IP Library › Patent Application 18984585

Patent Application

App. No. 18/984,585

ADENOSINE DEAMINASE BASE EDITORS AND METHODS FOR USE THEREOF

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Patent No.: US None
App. No.: 18/984,585

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Abstract

Adenosine deaminase base editors, compositions comprising the same, and methods for use thereof for altering a target nucleobase in a polynucleotide sequence. In embodiments of the disclosure, the base editors of the disclosure may be used to treat a disease or disorder, such as a hemoglobinopathy (e.g., sickle cell disease (SCD)).

Claims (163)

1 . A method for treating a hemoglobinopathy in a subject in need thereof, the method comprising:

a) contacting an isolated hematopoietic stem cell or progenitor thereof with two or more guide polynucleotides, or one or more polynucleotides encoding the guide polynucleotides, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase, or one or more polynucleotides encoding the base editor, wherein the adenosine deaminase comprises a combination of alterations relative to the following sequence of TadA*7.10 and has at least 85% sequence identity to the following sequence of TadA*7.10: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), wherein the combination of alterations comprises:

i) F149Y and V82T; or

ii) F149Y, T166I, and/or D167N, and the alteration V82T; and

wherein the two or more guide polynucleotides target the base editor to effect an alteration to a beta globin polynucleotide (HBB) that results in expression of a beta globin polypeptide having an alanine at position 6 (Hb G-Makassar), and wherein the two or more guide polynucleotides target the base editor to effect an alteration in a cluster of differentiation (CD117) polynucleotide that results in expression of a CD117 polypeptide with reduced binding to an antibody that selectively binds a wild type CD117 polypeptide, thereby generating an edited cell; and

(b) wherein the cell is in the subject or the method further comprises administering the edited cell to the subject.

2 . The method of claim 1 , wherein the combination of alterations comprises or further comprises:

I76Y, V82T, Y123H, Y147R, F149Y;

Q154R or F149Y, T166I, and D167N;

I76Y, Y123H, Y147D, and Q154R; and/or

I76Y, V82T, Y123H, Y147D, F149Y, Q154R, T166I, and D167N

3 . The method of claim 1 , wherein the adenosine deaminase comprises the following amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCDFYRMPRRVFNAQKKAQSSIN (SEQ ID NO: 519); and

wherein the two or more guide polynucleotides comprise a spacer nucleotide sequence selected from the group consisting of: UUCUCCACAGGAGUCAGGUG (SEQ ID NO: 445); ACUUCUCCACAGGAGUCAGG (SEQ ID NO: 446); GACUUCUCCACAGGAGUCAGG (SEQ ID NO: 447); CUUCUCCACAGGAGUCAGG (SEQ ID NO: 448); CUUCUCCACAGGAGUCAGAU (SEQ ID NO: 449); ACUUCUCCACAGGAGUCAGAU (SEQ ID NO: 450); GACUUCUCCACAGGAGUCAGAU (SEQ ID NO: 451); UCUGACUCCUGUGGAGAAGUCU (SEQ ID NO: 452); AGACUUCUCCACAGGAGUCAGA (SEQ ID NO: 453); and UCCACAGGAGUCAGAUGCAC (SEQ ID NO: 454); or a spacer sequence selected from those listed in Table 2; and, optionally, a scaffold with the following nucleotide sequence:

(SEQ ID NO: 317)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA

CUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.

4 . The method of 1, wherein the two or more guide polynucleotides comprise a spacer nucleotide sequence selected from the group consisting of:

(SEQ ID NO 660; CC128)

AAAUAUAAUAGCUGGCAUCA;

(SEQ ID NO: 722; CC200)

AUAAUAGCUGGCAUCACGGU;

(SEQ ID NO: 723; gRNA889)

CCACUAGCUUUCCAAACGGU;

(SEQ ID NO: 724; gRNA908)

GCUGAACUGAUAGUCAACGU;

(SEQ ID NO: 725; gRNA918)

UUUGACAAAGCCCGGAUCAG;

(SEQ ID NO: 726; gRNA923)

UGAAAGUGAGGCCAGGUACU;

(SEQ ID NO: 727; gRNA928)

AAACAGUCAGGUGAGUGAAU;

(SEQ ID NO: 728; gRNA929)

AACUACAGGAGAAAUAUAAU;

and

(SEQ ID NO: 729; gRNA944)

GAUUAAAAGGCACCGAAGGA.

5 . The method of claim 1 , wherein the two or more guide polynucleotides comprise a scaffold with a sequence selected from the group consisting of: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 521); GUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAG UCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmG mUmGmCmUsmUsmUsmU (SEQ ID NO: 522); mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU (SEQ ID NO: 443); and mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmC mCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAm GUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 444), wherein “N” represents any nucleotide, “mN” indicates a 2′-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS).

6 . A cell produced by the method of claim 1 .

7 . A pharmaceutical composition comprising an effective amount of the cell of claim 6 .

8 . An adenosine deaminase polypeptide comprising a combination of alterations relative to the following sequence of TadA*7.10: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), wherein the combination of alterations comprises F149Y and V82T, and wherein the adenosine deaminase has at least 85% sequence identity to TadA*7.10.

9 . The adenosine deaminase polypeptide of claim 8 , wherein the combination of alterations further comprises an alteration selected from the group consisting of 176Y, Y123H, Y147R, and Q154R.

10 . An adenosine deaminase polypeptide comprising the following amino acid sequence: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCRFYRMPRRVFNAQKKAQSSTD (SEQ ID NO: 518); or

comprising a combination of alterations to the following sequence of TadA*7.10: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), wherein the combination of alterations comprises F149Y, T166I, and/or D167N, and the alteration V82T, and wherein the adenosine deaminase has at least 85% sequence identity to TadA*7.10.

11 . The adenosine deaminase polypeptide of claim 10 , wherein the combination of alterations further comprises the following additional alterations: I76Y, Y123H, Y147D, and Q154R.

12 . A base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) and the adenosine deaminase of claim 10 .

13 . A base editor comprising: a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase, wherein the adenosine deaminase comprises a combination of alterations relative to TadA*7.10:

(SEQ ID NO: 1)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI

GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR

IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCY

FFRMPRQVFNAQKKAQSSTD,

wherein the combination of alterations comprises I76Y, V82T, Y123H, Y147R, F149Y, and Q154R, and wherein the adenosine deaminase has at least 85% sequence identity to TadA*7.10; or

the adenosine deaminase comprises a combination of alterations relative to TadA*7.10:

(SEQ ID NO: 1)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI

GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR

IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCY

FFRMPRQVFNAQKKAQSSTD,

wherein the combination of alterations comprises I76Y, V82T, Y123H, Y147D, F149Y, Q154R, T166I, and D167N, and wherein the adenosine deaminase has at least 85% sequence identity to TadA*7.10.

14 . A polynucleotide encoding the adenosine deaminase polypeptide of claim 10 .

15 . A method of altering a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with the base editor of claim 10 , thereby altering a nucleobase of the polynucleotide.

16 . A base editor system comprising the base editor of claim 13 , or one or more polynucleotides encoding the base editor, and a guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, that targets the base editor to effect an alteration to a polynucleotide associated with a genetic disorder.

17 . A base editor system comprising the base editor of claim 13 , or a polynucleotide encoding the base editor, and two or more guide polynucleotides, or one or more polynucleotides encoding the two or more guide polynucleotides, wherein one of the guide polynucleotides targets the base editor to effect an alteration to a beta globin polynucleotide (HBB) that results in expression of a beta globin polypeptide having an alanine at position 6 (Hb G-Makassar), and another of the guide polynucleotides targets the base editor to effect an alteration in a cluster of differentiation (CD117) polynucleotide that results in expression of a CD117 polypeptide with reduced binding to an antibody that selectively binds a wild type CD117 polypeptide.

18 . The base editor system of claim 17 , wherein:

a) one of the guide polynucleotides comprises a spacer sequence selected from the group consisting of:

(SEQ ID NO: 445)

UUCUCCACAGGAGUCAGGUG;

(SEQ ID NO: 446)

ACUUCUCCACAGGAGUCAGG;

(SEQ ID NO: 447)

GACUUCUCCACAGGAGUCAGG;

(SEQ ID NO: 448)

CUUCUCCACAGGAGUCAGG;

(SEQ ID NO: 449)

CUUCUCCACAGGAGUCAGAU;

(SEQ ID NO: 450)

ACUUCUCCACAGGAGUCAGAU;

(SEQ ID NO: 451)

GACUUCUCCACAGGAGUCAGAU;

(SEQ ID NO: 452)

UCUGACUCCUGUGGAGAAGUCU;

(SEQ ID NO: 453)

AGACUUCUCCACAGGAGUCAGA;

and

(SEQ ID NO: 454)

UCCACAGGAGUCAGAUGCAC;

b) another of guide polynucleotides comprises a spacer sequence selected from the group consisting of:

(SEQ ID NO 660; CC128)

AAAUAUAAUAGCUGGCAUCA;

(SEQ ID NO: 722; CC200)

AUAAUAGCUGGCAUCACGGU;

(SEQ ID NO: 723; gRNA889)

CCACUAGCUUUCCAAACGGU;

(SEQ ID NO: 724; gRNA908)

GCUGAACUGAUAGUCAACGU;

(SEQ ID NO: 725; gRNA918)

UUUGACAAAGCCCGGAUCAG;

(SEQ ID NO: 726; gRNA923)

UGAAAGUGAGGCCAGGUACU;

(SEQ ID NO: 727; gRNA928)

AAACAGUCAGGUGAGUGAAU;

(SEQ ID NO: 728; gRNA929)

AACUACAGGAGAAAUAUAAU;

and

(SEQ ID NO: 729; gRNA944)

GAUUAAAAGGCACCGAAGGA.

19 . A kit suitable for use in the method of any one of the above claims , wherein the kit comprises the base editor of claim 13 , or a polynucleotide encoding the base editor.

20 . The base editor of claim 13 , wherein the base editor comprises the following amino acid sequence:

(SEQ ID NO: 524)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA

LRQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP

GMNHRVEITEGILADECAALLCRFYRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSES

ATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK

HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL

NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG

LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD

AILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA

GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAI

LRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK

GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK

AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD

NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGI

RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

IKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ

ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV

LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN

YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI

MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG

FSKESILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI

TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELAL

PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV

LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSI

TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV;

wherein the base editor comprises the following amino acid sequence:

(SEQ ID NO: 525)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA

LRQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP

GMNHRVEITEGILADECAALLCDFYRMPRRVFNAQKKAQSSINSGGSSGGSSGSETPGTSES