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WO 2019/071054 Al 11 April 2019 (11.04.2019) W 1P O PCT

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WO 2019/071054 Al 11 April 2019 (11.04.2019) W 1P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/071054 Al 11 April 2019 (11.04.2019) W 1P O PCT (51) International Patent Classification: man [US/US]; C/o One Baylor Plaza, Houston, Texas C12N 15/09 (2006.01) C12Q 1/68 (2018.01) 77030 (US). HUANG, Su-Chen [US/US]; C/o One Baylor C12N 15/113 (2010.01) G06F 19/18 (201 1.01) Plaza, Houston, Texas 77030 (US). (21) International Application Number: (72) Inventor: LANDER, Eric S.; c/o 415 Main Street, Cam¬ PCT/US20 18/054476 bridge, Massachusetts 02142 (US). (22) International Filing Date: (74) Agent: SCHER, Michael B. et al.; Johnson, Marcou & 04 October 2018 (04. 10.2018) Isaacs, LLC, P.O. Box 691, Hoschton, Georgia 30548 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (26) Publication Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/568,306 04 October 2017 (04. 10.2017) U S 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, (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 415 Main Street, Cambridge, Massachusetts 02142 (US). MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, BAYLOR COLLEGE OF MEDICINE [US/US]; One OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, Baylor Plaza, Houston, Texas 77030 (US). SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (72) Inventors; and TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: RAO, Suhas S.P. [US/US]; C/o One Baylor (84) Designated States (unless otherwise indicated, for every Plaza, Houston, Texas 77030 (US). AIDEN, Erez Lieber- kind of regional protection available): ARIPO (BW, GH, (54) Title: METHODS AND COMPOSITIONS FOR ALTERING FUNCTION AND STRUCTURE OF CHROMATIN LOOPS AND/ OR DOMAINS VS1 (57) Abstract: Chromatin 3D stucture modulating agents in the context of the present invention are intended to interfere or manipulate the function of loop anchor motifs, such as CTCF motifs. In certain example embodiments, the present invention may block formation of all or essentially all loop anchor or chromatin domains or block formation of a loop anchor or chromatin domain at a targeted genomic location. For instance, the chromatin 3D structure modulating agent may bind a target region and mask a loop anchor motif, thereby preventing a loop anchor or chromatin domain from forming. The chromatin 3D structure modulating agent may bind a target region and cause a loop anchor of chromatin domain to form. [Continued on nextpage] W O 2019/071054 A l Illlll II lllll lllll lllll llll III III lllll lllll lllll lllll lllll llll llll llll llll 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)) — with sequence listing part of description (Rule 5.2(a)) METHODS AND COMPOSITIONS FOR ALTERING FUNCTION AND STRUCTURE OF CHROMATIN LOOPS AND/OR DOMAINS CROSS-REFERENCE TO RELATED APPLICTIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/568,306, filed October 4, 2017. The entire contents of the above-identified application are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under Grant Nos. PHY-1427654 granted by the National Science Foundation, OD008540, HG006193, HL130010 andHG009375 granted by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (BROD_2910WP_ST25.txt"; Size is 4 Kilobytes and it was created on September 27, 2018) is herein incorporated by reference in its entirety. TECHNICAL FIELD [0004] The present invention is in the field of genetic engineering and medicine. The present invention provides methods and tools for altering chromatin four-dimensional (4D) structure in a cell, in particular chromatin loop formation and structure over time. The present invention allows the altering the transcriptional activity of chromatin domains or genomic loci, including such domains and loci associated with a disease, such as cancer or a genetic disease, through use of such methods and tools. The present invention provides methods of treatment comprising altering chromatin 3D structure or gene expression within a chromatin domain. The present invention further provides methods of modulating chromatin loop formation to thereby interfere with higher-order chromatin structure, and ultimately control gene expression. BACKGROUND [0005] It has been suggested that the three-dimensional structure of nucleic acids in a cell may be involved in complex biological regulation, for example compartmentalizing the nucleus and bringing widely separated functional elements into close spatial proximity. Understanding how nucleic acids interact, and perhaps more importantly how this interaction, or lack thereof, regulates cellular processes, presents a new frontier of exploration. For example, understanding chromosomal folding and the patterns therein can provide insight into the complex relationships between chromatin structure, gene activity, and the functional state of the cell. Adding ribonucleic acids (RNAs) into the mix adds a further complexity. [0006] Typically, deoxyribonucleic acid (DNA) is viewed as a linear molecule, with little attention paid to the three-dimensional organization. However, chromosomes are not rigid, and while the linear distance between two genomic loci need may be vast, when folded, the special distance may be small. For example, while regions of chromosomal DNA may be separated by many megabases, they can also can be immediately adjacent in 3-dimensional space. Much the same way a protein can fold to bring sequence elements together to form an active site, from the standpoint of gene regulation, long-range interactions between genomic loci may for the same sort of active centers. For example, gene enhancers, silencers, and insulator elements might function across vast genomic distances. [0007] The existence of long-range interactions complicates efforts to understand the pathways that regulate cellular processes, because the interacting regulatory elements could lie at a great genomic distance from a target gene, even on another chromosome. In the case of oncogenes and other disease-associated genes, identification of long-range genetic regulators would be of great use in identifying the genomic variants responsible for the disease state and the process by which the disease state is brought about. [0008] The roughly two meters of DNA in the human genome is intricately packaged to form the chromatin and chromosomes in each cell nucleus. In addition to its structural role, this organization has critical regulatory functions. In particular, the formation of loops in the human genome plays an essential role in regulating genes. Applicants herein demonstrate the ability to create reliable maps of these loops, using an in situ Hi-C method for three-dimensional genome sequencing, and to control the formation of such loops, thereby altering gene expression. Hi-C characterizes the three-dimensional configuration of the genome by determining the frequency of physical contact between all pairs of loci, genome-wide. [0009] In order to control the regulatory function of chromatin folding, it would be required to provide methods for altering chromatin three dimensional (3D) structure in a cell, to remove or otherwise modify existing chromatin loop structures, or to introduce new chromatin loop structures where their presence is required or beneficial, for instance, in the context of treatment of disease conditions, such as cancer or genetic disease. However, to date, no such methods exist. The present invention aims to provide essential methods and tools for altering chromatin three dimensional (3D) structure. [0010] In order to associate the dynamics of chromatin loop structure to cellular processes in health and disease, the chromatin three dimensional (3D) structure from a large number of cells in different stages of development, from diseased and healthy subjects, and from a wide variety of cellular lineages and biological species need to be analysed and their genomes sequenced. Such studies are hampered by costs. There is therefore a need for further improvements in methods for de novo assembly of whole genomes and genomic fragments. The present invention aims to provide such improved methods. [0011] Further, while existing methods for assessing chromatin three dimensional (3D) structure are very suitable for indicating that two loci are spatially co-localized in the nucleus, it may be expected that there are multiple loci spatially co-localized in a living cell. Yet, methods that can indicate simultaneous co-localization of more than 2, such as up to 10 or more different loci are not available. The present invention aims to provide such methods. [0012] Many studies have shown that the insulator protein CTCF and the ring-shaped cohesin complex colocalize on chromatin (Wendt et al., 2008) and lie at the anchors of loops (Rao et al., 2014; Splinter et al., 2006) and the boundaries of contact domains (also called "topologically constrained domains", "topologically associated domains", or "physical domains") (Dixon et al., 2012; Lieberman-Aiden et al., 2009; Nora et al., 2012; Rao et al., 2014).
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