Prolonged Reduction of High Blood Pressure With an In Vivo, Nonpathogenic, Adeno-Associated Viral Vector Delivery of AT ₁ -R mRNA Antisense

Abstract

To produce a prolonged decrease m blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antIsense DNA for AT1-R AAV has many advantages over other viral vectors AAV does not stimulate mflammallon or immune reaction AAV enters nondtvlchng cells and does not replicate Therefore, it IS an appropriate choice for gene therapy Recombinant AAV was prepared with a cassette contaumg a cytomegalovtrus promoter and the cDNA for the AT, receptor inserted m the annsense dire&on The cassette was packaged ohgonucleotides, directed to either AT,-R mRNA or to anglotensmogen mRNA, slgmfscantly reduce blood pressure m hypertensive animals with a single mJection mto the bram I-3 Although the adn-nmstration of antisense m the brain proved that antlsense can reduce high blood pressure of neurogemc orlgm, it obviously 1s not an acceptable route for treatment of human hypertension To demonstrate that antisense acts via a systemic route of delivery, we have shown that antisense delivered mtravenously4 or mtra-arterially5 can also reduce blood pressure m hypertensive rats. Antisense AT1 mRNA significantly decreased the blood pressure m 2&idney, 1 clip rats, m which circulating remn-Ang levels are high 4 Anglotensmogen mRNA-directed antIsense ohgonucleotide, in a hposome carrier inJected mtravenously m SHR, also decreased hypertension 5 The uptake of antisense was predommantly m the hver, as shown by fluorescent-tagged antisense. A sm-nlar approach was taken by Tomlta et al," who prepared three angiotensmogen mRNA-directed antisense ohgonucleotides and delivered them m hposomes and Sendal virus by direct mJectlon mto the hepatlc portal vem They also noted a decrease m blood pressure m SHR While these results have been encouraging for the use of antisense as a poFrom the Department of Physiology, College of Medicine, Unlversity of Florlda, Gamesvllle, Fla, and Harvard Medical School (P W ), Boston, MassCorrespondence to Dr M I Pixlhps, Department of Physiology, College of Medicine, Utuverslty of Flonda, Gamesvllle, FL 32610 E-mad MIP@phys med ufl edu 0 1997 American Heart Assoclatron, Inc tential treatment of hypertension, the maximum effectlveness of a single injection lasts for 7 days. Although this 1s Impressively longer than the response to a single dose of any antlhypertenslve drug currently available, it 1s our hope that we can extend the effectiveness of the antisense approach by delivering antisense m a viral vector that will produce a prolonged reduction m blood pressure for weeks or months There are several vu-al vectors to choose from, mcludmg retrovlruses, adenovlrus, herpes virus, polo virus, and AAV All have disadvantages and some advantages, but the AAV offers the most attractive advantages and the fewest disadvantages AAV 1s safe to use It does not induce any pathogenic response and does not replicate mslde cells The AAV 1s a defective parvovlrus and cannot replicate m cells without the presence of wild-type adenovlrus 73 The AAV IS effective as a vector because it either mt...

[1] Gene therapy for hypertension: The preclinical data

“…However, unlike the reports of the effect of retrovirus delivery of an AT 1 R-AS in 5-day-old SHR "curing" hypertension, 32 we did not find a complete inhibition of the rise in blood pressure. 31,34 In rAAV-AGT-AS-treated SHR, measures of plasma AGT levels showed a corresponding lack of increase in AGT in the antisense-treated groups compared with the significant increase of AGT in the control animals. 34 Correlation of AGT versus blood pressure was significant (PϽ0.05) in the control-treated animals and not significant in the antisensetreated animals.…”

“…Similarly, the results with the rAAV-AT 1 -AS showed a reduction in hypertension development correlated with a consistent reduction in AT 1 Rs in vascular smooth muscle cells. 31 No toxicity was noted. 33 To prove the potential therapeutic value of rAAV, we have recently used a mouse model of hypertension that clearly depends on an overactive renin-angiotensin system.…”

“…27 In summary, AS-ODNs have proved to be useful in demonstrating in the preclinical setting because of the power of AS-ODN to target specific genes and reduce blood pressure for several days (or weeks) with a single administration. Laboratory data indicate that these effects are the result of rapid uptake of the AS-ODN into cells, 31 where they migrate to the nucleus and inhibit the production of protein, mostly likely through translational inhibition of messenger RNA. 38,39 This could occur by the hybridization of ODN with specific mRNA, preventing the passage of the mRNA through the ribosome.…”

Protection From Ischemic Heart Injury by a Vigilant Heme Oxygenase-1 Plasmid System

“…Our data and results from other studies show that a single injection of rAAV (serotype 2) expresses transgenes in tissue that can last for at least 6 to 18 months. 22 …”

Antisense Oligonucleotides in the Study of Central Mechanisms of the Cardiovascular Regulation

“…The antisense expression reduced the elevated blood pressure in SHR starting at 33 days of age and persisted throughout the observation period of 78 days. Similarly, Phillips et al (1997) observed a reduction in blood pressure in adult SHR treated intracerebrally or intravascularly with a recombinant adeno-associated virus containing antisense cDNA for AT1 receptor. The decrease in blood pressure lasted at least 2 months.…”