Abstract-Angiotensinogen (AGT), one of the major components in the renin-angiotensin system, has been linked to hypertension in humans and animals. We have previously systemically administered antisense oligonucleotides and plasmid vectors with DNA that targeted AGT and attenuated hypertension in spontaneously hypertensive rats. The aim of the present study was to prolong the effect of antisense treatment by the use of a recombinant adeno-associated viral (rAAV) vector targeted to AGT. Using a model of lifelong hypertension in which 5-day-old spontaneously hypertensive rats are treated, a single intracardiac injection of rAAV-AGT-antisense (rAAV-AGT-AS) delayed the onset of hypertension for 91 days and significantly attenuated hypertension in adulthood for up to 6 months. Key Words: adeno-associated virus Ⅲ hypertrophy, cardiac Ⅲ antisense Ⅲ gene therapy Ⅲ angiotensinogen A ntisense inhibition for gene therapy of hypertension is a novel approach to provide long-term control of hypertension. 1 Because the antisense strategy can target genes precisely, inhibition of specific proteins can be achieved. Although the genes involved in hypertension are largely unknown, currently used drugs control high blood pressure by inhibiting a few proteins such as angiotensin-converting enzyme (ACE), ␤ 1 -adrenoceptors (␤ 1 -AR), and angiotensin type 1 receptor (AT 1 -R). Therefore, specific gene targeting of these proteins has been our strategy with antisense oligodeoxynucleotides (AS-ODNs). Compared with current drugs, AS-ODNs are more specific and longer lasting and therefore may have certain advantages over current drug therapy. The longer duration of action could provide more consistently effective blood pressure control and thereby increase patient compliance. Despite all the drugs available, only Ϸ29% of all hypertensives have their high blood pressures controlled. Therefore, there is a need to explore a new generation of methods of treatment. Gene therapy, while oversold, still offers an approach that could produce prolonged benefits if based on scientific research. Whereas AS-ODN can reduce high blood pressure in rat models for weeks with a single dose, 1,2 even longer-lasting effects can be achieved with viral vectors. 1 Studies with the retrovirus used to deliver antisense to AT 1 -R or ACE have been shown to produce long-lasting effects in reducing high blood pressure when given in the first 5 days after birth. 3,4 Retroviruses, however, have disadvantages as a vector for therapeutic use. These include the possibility of tumorigenic activity, the vector entering the germ line, and the limitation that retroviruses only infect dividing cells. Adeno-associated virus (AAV) has the potential to provide stable, effective, and very long-lasting delivery of antisense, 5 even in nondividing cells. Because the previous studies have targeted the AT 1 -R for inhibition of the reninangiotensin system (RAS), the purpose of this study was to target angiotensinogen (AGT) mRNA with antisense and deliver it with AAV as a vector. AGT ha...

Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 +/- 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 +/- 5.2 mmHg compared with the control rats injected with saline (P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted.

Abstract.It is well known that the mammalian associate of Myc-1 (AMY-1) plays a significant role in spermatogenesis or cellular differentiation. A full-length complementary DNA (cDNA) encoding AMY-1 of Schistosoma japonicum (SjAMY-1) was identified and isolated from a cDNA library. The gene contained an open reading frame of 315 nucleotides, encoding 105 amino acids. Sequence analysis showed that SjAMY-1 shares 65.7% homology with Homo sapiens AMY-1 amino acids and contains a conserved domain from the AMY-1 family. In this study, we cloned and expressed a recombinant SjAMY-1 (rSjAMY-1) with a molecular size of 14 kDa. The native SjAMY-1 in soluble worm antigen was identified by anti-rSjAMY-1 sera in the Western blot analysis, which demonstrated the presence of this protein in the parasite. Immunofluorescence studies indicated a localization of SjAMY-1 in various tissues and organs including the tegument and subtegumental muscles in adult worms, the ventral sucker in cercariae and the internal structures of eggs. Given the key roles of mammalian AMY-1 in cell proliferation and differentiation, the characterization of SjAMY-1 may allow for a better understanding of the development of S. japonicum.

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P199 Introduction: We have previously shown that injection of antisense oligonucleotides (AS-ODNs) or plasmid producing antisense directed against angiotensinogen (AGT) mRNA, lowers BP in spontaneously hypertensive rats (SHR). The effects with ODNs, however, last ∼7 days. For more prolonged effects we have developed a recombinant adeno-associated viral vector (rAAV) to deliver AS-DNA targeted to AGT. Methods: Since captopril has been shown to inhibit development of hypertension in SHR, we hypothesized that AGT inhibition would prevent hypertension. Five day old SHR were injected with a single dose of 10 9 ipf (25 μl), intra-cardiacly, with AGT-AS-DNA (n=10) or control sense-AGT-DNA (n=6). Systolic BP was measured weekly from 6 and 26 weeks of age. At 14 weeks and 7 months, tissue DNA was analyzed for presence of the transgene. At 26 weeks, the left ventricle of the heart was dissected, weighed and compared to body weight, to determine the degree of hypertrophy (n=5/group). Every three weeks plasma was sampled for RIA of AGT. Results: The rats injected with rAAV-AGT-AS had lower BP than the control group from 10 weeks of age. The difference was significant at 10,11,12 and 13 weeks of age (p<0.05). The maximum difference was at 12 weeks (172 ± 7 for control versus 150 ± 8 mmHg for the treated group). Plasma AGT levels in the treated group did not correlate to BP, whereas plasma AGT in controls correlated to BP (p<0.05). The treated group had significantly lower BP than controls at 6 months (p<0.05). The presence of AS-transgene was confirmed by nested PCR on the DNA isolated from the heart, kidney and liver. The rAAV-AGT-AS significantly decreased the left ventricular hypertrophy measured at 26 weeks (LV/BW ratio: 3.18 ± 0.02 for control versus 2.92 ± 0.04 for the treated group, p<0.001). No signs of toxicity or body weight loss were observed. Conclusion: The study shows that AAV is a stable, effective vector to deliver AGT antisense and that AGT is an important factor in the development and maintenance of high BP in SHR. We conclude that the rAAV-AGT-AS vector shows promise for long-term attenuation of BP and heart hypertrophy.

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