MicroRNA to decrease vascular CaV1.2 in hypertension

描述（申请人提供）：有许多短效药物可用于治疗高血压。然而，大多数患者未能坚持每日多药抗高血压方案，副作用频繁。因此，只有少数高血压患者的血压恢复正常，并且只有副作用较少的长期靶向抗高血压治疗才能从根本上影响西方世界高血压的流行水平。本提案中的研究旨在通过使用含有增强型平滑肌特异性启动子（EnSM 221）的腺相关病毒（AAV）载体来递送针对血管L型（CaV 1.2）钙通道的外源性microRNA，从而开发一种新的长期治疗高血压的方法。值得注意的是，CaV1.2通道已经是用于降低血压的临床钙通道阻断药物的靶点。然而，我们的策略将使用AAV介导的CaV1.2通道microRNA的递送来实现钙通道表达的长期和血管特异性敲低。我们的目标包括：1）证明CaV1.2通道microRNA在高血压小鼠中降低血压至少8周而没有严重副作用的能力，2）在体内和体外证实CaV1.2通道的血管收缩功能在小肠系膜动脉中受到抑制，和3）证明血管CaV1.2通道表达、全细胞Ca 2+电流、并且肠系膜动脉细胞中K+通道表达和静息膜电位的恢复与由AAV递送至脉管系统的CaV1.2通道microRNA的抗高血压作用相关。这项研究的结果将有助于为使用靶向AAV和microRNA技术治疗短期治疗控制不佳的全身性高血压和其他慢性血管异常奠定基础。 公共卫生相关性：被称为高血压的高血压困扰着超过6000万美国人，并可能导致更虚弱的状况。虽然有许多短效药物（包括钙通道阻滞剂）治疗高血压，但大多数患者并没有忠实地服用药物，因此只有不到三分之一的患者血压得到充分控制。我们已经开发出一种基于小RNA的治疗剂（microRNA），它可以降低血管中钙通道的表达，而血管中钙通道也是所有抗高血压钙通道阻滞剂的作用部位。当与安全的腺相关病毒结合时，我们的治疗剂可以在数月内持续产生更多的microRNA。此外，我们的治疗剂与DNA序列偶联，该序列将选择性地仅在构成血管壁的血管平滑肌细胞中产生所需的microRNA。这可能会减少许多口服抗高血压药物的副作用，这些药物会扩散到全身。一个更持久的治疗，副作用更少，可能是非常有益的>40万美国人的高血压没有得到适当的管理。

DESCRIPTION (provided by applicant): There are many short-acting drugs available to treat hypertension. However, most patients fail to adhere to a daily, multi-drug antihypertensive regimen with frequent side effects. Thus, normal blood pressure is restored in only a minority of hypertensive patients, and only long-term, targeted antihypertensive therapies with fewer side effects will fundamentally impact the epidemic levels of hypertension in the Western world. The studies in this proposal seek to develop a novel, long-term therapy for hypertension by using an adeno-associated viral (AAV) vector containing an enhanced smooth muscle specific promoter (EnSM221) to deliver exogenous microRNA directed against vascular L-type (CaV1.2) calcium channels. Notably, CaV1.2 channels already are the target of clinical calcium channel blocking drugs used to lower blood pressure. However, our strategy will use AAV-mediated delivery of CaV1.2 channel microRNA to enable long- term and vascular-specific knockdown of calcium channel expression. Our aims include: 1) to demonstrate the ability of CaV1.2 channel microRNA to reduce blood pressure for at least 8 weeks without serious side effects in hypertensive mice, 2) confirm in vivo and in vitro that the vasoconstrictor function of CaV1.2 channels is suppressed in small mesenteric arteries, and 3) demonstrate that decreases in vascular CaV1.2 channel expression, whole-cell Ca2+ current, and restoration of K+ channel expression and resting membrane potential in mesenteric arterial cells correlate with the antihypertensive effect of CaV1.2 channel microRNA delivered by AAV to the vasculature. The results of this study will help to establish a foundation for using targeted AAV and microRNA technology to treat systemic hypertension and other chronic vascular abnormalities that are poorly controlled by short-term therapies. PUBLIC HEALTH RELEVANCE: High blood pressure known as hypertension afflicts over 60 million Americans and can lead to even more debilitating conditions. While there are numerous short-acting medications (including calcium channel blockers) to treat hypertension, most patients do not take their medications faithfully and consequently less than one third of patients have their blood pressure adequately controlled. We have developed a small RNA-based therapeutic (microRNA) which decreases expression of a calcium channel found in blood vessels that is also the site of action of all antihypertensive calcium channel blockers. When incorporated into a safe adeno associated virus, our therapeutic can continuously produce more microRNA over a period of months. In addition our therapeutic is coupled to a DNA sequence that will selectively generate the desired microRNA only in vascular smooth muscle cells that make up the wall of blood vessels. This is likely to reduce side effects encountered with many orally administered antihypertensive medications, which spread throughout the body. A longer lasting therapy with fewer side effects may be extremely beneficial for the >40 million Americans whose hypertension is not properly managed.