Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats.
BACKGROUND
Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension.


METHODS
A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group).


RESULTS
We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection.


CONCLUSIONS
These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.
Shenouda
Sherene M
SM
Hypertension and Atherosclerosis Section of the Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA. sherene@bu.edu
Johns
Conrado
C
Kintsurashvili
Ekaterina
E
Gavras
Irene
I
Gavras
Haralambos
H
D058669:Adrenergic alpha-2 Receptor Antagonists; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002490:Central Nervous System / Q000378:metabolism / Q000473:pathology; D016373:DNA, Antisense / Q000008:administration & dosage / Q000627:therapeutic use*; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006973:Hypertension / Q000628:therapy*; D007150:Immunohistochemistry; D007276:Injections, Intraventricular; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids; D051381:Rats; D017208:Rats, Wistar; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
C547772:Adra2b protein, mouse; D058669:Adrenergic alpha-2 Receptor Antagonists; D016373:DNA, Antisense; D018341:Receptors, Adrenergic, alpha-2; D049452:Green Fluorescent Proteins