GENE THERAPY FOR DYSLIPIDEMIA AND ATHEROSCLEROSIS

血脂异常是早发性动脉粥样硬化性心血管疾病（ASCVD）的主要危险因素。虽然许多血脂异常患者可以用现有药物有效治疗，但其他患者没有得到有效治疗，并且仍然处于过早ASCVD的极高风险中;经典的例子是纯合子家族性高胆固醇血症（FH）。因此，我们认为， 理解肝脏对含apoB脂蛋白的分泌和催化的调节以及HDL代谢的复杂途径对于开发靶向这些途径的新疗法是非常重要的。肝脏定向体细胞基因转移是一种有用的生物学工具，解决有关生理效应的假设 在肝脏中表达apoB脂蛋白和HDL代谢的特定基因;此外，它可能是治疗严重血脂异常的策略。在这个项目中，我们将利用基于新型腺相关病毒（AAV）假型（如项目1所述）的载体进行肝脏定向基因转移，以解决与特定基因产物及其相互作用对含载脂蛋白B的肝分泌调节的影响相关的问题。这些项目旨在利用基于新型AAV假型（如AAV 2/8）的载体的实质性优势，包括静脉内给药后稳定有效地转导肝细胞，而无明显炎症。基于AAV2/8的载体在表达水平方面（在我们的经验中超过一个数量级）优于基于AAV2的载体，并且优于基于腺病毒的载体，因为缺乏炎症（与腺病毒载体的严重炎症相比）和稳定的长期表达（与腺病毒载体的瞬时表达相比）。 向量）。具体目标1。通过AAV介导的cDNA基因转移实现过表达，通过AAV介导的RNAi基因转移实现表达降低，确定肝脏DGAT 1和DGAT 2表达在影响肝脏VLDL TG和apoB产生中的作用。检验肝脏MTP表达与肝脏DGAT表达相互作用影响肝脏VLDL产生的假设。具体目标2。验证AAV介导的LDL受体（LDLR）或VLDL受体（VLDLR）过表达增加分泌前apoB降解，降低肝脏VLDL甘油三酯和apoB产生速率的假设。确定AAV介导的LDLR和VLDLR过表达是否可以使VLDL甘油三酯和apoB过度产生的小鼠模型中的VLDL产生正常化。具体目标3。在LDLR/apobec-1双敲除小鼠中比较AAV介导的LDLR和VLDLR肝脏定向基因转移对既存动脉粥样硬化进展和消退的影响。目的比较腺相关病毒介导的LDL受体和VLDL受体基因转染对WHHL兔脂蛋白代谢和动脉粥样硬化的影响。具体目标4：目的：利用腺相关病毒介导的apoA-I基因在小鼠和家兔动脉粥样硬化模型中的肝脏定向转移，检测apoA-I的长期表达是否能预防动脉粥样硬化的进展和诱导其消退。检测apoA-I的长期稳定表达是否与动脉粥样硬化进展和消退相关的长期胆固醇降低有关。本研究的总体目标是利用AAV介导的肝脏定向基因转移：1）通过基因转移介导的过表达和基因敲减方法，在体内测试与DGAT 1和DGAT 2在调节肝脏VLDL TG和apoB产生中的作用相关的特定假设; 2）比较LDLR和VLDLR的基因转移对VLDL产生、对血浆脂蛋白和对血管生成的影响。 在两种不同的FH动物模型中的动脉粥样硬化;和3）产生原理证明，即AAV介导的apoA-I至肝脏的基因转移可以在小鼠和兔中诱导动脉粥样硬化的消退和预防动脉粥样硬化的进展。该项目将产生新的生物学信息，并将在纯合子家族性高胆固醇血症和低HDL胆固醇的相关动物模型中产生原理证明，这将支持基因转移作为这些疾病的潜在治疗方法的概念。

Dyslipidemia is a major risk factor for premature atherosclerotic cardiovascular disease (ASCVD). Although many patients with dyslipidemia can be treated effectively with existing drugs, others are not effectively treated and remain at exceptionally high risk of premature ASCVD; the classic example is homozygous familial hypercholesterolemia (FH). Therefore, understanding of the regulation of the secretion and catabolism of apoB-containing lipoproteins by the liver and the complex pathways of HDL metabolism is of major importance to the development of new therapies targeted toward these pathways. Liver-directed somatic gene transfer is a useful biological tool for addressing hypotheses regarding the physiological effects of expressing specific genes in the liver on apoB lipoprotein and HDL metabolism; furthermore, it could be a strategy for treating severe dyslipidemia. In this project, we will utilize liver-directed gene transfer using vectors based on novel adeno-associated virus (AAV) pseudotypes (as described in Project 1) to address questions related to the impact of specific gene products and their interactions on the regulation of hepatic secretion of apoB-containing lipoproteins. These projects have been designed to leverage the substantial advantages of vectors based on novel AAV pseudotypes, such as AAV2/8, including the stable and efficient transduction of hepatocytes after intravenous administration without apparent inflammation. AAV2/8-based vectors are a major advance over AAV2-based vectors with regard to levels of expression (more than an order of magnitude in our experience) and over adenoviral based vectors because of the lack of inflammation (compared to severe inflammation with adenoviral vectors) and the stable long-term expression (compared to transient expression with adenoviral vectors). Specific Aim 1. To determine the roles of hepatic DGAT1 and DGAT2 expression in affecting hepatic VLDL TG and apoB production through AAV-mediated gene transfer of cDNAs to achieve overexpression through AAV-mediated gene transfer of RNAi to achieve reduction of expression. To test the hypothesis that hepatic MTP expression interacts with hepatic DGAT expression in influencing hepatic VLDL production. Specific Aim 2. To test the hypothesis that AAV-mediated overexpression of the LDL receptor (LDLR) or VLDL receptor (VLDLR) increases presecretory apoB degradation, reducing the hepatic VLDL triglyceride and apoB production rate. To determine whether AAV-mediated LDLR and VLDLR overexpression can normalize VLDL production in mouse models of VLDL triglyceride and apoB overproduction. Specific Aim 3. To compare AAV-mediated liver-directed gene transfer of the LDLR and the VLDLR in LDLR/apobec-1 double knockout mice on progression and regression of pre-existing atherosclerosis. To compare AAV-mediated gene transfer of the LDL receptor and the VLDL receptor in WHHL rabbits on lipoprotein metabolism and atherosclerosis. Specific Aim 4: To use AAV-mediated liver-directed gene transfer of apoA-I to test whether long-term expression of apoA-I can prevent progression and induce regression of atherosclerosis and in murine and rabbit models of atherosclerosis. To test whether long-term stable expression of apoA-I is additive to long-term cholesterol reduction with regard to atherosclerosis progression and regression. The overall goals of this grant proposal are to use AAV-mediated liver-directed gene transfer: 1) to test specific hypotheses related to the role of DGAT1 and DGAT2 in the regulation of hepatic VLDL TG and apoB production in vivo through gene transfer-mediated overexpression and gene knockdown approaches; 2) to compare gene transfer of the LDLR and the VLDLR with regard to effects on VLDL production, on plasma lipoproteins, and on atherosclerosis in two different animal models of FH; and 3) to generate proof-of-principle that AAV-mediated gene transfer of apoA-I to liver can induce regression and prevent progression of atherosclerosis in mice and rabbits. This project will generate new biological information and will also generate proof of principle in relevant animal models of homozygous familial hypercholesterolemia and low HDL cholesterol that would support the concept of gene transfer as a potential therapeutic approach to these disorders.