HEART-SPECIFIC TARGETING OF BETA-GALACTOSIDASE BY THE VENTRICLE-SPECIFIC CARDIAC MYOSIN-LIGHT-CHAIN-2 PROMOTER USING ADENOVIRUS VECTORS

Adenoviruses are attractive vectors for gene transfer into cardiac mus cle. However, their promiscuous tissue tropism, which leads to an ecto pic expression of the transgene, is a considerable limitation. To rest rict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250 beta gal and AdMLC2-2100 beta gal) containi ng the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promot er (MlLC-2v), Our in vitro and in vivo data have evidenced that the 21 00-bp promoter allows stronger P-galactosidase activity than the 250-b p promoter and that the deleted promoter allows a weak P-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2V promoter of 250 pb con served its heart specificity in in ovo and in vivo when introduced int o the adenovirus genome, indicating that the specificity of this promo ter is neither altered by the inverted terminal repeat nor by the enha ncer of the Ela promoter, both of which located in the 5' flanking reg ion of the promoter, Systemic injections of both recombinant adenoviru ses into chicken embryos showed P-galactosidase expression mainly in t he right ventricle of the heart. We have confirmed the cardiac specifi city of both promoters in mammalian species after injection of both re combinant adenoviruses into the heart of adult rats in vivo. The compa rison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2V promoter is 80% less active than the 2100-bp MLC-2V promoter a nd has enabled us to conclude that the MLC-2v promoter of 2100 bp is t he most appropriate for efficient expression of a reporter gene or a t herapeutic cardiac gene (e,g,, SERCA2a or minidystrophin gene).