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US-20260125677-A1 - Compositions and Methods for Treating Alpha-1 Antitrypsin Deficiency

US20260125677A1US 20260125677 A1US20260125677 A1US 20260125677A1US-20260125677-A1

Abstract

Compositions and methods for introducing double-stranded breaks within the SERPINA1 gene are provided. Compositions and methods for reducing and eliminating mutant forms of α1-antitrypsin (AAT), such as seen in subjects having α1-antitrypsin deficiency (AATD), are provided.

Inventors

  • Shobu Odate
  • Walter Strapps
  • Reynald Michael Lescarbeau

Assignees

  • INTELLIA THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20251008

Claims (20)

  1. 1 .- 2 . (canceled)
  2. 3 . A method of treating alpha-1 antitrypsin deficiency (AATD), the method comprising administering a composition to a subject in need thereof, wherein the composition comprises a guide RNA comprising a guide sequence of SEO ID NO: 42 or a guide sequence that is at least 90% identical to SEO ID NO: 42, wherein the guide RNA is a single guide RNA (sgRNA) comprising 2′-O-methyl (2′-O-Me) modified nucleotides at the first three nucleotides at the 5′ end and/or 2′-O-Me modified nucleotides at the last three nucleotides at the 3′ end.
  3. 4 .- 243 . (canceled)
  4. 244 . The method of claim 3 , wherein the guide sequence is SEQ ID NO: 42.
  5. 245 . The method of claim 244 , wherein the guide RNA further comprises SEQ ID NO: 141 at the 3′ end of SEQ ID NO: 42.
  6. 246 . The method of claim 244 , wherein the sgRNA comprises mC*mA*mA*CCUCACGGAGAUUCCGGGUUUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 138), wherein a * indicates a phosphorothioate (PS) bond and a lower case “m” indicates that the nucleotide is 2′-O-Me modified.
  7. 247 . The method of claim 3 , wherein the sgRNA comprises a guide sequence that has the modifications of SEQ ID NO: 130 or wherein the sgRNA comprises SEQ ID NO: 130.
  8. 248 . The method of claim 3 , wherein the sgRNA comprises at least one further modification, wherein the at least one further modification comprises (a) a 2′-O-methyl (2′-O-Me) modified nucleotide; (b) a phosphorothioate (PS) bond between nucleotides; (c) a 2′-fluoro (2′-F) modified nucleotide; (d) a modification at one or more of the first five nucleotides at the 5′ end; (e) a modification at one or more of the last five nucleotides at the 3′ end; (f) PS bonds between the first four nucleotides; and/or (g) PS bonds between the last four nucleotides.
  9. 249 . The method of claim 248 , wherein the at least one modification comprises phosphorothioate (PS) bonds between the first four nucleotides and/or between the last four nucleotides.
  10. 250 . The method of claim 3 , wherein the guide RNA comprises a guide sequence that is at least 95% identical to SEQ ID NO: 42.
  11. 251 . The method of claim 250 , wherein the guide RNA further comprises SEQ ID NO: 141 at the 3′ end of the guide sequence.
  12. 252 . The method of claim 251 , comprising the modification pattern of SEQ ID NO: 130.
  13. 253 . The method of claim 3 , wherein the guide RNA is associated with a lipid nanoparticle (LNP).
  14. 254 . The method of claim 3 , wherein the composition further comprises an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.
  15. 255 . The method of claim 254 , wherein the guide RNA and the RNA-guided DNA binding agent or the nucleic acid encoding an RNA-guided DNA binding agent are associated with an LNP.
  16. 256 . The method of claim 255 , wherein the LNP comprises ((9Z, 12Z)-3-((4,4-bid(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate and/or PEG2k-DMG.
  17. 257 . The method of claim 254 , wherein the composition comprises the nucleic acid encoding the RNA-guided DNA binding agent, and the nucleic acid is an mRNA.
  18. 258 . The method of claim 257 , wherein the RNA-guided DNA binding agent is Cas9 from S. pyogenes.
  19. 259 . The method of claim 3 , wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.
  20. 260 . The method of claim 3 , wherein the guide RNA comprises a sequence selected from SEQ ID NOs: 130, 410-414, and 416-421, wherein each N in the sequence selected from SEQ ID NOs: 130, 410-414, and 416-421 is collectively replaced with a guide sequence of SEQ ID NO: 42 or a guide sequence that is at least 90% identical to SEQ ID NO: 42.

Description

This application is a Continuation of U.S. patent application Ser. No. 18/063,817, which was filed on Dec. 9, 2022, which is a Divisional of U.S. patent application Ser. No. 16/446,764, which was filed on Jun. 20, 2019, which is a Continuation of International Application No. PCT/US2017/067800, which was filed on Dec. 21, 2017 and claims the benefit of priority to U.S. Provisional Application No. 62/438,219, which was filed on Dec. 22, 2016, all of which are incorporated by reference in their entirety. The patent application is filed with a sequence listing in electronic format. The Sequence Listing is provided as a file entitled “01155-0005-01US_ST26,” which was created on Dec. 8, 2022, and which is 540,049 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Alpha-1 antitrypsin (AAT or A1AT) or serum trypsin inhibitor is a type of serine protease inhibitor (also termed a serpin) encoded by the SERPINA1 gene. AAT is primarily synthesized and secreted by hepatocytes, and functions to inhibit the activity of neutrophil elastase in the lung. Without sufficient quantities of functioning AAT, neutrophil elastase is uncontrolled and damages alveoli in the lung. Thus, mutations in SERPINA1 that result in decreased levels of AAT, or decreased levels of properly functioning AAT, lead to lung pathology. Moreover, mutations in SERPINA1 that lead to production of misformed AAT that does not exit the liver leads to liver pathology due to accumulation of AAT in the hepatocytes. Thus, insufficient and improperly formed AAT caused by SERPINA1 mutation leads to both lung and liver pathology. More than one hundred allelic variants have been described for the SERPINA1 gene. Variants are generally classified according to their effect on serum levels of AAT. For example, M alleles are normal variants associated with normal serum AAT levels, whereas Z and S alleles are mutant variants associated with decreased AAT levels. The presence of Z and S alleles is associated α1-antitrypsin deficiency (AATD or A1AD), a genetic disorder characterized by mutations in the SERPINA1 gene that leads to the production of abnormal AAT. There are many forms and degrees of AATD. The “Z-variant” is the most common, causing severe clinical disease in both liver and lung. The Z-variant is characterized by a single nucleotide change in the 5′ end of the 5th exon that results in a missense mutation of glutamic acid to lysine at amino acid position 342 (E342K). Symptoms arise in patients that are both homozygous (ZZ) and heterozygous (MZ or SZ) at the Z allele. The presence of one or two Z alleles results in SERPINA1 mRNA instability, and AAT protein polymerization and aggregation in liver hepatocytes. Patients having at least one Z allele have an increased incidence of liver cancer due to the accumulation of aggregated AAT protein in the liver. In addition to liver pathology, AATD characterized by at least one Z allele is also characterized by lung disease due to the decrease in AAT in the alveoli and the resulting decrease in inhibition of neutrophil elastase. The prevalence of the severe ZZ-form (i.e., homozygous expression of the Z-variant) is 1:2,000 in northern European populations, and 1:4,500 in the United States. A need exists to ameliorate the negative effects of AATD in both the liver and lung. The present invention provides compositions and methods using the CRISPR/Cas system to knock out the SERPINA1 gene thereby eliminating the production of mutant forms of AAT that are associated with liver symptoms in patients with AATD. SUMMARY Embodiment 01 A method of inducing a double-stranded break (DSB) within the SERPINA1 gene, comprising delivering a composition to a cell, wherein the composition comprises a guide RNA comprising a guide sequence selected from SEQ ID NOs: 5-129 or a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-129. Embodiment 02 A method of modifying the SERPINA1 gene comprising delivering a composition to a cell, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising a guide sequence selected from SEQ ID NOs: 5-129 or a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-129. Embodiment 03 A method of treating alpha-1 antitrypsin deficiency (AATD), comprising administering a composition to a subject in need thereof, wherein the composition comprises (i) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent and (ii) a guide RNA comprising a guide sequence selected from SEQ ID NOs: 5-129 or a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 5-129, thereby t