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WO 2018/119354 Al 28 June 2018 (28.06.2018) W ! P O P C T

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WO 2018/119354 Al 28 June 2018 (28.06.2018) W ! P O P C T (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 2018/119354 Al 28 June 2018 (28.06.2018) W ! P O P C T (51) International Patent Classification: KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, C12N 9/22 (2006.01) C12N 15/10 (2006.01) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, C12N 9/75 (2006.01) 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, (21) International Application Number: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. PCT/US20 17/068 105 (84) Designated States (unless otherwise indicated, for every (22) International Filing Date: kind of regional protection available): ARIPO (BW, GH, 22 December 2017 (22.12.2017) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (25) Filing Language: English UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (26) Publication Language: English EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (30) Priority Data: MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 62/438,869 23 December 2016 (23.12.2016) US TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (71) Applicant: PRESIDENT AND FELLOWS OF HAR¬ VARD COLLEGE [US/US]; 17 Quincy Street, Cam Published: bridge, MA 02138 (US). — with international search report (Art. 21(3)) (72) Inventors: MAIANTI, Juan, Pablo; 278 Endicott Avenue, — with sequence listing part of description (Rule 5.2(a)) #2, Revere, MA 0215 1 (US). LIU, David, R.; 3 Whitman Circle, Lexington, MA 02420 (US). (74) Agent: BAKER, C , Hunter; Wolf, Greenfield & Sacks, P.C., 600 Atlantic Avenue, Boston, MA 02210-2206 (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, (54) Title: GENE EDITING OF PCSK9 E32K D129G S127R GOF R46L (higher A53V R125H R104C E54A LDL-C) A68T Ί PreproPCSK9 R46L V 1 14A LOF A53V G106R (lower A68T AR97 ΐ E57K 93C LDL-C) T77I Ala68fsLeu82X N-glycosylation site Figure A (57) Abstract: Provided herein are systems, compositions, and methods of introducing loss-of- function mutations in to protein factors involved in the LDL-R-mediated cholesterol clearance pathway, e.g., PCSK9, APOC3, LDL-R, or IDOL. Loss-of-function mutations may be introduced using a CRISPR/Cas9-based nucleobase editor described in. Further provided herein are compositions and methods of treating conditions related to high circulating cholesterol levels. GENE EDITING OF PCSK9 RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 62/438,869, filed December 23, 2016, which is incorporated herein by reference. GOVERNMENT SUPPORT [0002] This invention was made with government support under grant number GM065865, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND OF THE INVENTION [0003] The liver protein Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a secreted, globular, auto-activating serine protease that acts as a protein-binding adaptor within endosomal vesicles to bridge a pH-dependent interaction with the low-density lipoprotein receptor (LDL-R) during endocytosis of LDL particles, preventing recycling of the LDL-R to the cell surface and leading to reduction of LDL-cholesterol clearance. Blocking or inhibiting the function of PCSK9 to boost LDL-R-mediated clearance of LDL cholesterol has been of significant interest in the pharmaceutical industry. However, current methods of generating PCSK9 protective variants and loss-of-function mutants in vivo have been ineffective due to the large number of cells that need to be modified to modulate cholesterol levels. Other concerns involve off-target effects, genome instability, or oncogenic modifications that may be caused by genome editing. SUMMARY OF THE INVENTION [0004] Provided herein are systems, compositions, kits, and methods for modifying a polynucleotide (e.g., DNA) encoding a PCSK9 protein to produce loss-of-function PCSK9 variants. Also provided herein are systems, compositions, kits, and methods for modifying a polynucleotide (e.g., DNA) encoding a LDLR, IDOL, or APOC3/C5 protein to produce loss- of-function mutants. The methodology for producing the mutatns relies on CRISPR/Cas9- based base-editing technology. The precise targeting methods described herein are superior to previously proposed strategies that create random indels in the PCSK9 genomic locus or other loci described herein using engineered nucleases. The methods also have a more favorable safety profile, due to the low probability of off-target effects. Thus, the base editing methods described herein have low impact on genomic stability, including oncogene activation or tumor suppressor inactivation. In some embodiments, the loss-of-function variants (e.g., PCSK9, LDLR, IDOL, or APOC3/C5 variants) generated using the methods described herein have a cardioprotective function. In some embodiments, the loss-of-function variants (e.g., PCSK9, LDLR, IDOL, or APOC3/C5 variants) generated using the methods described herein reduce LDL levels. In some embodiments, the loss-of-function variants (e.g., PCSK9, LDLR, IDOL, or APOC3/C5 variants) generated using the methods described herein reduce LDL cholesterol levels. In some embodiments, the loss-of-function variants (e.g., PCSK9, LDLR, IDOL, or APOC3/C5 variants) generated using the methods described herein lower overall cholesterol levels. In some embodiments, the loss-of-function variants (e.g., PCSK9, LDLR, IDOL, or APOC3/C5 variants) generated using the methods described herein increase HDL levels. [0005] Some aspects of the present disclosure provide methods of editing a polynucleotide encoding a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) protein, the method comprising contacting the PCSK9-encoding polynucleotide with (i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain; and (ii) a guide nucleotide sequence targeting the fusion protein of (i) to a target cytosine (C) base in the PCSK9-encoding polynucleotide, wherein the contacting results in deamination of the target C base by the fusion protein, resulting in a cytosine (C) to thymine (T) change in the PCSK9-encoding polynucleotide. [0006] In some embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is selected from the group consisting of nuclease inactive Cas9 (dCas9) domains, nuclease inactive Cpfl domains, nuclease inactive Argonaute domains, and variants and combinations thereof. In some embodiments, the guide nucleotide sequence- programmable DNA-binding protein domain is a nuclease inactive Cas9 (dCas9) domain. In some embodiments, the amino acid sequence of the dCas9 domain comprises mutations corresponding to a D10A and/or H840A mutation in SEQ ID NO: 1. In some embodiments, a Cas9 nickase is used. In some embodiments, the amino acid sequence of the Cas9 nickase comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1, and wherein the dCas9 domain comprises a histidine at the position corresponding to amino acid 840 of SEQ ID NO: 1. [0007] In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises a nuclease inactive Cpfl (dCpfl) domain. In some embodiments, the dCpfl domain is from a species of Acidaminococcus or Lachnospiraceae. [0008] In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises a nuclease inactive Argonaute (dAgo) domain. In some embodiments, the dAgo domain is from Natronobacterium gregoryi (dNgAgo). [0009] As a set of non limiting examples, any of the fusion proteins described herein that include a Cas9 domain can use another guide nucleotide sequence-programmable DNA binding protein, such as CasX, CasY, Cpfl, C2cl, C2c2, C2c3, and Argonaute, in place of the Cas9 domain. These may be nuclease inactive variants of the proteins. Guide nucleotide sequence-programmable DNA binding protein include, without limitation, Cas9 (e.g., dCas9 and nCas9), saCas9 (e.g., saCas9d, saCas9n, saKKH Cas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, Argonaute, and any of suitable protein described herein. In some embodiments, the fusion protein described herein comprises a Gam protein, a guide nucleotide sequence- programmable DNA binding protein, and a cytidine deaminase domain. [0010] In some embodiments, the cytosine deaminase domain comprises an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase is selected from the group consisting of APOBEC 1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced deaminase (AID), and pmCDAl. In some embodiments, the cytosine deaminase comprises the amino acid sequence of any one of SEQ ID NOs: 271-292 and 303. [0011] In some embodiments, the fusion protein of (a) further comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the cytosine deaminase domain is fused to the N-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. In some embodiments, the UGI domain is fused to the C-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain. [0012] In some embodiments, the cytosine deaminase is fused to the guide nucleotide sequence-programmable DNA-binding protein domain via an optional linker.
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