Methods of editing single nucleotide polymorphism using programmable base editor systems

Abstract

Provided herein are compositions and methods of using base editors comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain in conjunction with a guide polynucleotide. Also provided herein are base editor systems for editing nucleobases of target nucleotide sequences.

1 . A method of editing a SERPINA1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 Anti-Trypsin Deficiency (A1AD), the method comprising contacting the SERPINA1 polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with A1AD. 
     
     
         2 . The method of  claim 1 , wherein the contacting is in a cell, a eukaryotic cell, a mammalian cell, or a human cell. 
     
     
         3 . The method of  claim 1 , wherein the cell is in vivo or ex vivo. 
     
     
         4 . The method of  claim 1 , wherein the A⋅T to G⋅C alteration at the SNP associated with A1AD changes a lysine to a glutamic acid in the alpha-1 Anti-Trypsin (A1AT) polypeptide; or
 wherein the SNP associated with A1AD results in expression of an A1AT polypeptide having a lysine at amino acid position 342; or 
 wherein the base editor correction replaces the lysine at position 342 with a glutamic acid. 
 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the polynucleotide programmable DNA binding domain is a modified  Streptococcus pyogenes  Cas9 (SpCas9), or variants thereof; or
 wherein the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity.   
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of claim  8 , wherein the modified SpCas9 comprises the amino acid substitution D1332A and one or more of D1135M, S1137Q, G1218K, E1219F, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof; or
 wherein the modified SpCas9 comprises amino acid substitutions D1135M, S1137Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof.   
     
     
         11 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant. 
     
     
         15 - 17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the adenosine deaminase is a TadA deaminase. 
     
     
         19 . The method of  claim 18 , wherein the TadA deaminase is TadA*7.10. 
     
     
         20 . The method of  claim 1 , wherein the one or more guide RNAs comprises a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence comprising the SNP associated with A1AD; or
 wherein the base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an SERPINA1 nucleic acid sequence comprising the SNP associated with A1AD.   
     
     
         21 . The method of  claim 1 , wherein the base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an SERPINA1 nucleic acid sequence comprising the SNP associated with A1AD. 
     
     
         22 . A cell produced by introducing into the cell, or a progenitor thereof:
 a base editor, a polynucleotide encoding the base editor, to the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of the single nucleotide polymorphism (SNP) associated with alpha-1 Anti-Trypsin Deficiency (A1AD).   
     
     
         23 . The cell of  claim 22 , wherein the cell produced is a hepatocyte; or wherein the cell or progenitor thereof is an embryonic stem cell, induced pluripotent stem cell, or a hepatocyte. 
     
     
         24 . (canceled) 
     
     
         25 . The cell of  claim 23 , wherein the hepatocyte expresses an A1AT polypeptide. 
     
     
         26 . The cell of  claim 22 , wherein the cell is from a subject having A1AD. 
     
     
         27 - 50 . (canceled) 
     
     
         51 . A method of treating alpha-1 Anti-Trypsin Deficiency (A1AD) in a subject, the method comprising: administering to a subject in need thereof a cell of  claim 22 ; or
 a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of the single nucleotide polymorphism (SNP) associated with A1AD.   
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 51 , comprising delivering the base editor, or polynucleotide encoding the base editor, and the one or more guide polynucleotides to a cell of the subject. 
     
     
         54 . The method of  claim 51 , wherein the cell is a hepatocyte or wherein the cell is a progenitor of a hepatocyte. 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 54 , wherein the cell expresses an A1AT polypeptide comprising a mutation. 
     
     
         57 - 74 . (canceled) 
     
     
         75 . A method of producing a hepatocyte, or progenitor thereof, the method comprising:
 (a) introducing into an induced pluripotent stem cell or hepatocyte progenitor comprising a single nucleotide polymorphism (SNP) associated with alpha-1 Anti-Trypsin Deficiency (A1AD),   a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with A1AD; and   (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor into hepatocyte.   
     
     
         76 . The method of  claim 75 , comprising differentiating the induced pluripotent stem cell into a hepatocyte or progenitor thereof. 
     
     
         77 . The method of  claim 75 , wherein the induced pluripotent stem cell of step (a) comprises an E342K mutation. 
     
     
         78 . The method of  claim 75 , wherein the hepatocyte progenitor is obtained from a subject having A1AD. 
     
     
         79 - 97 . (canceled) 
     
     
         98 . The method of  claim 75 , wherein the base editor and the one or more guide polynucleotides form a complex in the cell; or
 wherein the base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence comprising the SNP associated with A1AD.   
     
     
         99 . (canceled) 
     
     
         100 . A base editor comprising:
 (i)   
       MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAE IMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLM DVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSE TPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIG EGWNRAIGLHDPTAHAEIIVIALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKK AQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYK VPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSN EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFmqPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAkfLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNE QKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT NLGAPrAFKYFDTTIaRKeYrSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSE FESPKKKRK (SEQ ID NO: 39); and
 (ii) an adenosine deaminase domain. 
 
     
     
         101 . A guide RNA comprising a nucleic acid sequence selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   5′- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
                 
                     
                 
                   CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′; 
                 
                     
                 
                   (SEQ ID NO: 3) 
                 
                   5′-ACCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGU 
                 
                     
                 
                   UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU 
                 
                     
                 
                   GCUUUU-3′; 
                 
                     
                 
                   (SEQ ID NO: 4) 
                 
                   5′-CCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUU 
                 
                     
                 
                   AAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG 
                 
                     
                 
                   CUUUU-3′; 
                 
                     
                 
                   (SEQ ID NO: 5) 
                 
                   5′-CAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUA 
                 
                     
                 
                   AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC 
                 
                     
                 
                   UUUU-3′; 
                 
                     
                 
                   (SEQ ID NO: 6) 
                 
                   5′-AUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAA 
                 
                     
                 
                   AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU 
                 
                     
                 
                   UUU-3′; 
                 
                     
                 
                   (SEQ ID NO: 7) 
                 
                   5′-UCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAA 
                 
                     
                 
                   AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU 
                 
                     
                 
                   UU-3′; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 8) 
                 
                   5′-CGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA 
                 
                     
                 
                   UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU 
                 
                     
                 
                   U-3′. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         102 . A protein nucleic acid complex comprising the base editor of  claim 100  and a guide RNA. 
     
     
         103 . A base editor system for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, wherein the base editor system comprises:
 (a) a base editor comprising:
 (i) a polynucleotide-programmable DNA-binding domain, and 
 (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and 
   (b) a guide polynucleotide in conjunction with the polynucleotide-programmable DNA-binding domain, wherein the guide polynucleotide targets the base editor to a target polynucleotide sequence at least a portion of which is located in the gene or its reverse complement;   wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation listed in Table 3A or Table 3B.   
     
     
         104 . A method for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene comprising:
 contacting a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complement, with a base editor comprising:
 (i) a polynucleotide-programmable DNA-binding domain in conjunction with a guide polynucleotide that targets the base editor to the target polynucleotide sequence, at least a portion of which is located in the gene or its reverse complement, and 
 (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and 
   editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence,   wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation.   
     
     
         105 . A method for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene comprising:
 contacting a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complement, with a base editor comprising:
 (i) a polynucleotide-programmable DNA-binding domain in conjunction with a guide polynucleotide that targets the base editor to the target polynucleotide sequence, at least a portion of which is located in the gene or its reverse complement, and 
 (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and 
   editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence,   wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation listed in Table 3A or Table 3B.   
     
     
         106 . A method of treating a genetic disorder in a subject by correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, the method comprising:
 administering a base editor, or a polynucleotide encoding the base editor, to a subject in need thereof, wherein the base editor comprises:
 (i) a polynucleotide-programmable DNA-binding domain, and 
 (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and 
   administering a guide polynucleotide to the subject, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence at least a portion of which is located in the gene or its reverse complement; and   editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence,   wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation listed in Table 3A or Table 3B and treating the genetic disorder.   
     
     
         107 . A method of producing a cell, tissue, or organ for treating a genetic disorder in a subject in need thereof by correcting a pathogenic single nucleotide polymorphism (SNP) in a gene of the cell, tissue, or organ, the method comprising:
 contacting the cell, tissue, or organ with a base editor, wherein the base editor comprises:
 (i) a polynucleotide-programmable DNA-binding domain, and 
 (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and 
   contacting the cell, tissue, or organ with a guide polynucleotide, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence at least a portion of which is located in the gene or its reverse complement; and   editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence,   wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation listed in Table 3A or Table 3B and producing the cell, tissue, or organ for treating the genetic disorder.   
     
     
         108 - 136 . (canceled) 
     
     
         137 . A method of editing a glucose-6-phosphatase (G6PC) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disorder Type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with GSD1a. 
     
     
         138 - 144 . (canceled) 
     
     
         145 . A cell produced by introducing into the cell, or a progenitor thereof:
 a base editor, a polynucleotide encoding the base editor, to the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) associated with glycogen storage disorder Type 1a (GSD1a).   
     
     
         146 - 150 . (canceled) 
     
     
         151 . A method of treating glycogen storage disorder Type 1a (GSD1a) or von Gierke Disease in a subject in need thereof, the method comprising administering to the subject the cell of  claim 145 . 
     
     
         152 . A method of producing a hepatocyte, or progenitor thereof, the method comprising:
 (a) introducing into an induced pluripotent stem cell or hepatocyte progenitor comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disorder Type 1a (GSD1a),   a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with GSD1a; and   (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor into hepatocyte.   
     
     
         153 - 160 . (canceled) 
     
     
         161 . A method of editing an alpha-L-iduronidase gene (IDUA) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with mucopolysaccharidosis type 1 (MPS1), the method comprising contacting the IDUA polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with MPS1. 
     
     
         162 - 167 . (canceled) 
     
     
         168 . A cell produced by introducing into the cell, or a progenitor thereof:
 a base editor, a polynucleotide encoding the base editor, to the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and   one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) associated with mucopolysaccharidosis type 1 (MPS1).   
     
     
         169 - 170 . (canceled) 
     
     
         171 . A method of treating mucopolysaccharidosis type 1 (MPS1) in a subject in need thereof, the method comprising administering to the subject the cell of  claim 168 .