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. 2024 Jul;81(7):1491-1499.
doi: 10.1161/HYPERTENSIONAHA.124.22878. Epub 2024 May 1.

Counteracting Angiotensinogen Small-Interfering RNA-Mediated Antihypertensive Effects With REVERSIR

Dien Ye #  1 , Edwyn O Cruz-López #  1 , Richard van Veghel  1 , Ingrid M Garrelds  1 , Anne Kasper  2 , Kelly Wassarman  2 , Ho-Chou Tu  2 , Ivan Zlatev  2 , A H Jan Danser  1
Affiliations

Counteracting Angiotensinogen Small-Interfering RNA-Mediated Antihypertensive Effects With REVERSIR

Dien Ye et al. Hypertension. 2024 Jul.

Abstract

Background: Small-interfering RNA (siRNA) targeting hepatic AGT (angiotensinogen) mRNA depletes AGT, lowering blood pressure for up to 6 months. However, certain situations may require a rapid angiotensin increase. The REVERSIR (RVR) - reverse siRNA silencing technology a potential approach to counteract siRNA effects.

Methods: Spontaneously hypertensive rats received 10 mg/kg AGT siRNA, and 3 weeks later were given AGT-RVR (1, 10, or 20 mg/kg). One week after AGT-RVR dosing, a redose of AGT siRNA assessed its post-AGT-RVR effectiveness for 2 weeks. Additionally, the impact of AGT-RVR after an equihypotensive dose of valsartan (4 mg/kg per day) was examined.

Results: Baseline mean arterial pressure (MAP) was 144±1 mm Hg. AGT siRNA reduced MAP by ≈16 mm Hg and AGT by >95%, while renin increased 25-fold. All AGT-RVR doses restored MAP to baseline within 4 to 7 days. Notably, 10 and 20 mg/kg restored AGT and renin to baseline, while 1 mg/kg allowed ≈50% AGT restoration, with renin remaining above baseline. A second AGT siRNA treatment, following 1 mg/kg AGT-RVR, reduced MAP to the same degree as the initial dose, while following 10 mg/kg AGT-RVR, it resulted in ≈50% of the first dose's MAP effect at 2 weeks. The valsartan-induced MAP reduction was unaffected by AGT-RVR.

Conclusions: In spontaneously hypertensive rats, angiotensinogen-RVR dose-dependently reversed AGT siRNA-induced AGT reduction, normalizing MAP. MAP normalization persisted even with 50% recovered AGT levels, likely due to upregulated renin maintaining adequate angiotensin generation. Post-AGT-RVR dosing, a second AGT siRNA dose lowered MAP again.

Keywords: RNA; angiotensinogen; blood pressure; hypertension; renin-angiotensin system.

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Conflict of interest statement

Disclosures A. Kasper, K. Wassarman, H.-C. Tu, and I. Zlatev are employees of Alnylam Pharmaceuticals. A.H. Jan Danser received a grant from Alnylam Pharmaceuticals, which has partially supported this work. The other authors report no conflicts.

Figures

Figure 1.
Figure 1.
AGT (angiotensinogen)-RVR reverses the antihypertensive and AGT-lowering effect of AGT small-interfering RNA (siRNA) in spontaneously hypertensive rats. A, Experimental set up. B, Mean arterial pressure (MAP) and delta MAP. C, Heart rate. D, Activity. E, Plasma AGT. F, Plasma renin. Data are mean±SEM of n=6–7. REVERSIR (RVR) indicates reverse siRNA silencing. *P<0.05, **P<0.01, ****P<0.0001 vs vehicle; ##P<0.01, ###P<0.001, ####P<0.0001 vs indicated group.
Figure 2.
Figure 2.
Reintroducing AGT (angiotensinogen) small-interfering RNA (siRNA) after AGT-RVR lowers blood pressure and AGT again in spontaneously hypertensive rats. A, Experimental set up. B, Mean arterial pressure (MAP), and delta MAP. C, Heart rate. D, Activity. E, Plasma AGT. F, Plasma renin. Data are mean±SEM of n=4. REVERSIR (RVR) indicates reverse siRNA silencing. &P<0.05, &&&P<0.001 vs indicated group.
Figure 3.
Figure 3.
AGT (angiotensinogen) mRNA and protein expression in liver and kidney of spontaneously hypertensive rats treated with AGT small interfering RNA (siRNA) and AGT-RVR. A, Hepatic AGT mRNA levels (normalized vs β-actin and β2-microglobulin). B, Renal AGT mRNA levels. C, Hepatic AGT protein levels (normalized vs GADPH [glyceraldehyde-3-phosphate dehydrogenase]). D, Renal AGT protein levels. E, Western blotting images of AGT in liver and kidney. Data are mean±SEM of n=6–7. ND indicates not done; and REVERSIR (RVR) indicates reverse siRNA silencing. ***P<0.001, ****P<0.0001 vs vehicle; ####P<0.0001 vs indicated group.
Figure 4.
Figure 4.
AGT (angiotensinogen) mRNA and protein expression in the liver and kidney of spontaneously hypertensive rats receiving a second dose of AGT small interfering RNA (siRNA) after AGT-RVR. A, Hepatic AGT mRNA levels (normalized vs β-actin and β2-microglobulin). B, Hepatic AGT protein levels (normalized vs GADPH [glyceraldehyde-3-phosphate dehydrogenase]). C, Renal AGT protein levels. D, Western blotting images of AGT in liver and kidney. Data are mean±SEM of n=4–7. REVERSIR (RVR) indicates reverse siRNA silencing. ****P<0.0001 vs vehicle; &P<0.05 indicated group.
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