Long-Acting RNAi Therapy for Atherosclerosis and Insulin Resistance

摘要 RNA干扰（RNAi）疗法能够使单个基因沉默，并对“坚不可摧”的基因进行药物治疗， 最近通过将小干扰RNA（siRNA）递送到肝脏用于遗传疾病而显示出临床成功。 然而，需要新的递送策略来扩大siRNA治疗的靶向可能性， 肝脏用于治疗其他疾病，如动脉粥样硬化性心血管疾病。因此，我们成立了一个 团队在siRNA递送和动脉粥样硬化方面具有互补的专业知识，并开发了靶向siRNA 沉默钙/钙调蛋白依赖性激酶-II γ（CaMKIIγ）的递送策略， 巨噬细胞的人和小鼠晚期动脉粥样硬化病变，并促进临床进展 危险的斑块我们发现，靶向siCamk 2g治疗通过减少坏死斑块的形成来改善斑块的稳定性。 核心区域和增加纤维帽厚度。尽管如此，由于siRNA介导的细胞凋亡的瞬时性， 基因沉默，siRNA治疗的关键挑战是作用持续时间短。在这个项目中，我们建议 i）探索一种新的siRNA递送策略，该策略可以显著延长CaMKIIγ沉默的持续时间， 动脉粥样硬化病变巨噬细胞;和ii）设计新的siCamk 2g平台，用于双细胞靶向， 肥胖引起的2型糖尿病和动脉粥样硬化的综合治疗。我们新的前期工作 确定了一种不同类型的合成脂质-聚乙二醇（脂质-PEG）生物材料， siRNA沉默及其血液循环。因此，我们假设新的脂质-PEG介导的长效 siCamk 2g疗法可以以低给药频率有效地靶向动脉粥样硬化和胰岛素抵抗。 目的一是合成一系列具有不同结构的脂质-PEG生物材料，系统地研究脂质-PEG生物材料的结构， 对siRNA作用持续时间和药代动力学的影响;并优化独特的siRNA递送平台 在已经建立动脉粥样硬化的小鼠模型中。巨噬细胞持续时间最长的主要候选者 将评估CaMKIIγ沉默抑制动脉粥样硬化的功效，重点是斑块 坏死、纤维帽厚度和红细胞增多症以及其他炎症消退终点。在目标2中，我们 基于以下事实，将长效siRNA疗法扩展到针对心脏代谢疾病的双细胞靶向， CaMKIIγ是肥胖诱导的胰岛素抵抗和损伤肝细胞的共同上游靶点， 巨噬细胞在动脉粥样硬化中的作用我们将在体外和体内反复优化双靶向siCamk 2g系统。 体内，包括在具有胰岛素抵抗和动脉粥样硬化组合的新小鼠模型中， 有效改善2型糖尿病，抑制动脉粥样硬化。我们期待这一工作的顺利完成， 该项目将导致对新的脂质-PEG化学如何控制siRNA递送的基本理解， 开发一种新型的长效RNAi治疗动脉粥样硬化和心脏代谢疾病。

ABSTRACT With the ability to silence individual genes and to drug the ‘undruggable’, RNA interference (RNAi) therapy has recently shown clinical success by delivering small interfering RNA (siRNA) to the liver for genetic diseases. However, new delivery strategies will be needed to expand the targeting possibilities of siRNA therapy beyond the liver for treatment of other diseases like atherosclerotic cardiovascular disease. We have therefore formed a team with complementary expertise in siRNA delivery and atherosclerosis, and developed a targeted siRNA delivery strategy to silence calcium/calmodulin-dependent kinase-IIγ (CaMKIIγ), a kinase that is activated in the macrophages of human and mouse advanced atherosclerotic lesions and promotes progression of clinically dangerous plaques. We showed that targeted siCamk2g treatment improved plaque stability by reducing necrotic core area and increasing fibrous cap thickness. Nevertheless, due to the transient nature of siRNA-mediated gene silencing, a critical challenge for siRNA therapy is the short duration of action. In this project, we propose to i) explore a novel siRNA delivery strategy that can dramatically extend the duration of CaMKIIγ silencing in atherosclerotic lesional macrophages; and ii) engineer the new siCamk2g platform for dual-cell targeting for integrated treatment of obesity-induced type 2 diabetes and atherosclerosis. Our new preliminary work has identified a distinct type of synthetic lipid-poly(ethylene glycol) (lipid-PEG) biomaterials that can markedly prolong siRNA silencing and its blood circulation. We thus hypothesize that the new lipid-PEG-mediated long-acting siCamk2g therapy could effectively target both atherosclerosis and insulin resistance with low dosing frequency. In Aim 1, we will synthesize a series of such distinct lipid-PEG biomaterials; systematically explore the lipid-PEG effects on the duration of action and pharmacokinetics of siRNA; and optimize the unique siRNA delivery platform in a mouse model with established atherosclerosis. The lead candidate with longest duration of macrophage CaMKIIγ silencing will be evaluated for efficacy in dampening atherosclerosis, with an emphasis on plaque necrosis, fibrous cap thickness, and efferocytosis and other inflammation resolution endpoints. In Aim 2, we will expand the long-acting siRNA therapy to dual-cell targeting for cardiometabolic disease, based upon the fact that CaMKIIγ is a common upstream target in both hepatocytes in obesity-induced insulin resistance and lesional macrophages in atherosclerosis. We will iteratively optimize the dual-targeting siCamk2g system in vitro and in vivo, including in a new mouse model with combined insulin resistance and atherosclerosis, in a manner to effectively improve type 2 diabetes and suppress atherosclerosis. We expect that successful completion of this project will lead to fundamental understanding of how the new lipid-PEG chemistry controls siRNA delivery and the development of a novel class of long-acting RNAi therapy for atherosclerosis and cardiometabolic disease.