Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension.

Abstract

Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT1 receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processig or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT1-receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT1 mRNA, there is efficient transfection into cells and an inhibition of AT1 receptor number. In in vivo tests, rAAV-AS AT1-receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT1-receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.