ANTISENSE INHIBITION AND ADENOASSOCIATED VIRAL VECTOR DELIVERY FOR REDUCING HYPERTENSION

Antisense oligodeoxynucleotides have been designed to inhibit the prod uction of specific proteins. In models of hypertension, we have target ed the renin-angiotensin system at the level of synthesis (angiotensin ogen) and the receptor (AT(1) receptor). The design of antisense oligo nucleotides requires choosing a site to inhibit mRNA processing or tra nslation. The strategy we use is to make three oligonucleotides of ant isense sequences, upstream and downstream from the AUG site and over t he AUG site. The oligonucleotides are tested in a screening test. Anti sense oligonucleotides to AT(1)-receptor mRNA and to angiotensinogen m RNA reduce blood pressure in spontaneously hypertensive rats when inje cted into the brain. They significantly reduce the concentration of th e appropriate protein. The oligonucleotides are also effective when ad ministered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To pr olong the action, direct injection of naked DNA and injection of DNA i n liposome carriers have been tested. Viral vectors have been develope d to deliver antisense DNA. The viral vectors available include retrov iruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safet y because it is nonpathogenic, has longevity because it integrates int o 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 AT( 1) mRNA, there is efficient transfection into cells and an inhibition of AT(1) receptor number. In in vivo tests, rAAV-AS AT(1)-receptor whe n injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT(1)-receptor decreas es blood pressure and slows the development of hypertension. While fur ther experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasib ility of AAV as a vector for antisense inhibition, which may ultimatel y be used in gene therapy for hypertension.