Over the past decade splice-blocking anti-sense oligonucleotides (SBASO) have been developed and approved for the treatment of uncommon human genetic diseases. This proposal describes a pilot study to validate the novel approach of SBASO against a novel target, myosin phosphatase (MP), as a novel therapy for US Veterans with hypertension (HTN) and its sequelae heart failure with preserved ejection fraction (HFpEF). HTN affects up to one-half of US Veterans and despite available therapies is still not optimally controlled in ~50%, accounting for much cardiovascular morbidity and mortality. HFpEF is increasing in prevalence but only a single therapy was recently shown to be modestly effective. MP by de-phosphorylating myosin causes vascular smooth muscle (VSM) relaxation; it is also the target of and integrates upstream vaso-dilator/constrictor signals thereby setting vascular tone and controlling blood pressure (BP). MP regulatory subunit Mypt1 has an alternative exon (E24) that tunes vascular smooth muscle sensitivity (VSM) to vasodilators (NO, ANP) signaling through cGMP. Skipping (exclusion) of E24 from mature Mypt1 mRNA codes for an isoform containing a C-terminal leucine zipper (LZ) motif required for cGMP-dependent kinase (PKG1 activation of MP and vasorelaxation. Inclusion of 31 nt E24 into mature Mypt1 mRNA shifts the reading frame and thus encodes an isoform of Mypt1 lacking the C-terminal LZ motif. This isoform of MP is not activated by cGMP. In small arteries of rodents and humans ~20-40% of the MP pool is the E24-/LZ+ isoform activated by cGMP signaling. HTN and HFpEF are driven by an imbalance of constrictor vs dilator signals resulting in increased systemic vascular resistance to blood flow. Here we propose to test the novel approach of treatment with SBASO to specifically suppress splicing of E24, thereby shifting expression to the naturally occurring Mypt1 E24-/LZ+ Mypt1 isoform. This will (re)-sensitize blood vessels to endogenous vasodilator signals (NO/ANP/cGMP) consequently lowering BP and improving blood flow in HTN and HFPEF. In proof-of-principle experiments we used the non-translatable Cre-Lox approach to achieve the same effect - this reduced BP in the AngII/ high salt model of HTN. Here we will test SBASOE24 against the same target. Preliminary data shows that injection of SBASOE24 is effective in suppression of E24 splicing and the hypertensive response to AngII. Experiments proposed here will validate efficacy of SBASOE24 in mouse models of HTN/HFpEF thus de-risking this product for further clinical and commercial development. Aim 1 will define the dose-response and kinetics of SBASOE24 in normal mice Mice will be administered: a) SBASOE24 over a 100-fold dose range and inhibition of the target measured by PCR/qPCR and Western blot; and b) administered the effective dose of 12.5 mg/kg qod x 3 and time to recovery to baseline of Mypt1 E24+/LZ- isoform measured over the ensuing weeks-months. Aim 2 will test SBASOE24 in ...