An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

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Abstract
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.