Granted Patent B2

US 12,622,931 · App. 17/430,665 · Granted May 12, 2026

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

Assignee: BEAM THERAPEUTICS INC.

A61K35/407A61K31/7088A61K38/465A61K48/0025C12N5/067C12N9/22C12N9/78C12N15/102C12N15/11C12N2310/20C12N2510/00

Abstract

Claims (101)

1 . A method of editing an alpha-1 antitrypsin polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency, the method comprising contacting the polynucleotide with one or more guide RNAs and a base editor comprising a polynucleotide programmable DNA binding domain and at least one base editor domain that comprises an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 compared to the TadA*7.10 amino acid sequence:

(SEQ ID NO: 3)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG

LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG

RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR

MPRQVFNAQKKAQSSTD,

wherein said guide RNA targets said base editor to effect an alteration of the SNP associated with alpha-1 antitrypsin deficiency.

2 . The method of claim 1 , wherein the adenosine deaminase variant further comprises alterations at amino acid position 82 and 166.

3 . The method of claim 1 , wherein the adenosine deaminase variant further comprises a V82S alteration.

4 . The method of claim 1 , wherein the adenosine deaminase variant further comprises a T166R alteration.

5 . The method of claim 1 , wherein the adenosine deaminase variant further comprises V82S and T166R alterations and, optionally, one or more of the following alterations: I76Y, Q154S, Y123H, and Q154R.

6 . The method of claim 1 , wherein the adenosine deaminase variant further comprises one of the following combinations of alterations: Q154R; Q154S; V82S+Q154S; V82S; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Q154R; Q154R+Y123H; Q154R+I76Y; Q154R+T166R; Y123H+Q154R+I76Y; and I76Y+V82S+Y123H+Q154R.

7 . The method of claim 1 , wherein the base editor domain comprises:

a) an adenosine deaminase variant monomer comprising the adenosine deaminase variant; or

b) an adenosine deaminase heterodimer comprising a wild-type adenosine deaminase domain and the adenosine deaminase variant.

8 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant.

9 . The method of claim 1 , wherein the contacting occurs in a hepatocyte or hepatocyte progenitor comprising a SNP associated with alpha-1 antitrypsin deficiency,

wherein the alteration of the SNP associated with alpha-1 antitrypsin deficiency is an A·T to G·C alteration,

and wherein when the hepatocyte progenitor is used in the contacting step, the method further comprises (b) differentiating the hepatocyte progenitor into a hepatocyte.

10 . A method of editing an alpha-1 antitrypsin polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency, the method comprising contacting an alpha-1 antitrypsin polynucleotide with one or more guide RNAs and a fusion protein comprising a polynucleotide programmable DNA binding domain comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 13, and at least one base editor domain comprising an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 compared to the TadA*7.10 amino acid sequence:

(SEQ ID NO: 3)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL

HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV

VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPR

QVFNAQKKAQSSTD.

11 . The method of claim 10 , wherein the adenosine deaminase variant further comprises alterations at amino acid position 82 and 166.

12 . The method of claim 10 , wherein the adenosine deaminase variant further comprises V82S and/or T166R alterations.

13 . The method of claim 10 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: I76Y, Q154S, Y123H, and Q154R.

14 . The method of claim 10 , wherein the adenosine deaminase variant further comprises one of the following combinations of alterations: Q154R; Q154S; V82S+Q154S; V82S; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Q154R; Q154R+Y123H; Q154R+I76Y; Q154R+T166R; Y123H+Q154R+I76Y; and I76Y+V82S+Y123H+Q154R.

15 . A base editor system comprising:

a) a base editor comprising a polynucleotide programmable DNA binding domain and at least one base editor domain that comprises an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 compared to the TadA*7.10 amino acid sequence:

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGG LVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITE GILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3); and

b) a guide RNA comprising a nucleic acid sequence selected from the group consisting of

(SEQ ID NO: 5)

5′-ACCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU

AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 6)

5′-CCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU

AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′;  

(SEQ ID NO: 7)

5′-CAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU

AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 8)

5′-AUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU

AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 9)

5′-UCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU

AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 10)

5′-CGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC

CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU-3′.

16 . A pharmaceutical composition or a kit comprising a base editor system comprising:

a) a base editor comprising a polynucleotide programmable DNA binding domain and at least one base editor domain that comprises an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 compared to the TadA*7.10 amino acid sequence:

(SEQ ID NO: 3)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI

GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR

IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCY

FFRMPRQVFNAQKKAQSSTD;

and

b) a guide RNA comprising a nucleic acid sequence selected from the group consisting of

(SEQ ID NO: 5)

5′-ACCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 6)

5′-CCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 7)

5′-CAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 8)

5′-AUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′;

(SEQ ID NO: 9)

5′-UCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′; and

(SEQ ID NO: 10)

5′-CGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC 

AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU 

GGCACCGAGU CGGUGCUUUU-3′;

wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, vehicle, or excipient.

17 . A base editor system comprising a polynucleotide programmable DNA binding domain and at least one base editor domain that comprises an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 of the TadA*7.10 amino acid sequence:

(SEQ ID NO: 3)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL

HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV

VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPR

QVFNAQKKAQSSTD

and a guide RNA, wherein said guide RNA targets said base editor to effect an alteration of a SNP associated with alpha-1 antitrypsin deficiency.

18 . The base editor system of claim 17 , wherein the adenosine deaminase variant further comprises a V82S alteration and/or a T166R alteration.

19 . The base editor system of claim 18 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: I76Y, Q154S, Y123H, and Q154R.

20 . A base editor system comprising one or more guide RNAs comprising a nucleic acid sequence complementary to an alpha-1 antitrypsin nucleic acid sequence containing a SNP associated with alpha-1 antiytrypsin deficiency, and a fusion protein comprising a polynucleotide programmable DNA binding domain comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 13, and at least one base editor domain comprising an adenosine deaminase variant comprising an amino acid sequence that is at least 90% identical to the following TadA*7.10 amino acid sequence and that further comprises an arginine or a threonine at amino acid position 147 of the TadA*7.10 amino acid sequence:

(SEQ ID NO: 3)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL

HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV

VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPR

QVFNAQKKAQSSTD.

21 . A cell comprising the base editor system of claim 20 .

Assignments (4)

Continuity (8)

References Cited (400)