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WO 2019/018440 Al 24 January 2019 (24.01.2019) W !P O PCT

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WO 2019/018440 Al 24 January 2019 (24.01.2019) W !P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2019/018440 Al 24 January 2019 (24.01.2019) W !P O PCT (51) International Patent Classification: CORPORATION [US/US]; 55 Fruit Street, Boston, Mass A61K 35/17 (2015.01) CI2N 5/071 (2010.01) achusetts 021 14 (US). A61P 1/00 (2006.01) G01N 33/50 (2006.01) (72) Inventors: BITON, Moshe; c/o 55 Fruit Street, Boston, (21) International Application Number: Massachusetts 021 14 (US). SHALEK, Alexander K.; c/ PCT/US2018/042554 o 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 (US). SMILLIE, Christopher; c/o 77 Massa (22) International Filing Date: chusetts Avenue, Cambridge, Massachusetts 02139 (US). 17 July 2018 (17.07.2018) HERBST, Rebecca H.; c/o 415 Main Street, Cam (25) Filing Language: English bridge, Massachusetts 02142 (US). REGEV, Aviv; c/o 415 Main Street, Cambridge, Massachusetts 02142 (US). OR- (26) Publication Language: English DOVAS-MONTANES, Jose; c/o 77 Massachusetts Av (30) Priority Data: enue, Cambridge, Massachusetts 02139 (US). XAVIER, 62/533,638 17 July 2017 (17.07.2017) US Ramnik; c/o 55 Fruit Street, Boston, Massachusetts 021 14 62/581,424 03 November 2017 (03 .11.20 17) US (US). 62/692,541 29 June 2018 (29.06.2018) US (74) Agent: SCHER, Michael B. et al; Johnson, Marcou & (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; Isaacs, LLC, P.O. Box 691, Hoschton, Georgia 30548 (US). 415 Main Street, Cambridge, Massachusetts 02142 (US). (81) Designated States (unless otherwise indicated, for every OF MASSACHUSETTS INSTITUTE TECHNOLOGY kind of national protection available): AE, AG, AL, AM, [US/US]; 77 Massachusetts Avenue, Cambridge, Mass AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, achusetts 02139 (US). THE GENERAL HOSPITAL (54) Title: CELL ATLAS OF THE HEALTHY AND ULCERATIVE COLITIS HUMAN COLON healthy UC ' - epithelial ' ·* ¾ ' i i -.'i' o FIG. 1 00 (57) Abstract: The present invention provides for a human cell atlas of the colon from healthy and diseased subjects. The atlas was o obtained by single sequencing of about 117,000 cells. The present invention discloses novel markers for cell types. Moreover, genes associated with disease are identified in the colon and colon specific cell types. The invention provides for diagnostic assays based on gene markers and cell composition, as well as target cell types that express genes associated with disease. Finally, disclosed are novel o cell types and methods of quantitating, detecting and isolating the cell types. o [Continued on nextpage] WO 2019/018440 Al llll II II 11III II I III II I II II II III II I II CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) — with sequence listing part of description (Rule 5.2(a)) CELL ATLAS OF THE HEALTHY AND ULCERATIVE COLITIS HUMAN COLON CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 62/533,638, filed July 17, 2017, 62/581,424, filed November 3, 2017 and 62/692,541, filed June 29, 2018. The entire contents of the above-identified applications are hereby fully incorporated herein by reference. STATEMENTREGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Grant Nos. OD020839, AI089992, CA217377, AI039671, All 18672, HG006193 and CA202820 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The subject matter disclosed herein is generally directed to a cell atlas of the human colon in healthy and disease states. The subject matter further relates to novel cell specific and disease specific markers. BACKGROUND [0004] Tissues function through the coordinated actions of diverse parenchymal, immune, and stromal cell types responding to local and distal cues. Breakdown in any cellular compartment can lead to disease, due to intrinsic cell dysfunction or through compensations by other cells to restore tissue homeostasis. This intricate interplay among cells makes it difficult to determine the causal cellular mechanisms that initiate or promote disease. In the past, measuring the contribution of each cellular compartment was hampered by the need to average bulk measurements across the entire tissue or to assess known cell subsets or selected genes a priori. [0005] The small intestinal mucosa is a complex system. The mucosa comprises multiple cell types involved in absorption, defense, secretion and more. These cell types are rapidly renewed from intestinal stem cells. The types of cells, their differentiation, and signals controlling differentiation and activation are poorly understood. The small intestinal mucosa also possesses a large and active immune system, poised to detect antigens and bacteria at the mucosal surface and to drive appropriate responses of tolerance or an active immune response. Finally, there is complex luminal milieu which comprises a combination of diverse microbial species and their products as well as derivative products of the diet. It is increasingly clear that a functional balance between the epithelium and the constituents within the lumen plays a central role in both maintaining the normal mucosa and the pathophysiology of many gastrointestinal disorders. Many disorders, such as irritable bowel disease, Crohn's disease, and food allergies, have proven difficult to treat. Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine, principally including Crohn's disease and ulcerative colitis, with other forms of IBD representing far fewer cases (e.g., collagenous colitis, lymphocytic colitis, diversion colitis, Behcet's disease and indeterminate colitis). Pathologically, Crohn's disease affects the full thickness of the bowel wall (e.g., transmural lesions) and can affect any part of the gastrointestinal tract, while ulcerative colitis is restricted to the mucosa (epithelial lining) of the colon and rectum. Some of the genetic factors predisposing one to IBD are known, as explored in Daniel B . Graham and Ramnik J . Xavier "From Genetics of Inflammatory Bowel Disease Towards Mechanistic Insights" TrendsImmunol. 2013 Aug; 34(8): 371-378 (incorporated herein). The manner in which these multiple factors interact remains unclear. [0006] A case in point is ulcerative colitis (UC), a major subtype of Inflammatory Bowel Disease (IBD), where inflammation begins in the rectum and extends proximally in a contiguous fashion. Left-sided disease is characterized by a sharp demarcation between inflamed and non- inflamed segments. Explaining the geographical restriction of UC remains a challenge. While endoscopic examination followed by histological analysis are the current standard of care, they fail to capture key aspects of the disease, including immune cell activation states, cell-specific cytokine profiles, and disease-related features of stromal cells, and do not distinguish the pathways associated with chronic inflammation from attempts at disease restitution. Furthermore, several genes mapped by genome-wide association studies (GWAS) as associated with disease risk suggests a critical role for the intestinal barrier in preventing IBD, with defects in autophagy, ER stress, oxidative stress, cell death, IL23R/Thl7 biology, immune tolerance, solute transport and perturbed innate and adaptive immune signaling [REF]. However, the cellular compartments and cell-cell interactions in which these GWAS genes and pathways act are often unknown. [0007] Single-cell RNA-Seq (scRNA-Seq) can help advance our understanding of complex disease by comprehensively mapping the cell types and states in the tissue, distinguishing changes in cell-intrinsic expression programs from cell-extrinsic changes in cell frequencies, and predicting how these connect through cell-cell interactions. When comparing healthy and disease tissue, this may help determine with precision: which cell populations are disrupted in disease? Which cell- intrinsic programs are changed? What cell-cell interactions affect cell recruitment during inflammation and disease resolution? What are the cells-of-action of disease risk genes? and, What are the effects of current therapies and how might therapeutic resistance arise? Unlike bulk profiling, which averages across cell types, scRNA-Seq can readily distinguish between cell- intrinsic changes in expression program and cell-extrinsic changes in cell frequencies and cell-cell interactions. Unlike most in situ methods, which require prior knowledge of the genes and proteins of interest, scRNA-Seq can in principle do so systematically across all cells and programs in the tissue ecosystem, including those that may not yet be known to present in the tissue. This approach has been demonstrated for tumors (see, e.g., Tirosh, et al., Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.
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