METHODS OF SUBSTITUTING PATHOGENIC AMINO ACIDS USING PROGRAMMABLE BASE EDITOR SYSTEMS

1 .- 38 . (canceled)

39 . A method of producing a cell for treating sickle cell disease or beta thalassemia in a subject in need thereof, the method comprising:

contacting the cell with a base editor, or mRNA encoding the base editor, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain or a cytidine deaminase domain, and a guide RNA (gRNA);

binding the gRNA in conjunction with the base editor to a target nucleotide sequence in the cell located in a regulatory region of an HBG1 and/or HBG2 gene; and

editing a nucleobase of the target nucleotide sequence by deaminating the nucleobase upon the binding of the gRNA to the target nucleotide sequence, thereby producing the cell for treating the sickle cell anemia or beta thalassemia by changing the nucleobase to another nucleobase, wherein the nucleobase is located in c. −114˜−102 of the HBG1 and/or HBG2 gene, and/or wherein the target nucleotide sequence comprises a nucleotide sequence selected from

(SEQ ID NO: 238; gRNA1)

CTTGACCAATAGCCTTGACAAGG, 

(SEQ ID NO: 240; gRNA4)

GCTATTGGTCAAGGCAAGGC,

and

(SEQ ID NO: 245; gRNA45)

AAGTTTGCCTTGTCAAGGCTATTGGT.

40 . The method of claim 39 , wherein the polynucleotide-programmable nucleotide-binding domain is a Cas9 nickase.

41 . The method of claim 40 , wherein the Cas9 nickase comprises a D10A amino acid substitution referenced to SEQ ID NO: 47.

42 . The method of claim 39 , wherein the adenosine deaminase is a TadA deaminase comprising an amino acid sequence that is at least 85% identical to the following amino acid sequence:

SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR

VIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID

ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAG

SLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPR

QVENAQKKAQSSTD

(Positions 2 to 167 of SEQ ID NO: 152).

43 . The method of claim 39 , wherein the cell is a mammalian cell.

44 . The method of claim 43 , wherein the cell is a CD34 + cell.

45 . The method of claim 39 , wherein the gRNA comprises a nucleotide sequence selected from CUUGACCAAUAGCCUUGACA (SEQ ID NO: 176; gRNA1), GCUAUUGGUCAAGGCAAGGC (SEQ ID NO: 177; gRNA4), AAGUUUGCCUUGUCAAGGCU (SEQ ID NO: 223; gRNA45), GCCUUGACAAGGCAAACUUG (SEQ ID NO: 224), UUGACAAGGCAAACUUGACC (SEQ ID NO: 225), CAAGGCUAUUGGUCAAGGCA (SEQ ID NO: 209), CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 210), GUUUGCCUUGUCAAGGCUAU (SEQ ID NO: 211), UGGUCAAGUUUGCCUUGUCA (SEQ ID NO: 212), CUUGCCUUGACCAAUAGCCU (SEQ ID NO: 217), and UAGCCUUGACAAGGCAAACU (SEQ ID NO: 218).

46 . The method of claim 39 , wherein the first and last three bases of the gRNA are modified with 2′OMe.

47 . The method of claim 39 , wherein the mRNA encoding the base editor is N1MePseudoU modified mRNA.

48 . The method of claim 39 , wherein the subject comprises a β-globin (HBB) protein associated with sickle cell disease.

49 . The method of claim 48 , wherein the HBB protein associated with sickle cell disease has a valine at amino acid position 7 referenced to SEQ ID NO: 37.

50 . The method of claim 39 , wherein the editing alters a binding pattern of at least one protein to the promoter of the HBG1 and/or HBG2 gene.

51 . A method for treating sickle cell disease or beta thalassemia in a subject in need thereof, the method comprising administering to the subject the cell produced by the method of claim 39 .

52 . A method of producing a cell for treating sickle cell disease or beta thalassemia in a subject in need thereof, the method comprising:

contacting the cell with a base editor, or mRNA encoding the base editor, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain, and a guide RNA (gRNA);

binding the gRNA in conjunction with the base editor to a target nucleotide sequence in the cell located in a regulatory region of an HBG1 and/or HBG2 gene; and

editing a nucleobase of the target nucleotide sequence by deaminating the nucleobase upon the binding of the gRNA to the target nucleotide sequence, thereby producing the cell for treating the sickle cell anemia or beta thalassemia by changing the nucleobase to another nucleobase, wherein the nucleobase is located in c. −114˜−102 of the HBG1 and/or HBG2 gene, and/or wherein the target nucleotide sequence comprises a nucleotide sequence selected from CTTGACCAATAGCCTTGACAAGG (SEQ ID NO: 238; gRNA1), GCTATTGGTCAAGGCAAGGC (SEQ ID NO: 240; gRNA4), and AAGTTTGCCTTGTCAAGGCTATTGGT (SEQ ID NO: 245; gRNA45).

53 . The method of claim 52 , wherein the polynucleotide-programmable nucleotide-binding domain is a Cas9 nickase.

54 . The method of claim 52 , wherein the first and last three bases of the gRNA are modified with 2′OMe.

55 . The method of claim 52 , wherein the mRNA encoding the base editor is N1MePseudoU modified mRNA.

56 . A method of producing a cell for treating sickle cell disease or beta thalassemia in a subject in need thereof, the method comprising:

contacting the cell with a base editor, or mRNA encoding the base editor, wherein the base editor comprises a Cas9 nickase and an adenosine deaminase domain, and a guide RNA (gRNA);

binding the gRNA in conjunction with the base editor to a target nucleotide sequence in the cell located in a regulatory region of an HBG1 and/or HBG2 gene; and

editing a nucleobase of the target nucleotide sequence by deaminating the nucleobase upon the binding of the gRNA to the target nucleotide sequence, thereby producing the cell for treating the sickle cell anemia or beta thalassemia by changing the nucleobase to another nucleobase, wherein the gRNA comprises the nucleotide sequence CUUGACCAAUAGCCUUGACA (SEQ ID NO: 176).

57 . The method of claim 56 , wherein the first and last three bases of the gRNA are modified with 2′OMe.

58 . The method of claim 56 , wherein the mRNA encoding the base editor is N1MePseudoU modified mRNA.