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天服用多种药物的降压方案，副作用频繁。因此，只有少数高血压患者可以恢复正常血压，只有副作用较少的长期、有针对性的降压治疗才能从根本上影响西方世界的高血压流行水平。这项研究旨在通过使用腺相关病毒载体(含有增强型平滑肌特异性启动子(EnSM221))来携带针对血管L型(CaV1.2)钙通道的外源microRNA来开发一种新的、长期的高血压治疗方法。值得注意的是，CaV1.2通道已经成为临床用来降低血压的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的传递来实现对钙通道表达的长期和血管特异性的抑制。我们的目标包括：1)证明CaV1.2通道microRNA在高血压小鼠中至少有8周的降压能力，并且没有严重的副作用；2)在体内和体外证实CaV1.2通道的血管收缩功能在小的肠系膜动脉受到抑制；3)证实血管CaV1.2通道表达的减少，全细胞钙电流，以及肠系膜动脉细胞K+通道表达的恢复和静息膜电位的恢复与AAV传递的CaV1.2通道microRNA的降压作用相关。这项研究的结果将有助于为使用靶向AAV和microRNA技术治疗短期治疗效果不佳的系统性高血压和其他慢性血管异常奠定基础。 与公共健康相关：被称为高血压的高血压困扰着6000多万美国人，并可能导致更令人虚弱的疾病。虽然有许多短效药物(包括钙通道阻滞剂)来治疗高血压，但大多数患者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了充分的控制。我们已经开发出一种基于小RNA的治疗药物(MicroRNA)，它可以减少血管中发现的钙通道的表达，这也是所有抗高血压钙通道阻滞剂的作用部位。当整合到安全的腺相关病毒中时，我们的治疗方法可以在几个月内持续产生更多的microRNA。此外，我们的治疗方法与DNA序列相连，该DNA序列将仅在构成血管壁的血管平滑肌细胞中选择性地产生所需的microRNA。这可能会减少许多口服降压药的副作用，这些药物会扩散到全身。一种持续时间更长、副作用更少的疗法可能对4000万高血压没有得到适当控制的美国人非常有利。

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.