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

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. baculovirus ͉ gene therapy

Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

“…HEK 293-or HeLa-derived C12 cells were cultivated as adherent monolayers at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 2.5 g/liter glucose, 100 g/ml streptomycin, 100 U/ml penicillin (PAA), and 10% (vol/vol) fetal calf serum (FCS) (Gibco). Sf9 cell lines for recombinant AAV (rAAV) production expressing Rep and Cap of various serotypes were described previously (20). Sf9 cells were maintained in suspension culture under constant agitation with serum-free Spodopan medium (Pan-Biotech) supplemented with 200 g/ml streptomycin, 200 U/ml penicillin, and 250 ng/ml amphotericin B (Invitrogen) at 27°C.…”

“…Sf9 cell-derived AAV rep-cap-expressing cell lines (20,22) were continuously held in suspension culture within the logarithmic growth phase prior to infection with the recombinant baculovirus Bac-rAAV-GFP (multiplicity of infection [MOI] ϭ 5). The infected cells were incubated for 72 h at 27°C under constant agitation.…”

OneBac 2.0:Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

“…Upon infection the integrated rep and cap genes are highly amplified for efficient gene expression. 16 For AAV cap expression in insect cells, the individual VP proteins are translated from a single transcript. VP1 is initiated at a mutated start codon (ACG), while VP2 and VP3 are translated from their authentic start codons to yield the prototypic 1:1:10 ratio.…”

Large-Scale Adeno-Associated Viral Vector Production Using a Herpesvirus-Based System Enables Manufacturing for Clinical Studies

“…Yields reported using baculoviral helpers are comparable to HSV complementation, with successful baculovirus production of rAAV scale-up to 10-, 40-, and 100-liter bioreactors accomplished (Negrete et al, 2007a,b;Cecchini et al, 2009). The method has been improved by Zolotukhin and colleagues by developing packaging cell lines that contain AAV rep and cap integrated into the host insect cell chromosome, permitting multiple serotypes to be produced by a single baculovirus infection of serotype-specific cell lines (Aslanidi et al, 2009). The final hurdles for baculovirus rAAV manufacture needing to be addressed are the apparent instability of the baculoviral constructs during serial passage amplification and the significantly higher particle-to-infectivity ratios reported (>1000) as compared with HSV-derived rAAV.…”