Development of a FLP/frt system for generating helper-dependent adenoviralvectors

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequenc es have a large cloning capacity and have been reported to provide long-ter m transgene expression in vivo with negligible toxicity, making them attrac tive vectors for gene therapy. Currently, the most efficient means of gener ating HD vectors involves co-infecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by IoxP sites . Cre-mediated excision of the packaging signal renders the helper virus ge nome unpackageable but still able to replicate and to provide helper functi ons for HD vector propagation. HD vector titer is increased by serial coinf ections. Typically helper virus contamination is less than or equal to1% pr e- and less than or equal to0.1% postpurification by CsCI banding. While th ese contamination levels are low, further reduction is desirable. Alternati ve methods of selection against the helper virus may achieve this goal, esp ecially when combined with Cre/IoxP. We describe the development of a syste m for generating HD vectors based on site-specific recombination between fr t sites catalyzed by FLP recombinase and show by direct comparison that the FLP/frt and Cre/IoxP systems are equivalent with respect to HD vector ampl ification efficiency and helper virus contamination levels. Availability of a second recombinase system for HD vector production will enhance the util ity and flexibility of NO vectors.