Adenosine deaminase base editors and methods of using same to modify a nucleobase in a target sequence

Abstract

The disclosure provides compositions comprising novel adenosine base editors (e.g., ABE8) that have increased efficiency and methods of using these adenosine deaminase variants for editing a target sequence.

1 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 of the following sequence and having at least 85% identity to the following sequence: 
       
         
           
                 
                 
               
                     
                   MSEVEFSHEYWMRHALTLAKRARDEREVPVGA 
                 
                     
                     
                 
                     
                   VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                 
                     
                     
                 
                     
                   QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIH 
                 
                     
                     
                 
                     
                   SRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
                 
                     
                     
                 
                     
                   RVEITEGILADECAALLCYFFRMPRQVFNAQK 
                 
                     
                     
                 
                     
                   KAQSST. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . The fusion protein of  claim 1 , wherein the adenosine deaminase variant comprises alterations at amino acid position 82 and 166. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The fusion protein of  claim 1 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, and Q154R. 
     
     
         7 - 54 . (canceled) 
     
     
         55 . A fusion protein comprising:
 an adenosine deaminase variant domain, wherein the adenosine deaminase variant domain comprises the amino acid sequence of:   
       
         
           
                 
                 
               
                     
                   MSEVEFSHEYWMRHALTLAKRARDEREVPVG 
                 
                     
                     
                 
                     
                   AVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                 
                     
                     
                 
                     
                   LRQGGLVMQNYRLIDATLYVTFEPCVMCAGA 
                 
                     
                     
                 
                     
                   MIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
                 
                     
                     
                 
                     
                   GMNHRVEITEGILADECAALLCYFFRMPRQV 
                 
                     
                     
                 
                     
                   FNAQKKAQSST, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       or a variant thereof, wherein the amino acid sequence comprises at least one alteration,
 and a Cas9 or a Cas12 polypeptide, wherein the adenosine deaminase variant domain is inserted within the Cas9 or the Cas12 polypeptide. 
 
     
     
         56 . The fusion protein of  claim 55 , wherein the adenosine deaminase variant domain comprises alterations at amino acid position 82 and/or 166. 
     
     
         57 . The fusion protein of  claim 55 , wherein the at least one alteration comprises: V82S, T166R, Y147T, Y147R, Q154S, Y123H, and/or Q154R. 
     
     
         58 . The fusion protein of  claim 55 , wherein the adenosine deaminase variant domain comprises one of the following combination of alterations: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     
     
         59 - 63 . (canceled) 
     
     
         64 . The fusion protein of  claim 55 , wherein the adenosine deaminase variant domain is inserted within a flexible loop, an alpha helix region, an unstructured portion, or a solvent accessible portion of the Cas9 or Cas12 polypeptide. 
     
     
         65 . The fusion protein of  claim 64 , wherein the flexible loop comprises a part of an alpha helix structure of the Cas9 or Cas12 polypeptide. 
     
     
         66 . The fusion protein of  claim 55 , wherein the adenosine deaminase variant domain is flanked by a N-terminal fragment and a C-terminal fragment of the Cas9 polypeptide. 
     
     
         67 . The fusion protein of  claim 65 , wherein the fusion protein comprises the structure:
 NH 2 -[N-terminal fragment of a Cas9]-[adenosine deaminase variant]-[C-terminal fragment of a Cas9]-COOH,   wherein each instance of “]-[” is an optional linker.   
     
     
         68 . The fusion protein of  claim 67 , wherein the N-terminal fragment or the C-terminal fragment of the Cas9 or Cas12 polypeptide binds a target polynucleotide sequence. 
     
     
         69 . The fusion protein of  claim 64 , wherein a C-terminus of the N terminal fragment or an N-terminus of the C terminal fragment comprises a part of a flexible loop of the Cas9 or the Cas12 polypeptide. 
     
     
         70 - 72 . (canceled) 
     
     
         73 . The fusion protein of  claim 69 , wherein:
 the N-terminal fragment or the C-terminal fragment comprises a RuvC domain;   the N-terminal fragment or the C-terminal fragment comprises a HNH domain;   neither the N-terminal fragment nor the C-terminal fragment comprises an HNH domain; or   neither the N-terminal fragment nor the C-terminal fragment comprises a RuvC domain.   
     
     
         74 - 79 . (canceled) 
     
     
         80 . The fusion protein of  claim 69 , wherein the adenosine deaminase variant domain is inserted within a flexible loop of the Cas9 polypeptide. 
     
     
         81 . The fusion protein of  claim 80 , wherein the flexible loop comprises a region selected from the group consisting of amino acid residues at positions 530-537, 569-579, 686-691, 768-793, 943-947, 1002-1040, 1052-1077, 1232-1248, and 1298-1300 as numbered in the Cas9 reference sequence, or corresponding amino acid positions thereof. 
     
     
         82 - 103 . (canceled) 
     
     
         104 . A fusion protein comprising the structure:
 NH 2 -[TadA*8]-[Cas9]-[cytidine deaminase]-COOH; or   NH 2 -[cytidine deaminase]-[Cas9]-[TadA*8]-COOH,   
       wherein each instance of “]-[” is an optional linker; or
 NH 2 -[Cas9(TadA*8)]-[cytidine deaminase]-COOH; 
 NH 2 -[cytidine deaminase]-[Cas9(TadA*8)]-COOH; 
 NH 2 -[Cas9(cytidine deaminase)]-[TadA*8]-COOH; or 
 NH 2 -[TadA*8]-[Cas9(cytidine deaminase)]-COOH, 
 
       wherein each instance of “]-[” is an optional linker; or
 NH 2 -[Cas12(adenosine deaminase)]-[cytidine deaminase]-COOH; 
 NH 2 -[cytidine deaminase]-[Cas12(adenosine deaminase)]-COON; 
 NH 2 -[Cas12(cytidine deaminase)]-[adenosine deaminase]-COOH; or 
 NH 2 -[adenosine deaminase]-[Cas12(cytidine deaminase)]-COOH, 
 
       wherein each instance of “]-[” is an optional linker 
     
     
         105 - 108 . (canceled) 
     
     
         109 . A polynucleotide encoding the fusion protein of  claim 1 . 
     
     
         110 . An expression vector comprising the polynucleotide of  claim 109 . 
     
     
         111 - 113 . (canceled) 
     
     
         114 . A cell comprising the fusion protein of  claim 1 . 
     
     
         115 . The cell of  claim 114 , wherein the cell is a bacterial cell, plant cell, insect cell, a human cell, or mammalian cell. 
     
     
         116 . A base editor comprising the fusion polypeptide of  claim 1  in a complex with one or more guide polynucleotides. 
     
     
         117 . A pharmaceutical composition comprising the fusion protein of  claim 1 , and a pharmaceutically acceptable excipient. 
     
     
         118 . A kit comprising the fusion protein of  claim 1 . 
     
     
         119 . A method for base editing comprising contacting a polynucleotide sequence with the fusion protein of  claim 1 , wherein the adenosine deaminase variant domain of the fusion protein deaminates a nucleobase in the polynucleotide, thereby editing the polynucleotide sequence. 
     
     
         120 . (canceled) 
     
     
         121 . A method of editing a target polynucleotide, the method comprising contacting the target polynucleotide with the base editor of  claim 116  to effect an A⋅T to G⋅C alteration in the target polynucleotide. 
     
     
         122 - 124 . (canceled) 
     
     
         125 . A method of treating a genetic defect in a subject, the method comprising administering to the subject a base editor comprising or consisting essentially of the fusion protein of  claim 1 , or a polynucleotide encoding said base editor and one or more guide polynucleotides that direct the base editor to deaminate a target nucleobase in a target nucleotide sequence of the subject, thereby treating the genetic defect. 
     
     
         126 . The method of  claim 125 , wherein the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   a) 
                 
                     
                   GACCUAGGCGAGGCAGUAGG; 
                 
                     
                     
                 
                     
                   b) 
                 
                     
                   CCAGUAUGGACACUGUCCAAA; 
                 
                     
                     
                 
                     
                   c) 
                 
                     
                   CAGUAUGGACACUGUCCAAA; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   d) 
                 
                     
                   AGUAUGGACACUGUCCAAAG. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         127 . The method of  claim 126  wherein the gRNA comprises the nucleic acid sequence: 
       
         
           
                 
                 
               
                     
                   GUUUUUGUACUCUCAAGAUUUAAGUAACUGU 
                 
                     
                     
                 
                     
                   ACAACGAAACUUACACAGUUACUUAAAUCUU 
                 
                     
                     
                 
                     
                   GCAGAAGCUACAAAGAUAAGGCUUCAUGCCG 
                 
                     
                     
                 
                     
                   AAAUCAACACCCUGUCAUUUUAUGGCAGGGU 
                 
                     
                     
                 
                     
                   G. 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         128 - 133 . (canceled) 
     
     
         134 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 and/or 166 of 
       
         
           
                 
                 
               
                     
                   MSEVEFSHEYWMRHALTLAKRARDEREVPVG 
                 
                     
                     
                 
                     
                   AVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                 
                     
                     
                 
                     
                   LRQGGLVMQNYRLIDATLYVTFEPCVMCAGA 
                 
                     
                     
                 
                     
                   MIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
                 
                     
                     
                 
                     
                   GMNHRVEITEGILADECAALLCYFFRMPRQV 
                 
                     
                     
                 
                     
                   FNAQKKAQSST, 
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       or an alteration corresponding to positions 82 and/or 166 in a variant of 
       
         
           
                 
                 
               
                     
                   MSEVEFSHEYWMRHALTLAKRARDEREVPVG 
                 
                     
                     
                 
                     
                   AVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                 
                     
                     
                 
                     
                   LRQGGLVMQNYRLIDATLYVTFEPCVMCAGA 
                 
                     
                     
                 
                     
                   MIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
                 
                     
                     
                 
                     
                   GMNHRVEITEGILADECAALLCYFFRMPRQV 
                 
                     
                     
                 
                     
                   FNAQKKAQSST.