcc ttatgtcttc 70440

cataggaaaa taatagacaa agtacaaaga tctgcttaaa actgaatgta agaagtggct 70500

taggtggatt ttgccggctt ttgcaataga ttgtatacat tttttaaaat ttttatttat 70560

tttattttat tttttgagac gaagccttgt tctgtcaccc aggctggagt gcaatggtgc 70620

aatctcggct cactgcaacc tccgcctccc aggttcaagc gattctgctg cctcagcctt 70680

ctgagtagct gggattacag gcatccgcca tcacgcccag ctaatttttg catttttgta 70740

gagacgggtt tcaccatgtt agccagactg gtcttgaact cctgacctca ggtgatccac 70800

ccacctcagc ctcccaaagt gctgggatta caggtgtgag ccaccgcacc tggccacatt 70860

tttattttaa tagctgctag ggctcaagac ccaaaaactt ctttcaaaac aaattactta 70920

cctatcttgt gctaggactt gtctagacat cttcttcaat ctttaaaaca acccatgaga 70980

taagtgttac gcatctattt tataatgagg aaactgaaac ttagagtagt tgaggaaact 71040

tttcaaggtc atagagctgc taagtgacag actaaaattc aaatcctttt ctttcaatgt 71100

cctggagtct attgtctttc ttttatacaa accagctccc atttcagttg ttagcatgac 71160

tattatcgta ttgatgagct tgcctaaacg ttttaggcgt aaaaaaattg agatctggtt 71220

gtacaatggg tgaacataac tctaaataga aatttcgagt tgaaaacttt taggcatgac 71280

tatttcacca ttgaccactg acagaccatt tatctccttg tgtagaaatc aggcaggaat 71340

agtataagaa cttttgcagg ttgtattgct attctttgca ccttcattta tttaatattc 71400

attaagcaac tcctgtgtac gaggcactgt tctgggtact ggggaatcag cagtgaaaac 71460

aatgagcaaa gcccctgtct tcatggagct tctattctag ccagacaggg cagaaaaaca 71520

gcaaacaaaa caagaagaaa agtcaggtgg tggtgaagtg tcgtaaagaa acatgaagtg 71580

ggtaggcatg gtggctcaca ttttgtaatc ccagcacttt gggaggccaa ggcaggcaga 71640

ttgcttgagt ccaggagttt gagaccagcc tgcacaacat ggcaaaaccc catttttaca 71700

aaaaatacaa aaattagatg ggtgtggtgt catgtacctg tagtcccagc tacttgggag 71760

gctgaggtgg gaggatcaac tgagcaccag agatagaggc tgcagtgagc tccactgcat 71820

tcccgcctgg gcaacagagc aagaccctgt ctcaggaaaa aaaaaaaaaa gaggaaaaag 71880

aagaaaaaga aaaagaaaca tgaagaaagg taagggcact ctgaattatc aatcaattgc 71940

aagccaagtg cttaggttca gtacagttcc ctaattatag atgcctacac agacctacct 72000

acaccttgat atttctgtgg gatcagtgga ggttaggaac tcatggcagc tctgttagat 72060

aagtattatt atctctattt tccaaaagag aaaacctgag actcagcaag ttcataatta 72120

tgccccaagg tcacagagct gataagaggc agagtttaat tcaaacccag gtatatcagg 72180

ccacgctctt ggtcattctg ctctactgct tagacccctt tgccgagcac tgtgttgacc 72240

tgagggctgt ctatcctctt ccaggcacta attaatgccc aagggtgtgt ggcagccagt 72300

acccccaatt agttgcttca aatagtcaga aatagctctt tagccctggg aaaacattaa 72360

tttcatatcc catcaagatt ttgcagtgag ctagagttac tcagactcct gcctgctgat 72420

tggctttaga agagggctgg gcagcctgga gaccccggta tgtgaaataa ctagggtggg 72480

gcaaacacag tgtctgccac atggtaagta cccaataaac atttgctgaa ggaagggagg 72540

gagtgaggaa ggaggatgga agggaagcca atacccaaca tggctctaga acaaattcca 72600

atgtgataag taatgcctca actatcttct atatttgaaa atagggcttt ttcatgtacc 72660

agggagaaag catgatgagc ctggtgggta atatgtgttg aataaattat attaattatt 72720

taaatatttt aggagattaa ctcaactttg acatgcaaga aaagcattgg ttttgtttgt 72780

ttgtttgttt gtttttgaga cacagtctgg ctctgtcgcc caggctggag tgcagcggca 72840

cgacctcagc tcactgcaac ctccacctcc caggctcaag ccatcttcct accttggcct 72900

cccaagtagc ggggactaca ggcacatgcc accacacccg gctaattttt gtaatttttg 72960

tagagatgga gttctcactt tgttgcccag gttggtctca aacttctgag ctcaagtgat 73020

cctcccacct cggcctctca aagtgctggg attataggcg tgagtcactg tgcccagcca 73080

gcattgtttt ttaactaagt gtgagtgcag cataaaggca attttgattt cttttttatt 73140

ttttattttt atcttaactt gccagtgtat gatttctttc ttatcacgta tattgctcta 73200

agtaaagtgg atgtcctttc tcagcaggaa aaatttcatg agcaatagat tttgaagaat 73260

gatgacaaat tgcacacatt ctatctcaca caaaaagtta aaaaaaatca gtatgattgt 73320

aaccagcttt agacattgtt acagcaattg ggaattctca cctgtgtcag acaagccaaa 73380

tgaagctcac cactaagaat ttatacgaaa tttgcatgca caagccgacc acatttgcca 73440

gagatgcact tctaaaaacc cactgacatc agatacatgt agcccaactt tctcaaacaa 73500

aaagttgttt cctggggtag ttgtgcactc tggaaaaaca gtcactctgt ggcctaaagt 73560

aaaggttaat tttgcttccc cccacccttt ctcctttgag acctttgctt tgagcagagt 73620

aaagagaata gtaattctgg tatcaaatga agactaatgc ttggttaaaa ttatttttct 73680

ttcctttcat tagacaacag aggagacatt ggacttttat tgggaatgat cgtctttgct 73740

gttatgttgt caattctttc tttgattggg atatttaaca gatcattccg aactgggtag 73800

gtttttgcag aatttctgtt ttctgattta gactacatgt atatgtatca ccaaaattta 73860

gtcatttcag ttgtttacta gaaaaatctg ttaacatttt tattcagata aaggaaaata 73920

aaaagaacaa tgtttaataa gtacttaccc atgccaaact ctctacaaat gtctttcctt 73980

taatcctcaa aatgaccctg ccagaaaagc ttcctggcct attttacagg tgacttaaat 74040

gaggcttaaa gaggctaagt cctcagccca gaatcactga acagtaagcc ctgagtccaa 74100

acacagctga tatcaaagcc catttctctc ctttactaca cggcgttttc cattgtgcct 74160

caaatttacc tacagtgcct agattcttga gagtggtgaa gtcacaaatt gccttgtgtt 74220

aaaagaaaaa cttcagccaa attaaattta aaggagttta attgagcaat gaatgtgcga 74280

attgggcagc ccccagaatt acagcagatt cagaaagact ccagggttgc cttgtggtca 74340

gagcaaatgt atagacaaaa aaaaaagtga cacatagaaa ttggcagtga ggtacagaaa 74400

cagctggata ggttatggat tggcatttgc cttatttgaa cacagtttga acacttagca 74460

gtctatgagt ggttggagta tggctgctgg gattggccaa gactcagcta ttgttacggg 74520

cacatactac taagttaggt tttcaatctt gtctgcctat taggctaggt tacagttcat 74580

ccacaaggac ttgaatatag aagtatggag tccttctcag atcatattta gtttgcttta 74640

ataattcccc ccttttggtg attttctcaa ttttgagaga ttgaccaaaa ctttagtcat 74700

tgatgtcact atcaccattg taaatgtact tatatggtct ttaaacccac tgggaaacag 74760

tagaacagtg ggttctgtaa ggtggcaaca aggactgagt agaaggtacc tccttatgct 74820

ggaacatcct gtttatagga gaaaaacaaa acctggtctg ttctaggatc tatgtgtttc 74880

cttaaagtct tagtttgact ttgtcacatt tagcatgaac aactccattt tggtttggtt 74940

tggtctgttg gagtctagtg catgagctca gtccaaaaca atggcctccc ataatttcgt 75000

tttaaaaaaa aaatccccct ttttggtcag gttctcactt aggtgacagt gtgaccaaaa 75060

cctagggcct cagtgccact ctcagttacc atcattttgg gtttctgttc tcagcatgta 75120

attcataggt tatggtgtcc tgatggtcac acatttcttt tagctcttgt tattccagtt 75180

gaagagagac catttgacat tctggagatg gctgcttgta aacatttaaa acctttgaga 75240

gaatacagca taccagggag attactatta ggactattgg gagaataata ctaagagttt 75300

gggttatgtt ccttacccag ggtcctcata aaccaaacca aaccatctaa aaatcaaata 75360

gatcaaagaa tgagctacat gaagagtcta ctcacttaag tgattttttt cattaatccc 75420

ctacaactga atcactataa tacccgatgt tttctccata catcataagt gccagcagct 75480

gtttagccaa tacttttcgg tttggccaat tctattattt cgcataactt tcacaagaga 75540

atttaaagtt ttttgtgtaa ccataggctt tacagtagaa tctgctatag aacctatcat 75600

gagggataca tttctaatca gtgcctcttt tacttcaaac tatggaaaaa agacctaaca 75660

aatgatgccc tgctagaaga gtgaaggcct cctggcaatg ttctctttag cccatgatgt 75720

ggcttaagag gaatgaatca atgttctgtt tctgactgac tatgaggcaa tatatgaacc 75780

cttaaaattt ctcacctgca ttgggccttc atcttttatc catcaaagta taacattatc 75840

catgtataag gctggctgca aaatccttca caaataaaca tataccccat aatacacaca 75900

taacagaccc ccttttcact tctattgttc atagaggcat aagcaagaaa aaaaatattc 75960

aaagttaaga gtctcatgat agtagagaag tcttaatcca tgattttagg ataagctgtt 76020

cacattaagg acgctgtctt cttgggagaa gctgtcctgg ttagctttac cttaagggtt 76080

ccgatgggtg tggagggacc ctcctcagtt gtgagattat gaaagttgtg ggatctctga 76140

aaccaaaagt tcaaggtccc aaagttttgc tgtagtgtgg atggcaagga cagtctttct 76200

ctaatgttct cagaagattc attctttggg ttctagattg tgaaggggtt gattgtcctc 76260

agtgaaccat aaacagcttt ttttaactat gtaaaaatgt actgcagcat aataatctac 76320

tattataaca tcagtcttct tgcatgggaa agctttcata caaccagaaa acatgcattg 76380

aaagtgacaa ttgaatgaaa tcccttataa atatttaaat gtccaatcag gtagccaaat 76440

gtacctgaag ctttgattgt tttcccagga atatgggttt gacaagccaa atattgttta 76500

taactatttt agtagtttat aagtcaccac acaaacatat ttaatttgga tcattttatc 76560

ttttccatta caagtcgtaa aatgcagaac ttttaataat gaaagcctta aggactctgg 76620

aaggataagg tggctgtctt ggttctccac gagtccatgc ttaaaaatgg acttatgtcc 76680

tcttgaatac cagttgtttc tccaatttag gtgcatagca ctgataactg atgggttatc 76740

ataggtaatt tggcttagat catggagttt attcaaattg tatatctaaa caattttagt 76800

attggctgat ttagcatgcg gatttctctt tttttttctt atactgtaag ttctgggata 76860

catgtgcaga acatgcaggt tcgttacata ggtatacatg tgccatggtg gtttgctgca 76920

cccatcaacc cgtcatctac attaggaatt tctcctaatg ctatccctcc cccagcctcc 76980

taccccttga caggccccaa tgtgtgatgt tcccctcctt gtgtccgtgt gttctcattg 77040

ttcagctccc acttatgagt gagaacatgt ggtgtttggt tttctgttct tgtgttagtt 77100

tgctgagaat gacggtttcc agattcatcc atgtccctgc aaaggacatg aactcatcct 77160

tttttatggc tgcatagtat tcaatgatgt atgtgtgcca cattttcttt atccaatcta 77220

tctttgatgt gcatttgggt tggttccaaa tctttgctat tgtgaacagt gctgcaataa 77280

acatacatgt gcatgtgtct ttatagtaga atgatttata atcctttggg tatataccca 77340

gcaatgggat tgctgggtca aatggtattt ctgatcctag atccttgagg aattgccaca 77400

ctgtcttcca caatggttga actcatttac actcccacca acagtgtaaa agcattccta 77460

tttctccaca gccttgccag cgtctcttgt ttcctgactt tttaatgatc tccattctaa 77520

ctggcatgag atggtatctc attgtggttt tgatttgcat ttctctaatg actagtgatg 77580

acgagctttt tttcatatgt ttgttggccg cataaatgtc ttcttttgag aagtgtctgt 77640

tcatatcctt cacccacttt ttgatgggtt ggtttgtttg ttttcttgta aatttgttta 77700

agttccttgt agattctgga tattagccct ttgtcagatg ggtagattgc aaaaattttc 77760

tcccatactc taggttgcct gttcactttg atgatagttt cttttgctgt gcagaagctc 77820

tttagtttaa ttagatccca tttgtcaatt ttggcttttg ttgccattgt ttttagtgtt 77880

ttcatcatga actctttgcc catgcctatg tcctgaatgg tattatctag gtattcttct 77940

aggtttttta tggttattag gtcttatgtt taagtattga atccatcttg agttaatttt 78000

tgtataaggt gcaaggaagg gatccagttt cagccttatg catatggcta gccagtttcc 78060

caacacaatt tattaaatag ggaatccttt ccctattgct cgtttttgtc aggtttgtca 78120

aagatcagat ggttgtagat gtgtggtgtt atttctgagg tctctgttct gttccattgg 78180

tctatatatc tgttttggta ccagtatcct gctgtttttg ttactgtaac cttgtagtat 78240

agtttgaagt cagatagcgt gatgcctcca ggtttgttct ttttgcttag aattgtcttg 78300

gctatgtggg ctcttttttg attccatatg aaatttaaag tagttttttc caattctgtg 78360

aagaaagtca atggtagctt gatggggata gcattgaatc tataaattac tttgggcaat 78420

atggccattt tcatgatatt gattcttcct acccatgagc atggaatgtt tttccatttg 78480

tttgtgtcct ctcttatttc cttgagcagt ggtttgtagt tctttttgaa gaggtctttc 78540

acatcccttg taagttgtat tcctagttat tttattctct ttgtagcaat tgtgaatggg 78600

agttcactca tgatttggct cactgtttgc ctgttattgg cgtataggaa tgcttctgat 78660

ttttgcacat taattttgta tcctgagagt ttgctgaagt tgcttatcag tgtgaggagt 78720

ttttgggctg agatgatggg gttttctaaa tatacagtca tgtcatctgc agagacaatt 78780

tgacatcctc ttttcctaat tgaataccct ttatttcttt ctcttgcctg attgccctgg 78840

ccagaacttc caatactatg ttgaatagga gtgcggagag acagcatcct tgtcttgtgc 78900

tggttttcaa agggaatgct tccagttttt gtccattcag tatgatattg tctgtgggtt 78960

tgtcataaat agttcttatt attttgagat acattccatc aatacctagt tcattgagag 79020

tttttagcat gaagggctgt tgaattttgt caaaggcctt ttctgcatct attcaggtta 79080

tcatgtggtt ttcatcatta gttctgttta tgtgatggat tacgtttatt catttgccta 79140

tgttgaacca gccttgcatc ccaggaataa agccagcttg attgtggtgg ataagctttt 79200

tgatgtgctg ctggattccg tttgccagta ttttattgag gattttcaca tcgatgttta 79260

tcagggatat tggcctaaaa ttttcttttt tttgttgtgt ctctgccagg ttttggtatc 79320

agaatgatgc tggcctcata aaatgagtta gggagtattc cctctttttc tactgtttgg 79380

aacagtttca gaaggaatgg taccagctcc tctttgtacc tctggtagaa tgtggctgtg 79440

aatccgtctg gtcctggact ttttttgatt ggtaggctat taattactgc ctcaatttca 79500

gaactggttt ttggtctatt cagggatttc acttcttcct ggtttagtgt tgggaaggtg 79560

tatgtgtcca ggaatttatc catttcttct agattttcta gtttatttgc acagaggtat 79620

ttatagtatt ctctgatgat agtttatatt tctgtgggat tggtggtgat atctccttta 79680

tcatttttta ttgcatctat ttgattcttc tctcttttct tctttattag tctggctagc 79740

agtctatcta ttttgttgat cttttcaaaa aaccagctcc tggattcatt gatttttttg 79800

aagagttttt catgtctctg tctccttcag tgcttctctg atcttagtta cttcttgtct 79860

tctgctagct tttgaatttg tttgctcttg cttctctaat tcttttaatt gtgatgttag 79920

ggtgtcaatt ttagatcttt ccagctttgt gatgtgggca tttagtgcta taaatttcct 79980

tctacacact gctataaatg tgtcccagag attctggtac attgtgtgtt ttttctcatt 80040

ggtttcaaag aacatcttta tttctgcctt cattttgtta tttacccagt agtcactcag 80100

gagcagattg ttcagtttcc atgtagttgt gcagttttga gtgagtttct taatcctgag 80160

ttctaatttg atttcactgt ggtctgagag acagtttgtt atgatttcca ttcttttgca 80220

tttgctgagg agtgttttac ttacaattat gtgatcaatt ttagagtatg tgtgatgtag 80280

tcctgagaag aatgtatatt ctgttgattt ggggtggaca gttctgtaga tgtctatagg 80340

tcccacttgg tccagagctg agttcaagtc ctggatatcc ttgttaattt tctgtctcgc 80400

tgatctgtct aatattgaca gtggggtgtt aaagtctccc actattattg tgtgggagtc 80460

taagtctctt tgtaggtctc taagaactga ctttataaat ctggatgctt ctgtattggg 80520

tgcatatata tttaggatag ttagctcttc ttgttgcatt tatcctttta tcattatgta 80580

atgcccttct ttgtctcttt tgatctttgt tggtttaaag tctgttttat cagagactag 80640

gattgcaacc cctgcttttt tttttctttc cattttcttg gtaaatcttc cttcatccct 80700

ttattttgtg tgtgtttttg cacatgagat gggtctcctg aatacagcac tactgatggg 80760

tcttattctt tatccaattt gccagtctgt gtcttttaat tggggcattt agcccattta 80820

cttttttttt ttttttgaga tggagtttca cttttgttgc ccagactgga gtgcaatggc 80880

acgatcttgg ctccctgcaa cctctgcctc ccaggtgcaa gggattctcc tgcctcagcc 80940

tccctagtag ctgggattac aggcatgtgc caccatgcct ggctaatttt gtatttttag 81000

tagagatgga gtttctccat gttggtcagg taggtctcaa actcctgacc tcaggtgatc 81060

cgcccacctt ggcctcccaa agtgctggga ttacaggcat gagccaccgt gcccagccta 81120

gtccatttac atttaaggtt aatattgtta tctgtgagtt tgatcttgtc attatgatgt 81180

tagctggtta ttttgcccat tagttgatgc ggtttcttca tagtgttaat ggtctttaca 81240

atttggtatg tttttgcagt ggctggtact ggttcctttc catgtttagt gcttccttca 81300

ggagctcttg taaggcaggc ctggtggtga cagaatctct cagcatttgc ttgtctggaa 81360

aggattttat ttctccttcg cttatgaagc ttagtttggc tggatatgaa attctaggtt 81420

gaaaattctt tcctttaaga atgttgaaca ttgaccccca ctgtcttctg gcttgtgggg 81480

tttctgctga gacatctgct tttagtctga tgggcttccc tttgtaggta accagacctt 81540

tttctctggc tgcccttaac attttttcct tcatttcaac gtcggtgtat ctgatgagtt 81600

gtgtcttagg gttcctgttc tccaggaata tctttgtggt gttctctata tttcctgaat 81660

ttaaagtttg gcctgttttg ctaggttggg gaagttctcc tggataatat cctgaaatgt 81720

gttttacaac ttggttccat tcttcttgtc acttttaggt acaccgttca aacatagatt 81780

tggtcttttc acatattccc atatttcttg gaggctttgt ttgattcttt tcattctttt 81840

ttctctgatc ttgtcttctt gctttatttc attgagttaa tcttcaatct ctggtattct 81900

ttcttccact tgattgactc agctattgat acttgtgtat gcttcacgaa gttcttgtgc 81960

tgtgtttttc agcaccatca ggtcatttat gttcttctct accctggtta ttctagttag 82020

caattcgtct ttttttcaag gttcttagct tccttgcatt gggttagaac atgctgcttt 82080

aactcagagg agtttgttat tacccacctt ctaaagtcta cttctgtcaa ttcatcaaac 82140

tcattctcca tccagttttg ttcccttgct ggcgaggagt tgtgatcctt tggaggggaa 82200

gaggcattct ggtttttgga aatttcagct tttttgtgct ggtttctccc aatcaagcca 82260

gtggatctta gcttgctggg ctctgtgggg gtgggaccca ctgagccagg caccggaggg 82320

aatctcctga tctgctggtt gtgaagaccg tggggaaagc acagtatctg ggccagagta 82380

tactgttcct ccccgtacag tctgtcatgg cttcttttga ctaggaaagg gaaatccccc 82440

agccccttgt gcttcctggg tgagacgatg ccccaccctg cttcagcccc ccctccatga 82500

gctgtaccca ctatccaacc agtcccaatg ggatgaactt ggtacctcag atggaaatgc 82560

agaaataacc cgccttctgc gttgatctct ctgggagctg cagaccagac ctgttcctat 82620

tcagccacct tgccagctct ccagcatgct gatttttcac gctgatttag catgaaaatc 82680

tggcatagta ttttcttggt atttaattaa tttttgttct actttggtta gcagtcttat 82740

aaaccagtta gtctttttat taaagtttta gggattctta ccgagtctaa atgatacgaa 82800

tttaaagtta ttagaaacct gtattcaaga gtgcttttta gcatcctttt tatcctttca 82860

tgaactttct aaaagatacc atattctagg attttccgtg tttgtgaagt tttcagaaat 82920

tgcattagca ttaagtaatt aacttaatgt aatgacttta aacagtgata tttaaaaaca 82980

caattcacaa ggaaatgtgg ttatctctgt ggtctgtgat aaattaacat aataacctta 83040

attatgattg aaaggatata ctcagacatt agaattttag aaatcctata caattttgga 83100

acatatatta atattattta acctgaagaa gattaaacat tgtttttatt ttgacaatcc 83160

catgtaacta aacatgtcag ataatcctat ttacctctcc tttggatgct ccaagggccc 83220

tctgtagcat ccaaaatttg ggggttagaa agacaatttt gaagctgaaa tttcattttg 83280

ggaagcctat aaaatatgtt aaaggtttaa aatacttgat ataatgctta accagtttga 83340

ccatgaggtg aaattcttgt aaacattttg taacccttta caaatttttg ttaaaaagca 83400

gatcagtgct ctaagaaaaa catgttgtgc ttttatttca attttaattt atagaaaaac 83460

ttagcaatac ccctttatct ttagccaatg tccacacaga atttcttttt ataagattaa 83520

cttttcataa accttccaca atgtattcaa gcctttagct ttattttaat ctaatttaaa 83580

acaatatttt aaccctctaa cctagacaaa aatttacatt cctgtgcctt cttataacct 83640

tttagtaaaa acgcatttta gtttcctgac acaccttgca tgtaaatcta ttttcagtag 83700

tctggattac atgttgtaat ggtaactgtt agcaactttt aattttggtg cctaaatttc 83760

ctttcatgta tcctttcatg acttatgcag accatctatg acatgcttag actttctgac 83820

ttgtcctaat catccctctt tttaagcaac cagttatttt actttaggac aagaatttac 83880

catacaactt tctttcttat ataaaatcta ttttctttat aatctttttt gcatagctag 83940

gggagcatgg ctaattccac atgtccccag gccttattga gaatctaatg cctccaagat 84000

aggtaaattg aacaattttc aaaagtcaaa gcagtttatg acctgaaagc attagtaaac 84060

ctaatatctg acctgtctaa tttagacaaa atgtctttat tttaccaata atcgttaaag 84120

ccatttttat ttcccaaaga ttactaaagt tacttgaact aagacattac agtttttatt 84180

ttttcttcca aaagtgtttt atctaagcac ttatttttct ttaagccaat tacttagagt 84240

tcttttatat gaacatcaca cacacaacac atatataact acacagagaa caagatccag 84300

tagttgtagg atttttcatt tgccagtttc ttcattggat tactgacctt ggggtggagc 84360

ccatcaagaa atggctagga aaacatgcag tttctggggt ctaataagca ggcacagctg 84420

gaaggcaaaa tggatttcga gagagatcta tttgctttta attcttgagg ttccatgagg 84480

aaaacagagg gttttttcca aaacaggatc agtggcacct tctttatttt tcccaaggag 84540

tcctaggcta tcagaagtta tcttagggcc tctcatgtgt gccttaagag tggcaagaca 84600

aaatggagaa aaataattca gtcgactgag aagaaaaaac ctttttcagc aaaacaagat 84660

ccacaaaaag gaaagacata aatgtggcca ggcgcagtgg ctcatgcctg taattccagc 84720

actttgggag gccgagctag gcagatcacg aggtcagcag atcgagacca tcctggctaa 84780

cacggtgaaa ccccgtctct actaaaaata caacaaatta gccaggtgtg gtggcaggca 84840

cctgtagtcc cagctacttg ggaggctgag gcaggagaat ggtgtgaacc caggaggtgg 84900

agcttgcagt gagctgagat catgccactg cactccagcc tgggtgacag agcgagactc 84960

tgtctcaaaa aaaaaaaaaa aggaaaagac ataaaggcct tttaaatata cctatagctt 85020

ggatatccac ttttaattaa gctgactctt aactatagtg ctctttaaaa aatcctttta 85080

aatctcttgt tacccaactt tagccatgcc aagtggcctg tatttctggc ttttgaactt 85140

tacctcccag gtgctcagag aaaggaaaat tcaagacaat tcatggaagg gaagagaatc 85200

agcaaatgat aaagttccca caggtatcaa accagaaagg actcattcct taacccagga 85260

attgaaccca ggccaccact gtgaaagtaa aaaactttag ctactgagct acagtactgg 85320

gtagtctcca ttgtgcttcc cagaagggct ctaaagtact taattttgag cttgcaaaag 85380

cttttaacta ctcaacttaa tttttagagc taactgtgac atgaacccta aaattcctgt 85440

tcccttgaag gcagagacca agaaaaagta ccaccacgtg gttacaaagt caagctccca 85500

agaatgtaaa acaagatgga gacctcatcc agtttttttt taatgcactt cagtgtgttg 85560

ttgttcattt ggaatgttcc attgtaagtt atctttagta aaattttgct atttctgtaa 85620

gactttgctg cctcccaagc ctaatgtata aactggaagg aactctgctt ttcagaaatt 85680

aaggatctca tttttaccta aaatattggc tttgctttca ggttcccttg atgaacttag 85740

ccaatgattt ttcctaccta agtgtgcaag aaaaatgaaa caaaggtgta gaaaaccaaa 85800

aatccctgtg aatttccaaa tgccaaattt tacaacccct gcaatactac catttactac 85860

cagtttcttt ctgacctagt caaatctaag aggcctctaa ttggatccta gcctgttaat 85920

tactgatcaa atccaatcct ggacccagtc cagtttctgt tgtgacttct gaacccagtt 85980

tggaacagga atttgctcaa agaaacttgg agagttcaaa acacaaatct gtggagctct 86040

gaaatccaaa agagaacttg ccatgatccc cagccactct gagagatcaa aggacacaag 86100

taggtccaac aggttccttg cttgttcact cagtgctcct gggggtcgtt agaaggtcta 86160

attcatatcc cgcttctgac accatctagt aaaagaaaaa cttcagccaa tttaaaggag 86220

tttaattgag caatgaacaa tttgggaatt tggcagcaga atcacagcag attcagagaa 86280

actctaggga tgccttgtgg tcagaacaaa tttatagaca aaaaaaaaaa aaaagagaga 86340

agtgacatac agaaattggc agtgatgtac agaaacagct ggttggttac aggttggcat 86400

ttgtcttatt tgaacacagt ttgaacattt agcagtctat gagtggttga agtatggtcc 86460

actgggattg gccaagactc agttactgtt acaggcacat actcctaagt caggttttca 86520

ctcttgtctg cctgttaagt taggttacag ttcatccaca gggattcaaa tatagaggta 86580

tgaagtcctt ctcaggccat atttagtttg ctttaacact tgaattccac ccaaacaaat 86640

cagcttgaat cagtgtgaag ggaagtgtga caatttttaa atttgtcaat tccttaccaa 86700

ttgttttagt gttttagcat tcagccccaa agtcccttac tcttggcccc cttcagtttt 86760

ttccctcctg tgatggtatt agtaaagatc tatggttagt tatttaaaat gagactttga 86820

gaaaagcaag acccttggat ttctaaattt tactgatgca ttgagtattt ctaagctgct 86880

cgatagatta gagttgtttg gtgtggcagt tccccagtgt gtccagttgc tcacaaattt 86940

tgacttgaat gttctttgcc gaattggcac tgagtttctc cttcttgcca tcatttgctt 87000

catgaaataa tctttctttc gtttacattt ataatcaagt gcagtagaaa gattttaaat 87060

gagctattat aaagtctact aatgatttct tatctacata ggttttttgc tcagaactta 87120

atatttcaaa atttaaatta cacattaata aacatattcc taataccctt gtaaggaagg 87180

caaactaata cggaatttta tttgaggctg ttttaaaata tacttgatta tgaagtccct 87240

tgaaatattt taatgttcaa attaataccc cataaaaaaa tcaatatttg tgttattatt 87300

taaacatgcc ctagtgtaaa agtttaagta acactaagtt cataactaag aatagcctaa 87360

aaactaattt tcagttcata acaaggaatt agggcaattc tttgctcatt taattattta 87420

aataagtact ctttttaaaa agataaaaca agacataatt ccaatatatt tattttacat 87480

ttgaacagat acttttattt aatacaacta ttcattatct ataaaagcct tagttctgaa 87540

acagaagtga gcaacagaaa gcaaaatatt ttatttattt atttatttat tattattttt 87600

tgagacagag tctcgctcta tcatccatac tggagtgcag tggtgcaatc tcggctcact 87660

gcaacctcca tctcctgggt tcaagtgatt ctcatgcctc agcctcccaa gtagctagga 87720

atacaggcac acaccaccat ttccaactaa tttttatatt tttggtggag acgggatttc 87780

accatgttgg ccaggctgct cttgagctct tggcctcaag tgatctgcct gtctttgcct 87840

cccaaagtgt tgggattaca ggtgtgagcc aacatgcccg gcccaaaaca ttttaaatat 87900

aataaaacca aaagtgggta aatatatata catatatata tatatattct cactgtatca 87960

gtgtactgta taaaaacttt actccaatag cctgaattta ctaatttggt ctaattttaa 88020

atacagaaga gagtgtgaat tagtaaacaa aacaacaaca aaaaaaatgg aatcataaaa 88080

cattgtataa agatgagtaa agaaataata tttaccctaa agtccaaggt caaattgggc 88140

tagcaaagtt cactcagcca ttttggttgt tatttagtac aggtcctttg agaaccaatt 88200

taaacaaata agtctgatcc gttgctaaca ggatccattg ttaatatgta aaaaggtgtc 88260

tgagatcagt ttagtctctt ctaagttctt ggtacaacct ttctgcccaa accaatttgt 88320

actgtcaatg cagagagtgt ttttaaacac aatggcagtg ctaatagata gcttggggga 88380

aaaagtggga tgtttatgac ttctaccttt tctttccaaa gccccacccc actgatcctt 88440

aaaataagca catggtctag gtagaaacca gttctcacta tgccacacaa ctgcaagaca 88500

tgttccccag cctccttctc catccgcatc aagaaagttt ggagctgtca ccatcattac 88560

aaccctattt gaaataatta caaaatctag actcagaaca gcctaaattt tagggcttta 88620

ttatataaca ttctcttttt aaatatgcgg tagttacggt caccttggaa agttctacaa 88680

aatatccctt aagttttttg aactttccca catgggaatc ttctggttat gagagtttgc 88740

tctatttaat atgtgtacgg tttcactgct agggtggttc tcccacttat cttgaatcta 88800

gtgtgagtgt tttgaaatgt ttctgctcat tgaaaagaag cagagcaata gagatgagag 88860

gaaaatctga aaagataatg acacaatttc ccacttaatt ttcattaagt aagagatgaa 88920

aactttagcc tcggcatcag gaagtttgat ttctttaatt aatttttttt ttgagtcagg 88980

gtctcactct gttgcccaga gtgagtgcag tggcatggtc acagctcact acagccttga 89040

cctcccaggc tcaagcgatc tttccacctc agcctcccaa gtagctggga ccacaggcat 89100

gcaccaccac acccagctaa ttttttaata ttctgtagag acagggtctt gctatgttgc 89160

ccaggctggt ctcgaactcc tggactcaag caatcctccc acctcagcct cccaaagtgc 89220

tgggattaca ggcatgagcc actgtgcccg acctaggaaa tttgattttt aatatacatt 89280

ttattctagt tgacttccta atctcctata tgattgcctg cttcttctaa cgtgtcattt 89340

ttgtttttta ggattaaaag aaggatctta ttgttaatac caaagtggct ttatgaagat 89400

attcctaata tgaaaaacag caatgttgtg aaaatgctac aggtaaccta acatcatcca 89460

acaaaaactg agtggcaatt gagaccaaga gtgcagtcta ataattagaa tagaatttcc 89520

attcaaataa ataaatgtgc acacatatta acataattat atagacatgt gtaaagacaa 89580

aactgtacct gtttcaaatc atacttaaaa gagatgagac aatagagttg ggggaaagaa 89640

aacagagaag aaataaagaa acaaacctgt tcaaaagaga aaagtgaagt ggaagattgt 89700

gggccttgtg cacgagtcac ctggtgacca aacaatggta gatgtcttca ggaacaaagg 89760

gagtagggaa gaaaagctca atgaaatgag cctcaaagat tcaaagggct tctgtttcac 89820

atgagatgga gagccaggtg gagcccaaga agggcatcat tttactcttt aaacccagtg 89880

gaatttgaga aaagcaatgt gagagaactg tgaacaatag tgtcacaggg gtgggctagg 89940

tttccattct ggcaaagaga aggccacaca ccaggaagcc cctgagggta cagggacatt 90000

actgattata aaggagggaa ggaacaagct atgtgtgttc ctgataaccc ctggccctcg 90060

ggattggctg tcaaggggct caaaacccag tccaagggac aaacacatca tccaagcctt 90120

gcaatgcagt gatgtaagtg caatgataga aatgagccca aacatgtgat gggagtggaa 90180

aggaaacact gaaaggagag gagctgctca ggagaagcag ggagaattca aagtcagggg 90240

gacccctggg ggttagaact gcatgttggg taagaagatg aggtgtttgt aaaacaaata 90300

gaagtggctc tgtttcaagc tctggtaagc ctattagcta actctttccc caacctcatg 90360

tcatctgaac aaagggtttc taggctaaaa ataaaatact ttttaaaagt tcaaaaacaa 90420

ctggtcaaca gaatagagtc tgagttctgt aacacaagac ttctgtgatc tgatccactc 90480

accattccag ctttactcca gccactcctc acgtcaagat ttttgatgaa gcaataccca 90540

atgtgctgtt cttacccaaa cctggcaagc tccctaagga ggctggggct gaagaaaatt 90600

ggaatgacac ttcatgtctg tttattcaag atctccttct ttgctaaact cctttcctat 90660

tctgggctcc tggaattaga ggcaaacctt agggcttccc tcagataagg ttttgttttc 90720

tgcccgagag actaaggaat gcttagtcgg cttgggtggc ttaaacaaca aacatttatt 90780

tgtcacagtg ctggaggctg ggaagtccat gatcaaggag ccagtcaatt cacttgctag 90840

tgagggctgt cttgtggaca gctgccttct cacatggcag aggaagagat catctctttc 90900

ctgtctcttc ttcactaatc ccattcatga aggctccgcc ctcatgacct aattaccacc 90960

caaagtctcc actttcaaat aacatcacat tggcttcaat gtgagggggg ctggggagag 91020

ggcacacaaa cgtttagtcc gtaacagggg gctataatat tataattgtg ttgggtgaca 91080

gtttgcccag gaaggccagc catctgttgg aaggcctcca gtccatttat tgcccagaaa 91140

agtcatcagg ctaaaacctt cacccaccct ccttatcatc acaaacatgt ctgaattgtg 91200

agtagcttta tttatttatt tgttgttttt tgttttgaga cagagtcttg ctctgtcacc 91260

caggctagag tgcagtggca tgatctcagc tcactgcaac ctccgcctcc cgggctcaag 91320

caattctcct gtctcagcct cctgagtagc tgggattaca ggcttgcgct actacgcctg 91380

gctgattttt gtatttttag tagagacagg gtttcaccat gttggccagg ctggtcttga 91440

actcctgatc tcaagtgatc tgcctgcctt ggcctcaaag tgctgggatt acaggcctta 91500

tccaccttta gaacctcggc atcaagcatt tgtcacacac tcattctcgg tatgtgagca 91560

aaataggatt ctccccaacc tttttttatt taagcaacaa agtattttct tgtaacaaga 91620

aatattaata ttaatattct tgcctcagaa gaccaacttt taaaaggccc taaaattaag 91680

taataagtaa taagttaata tataagactc aacttttctc tttaaaactt ttaaaccaag 91740

aaatgtgctt aagaagtgga ttatttttac tattcctttt cttcagtctt ctcatgcaaa 91800

tttaataaag atttggaagc aagcaggata acaaaagaca aaaggaggaa atcatatggg 91860

aagataaaca atatgagaaa atggagggag aaaggaaagt tgaaagaaag caaaattcct 91920

atcctcattc aattatccag ttggttcttt taaatgtctt ttgcaaaata aaatgatgtc 91980

tttttttcct aggaaaatag tgaacttatg aataataatt ccagtgagca ggtcctatat 92040

gttgatccca tgattacaga gataaaagaa atcttcatcc cagaacacaa gcctacagac 92100

tacaagaagg agaatacagg acccctggag acaagagact acccgcaaaa ctcgctattc 92160

gacaatacta cagttgtata tattcctgat ctcaacactg gatataaacc ccaaatttca 92220

aattttctgc ctgagggaag ccatctcagc aataataatg aaattacttc cttaacactt 92280

aaaccaccag ttgattcctt agactcagga aataatccca ggttacaaaa gcatcctaat 92340

tttgcttttt ctgtttcaag tgtgaattca ctaagcaaca caatatttct tggagaatta 92400

agcctcatat taaatcaagg agaatgcagt tctcctgaca tacaaaactc agtagaggag 92460

gaaaccacca tgcttttgga aaatgattca cccagtgaaa ctattccaga acagaccctg 92520

cttcctgatg aatttgtctc ctgtttgggg atcgtgaatg aggagttgcc atctattaat 92580

acttattttc cacaaaatat tttggaaagc cacttcaata ggatttcact cttggaaaag 92640

tagagctgtg tggtcaaaat caatatgaga aagctgcctt gcaatctgaa cttgggtttt 92700

ccctgcaata gaaattgaat tctgcctctt tttgaaaaaa atgtattcac atacaaatct 92760

tcacatggac acatgttttc atttcccttg gataaatacc taggtagggg attgctgggc 92820

catatgataa gcatatgttt cagttctacc aatcttgttt ccagagtagt gacatttctg 92880

tgctcctacc atcaccatgt aagaattccc gggagctcca tgccttttta attttagcca 92940

ttcttctgcc tcatttctta aaattagaga attaaggtcc cgaaggtgga acatgcttca 93000

tggtcacaca tacaggcaca aaaacagcat tatgtggacg cctcatgtat tttttataga 93060

gtcaactatt tcctctttat tttccctcat tgaaagatgc aaaacagctc tctattgtgt 93120

acagaaaggg taaataatgc aaaatacctg gtagtaaaat aaatgctgaa aattttcctt 93180

taaaatagaa tcattaggcc aggcgtggtg gctcatgctt gtaatcccag cactttggta 93240

ggctgaggta ggtggatcac ctgaggtcag gagttcgagt ccagcctggc caatatgctg 93300

aaaccctgtc tctactaaaa ttacaaaaat tagccggcca tggtggcagg tgcttgtaat 93360

cccagctact tgggaggctg aggcaggaga atcacttgaa ccaggaaggc agaggttgca 93420

ctgagctgag attgtgccac tgcactccag cctgggcaac aagagcaaaa ctctgtctgg 93480

aaaaaaaaaa aaaa 93494

<210> 3

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 3

atagaacaac agctcggatt 20

<210> 4

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 4

acaacaacta cacgtccatc 20

<210> 5

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 5

cccttaagca ctgccgacca 20

<210> 6

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 6

tgtgtcattt atgaatactc 20

<210> 7

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 7

accatctgaa gagcacataa 20

<210> 8

<211> 20

<212> DNA

<213> mouse (Mus musculus)

<400> 8

atctccactg actcactgca 20

<210> 9

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 9

auagaacaac agcucggauu 20

<210> 10

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 10

acaacaacua cacguccauc 20

<210> 11

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 11

cccuuaagca cugccgacca 20

<210> 12

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 12

ugugucauuu augaauacuc 20

<210> 13

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 13

accaucugaa gagcacauaa 20

<210> 14

<211> 20

<212> RNA

<213> mouse (Mus musculus)

<400> 14

aucuccacug acucacugca 20

<210> 15

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 15

gtgcagtaca tagaagcatg 20

<210> 16

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 16

acaacaacta cacgtccatc 20

<210> 17

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 17

ctacatagac acaaaatacg 20

<210> 18

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 18

accagctgaa gagtatgtaa 20

<210> 19

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 19

ccctttacat actcttcagc 20

<210> 20

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 20

acttcatcag gaatatctgg 20

<210> 21

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 21

gugcaguaca uagaagcaug 20

<210> 22

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 22

acaacaacua cacguccauc 20

<210> 23

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 23

cuacauagac acaaaauacg 20

<210> 24

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 24

accagcugaa gaguauguaa 20

<210> 25

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 25

cccuuuacau acucuucagc 20

<210> 26

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 26

acuucaucag gaauaucugg 20

<210> 27

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 27

tgtcaattct ttctttgatt 20

<210> 28

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 28

tttaacagat cattccgaac 20

<210> 29

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 29

ttaacagatc attccgaact 20

<210> 30

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 30

cagatcattc cgaactgggt 20

<210> 31

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 31

ctgcaaaaac ctacccagtt 20

<210> 32

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 32

ugucaauucu uucuuugauu 20

<210> 33

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 33

uuuaacagau cauuccgaac 20

<210> 34

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 34

uuaacagauc auuccgaacu 20

<210> 35

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 35

cagaucauuc cgaacugggu 20

<210> 36

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 36

cugcaaaaac cuacccaguu 20

<210> 37

<211> 178

<212> PRT

<213> human (Homo sapiens)

<400> 37

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<210> 38

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 38

gaaccaagac ccagacatca 20

<210> 39

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 39

caaggcgcat gtgaactccc 20

<210> 40

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 40

aaggcgcatg tgaactccct 20

<210> 41

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 41

aggcgcatgt gaactccctg 20

<210> 42

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 42

ggcgcatgtg aactccctgg 20

<210> 43

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 43

gggagaacct gaagaccctc 20

<210> 44

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 44

gcctcagcct gagggtcttc 20

<210> 45

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 45

gggtcttcag gttctccccc 20

<210> 46

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 46

ggtcttcagg ttctccccca 20

<210> 47

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 47

gaaccaagac ccagacauca 20

<210> 48

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 48

caaggcgcau gugaacuccc 20

<210> 49

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 49

aaggcgcaug ugaacucccu 20

<210> 50

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 50

aggcgcaugu gaacucccug 20

<210> 51

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 51

ggcgcaugug aacucccugg 20

<210> 52

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 52

gggagaaccu gaagacccuc 20

<210> 53

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 53

gccucagccu gagggucuuc 20

<210> 54

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 54

gggucuucag guucuccccc 20

<210> 55

<211> 20

<212> RNA

<213> human (Homo sapiens)

<400> 55

ggucuucagg uucuccccca 20

Claims (30)

1. A method of treating a subject having an autoimmune disorder, the method comprising

(a) Reducing the amount of one or more genetic variants associated with susceptibility to the autoimmune disorder ("one or more susceptibility genetic variants") in one or more cells of the subject; and/or

(b) Increasing the amount of one or more genetic variants ("one or more protective genetic variants") that protect the autoimmune disorder in one or more cells of the subject.

2. The method of claim 1, comprising reducing the amount of susceptibility genetic variants in one or more immune cells and/or one or more hematopoietic stem cells of the subject and increasing the amount of protective genetic variants in one or more immune cells and/or one or more hematopoietic stem cells of the subject.

3. A method of treating a subject having an autoimmune disorder, the method comprising

(a) Reducing the number of cells in the subject having one or more genetic variants associated with susceptibility to the autoimmune disorder; and/or

(b) Increasing the number of cells in the subject that have one or more genetic variants that protect against the autoimmune disorder.

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

5. The method of claim 2 or 4, wherein the immune cells comprise one or more of leukocytes, phagocytes, macrophages, neutrophils, dendritic cells, innate lymphoid cells, eosinophils, basophils, natural killer cells, B cells, and T cells.

6. The method of any one of claims 1-5, comprising administering to the subject

Immune cells and/or hematopoietic stem cells comprising the protective genetic variant; and/or

Immune cells and/or hematopoietic stem cells containing the protective genetic variant and not containing the susceptibility genetic variant.

7. The method of any one of claims 1-6, wherein the proportion of protective protein variant to susceptible protein variant in the subject is increased.

8. The method of any one of claims 1-7, further comprising obtaining immune cells and/or hematopoietic stem cells from a first subject,

altering the obtained immune cells and/or hematopoietic stem cells to reduce the amount of the susceptibility genetic variant and/or to increase the amount of the protective genetic variant, and

administering the altered immune cells and/or hematopoietic stem cells to a subject in need of treatment.

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

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

11. The method of claim 9 or 10, wherein the altered immune cells and/or hematopoietic stem cells are administered intravenously or via bone marrow transplantation.

12. The method of claim 11, further comprising depleting at least a portion of hematopoietic stem cells in the subject prior to administering the immune cells and/or hematopoietic stem cells.

13. The method of claim 12, wherein the ablating comprises administering chemotherapy or radiation to the subject; administering an anti-c-Kit monoclonal antibody to the subject; and/or administering a CD47 blocker to the subject.

14. The method of any one of claims 1-13, comprising administering a genetic modifier to the subject, wherein the genetic modifier (a) reduces the amount of the susceptibility genetic variant in one or more cells in the subject, and/or (b) increases the amount of the protective genetic variant in one or more cells in the subject.

15. The method of claim 14, wherein the genetic modification agent comprises a nuclease.

16. The method of claim 15, wherein the nuclease is (1) a class 2 regularly interspaced clustered short palindromic repeats (CRISPR) -associated nuclease, (2) a Zinc Finger Nuclease (ZFN), (3) a transcription activator-like effector nuclease (TALEN), or (4) a meganuclease.

17. The method of claim 16, wherein the nuclease comprises Casl, CaslB, Cas, CaslO, Cpf, Cyl, Csy, Csel, Cse, Ccll, Csc, Csa, Csn, Csm, Cmrl, Cmr, Cbl, Csb, Csxl, Csfl, Csf, or Csf.

18. The method of claim 16, wherein the nuclease comprises Cas9 or Cpf 1.

19. A method of treating a subject having an autoimmune disorder, the method comprising editing DNA in immune cells and/or hematopoietic stem cells in the subject to:

(a) reducing the amount of one or more genetic variants associated with: (i) resistance to a particular drug used to treat the autoimmune disorder or (ii) a bacterial distribution in the gut of the subject that is associated with increased susceptibility to the autoimmune disorder; and/or

(b) Increasing the amount of one or more genetic variants associated with: (i) increased sensitivity to a particular drug used to treat the autoimmune disorder or (ii) bacterial distribution in the gut of a subject that protects against the autoimmune disorder.

20. The method of any one of claims 1-19, wherein the genetic variant is an IL23 variant, CARD variant, NOD/2 variant, PTPN variant, NADPH oxidase complex gene variant, TTC7 variant, XIAP variant, IL-10RA variant, IL-10RB variant, RPL variant, CPAMD variant, PRG variant, hetr variant, ATG16L variant, TNFsf variant, MHCII variant, ELF variant, HLA-DB 01:03 variant, HLA-BTNL variant, ARPC variant, IL12 variant, STAT variant, IRGM variant, IRF variant, TYK variant, STAT variant, IFNGR variant, ringr variant, RIPK variant, LRRK variant, C13orf variant, ECM variant, NKX-3 variant, TNF JAK variant, tpt variant, NUDT variant, LOC 44mt variant, prlgm variant, clgi variant, CLCA variant, cldm variant, RPL variant, and/hci variant, 2q24.1 variant, LY75 variant, or a combination thereof.

21. The method of any one of claims 1-20, wherein the autoimmune disorder comprises inflammatory bowel disease, wherein the protective genetic variant encodes one or more of a R381Q mutation, a G149R mutation, and a V362I mutation in IL23R protein.

22. The method of claim 21, wherein the protective genetic variant comprises a G to a mutation at rs 11209026.

23. The method of any one of claims 1-22, wherein the susceptibility genetic variant and the protective genetic variant are determined based on one or more of:

(a) a phenotype of one or more family members, sequencing of a set of genes in one or more family members, sequencing of a full exome in one or more family members, and/or sequencing of a full genome of one or more family members;

(b) computer modeling of cellular signaling and/or immune system responses;

(c) machine modeling of mutations affecting phenotype, such as using linear and/or non-linear regression models, neural networks;

(d) data describing gene expression and/or gene signaling; and

(e) animal models (e.g., pigs).

24. An isolated immune cell or hematopoietic stem cell in which cellular DNA has been modified by gene editing to (a) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and/or (b) increasing the amount of one or more genetic variants that protect against the autoimmune disorder.

25. 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) reduce the amount of one or more genetic variants associated with susceptibility to an autoimmune disorder; and (b) increasing the amount of one or more genetic variants that protect against the autoimmune disorder.

26. 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 within the genomic DNA of a cell that encodes a genetic variant associated with susceptibility to an autoimmune disorder, and (iii) a DNA repair template that encodes a genetic variant that is unrelated to susceptibility to an autoimmune disorder.

27. 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 within the genomic DNA of a cell that encodes a genetic variant that does not protect against an autoimmune disorder, and (iii) a DNA repair template that encodes the genetic variant that protects against an autoimmune disorder at the location of the target sequence.

28. A composition, comprising:

(a) (ii) a guide RNA or nucleic acid encoding the guide RNA that hybridizes to a target sequence within the genomic DNA of a cell that encodes a genetic variant associated with susceptibility to an autoimmune disorder, and (iii) a DNA repair template that encodes a genetic variant not associated with susceptibility to an autoimmune disorder; and

(b) (ii) a guide RNA or a nucleic acid encoding the guide RNA that hybridizes to a target sequence within the genomic DNA of a cell, the target sequence encoding a genetic variant that does not protect against an autoimmune disorder, and (iii) a DNA repair template encoding the genetic variant that protects against an autoimmune disorder at the location of the target sequence.

29. The composition of any one of claims 26-28, comprising two or more guide RNAs, wherein the guide RNAs collectively hybridize to more than one target sequence.

30. The composition of any one of claims 26-29, wherein the genetic variant is an IL23 variant, CARD variant, NOD/2 variant, PTPN variant, NADPH oxidase complex gene variant, TTC7 variant, XIAP variant, IL-10RA variant, IL-10RB variant, RPL variant, CPAMD variant, PRG variant, hetr variant, ATG16L variant, TNFsf variant, MHCII variant, ELF variant, HLA-DB 01:03 variant, HLA-BTNL variant, ARPC variant, IL12 variant, STAT variant, IRGM variant, IRF variant, TYK variant, STAT variant, IFNGR variant, ringr variant, RIPK variant, LRRK variant, C13orf variant, ECM variant, NKX-3 variant, TNF JAK variant, tpt variant, NUDT variant, LOC 44mt variant, prlgm variant, clgi variant, CLCA variant, cldm variant, RPL variant, and/hca variant, 2q24.1 variant, or LY75 variant, or a combination thereof.