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Dual O G to T*A and A*T to G*C Base Editor

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Dual O G to T*A and A*T to G*C Base Editor ( (51) International Patent Classification: (74) Agent: UJWAL, Rachna; Wilson Sonsini Goodrich & C12N 15/10 (2006.0 1) C12N 9/78 (2006.0 1) Rosati, 650 Page Mill Road, Palo Alto, California 94304 C12N 9/22 (2006.01) (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 19/04493 5 kind of national protection av ailable) . AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (22) International Filing Date: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 02 August 2019 (02.08.2019) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (25) Filing Language: English 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, (26) Publication Language: English MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (30) Priority Data: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 62/714,550 03 August 2018 (03.08.2018) US 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. (71) Applicant: BEAM THERAPEUTICS INC. [US/US]; 2nd Floor, 26 Landsdowne Street, Cambridge, Massachu¬ (84) Designated States (unless otherwise indicated, for every setts 02139 (US). kind of regional protection available) . ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (72) Inventors: GAUDELLI, Nicole; 2nd Floor, 26 Lands¬ UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, downe Street, Cambridge, Massachusetts 02139 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EVANS, John; 2nd Floor, 26 Landsdowne Street, Cam¬ EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, bridge, Massachusetts 02139 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (54) Title: MULTI-EFFECTOR NUCLEOBASE EDITORS AND METHODS OF USING SAME TO MODIFY A NUCLEIC ACID TARGET SEQUENCE FIG. 1 Dual O G to T*A and A*T to G*C base editor (57) Abstract: The invention features a multi-effector nucleobase editor capable of inducing changes at multiple different bases within a target nucleic acid and methods of using such editors. [Continued on next page] ||| ||||| ||||| ||||| |||| 11| ||| ||||| ||||| ||||| ||||| ||||| |||| limn nil nil nil Declarations under Rule 4.17: as to applicant's entitlement to apply for and be granted a patent (Rule 4.17(H)) as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(iii)) 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)) MULTI-EFFECTOR NUCLEOBASE EDITORS AND METHODS OF USING SAME TO MODIFY A NUCLEIC ACID TARGET SEQUENCE CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application Number 62/714,550, filed on August 3, 2018, the entire contents of which are hereby incorporated by reference herein. BACKGROUND Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases. Currently available base editors include cytidine base editors (e.g., BE4) that convert target C G to Τ·Α and adenine base editors (e.g., ABE7. 10) that Α Τ convert target · to G*C . There is a need in the art for base editors capable of inducing novel types of modifications within a target sequence. SUMMARY OF THE DISCLOSURE As described below, the present invention features multi-effector nucleobase editors capable of inducing changes at multiple different bases within a target nucleic acid and methods of using such editors. In one aspect, the invention features a multi-effector nucleobase editor polypeptide comprising an adenosine deaminase, a cytidine deaminase, and/or a DNA glycosylase domain, where the aforementioned domains are fused to a polynucleotide binding domain, thereby forming a nucleobase editor capable of inducing changes at multiple different bases in a nucleic acid molecule. In one embodiment, the polypeptide further comprises one or more Nuclear Localization Signals (NLS). In another embodiment, the NLS is a bipartite NLS. In another embodiment, the polypeptide comprises an N-terminal NLS and a C- terminal NLS. In another embodiment, the polypeptide further comprises one or more Uracil DNA glycosylase inhibitors (UGI). In another embodiment, the adenosine deaminase is a TadA deaminase. In another embodiment, the TadA deaminase is a modified adenosine deaminase that does not occur in nature. In another embodiment, the polypeptide comprises two adenosine deaminases that are the same or different. In another embodiment, the two adenosine deaminases are capable of forming hetero or homodimers. In another embodiment, the adenosine deaminase domains are wild-type TadA and TadA7.lO. In another embodiment, the domain having nucleic acid sequence specific binding activity is a nucleic acid programmable DNA binding protein (napDNAbp). In another embodiment, the napDNAbp domain comprises a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9. In another embodiment, the napDNAbp is selected from the group consisting of Cas9, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, and Casl2i or active fragments thereof. In certain embodiments, the napDNAbp domain contains a Cas9 domain, a Casl2a domain, a Casl2b domain, a Casl2c domain, a Casl2d domain, a Casl2e domain, a Casl2f domain, a Casl2g domain, Casl2h domain, Casl2i domain, or an argonaute domain. In another embodiment, the napDNAbp domain comprises a catalytic domain capable of cleaving the reverse complement strand of the nucleic acid sequence. In another embodiment, the napDNAbp domain does not comprise a catalytic domain capable of cleaving the nucleic acid sequence. In another embodiment, the Cas9 is dCas9 or nCas9. In another embodiment, the cytidine deaminase is Petromyzon marinus cytosine deaminase 1 (pCDM), or Activation-induced cytidine deaminase (AICDA). In another embodiment, the polypeptide further comprises an abasic nucleobase editor. In another embodiment, UGI is derived from Bacillus subtilis bacteriophage PBS1 and inhibits human UDG activity. In another aspect, the invention features a multi-effector nucleobase editor polypeptide comprising one or more Nuclear Localization Signal (NLS), a napDNAbp, a Uracil DNA glycosylase inhibitor, an adenosine deaminase, and a cytidine deaminase. In one embodiment, the polypeptide comprises two NLS. In one embodiment, one NLS is a bipartite NLS. In another embodiment, the polypeptide comprises two Uracil DNA glycosylase inhibitors. In another embodiment, the polypeptide comprises two adenosine deaminases and a cytidine deaminase, or an abasic nucleobase editor and a cytidine deaminase, or an abasic nucleobase editor and an adenosine deaminase. In one aspect, the invention features a Multi-Effector Nucleobase Editor polypeptide comprising the following domains A-C, A-D, or A-E: NH2-[A-B-C]-C00H, NH2-[A-B-C-D]-COOH, or NH2-[A-B-C-D-E]-COOH wherein A and C or A, C, and E, each comprises one or more of the following: an adenosine deaminase domain or an active fragment thereof, a cytidine deaminase domain or an active fragment thereof, a DNA glycosylase domain or an active fragment thereof; and wherein B or B and D, each comprises one or more domains having nucleic acid sequence specific binding activity. In one embodiment, the Multi-Effector Nucleobase Editor polypeptide of the previous aspect contains: H 2-[A n-Bo-Cn]-COOH, H 2-[A n-Bo-Cn-D 0]-COOH, or H 2-[A n-Bo-Cp-Do-Eq]-COOH; wherein A and C or A, C, and E, each comprises one or more of the following: an adenosine deaminase domain or an active fragment thereof, a cytidine deaminase domain or an active fragment thereof, a DNA glycosylase domain or an active fragment thereof; and wherein n is an integer: 1, 2, 3, 4, or 5, wherein p is an integer: 0, 1, 2, 3, 4, or 5; wherein q is an integer 0, 1, 2, 3, 4, or 5; and wherein B or B and D each comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5. In one embodiment, the polypeptide contains one or more nuclear localization sequences. In one embodiment, the polypeptide contains at least one of said nuclear localization sequences is at the N-terminus or C-terminus. In one embodiment, the polypeptide contains the nuclear localization signal is a bipartite nuclear localization signal. In one embodiment, the polypeptide contains one or more domains linked by a linker. In one embodiment, the adenosine deaminase is a TadA deaminase. In one embodiment, the TadA is a modified adenosine deaminase that does not occur in nature. In another embodiment, the polypeptide comprises two adenosine deaminase domains that are the same or different. In one embodiment, the two adenosine deaminase domains are capable of forming hetero or homodimers. In one embodiment, the adenosine deaminase domains are wild-type TadA and TadA7. 10. In one embodiment, the polypeptide contains a domain having nucleic acid sequence specific binding activity is a nucleic acid programmable DNA binding protein (napDNAbp). In one embodiment, the napDNAbp domain comprises a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9.
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