Proviral plasmids and production of recombinant adeno-associated virus

US9896665B2 · kind B2 · utility

Assignee

• The Trustees of the University of Pennsylvania · US

Inventors

• Jean Bennett · Via Enrico Fermi, US
• Jeannette Bennicelli · Philadelphia, US

Key dates

• Filing date: Dec 28, 2015
• Grant date: Feb 20, 2018
• Expiry date: Dec 28, 2035

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N2830/90
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Proviral plasmids contain a modular gene expression cassette with one or a combination of (i) a wildtype 5′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the ITR; (ii) a promoter flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence; (iii) a polylinker sequence that permits insertion of a gene coding sequence without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, the aforementioned promoter; (iv) a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of the polyA sequence; and (v) a wildtype 3′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3′ ITR. These plasmids enable rapid manipulation of the components of the cassette, such as rapid mutation and/or replacement of any component, and thereby increase the efficiency of recombinant viral vector, such as rAAV, production.