Long-Acting RNAi Therapy for Atherosclerosis and Insulin Resistance

摘要 RNA干扰(RNAi)疗法能够沉默单个基因，并用药物治疗“无法下药”的药物。 最近通过将小干扰RNA(SiRNA)输送到肝脏治疗遗传病，显示出临床上的成功。 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 在siRNA传递和动脉粥样硬化方面具有互补专业知识的团队，并开发了靶向siRNA 沉默钙/钙调蛋白依赖性激酶-IIγ(CaMKIIγ)的传递策略 人和小鼠巨噬细胞加重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siCamk2g治疗通过减少坏死灶改善了斑块的稳定性。 核心区和增加纤维帽厚度。然而，由于siRNA介导的瞬时性质， 基因沉默，siRNA治疗的一个关键挑战是作用时间短。在这个项目中，我们建议 探索一种新的siRNA传递策略，该策略可以显著延长CaMKIIγ沉默的持续时间 动脉粥样硬化性病变巨噬细胞；以及ii)设计新的siCamk2g平台，用于双细胞靶向 肥胖型2型糖尿病和动脉粥样硬化的综合治疗。我们新的前期工作已经 鉴定了一种独特的合成脂质-聚乙二醇类生物材料，这种材料可以显著延长 SiRNA沉默及其血液循环。因此，我们假设新的脂质-聚乙二醇介导的长效 SiCamk2g治疗能有效地针对动脉粥样硬化和胰岛素抵抗，且给药频率低。 在目标1中，我们将合成一系列不同的脂质-聚乙二醇生物材料；系统地探索脂质-聚乙二醇 对siRNA作用时间和药代动力学的影响；并优化独特的siRNA传递平台 在已经建立的动脉粥样硬化的小鼠模型中。巨噬细胞持续时间最长的候选人 CaMKIIγ沉默在抑制动脉粥样硬化方面的有效性将被评估，重点是斑块。 坏死、纤维帽增厚、泡沫化和其他炎症消退终点。在目标2中，我们将 根据以下事实，将长效siRNA疗法扩展到心脏代谢疾病的双细胞靶向治疗 CaMKIIγ是肥胖诱导的胰岛素抵抗和皮损性肝细胞共同的上游靶点 动脉粥样硬化中的巨噬细胞。我们将在体外和体内迭代优化双靶向siCamk2g系统 体内，包括在胰岛素抵抗和动脉粥样硬化合并的新小鼠模型中，以一种方式 有效改善2型糖尿病，抑制动脉粥样硬化。我们期待着这一工作的圆满完成 该项目将导致对新的脂类-聚乙二醇化学如何控制siRNA传递和 开发一类针对动脉粥样硬化和心脏代谢性疾病的长效RNAi疗法。

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.