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 adenovirus es (AdVs) expressing a 6.3 kb partial dystrophin cDNA (Becker) under t he control of either the CMV early or the RSV LTR promoter/enhancer in combination with various polyadenylation sequences (polyA), were deve loped 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 cassett e inserts was established. Here we focus on some aspects of stability and safety of such AdVs as gene therapeutic tools based on relevant mo lecular biological methods. Furthermore, the quality of our best AdV-m inidystrophin construct was validated following its large scale produc tion and purification as well as its delivery in mdx mice. These resul ts are of interest for establishing other AdVs, where the combined len gth of a tissue specific promoter, the gene of interest and the polyA sequences reach the upper limit of the packaging capacity of first gen eration AdVs. (C) 1997 Elsevier Science B.V.