Gene editing methods for treating alpha-1 antitrypsin (AAT) deficiency
Disclosed are engineered meganucleases that bind and cleave a recognition sequence within a serine peptidase inhibitor, Clade A, Member 1 (SERPINA1) gene, which encodes alpha-1 antitrypsin (AAT). Further disclosed are donor polynucleotides that encode functional AAT proteins. The present disclosure also encompasses methods of using such engineered meganucleases and donor polynucleotides to make genetically-modified cells and use of such compositions for treatment of AAT deficiency.
1 . An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 9 within a SERPINA1 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 17-22.
2 . An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 11 within a SERPINA1 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 41-46.
3 . An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 13 within a SERPINA1 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 65-71.
4 . An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 15 within a SERPINA1 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region, wherein said engineered meganuclease comprises the amino acid sequence of any one of SEQ ID NOs: 93-100.