RECOMBINANT TRUNCATED DYSTROPHIN MINIGENES - CONSTRUCTION, EXPRESSION, AND ADENOVIRAL DELIVERY

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder for whi ch there is currently no effective treatment, Although clinical applic ation of adenoviral vector-mediated gene transfer has not been fully d eveloped, it shows promise for the treatment of DMD, One significant p roblem posed by adenoviral vector-mediated gene transfer for DMD is th at currently available adenoviral vectors cannot accommodate the entir e 14-kb dystrophin cDNA, To address this problem, we selectively delet ed regions of the murine dystrophin cDNA to produce truncated construc ts, We created three constructs, each with an in-frame deletion of a s egment (3.0, 4.4, and 5.7 kb) of the spectrin-like repeat region of dy strophin, As an additional modification, we removed the majority of th e 3' untranslated region of the cDNA in expression vectors encoding so me of these truncated constructs, Comparative quantitative expression studies after transfection into COS and C2C12 mouse muscle cells demon strate variations in the level of expression with different deletions in the spectrin-like repeat region, Furthermore, deletion of the 3' un translated region was tested for one recombinant construct and resulte d in a reduction in the level of expression in both cell culture syste ms, Toward the ultimate goal of gene transfer therapy for DMD, we crea ted an adenoviral vector from one of our truncated constructs, Using t his vector, we demonstrated truncated dystrophin expression in vitro i n primary mdx (dystrophin-deficient) muscle cells and in vivo in mdx m ouse muscle, In vivo, recombinant dystrophin was properly localized to the muscle membrane.