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US-12622931-B2 - Compositions and methods for treating alpha-1 antitrypsin deficiency

US12622931B2US 12622931 B2US12622931 B2US 12622931B2US-12622931-B2

Abstract

The present invention features compositions and methods for editing deleterious mutations associated with alpha-1 anti-trypsin (A1AT) deficiency. In particular embodiments, the invention provides methods for correcting mutations in an A1AT polynucleotide using an adenosine deaminase base editor. ABE8, having unprecedented levels of efficiency.

Inventors

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

Assignees

  • BEAM THERAPEUTICS INC.

Dates

Publication Date
20260512
Application Date
20200213

Claims (20)

  1. 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. 2 . The method of claim 1 , wherein the adenosine deaminase variant further comprises alterations at amino acid position 82 and 166.
  3. 3 . The method of claim 1 , wherein the adenosine deaminase variant further comprises a V82S alteration.
  4. 4 . The method of claim 1 , wherein the adenosine deaminase variant further comprises a T166R alteration.
  5. 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. 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. 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. 8 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant.
  9. 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. 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. 11 . The method of claim 10 , wherein the adenosine deaminase variant further comprises alterations at amino acid position 82 and 166.
  12. 12 . The method of claim 10 , wherein the adenosine deaminase variant further comprises V82S and/or T166R alterations.
  13. 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. 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. 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. 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. 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. 18 . The base editor system of claim 17 , wherein the adenosine deaminase variant further comprises a V82S alteration and/or a T166R alteration.
  19. 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. 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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT International Application No. PCT/US2020/018195, filed Feb. 13, 2020, which claims the benefit of U.S. Provisional Application Nos. 62/805,238, filed Feb. 13, 2019; 62/805,271, filed Feb. 13, 2019; 62/852,224, filed May 23, 2019; 62/852,228, filed May 23, 2019; 62/931,722, filed Nov. 6, 2019; 62/941,569, filed Nov. 27, 2019; and 62/966,526, filed Jan. 27, 2020, the contents of all of which are incorporated by reference herein in their entireties. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 27, 2020, is named 180802-041902PCTSequenceListing.txt and is 818,399 bytes in size. INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties. BACKGROUND OF THE DISCLOSURE In healthy individuals, alpha-1 antitrypsin (A1AT) is produced by hepatocytes within the liver and secreted into systemic circulation where it functions as a protease inhibitor. It is a particularly good inhibitor of neutrophil elastase, thus protecting tissues and organs such as the lung from elastin degradation. In patients with Alpha-1 Antitrypsin Deficiency (A1AD), mutations in the gene that encodes A1AT lead to diminished protein production. Consequently, elastin in the lung is degraded more readily by neutrophil elastase, and over time the loss in lung elasticity develops into chronic obstructive pulmonary disease (COPD). The most common pathogenic A1AT variant is a Guanine to Adenine mutation resulting in a glutamate to lysine substitution at amino acid 342. This substitution causes the protein to misfold and polymerize within hepatocytes, and ultimately the toxic aggregates can lead to liver injury and cirrhosis. While the liver toxicity could be addressed by a gene knockout (CRISPR/ZFN/TALEN) or gene knockdown (siRNA), neither approach addresses the pulmonary pathology. Although pulmonary pathology may be addressed with protein replacement therapy, this therapy also fails to address the liver toxicity. Gene therapy also would be inadequate to address the A1AT genetic defect. Because the liver of patients with A1AD is already under a severe disease burden caused by the endogenous A1AT, gene therapy that increases A1AT in the liver would be counterproductive. Therefore, there is a need for a method of treating patients with A1AD that addresses both the lung pathology and the liver toxicity. SUMMARY As described below, the present invention features compositions and methods for editing deleterious mutations associated with Alpha-1 Antitrypsin Deficiency (A1AD). In particular embodiments, the invention provides methods for treating A1AD using a modified adenosine deaminase termed “ABE8,” which has unprecedented levels (e.g., >60-70%) of efficiency and specificity, to correct mutations associated with A1AD. In one aspect, the invention provides a method of editing a alpha-1 antitrypsin polynucleotide containing a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency, the method involving contacting the polynucleotide with one or more guide RNAs and a base editor containing a polynucleotide programmable DNA binding domain and at least one base editor domain that is an adenosine deaminase variant containing an alteration at amino acid position 82 or 166 of (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. In another aspect, the invention provides a method of editing an alpha-1 antitrypsin polynucleotide containing a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency, the method involving contacting a alpha-1 antitrypsin polynucleotide with one or more guide RNAs and a fusion protein containing a polynucleotide programmable DNA binding domain containing the following sequence: (SEQ ID NO: 4)EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD PKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYF DTTIARKEYRSTKEVLDATLIHQSITGL