OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR/CAS9 WITH TRIPLE GUIDE SEQUENCES

摘要

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 12 July 2018 (12.07.2018) WIPO I PCT omit VIII °nolo VIII ois (10) International Publication Number WO 2018/129296 Al (51) International Patent Classification: C12N 15/113 (2010.01) Cl 2N 9/22 (2006.01) C12N 15/864 (2006.01) (21) International Application Number: PCT/US2018/012558 (22) International Filing Date: 05 January 2018 (05.01.2018) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/442,606 05 January 2017 (05.01.2017) US 62/544,449 11 August 2017 (11.08.2017) US 62/596,298 08 December 2017 (08.12.2017) US (71) Applicant: THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM [US/US]; 210 West 7th Street, Austin, TX 78701 (US). (72) Inventors: AMOASII, Leonela; 8610 Southwestern Blvd., Apt. 415, Dallas, TX 75206 (US). OLSON, Eric; 3219 Southwestern Blvd., University Park, TX 75225 (US). (74) Agent: HIGHLANDER, Steven, L.; Parker Highlander PLLC, 1120 S. Capital Of Texas Highway, Blvd. One, Suite 200, Austin, TX 78746 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 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)) = (54) Title: OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR/CAS9 WITH TRIPLE GUIDE SEQUENCES A AAV9-Cas9 polyA SpCas9 B s RNA-51-SA2 sgRNA-51-SA2 sgRNA-51-SA2 AAV9-sg RNA K.CKaa FIGS. 9A-B (57) : CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Here, using three promoters to drive expression of the same DMD guide RNA, a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a deletion in exon 50, and in a AEx50-MD Dog. W O 20 18/ 129 296 Al