Long-term inhibition of the central alpha 2B-adrenergic receptor gene via recombinant adeno-associated virus-delivered antisense DNA in hypertensive rats

The sympathetic nervous system (SNS) elicits its effects by controlling the release of catecholamines, whose actions are mediated by their binding to adrenergic receptors (AR). It has been demonstrated that salt-induced hypertension is mediated via the alpha2B-AR subtype. Previous studies by our laboratory in alpha2B-AR gene knockout (KO) mice established that blood pressure (BP) did not rise in these mice with salt loading as it did for the corresponding alpha2A-AR and alpha2C-AR KO mice. Additionally, in rats with salt-induced hypertension BP decreased transiently with antisense (AS) treatment targeting the alpha2B-AR gene in the central nervous system (CNS). The objective of the present experiments was to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA in order to prolong alpha2B-AR inhibition and hence reversal of salt-dependent hypertension. To do so, alpha2B-AS fragments were designed and tested in vitro for their ability to inhibit endogenous protein production. Studies demonstrated one AS fragment (432bp in length) produced the greatest decrease, 92% inhibition of alpha 2B-AR protein levels in transfected cells. To develop a delivery system that would prolong inhibition in vivo, a recombinant adeno-associated virus (rAAV) encoding the AS fragment was generated and tested in vitro to demonstrate viability and functionality. It showed 55% decrease in protein expression in infected cells. Vector preparations were injected ICV into subtotally nephrectomized, salt-fed rats. Salt-induced hypertension was demonstrated by a rise in systolic BP from a baseline of 120 +/- 10 to 184 +/- 12 mmHg. Hypertensive rats injected with rAAV-alpha 2B-AS showed an average 35 +/- 12 mmHg fall in BP, lasting without diminishing for at least 16 days, whereas control rAAV-GFP injected rats showed a continuing rise in BP. Analysis of rAAV-alpha2B-AS treated rats demonstrated 45--65% decrease in protein levels in key regulatory regions of the brain. Neither group had signs of immunological response to the virus injection. These findings indicate that the construct, when given ICV, could reach multiple sites of the central nervous system relevant to blood pressure regulation and safely inhibit for a period of weeks the central alpha 2B-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.