Compositions and methods for treating hemoglobinopathies

Patent metadata

Field	Value
Publication number	US-11142760-B2
Application number	US-202117214461-A
Country	US
Kind code	B2
Filing date	Mar 26, 2021
Priority date	Feb 13, 2019
Publication date	Oct 12, 2021
Grant date	Oct 12, 2021

Abstract

The present invention features compositions and methods for editing deleterious mutations associated with hemoglobinopathies, such as sickle cell disease (SCD). In particular embodiments, the invention provides methods for correcting mutations in a beta globin polynucleotide using modified adenosine base editors termed “ABE8” having unprecedented levels (e.g., >60-70%) of efficiency.

First claim

What is claimed is:

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.

Assignees

• Beam Therapeutics Inc

Inventors

• Slaymaker Ian
• Gaudelli Nicole
• Yu Yi
• Zetsche Bernd
• Born David A
• Lee Seung-Joo
• Packer Michael

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N2310/315

  Phosphorothioates · CPC title

• C12N2310/321

  2'-O-R Modification · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title