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US-20260125690-A1 - COMPOSITIONS AND METHODS FOR TREATING ALPHA-1 ANTITRYPSIN DEFICIENCY

US20260125690A1US 20260125690 A1US20260125690 A1US 20260125690A1US-20260125690-A1

Abstract

Compositions and methods for expressing alpha 1 antitrypsin (AAT) in a host cell are provided. Also provided are compositions and methods for treating subjects having alpha 1 antitrypsin deficiency (AATD).

Inventors

  • Jonathan Douglas Finn
  • Hon-Ren Huang
  • ANTHONY FORGET
  • Xin Xie

Assignees

  • INTELLIA THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20250923

Claims (20)

  1. 1 .- 123 . (canceled)
  2. 124 . A method of expressing heterologous alpha-1 antitrypsin (AAT) in a subject, the method comprising administering to the subject: (a) a nucleic acid construct comprising a heterologous AAT protein coding sequence; (b) a Cas9 nuclease; and (c) a guide RNA (gRNA) comprising a sequence that targets the albumin locus at a genomic coordinate listed in Table 1, thereby expressing heterologous AAT from the albumin locus of the subject.
  3. 125 . A method of introducing a nucleic acid encoding alpha-1 antitrypsin (AAT) to a cell or a population of cells, the method comprising delivering to the cell or population of cells: (a) a bidirectional nucleic acid construct comprising: (i) a first segment comprising a first coding sequence for an AAT polypeptide; and (ii) a second segment comprising a reverse complement of a second coding sequence for the AAT polypeptide; (b) a Cas9 nuclease; and (c) a gRNA that targets the albumin locus at a genomic coordinate listed in Table 1.
  4. 126 . A bidirectional nucleic acid construct comprising: (a) a first segment comprising a first coding sequence for an alpha-1 antitrypsin (AAT) polypeptide; and (b) a second segment comprising a reverse complement of a second coding sequence for the AAT polypeptide, wherein the bidirectional nucleic acid construct does not comprise: (i) a promoter that drives the expression of the first coding sequence; (ii) a promotor that drives the expression of the second coding sequence; and (iii) a homology arm.
  5. 127 . The bidirectional nucleic acid construct of claim 126 , wherein the second coding sequence adopts a different codon usage from the first coding sequence.
  6. 128 . The bidirectional nucleic acid construct of claim 126 , wherein the second segment is 3′ of the first segment.
  7. 129 . The bidirectional nucleic acid construct of claim 126 , further comprising polyadenylation sequences at the 3′ end of each of the first coding sequence and the second coding sequence.
  8. 130 . The bidirectional nucleic acid construct of claim 126 , wherein the polyadenylation sequences comprise polyadenylation signal sequences or polyadenylation tail sequences.
  9. 131 . The bidirectional nucleic acid construct of claim 126 , wherein the construct comprises a splice acceptor site.
  10. 132 . The bidirectional nucleic acid construct of claim 126 , wherein the construct comprises a first splice acceptor site 5′ of the first segment and a second splice acceptor site 3′ of the second segment.
  11. 133 . The bidirectional nucleic acid construct of claim 126 , wherein the construct comprises one or more of the following terminal structures: hairpin, loops, inverted terminal repeats (ITR), or toroid.
  12. 134 . A method of expressing heterologous alpha-1 antitrypsin (AAT) in a subject, the method comprising administering to the subject: (a) the bidirectional nucleic acid construct of claim 126 ; (b) a Cas9 nuclease; and (c) a guide RNA (gRNA) that targets intron 1 of the human albumin locus, thereby expressing heterologous AAT from the albumin locus of the subject.
  13. 135 . The method of claim 134 , wherein the bidirectional nucleic acid construct is administered to the human subject in an adeno-associated virus (AAV) vector.
  14. 136 . The method of claim 135 , wherein the AAV vector is AAV2, AAV3, AAV3B, AAV5, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, or AAVLK03.
  15. 137 . The method of claim 134 , wherein the gRNA is a single guide RNA (sgRNA).
  16. 138 . The method of claim 134 , wherein the Cas9 nuclease is administered as a protein or an mRNA encoding the Cas9 nuclease.
  17. 139 . The method of claim 134 , wherein the Cas9 nuclease is an S. pyogenes Cas9 or a variant thereof.
  18. 140 . The method of claim 134 , wherein the gRNA comprises a sequence that targets the albumin locus at a genomic coordinate listed in Table 1.
  19. 141 . A vector comprising the bidirectional nucleic acid construct of claim 134 .
  20. 142 . The vector of claim 141 , wherein the vector is an AAV vector.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Continuation of U.S. application Ser. No. 16/657,967, filed on Oct. 18, 2019. U.S. application Ser. No. 16/657,967 claims the benefit of priority from U.S. Provisional Application No. 62/747,522, filed on Oct. 18, 2018. The entire contents of these applications are incorporated herein by reference in their entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Sep. 22, 2025, is named “ILH-02302.xml” and is 976,007 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein 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 leads to liver pathology due to accumulation of AAT in the hepatocytes. Thus, insufficient and improperly formed AAT caused by SERPINA1 mutation can lead to 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 with al-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. The other common mutation is the S-variant, which results in a protein that is degraded intracellularly before secretion. Compared to the Z-variant, the S-variant causes milder reduction in serum AAT and lower risk for lung disease. A need exists to ameliorate the negative effects of AATD in both the liver and lung. The present disclosure provides compositions and methods for expressing heterologous AAT at a human genomic locus, such as an albumin safe harbor site, thereby allowing secretion of heterologous AAT and alleviating the negative effects of AATD in the lung. The present disclosure also provides compositions and methods to knock out the endogenous SERPINA1 gene thereby eliminating the production of mutant forms of AAT that are associated with liver symptoms in patients with AATD. The invention combines knock out of an endogenous SERPINA1 allelle with insertion of heterologous AAT at a safe harbor site to restore AAT function in a cell or an organism. In particular, provided herein are guide RNAs for use in targeted insertion of a nucleic acid sequence encoding AAT into a human safe harbor site, such as intron 1 of an albumin safe harbor site. Also provided are donor constructs (e.g., bidirectional constructs), comprising a sequence encoding AAT, for use in targeted insertion into a human safe harbor site, such as intron 1 of an albumin safe harbor site. In some embodiments, the guide RNA disclosed herein can be used in combination with a RNA-guided DNA binding agent (e.g., Cas nuclease) and a donor construct comprising a sequence encoding AAT (e.g., bidirectional construct). In some embodiments, the donor construct (e.g., bidirectional construct) can be used with any one or more gene editing systems (