US 2020/0399626 A1 · USES OF ADENOSINE BASE EDITORS

Abstract

The disclosure provides methods and compositions for treating blood diseases/disorders, such as sickle cell disease, hemochromatosis, hemophilia, and beta-thalassemia. For example the disclosure provides therapeutic guide RNAs that target the promotor of HBG1/2 to generate point mutations that increase expression of fetal hemoglobin. As another example, the disclosure provides therapeutic guide RNAs that target mutations in HBB, Factor VIII, and HFE to treat sickle cell disease, beta-thalassemia, hemophilia and hemochromatosis. The disclosure also provides fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA. In some embodiments, the fusion proteins are in complex with nucleic acids, such as guide RNAs (gRNAs), which target the fusion proteins to a DNA sequence (e.g., an HBG1 or HBG2 protmoter sequence, or an HFE, GBB, or F8 gene sequence). Such complexes may be useful for increasing expression of fetal hemoglobin or correcting a poing mutation (e.g., C282Y) in HFE.

Claims49

Dependent claims

A method for deaminating an adenosine (A) nucleobase in a sense or antisense strand of a promoter of an HBG1 or HBG2 gene, the method comprising contacting the promoter with a base editor and a guide RNA bound to the base editor, wherein the guide RNA (gRNA) comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter of at least one of the HBG1 and HBG2 gene.

A complex comprising (i) a fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) and (ii) an adenosine deaminase, and a guide RNA, wherein the guide RNA comprises a guide sequence comprising the nucleic acid sequence

(SEQ ID NO:
254)
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
255)
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
256)
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
257)
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
258)
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
259)
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
260)
5′-AUGCAAAUAUCUGUCUGAAACGG-3′;
(SEQ ID NO:
261)
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
262)
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
263)
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
264)
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
265)
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
266)
5′-UAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
267)
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′;
(SEQ ID NO:
268)
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′;
(SEQ ID NO:
269)
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′;
(SEQ ID NO:
270)
5′-CUUGACCAAUAGCCUUGACAAGG-3′;
(SEQ ID NO:
271)
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
272)
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
273)
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
274)
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
275)
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
276)
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
277)
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′;
(SEQ ID NO:
278)
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′;
(SEQ ID NO:
279)
5′-UUGACCAAUAGCCUUGACAAGGC-3′;
[[or]]
(SEQ ID NO:
280)
5′-UAGCCUUGACAAGGCAAACUUGA-3′;
(SEQ ID NO:
281)
5′-UUCUCCACAGGAGUCAGAUGCAC-3′;
(SEQ ID NO:
282)
5′-UCUCCACAGGAGUCAGAUGCACC-3′;
(SEQ ID NO:
283)
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
284)
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
285)
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
286)
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
287);
5′-CCACAGGAGUCAGAUGCACCAUGG-3′
(SEQ ID NO:
288)
5′-CACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
289)
5′-ACUUCUCCACAGGAGUCAGAUGC-3′;
(SEQ ID NO:
290)
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′;
(SEQ ID NO:
291)
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′;
(SEQ ID NO:
292)
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′;
(SEQ ID NO:
293)
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′;
(SEQ ID NO:
294)
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′;
(SEQ ID NO:
295)
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′;
(SEQ ID NO:
296)
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′.
(SEQ ID NO:
862)
5′-AUAUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
863)
5′-UAUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
864)
5′-AUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
865)
5′-UACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
866)
5′-ACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
867)
5′-CGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
868)
5′-GUACCAGGUGGAGCACCC-3′;
or
(SEQ ID NO:
869)
5′-UACCAGGUGGAGCACCC-3′.

152 more elements

A single guide RNA (sgRNA) comprising the nucleic acid sequence

(SEQ ID NO:
846)
5′-UCAUGUGGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
847)
5′-CAUGUGGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
848)
5′-AUGUGGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
849)
5′-UGUGGGGAAGGGGCCCCCAAG-3′.
(SEQ ID NO:
850)
5′-GUGGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
851)
5′-UGGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
852)
5′-GGGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
853)
5′-GGGAAGGGGCCCCCAAG-3′;
(SEQ ID NO:
862)
5′-AUAUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
863)
5′-UAUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
864)
5′-AUACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
865)
5′-UACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
866)
5′-ACGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
867)
5′-CGUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
868)
5′-GUACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
869)
5′-UACCAGGUGGAGCACCC-3′;
(SEQ ID NO:
254)
5′-GACAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
255)
5′-ACAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
256)
5′-CAGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
257)
5′-AGAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
258)
5′-GAUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
259)
5′-AUAUUUGCAUUGAGAUAGUGUGG-3′;
(SEQ ID NO:
260)
5′-AUGCAAAUAUCUGUCUGAAACGG-3′;
(SEQ ID NO:
261)
5′-GCAAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
262)
5′-CAAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
263)
5′-AAAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
264)
5′-AAUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
265)
5′-AUAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
266)
5′-UAUCUGUCUGAAACGGUCCCUGG-3′;
(SEQ ID NO:
267)
5′-AGAUAUUUGCAUUGAGAUAGUGU-3′;
(SEQ ID NO:
268)
5′-ACAGAUAUUUGCAUUGAGAUAGU-3′;
(SEQ ID NO:
269)
5′-GUGGGGAAGGGGCCCCCAAGAGG-3′;
(SEQ ID NO:
270)
5′-CUUGACCAAUAGCCUUGACAAGG-3′;
(SEQ ID NO:
271)
5′-CUUGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
272)
5′-UUGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
273)
5′-UGUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
274)
5′-GUCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
275)
5′-UCAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
276)
5′-CAAGGCUAUUGGUCAAGGCAAGG-3′;
(SEQ ID NO:
277)
5′-UUGUCAAGGCUAUUGGUCAAGGC-3′;
(SEQ ID NO:
278)
5′-CUUGUCAAGGCUAUUGGUCAAGG-3′;
(SEQ ID NO:
279)
5′-UUGACCAAUAGCCUUGACAAGGC-3′;
(SEQ ID NO:
280)
5′-UAGCCUUGACAAGGCAAACUUGA-3′;
(SEQ ID NO:
281)
5′-UUCUCCACAGGAGUCAGAUGCAC-3′;
(SEQ ID NO:
282)
5′-UCUCCACAGGAGUCAGAUGCACC-3′;
(SEQ ID NO:
283)
5′-UUCUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
284)
5′-UCUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
285)
5′-CUCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
286)
5′-UCCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
287)
5′-CCACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
288)
5′-CACAGGAGUCAGAUGCACCAUGG-3′;
(SEQ ID NO:
289)
5′-ACUUCUCCACAGGAGUCAGAUGC-3′;
(SEQ ID NO:
290)
5′-UCUCCACAGGAGUCAGAUGCACCAUGGU-3′;
(SEQ ID NO:
291)
5′-ACUCCUAAGGAGAAGUCUGCCGU-3′;
(SEQ ID NO:
292)
5′-UUGGUGGUAAGGCCCUGGGCAGG-3′;
(SEQ ID NO:
293)
5′-UGGUGGUAAGGCCCUGGGCAGGU-3′;
[[or]]
(SEQ ID NO:
294)
5′-UGGUAAGGCCCUGGGCAGGUUGG-3′;
(SEQ ID NO:
295)
5′-AGCAUUGUAUAUUCUCUGUGAGG-3′;
or
(SEQ ID NO:
296)
5′-AAAGCAUUGUAUAUUCUCUGUGA-3′.

176 more elements

Dependencies are inferred from the claim text.

Uses of adenosine base editors

Life of this patent

Uses of adenosine base editors

Life of this patent

Abstract

Claims49