Heart-Specific Targeting of β -Galactosidase by the Ventricle-Specific Cardiac Myosin Light Chain 2 Promoter Using Adenovirus Vectors

Abstract

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250βgal and Ad-MLC2-2100βgal) containing the β-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger β-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak β-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 conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the E1a promoter, both of which located in the 5′ flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed β-galactosidase expression mainly in the right ventricle of the heart. We have confirmed th cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison 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 and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

Overview summary

We have evaluated the potential of replication-defective adenoviruses to restrict expression in the cardiac muscle through the use of cardiac-specific promoters. We have constructed two adenoviruses harboring the β-galactosidase reporter gene under the control of two different versions of the rat ventricle-specific cardiac myosin light chain 2 promoter (MLC-2v) of 2100 and 250 bp. We have shown in this work that a highly deleted MLC-2v promoter cloned into the adenovirus genome retains its cardiac specificity in ovo and in vivo. Systemic injections of both recombinant adenoviruses into chicken embryos showed β-galactosidase expression mainly in the right ventricle, with an efficient gene transfer into myocardial cells. We have confirmed the ventricular specificity of both promoters in mammalian species after injection in vivo of both recombinant adenoviruses into the heart of adult rats. Nevertheless, our in vitro and in vivo data have shown that the 2100-bp promoter is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene.