Antisense inhibition of AT1 receptor in vascular smooth muscle cells using adeno-associated virus-based vector.

Vascular smooth muscle cells (VSMCs) are the main peripheral target for vasoconstriction and growth-promoting activity of angiotensin II (Ang II), acting through angiotensin type 1 receptors (AT1-R). Current antihypertension treatments include daily reductions in the effects of Ang II. To decrease an effect of Ang II in a prolonged fashion, we have developed an adeno-associated virus (AAV) vector with antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV is nonpathogenic, does not stimulate inflammation or immune reaction and enters nondividing cells, and provides stable long-term gene expression. To test AAV in VSMCs, we constructed and tested plasmid AAV (pAAV) and recombinant AAV (rAAV) with AT1-R antisense DNA. rAAV was constructed with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1-R inserted in the antisense direction. The cassette was packaged into the virion. Transfection of VSMCs with the pAAV antisense to AT1-R produced a significant reduction in the amount of AT1-R (P<0.01). Transduction of VSMCs with the rAAV-AT1-R-AS at MOI of 5 also showed significant reduction of AT1-R and long-lasting expression of the transgene for at least 8 weeks. The reduction of AT1-R number in VSMCs was concomitant with a decrease in the Ang II-stimulated increase of intracellular calcium. The results show that AAV vector delivers AT1-R antisense to inhibit AT1-R in VSMCs. For the purpose of gene therapy for hypertension, it is necessary to demonstrate the effectiveness of a vector system in VSMCs. This study provides support for the potential use of AAV AT1-R antisense in VSMCs.