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

Patent metadata

Field	Value
Publication number	US-11155803-B2
Application number	US-202017127630-A
Country	US
Kind code	B2
Filing date	Dec 18, 2020
Priority date	Feb 13, 2019
Publication date	Oct 26, 2021
Grant date	Oct 26, 2021

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.

First claim

What is claimed is:

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

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI

GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS

RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLC

YFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4).

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

3. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises a T166R alteration.

4. 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.

5. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises an alteration or combination of alterations selected from the group consisting of:
Y147T and Q154R;
Y147T and Q154S;
Y147R and Q154S;
Q154S;
Y147R;
Q154R;
Y123H;
I76Y;
Y123H, and Y147T;
Y123H, and Y147R;
Y123H, and Q154R;
Y147R, Q154R, and Y123H;
Y147R, Q154R, and I76Y;
Y147R, Q154R, and T166R;
Y123H, Y147R, Q154R, and I76Y;
Y123H, Y147R, and Q154R; and
I76Y, Y123H, Y147R, and Q154R.

6. The fusion protein of claim 1 , wherein the fusion protein further comprises a second adenosine deaminase variant or a second adenosine deaminase.

7. The fusion protein of claim 1 , wherein the fusion protein is ABE8.5-m, 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, or ABE8.24-m.

8. The fusion protein of claim 6 , wherein the second adenosine deaminase comprises a wild-type adenosine deaminase.

9. The fusion protein of claim 8 , wherein the fusion protein is ABE8.5-d, 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.

10. The fusion protein of claim 1 , wherein the adenosine deaminase variant is TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.22, TadA*8.23, or TadA*8.24.

11. The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises 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 adenosine deaminase.

12. The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises the following sequence:

EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK

GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD

WDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKG

NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ

ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP

RAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD
GG

SGGSGGSGGSGGSGGSGGM
DKKYSIGLAIGTNSVGWAVITDEYKVPSKK

FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC

YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH

EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN

SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA

QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD

NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY

DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK

FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL

RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET

ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY

NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK

KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI

VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN

GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG

DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE

NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY

LQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR

GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK

AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL

Assignees

• Beam Therapeutics Inc

Inventors

• Gaudelli Nicole
• Packer Michael
• Slaymaker Ian
• Yu Yi
• Zetsche Bernd
• Gehrke Jason Michael
• Petrossian Natalie
• Messana Angelica
• Aratyn Yvonne
• Gregoire Francine
• Lung Genesis
• Berkovitch Shaunna
• Born David A
• Lee Seung-Joo

Classifications

• C12Y305/04005

  Cytidine deaminase (3.5.4.5) · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · 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

• C12Y305/04004

  Adenosine deaminase (3.5.4.4) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title