An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells.
Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa californica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system.
Aslanidi
George
G
Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.
Lamb
Kenneth
K
Zolotukhin
Sergei
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000096:biosynthesis*; D007313:Insecta; D008969:Molecular Sequence Data