USES OF ADENOSINE BASE EDITORS
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.
1 .- 64 . (canceled)
65 . A method for deaminating an adenosine (A) nucleobase in a sense or antisense strand of an HFE or F8 gene, the method comprising contacting the HFE or F8 gene with a base editor and a guide RNA (gRNA) bound to the base editor, wherein the gRNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the HFE or F8 gene.
66 .- 92 . (canceled)
93 . A method for deaminating an adenosine (A) nucleobase in a sense or antisense strand of an HBB gene, the method comprising contacting the HBB gene with a base editor and a guide RNA (gRNA) bound to the base editor, wherein the gRNA comprises a guide sequence that is complementary to a target nucleic acid sequence in the HBB gene.
94 . The method of claim 93 , wherein the HBB gene comprises a C to T mutation.
95 . The method of claim 94 , wherein deaminating an adenosine nucleobase complementary to the T corrects the C to T mutation.
96 . The method of claim 94 , wherein the HBB gene encodes a protein comprising a Glu to Val mutation or a Glu to Lys mutation.
97 . The method of claim 96 , wherein the Glu to Val mutation or the Glu to Lys mutation is at amino acid position 6 of the amino acid sequence
(SEQ ID NO: 340)
VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLST
PDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDP
ENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH
98 . The method of claim 97 , wherein deaminating an adenosine nucleobase complementary to the T corrects the Glu to Val mutation or the Glu to Lys mutation.
99 . The method of claim 93 , wherein the guide sequence comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleic acids that are 100% complementary to the target nucleic acid sequence of the HBB gene.
100 . The method of claim 93 , wherein the base editor nicks the target nucleic acid sequence.
101 . The method of claim 93 , wherein the target nucleic acid sequence in the HBB gene comprises:
(SEQ ID NO: 324)
5′-GTGCATCTGACTCCTGTGGAGAA-3′;
(SEQ ID NO: 325)
5′-GGTGCATCTGACTCCTGTGGAGA-3′;
(SEQ ID NO: 326)
5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′;
(SEQ ID NO: 327)
5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′;
(SEQ ID NO: 328)
5′-CCATGGTGCATCTGACTCCTGTGGAG-3′;
(SEQ ID NO: 329)
5′-CCATGGTGCATCTGACTCCTGTGGA-3′;
(SEQ ID NO: 330)
5′-CCATGGTGCATCTGACTCCTGTGG-3′;
(SEQ ID NO: 331)
5′-CCATGGTGCATCTGACTCCTGTG-3′;
(SEQ ID NO: 332)
5′-GCATCTGACTCCTGTGGAGAAGT-3′;
(SEQ ID NO: 333)
5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′;
(SEQ ID NO: 334)
5′-ACGGCAGACTTCTCCTTAGGAGT-3′;
(SEQ ID NO: 335)
5′-CCTGCCCAGGGCCTTACCACCAA-3′;
(SEQ ID NO: 336)
5′-ACCTGCCCAGGGCCTTACCACCA-3′;
or
(SEQ ID NO: 337)
5′-CCAACCTGCCCAGGGCCTTACCA-3′
102 . The method of claim 101 , wherein the target nucleic acid sequence comprises 5′-GTGCATCTGACTCCTGTGGAGAA-3′ (SEQ ID NO: 324),
wherein deamination of the A nucleobase that is complementary to the T at position 17 of SEQ ID NO: 324 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-GGTGCATCTGACTCCTGTGGAGA-3′ (SEQ ID NO: 325), wherein deamination of the A nucleobase that is complementary to the T at position 18 of SEQ ID NO: 325 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTGGAGAA-3′ (SEQ ID NO: 326), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTGGAGA-3′ (SEQ ID NO: 327), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTGGAG-3′ (SEQ ID NO: 328), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTGGA-3′ (SEQ ID NO: 329), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTGG-3′ (SEQ ID NO: 330), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCATGGTGCATCTGACTCCTGTG-3′ (SEQ ID NO: 331), wherein deamination of the A nucleobase that is complementary to the T at position 22 of SEQ ID NO: 331 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-GCATCTGACTCCTGTGGAGAAGT-3′ (SEQ ID NO: 332), wherein deamination of the A nucleobase that is complementary to the T at position 15 of SEQ ID NO: 332 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-ACCATGGTGCATCTGACTCCTGTGGAGA-3′ (SEQ ID NO: 333), wherein deamination of the A nucleobase that is complementary to the T at position 23 of SEQ ID NO: 333 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-ACGGCAGACTTCTCCTTAGGAGT-3′ (SEQ ID NO: 334), wherein deamination of the A nucleobase that is complementary to the T at position 17 of SEQ ID NO: 334 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-CCTGCCCAGGGCCTTACCACCAA-3′ (SEQ ID NO: 335), wherein deamination of the A nucleobase that is complementary to the T at position 15 of SEQ ID NO: 335 results in a T to C mutation in the target nucleic acid sequence;
the target nucleic acid sequence comprises 5′-ACCTGCCCAGGGCCTTACCACCA-3′ (SEQ ID NO: 336), wherein deamination of the A nucleobase that is complementary to the T at position 16 of SEQ ID NO: 336 results in a T to C mutation in the target nucleic acid sequence; and/or
the target nucleic acid sequence comprises 5′-CCAACCTGCCCAGGGCCTTACCA-3′ (SEQ ID NO: 337), wherein deamination of the A nucleobase that is complementary to the T at position 19 results in a T to C mutation in the target nucleic acid sequence.
103 .- 107 . (canceled)
108 . The method of claim 93 , wherein the guide sequence of the gRNA comprises at least 15 contiguous nucleotides of a sequence selected from the group consisting of:
(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′.
109 . The method of claim 108 , wherein the guide sequence of the gRNA further comprises a G at the 5′ end.
110 . The method of claim 93 , wherein deaminating the adenosine nucleobase in the HBB gene results in a T-A base pair in the HBB gene being mutated to a C-G base pair in the HBB gene.
111 . The method of claim 93 , wherein deaminating the adenosine nucleobase in the HBB gene results in correcting a sequence associated with sickle cell disease.
112 . The method of claim 93 , wherein deaminating the adenosine nucleobase in the HBB gene results in correcting a sequence associated with Hb C beta-thalassemia.
113 . The method of claim 93 , wherein deaminating the adenosine nucleobase in the HBB gene leads to an increase function of beta-globin protein transcribed from the HBB gene.
114 . The method of claim 93 , wherein deaminating the adenosine nucleobase in the HBB gene leads to an increase in beta-globin stability or half life.
115 . The method of claim 93 , wherein the HBB gene is in a cell.
116 . (canceled)
117 . The method of claim 115 , wherein the cell is in a subject and the subject has sickle cell disease, beta-thalassemia or Hb C beta-thalassemia (Hemoglobin C disease).
118 .- 123 . (canceled)
124 . The method of claim 94 , wherein the HBB gene encodes a protein comprising a Glu to Lys mutation.
125 . The method of claim 124 , wherein the Glu to Lys mutation is at amino acid position 26 of the amino acid sequence
(SEQ ID NO: 340)
VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDL
STPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKL
HVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHK
YH.
126 . The method of claim 125 , wherein deaminating an adenosine nucleobase complementary to the T corrects the Glu to Lys mutation.
127 . The method of claim 124 , wherein the guide sequence comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleic acids that are 100% complementary to the target nucleic acid sequence of the HBB gene.
128 .- 264 . (canceled)
265 . A guide RNA (sgRNA) comprising at least 15 contiguous nucleotides of the nucleic acid sequence
(SEQ ID NO: 289)
5′- ACUUCUCC A CAGGAGUCAGAUGC -3′;
266 .- 272 . (canceled)