Granted Patent B2

US 11,142,760 · App. 17/214,461 · Granted Oct 12, 2021

Inventors: Ian Slaymaker (Cambridge, MA); Nicole Gaudelli (Cambridge, MA); Yi Yu (Cambridge, MA); Bernd Zetsche (Cambridge, MA); David A. Born (Cambridge, MA); Seung-Joo Lee (Cambridge, MA); Michael Packer (Cambridge, MA)

Assignee: Beam Therapeutics Inc.

C12N15/11A61K35/18C07K14/4717C12N5/0641C12N9/22C12N9/78C12N15/907C12Y305/04004C12N2310/20C12N2510/00

Abstract

Claims (161)

1. A method for editing a beta globin (HBB) polynucleotide associated with sickle cell disease, the method comprising contacting a cell comprising a single nucleotide polymorphism (SNP) in the beta globin (HBB) polynucleotide with a guide RNA and a fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain comprising a serine (S) at amino acid position 82 of the following amino acid sequence and having at least 85% sequence identity to the following amino acid sequence

(SEQ ID NO: 2)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR

AIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAM

IHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC

AALLCYFFRMPRQVFNAQKKAQSSTD,

wherein said guide RNA targets said polynucleotide programmable DNA binding domain to the SNP in the beta globin (HBB) polynucleotide.

2. The method of claim 1 , wherein the adenosine deaminase domain further comprises an alteration at amino acid position 166.

3. The method of claim 2 , wherein the alteration at amino acid position 166 is T166R.

4. The method of claim 1 , wherein the adenosine deaminase domain further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, and Q154R.

5. The method of claim 1 , wherein the adenosine deaminase domain comprises a combination of alterations selected from the group consisting of:

Y147T and Q154R;

Y147T and Q154S;

Y147R and Q154S;

V82S and Q154S;

V82S and Y147R;

V82S and Q154R;

V82S and Y123H;

I76Y and V82S;

V82S, Y123H, and Y147T;

V82S, Y123H, and Y147R;

V82S, Y123H, and Q154R;

Y147R, Q154R, and Y123H;

Y147R, Q154R, and I76Y;

Y147R, Q154R, and T166R;

Y123H, Y147R, Q154R, and I76Y;

V82S, Y123H, Y147R, and Q154R; and

I76Y, V82S, Y123H, Y147R, and Q154R.

6. The method of claim 1 , wherein the adenosine deaminase domain comprises V82S and T166R.

7. The method of claim 1 , wherein the fusion protein comprises a heterodimer comprising the adenosine deaminase domain of claim 1 and a wild-type adenosine deaminase domain.

8. The method of claim 1 , wherein the adenosine deaminase domain comprises Y147T and Q154S.

9. The method of claim 1 , wherein an A•T to G•C alteration at the SNP results in expression of an HBB polypeptide having an alanine at amino acid position 6.

10. The method of claim 1 , wherein the programmable DNA binding domain comprises a Cas9 domain.

11. The method of claim 10 , wherein the Cas9 domain comprises a dead Cas9 (dCas9) or a nickase Cas9 (nCas9).

12. The method of claim 11 , wherein the Cas9 domain is capable of programmable DNA binding and has at least 85% amino acid sequence identity to a Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), or a Streptococcus thermophilus 1 Cas9 (St1Cas9).

13. The method of claim 11 , wherein the Cas9 domain comprises the following amino acid sequence:

(SEQ ID NO: 1)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG

ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF

HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD

KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK

NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL

PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK

LNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE

KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS

FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF

LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN

ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLK

TYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSD

GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK

GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI

EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL

SDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY

WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV

AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN

YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI

GKATAKYFFYSNIMNFEKTEITLANGEIRKRPLIETNGETGEIVWDKGR

DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD

PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK

NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE

LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS

EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA

FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD.

14. The method of claim 1 , wherein the fusion protein is selected from ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.22-m, ABE8.23-m, ABE8.24-m, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.

15. The method of claim 1 wherein the adenosine deaminase domain comprises a truncated TadA8 comprising a deletion of 1, 2, 3, 4, 5,6, 7, or 8 N-terminal or C-terminal amino acid residues relative to the full length TadA8.

16. The method of claim 1 , wherein the adenosine deaminase domain has at least 90% sequence identity to the amino acid sequence.

17. A base editing system comprising the fusion protein of claim 1 and a guide RNA comprising a nucleic acid sequence selected from the group consisting of

(SEQ ID NO: 4)

CUUCUCCACAGGAGUCAGAU;

(SEQ ID NO: 5)

ACUUCUCCACAGGAGUCAGAU;

and

(SEQ ID NO: 6)

GACUUCUCCACAGGAGUCAGAU.

18. A method for editing a beta globin (HBB) polynucleotide associated with sickle cell disease, the method comprising contacting a cell comprising a single nucleotide polymorphism (SNP) in the beta globin (HBB) polynucleotide with a guide RNA and a fusion protein comprising a polynucleotide programmable DNA binding domain comprising the following amino acid sequence:

(SEQ ID NO: 3)

EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK

GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD

WDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKG

NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ

ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP

RAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGG

SGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKK

FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC

YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH

EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN

SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA

QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD

NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY

DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK

FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL

RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET

ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY

NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK

KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI

VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN

GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG

DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE

NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY

LQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR

GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK

AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL

VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG

DYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV,

and at an adenosine deaminase domain comprising a serine (S) at amino acid position 82 of the following amino acid sequence and having at least 85% sequence identity to the following amino acid sequence

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEI MALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMD VLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 2).

19. A cell or a progenitor thereof, comprising:

a fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain described in claim 1 or a polynucleotide encoding said fusion protein; and

one or more guide polynucleotides that target the fusion protein to effect an A•T to G•C alteration of the SNP in the HBB polynucleotide.

20. The cell of claim 19 , wherein the cell is a hematopoietic stem cell, a common myeloid progenitor, proerythroblast, erythroblast, reticulocyte, or erythrocyte.

21. The cell of claim 19 , wherein the cell is a mammalian cell or a human cell.

22. A method of treating sickle cell disease in a subject comprising administering to said subject a cell comprising:

a fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain described in claim 1 or a polynucleotide encoding said fusion protein; and

one or more guide polynucleotides that target the fusion protein to effect an A•T to G•C alteration of the SNP in the HBB polynucleotide.

23. A population of cells comprising:

a fusion protein comprising the polynucleotide programmable DNA binding domain and the adenosine deaminase domain described in claim 1 or a polynucleotide encoding said fusion protein; and

one or more guide polynucleotides that target the fusion protein to effect an A•T to G•C alteration of the SNP in the HBB polynucleotide.

24. A method of producing a red blood cell, or progenitor thereof, comprising:

(a) introducing into a red blood cell progenitor comprising an HBB polynucleotide associated with sickle cell disease:

i) a fusion protein, or a polynucleotide encoding said fusion protein, wherein said fusion protein comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain described in claim 1 ; and

ii) one or more guide polynucleotides, wherein said one or more guide polynucleotides target said fusion protein to effect an A•T to G•C alteration of an SNP in the HBB polynucleotide; and

(b) differentiating the red blood cell progenitor into a red blood cell.

25. A method for treating sickle cell disease (SCD) in a subject, the method comprising: administering to the subject the fusion protein described in claim 1 , or a polynucleotide encoding the fusion protein; and a guide polynucleotide to target the fusion protein to effect an A•T to G•C alteration of a single nucleotide polymorphism (SNP) associated with SCD, thereby treating SCD in the subject.

26. The method of claim 25 , wherein the guide polynucleotide is a guide RNA.

27. The method of claim 26 , wherein the alteration of the SNP replaces a target nucleobase with a non-wild type nucleobase, and wherein the alteration of the target nucleobase ameliorates symptoms of sickle cell disease.

28. A pharmaceutical composition comprising a base editing system comprising the fusion protein of claim 1 , a guide RNA, and a pharmaceutically acceptable carrier, vehicle, or excipient.

29. A base editor system comprising a fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain comprising a serine (S) at amino acid position 82 of the following amino acid sequence and having at least 85% sequence identity to the following amino acid sequence

(SEQ ID NO: 2)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR

AIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAM

IHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC

AALLCYFFRMPRQVFNAQKKAQSSTD

and a guide RNA, wherein said guide RNA targets said fusion protein to effect an alteration of a single nucleotide polymorphism (SNP) in a beta globin (HBB) polynucleotide.

30. The base editor system of claim 29 , wherein the programmable DNA binding domain comprises the following amino acid sequence:

(SEQ ID NO: 3)

EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM

PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKV

EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR

MLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQI

SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEY

RSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGL

AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR

RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ

LFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD

LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSA

SMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE

KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI

LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN

EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL

KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED

REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA

NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL

YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP

SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV

AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV.

Assignments (3)

Continuity (10)

Cited By (10)

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