Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA.

Human, serotype 5 (Ad 5), replication-defective recombinant adenoviruses (AdVs) expressing a 6.3 kb partial dystrophin cDNA (Becker) under the control of either the CMV early or the RSV LTR promoter/enhancer in combination with various polyadenylation sequences (polyA), were developed for gene transfer studies aimed at Duchenne muscular dystrophy. Based on previous experience, a strategy for generation, screening and validation of AdVs with relatively large size gene expression cassette inserts was established. Here we focus on some aspects of stability and safety of such AdVs as gene therapeutic tools based on relevant molecular biological methods. Furthermore, the quality of our best AdV-minidystrophin construct was validated following its large scale production and purification as well as its delivery in mdx mice. These results are of interest for establishing other AdVs, where the combined length of a tissue specific promoter, the gene of interest and the polyA sequences reach the upper limit of the packaging capacity of first generation AdVs.