Coupled Gene Delivery and Protein Transduction for the Reduction of Atheroscleros

描述（由申请人提供）：拟议的研究将开发一种新的治疗方法，作为降低动脉粥样硬化危险因素LDL的原理证明。大约10%有心血管和外周血管疾病风险的人群由于不良的副作用或有限的反应而无法接受当前降脂疗法的治疗剂量。肥胖和II型糖尿病的临床管理面临着由肝内脂质动员进入血液循环引起的肝纤维化和动脉粥样硬化风险引起的并发症。编辑酶（apobec1）基因治疗通过表达和分泌含极低密度脂蛋白（VLDL）的载脂蛋白48诱导人肝脏载脂蛋白B (apoB) mRNA编辑和降低血清LDL的理论价值早已被认识。目前面临的挑战是如何在治疗性地递送apobec1的同时，将酶活性不调节引起的副作用风险降到最低。通过TAT对apobec1的蛋白转导，在原代肝细胞中实现了可控的编辑活性。一种新的酶替代疗法已经被设计出来，涉及原位产生apobec1嵌合体，这种嵌合体从产生细胞分泌，只有在转导到邻近的肝细胞后才能激活apoB mRNA编辑。受感染肝细胞分泌APOBEC限制了其在产生细胞中的积累，消除了过度编辑造成脱靶效应的风险。嵌合apobec1构建体将优化体外分泌转导，并在急性动物研究中评估其治疗效果。具体目的是：(1)构建APOBEC嵌合体，优化其在受体细胞中的分泌、转导和诱导活性；(2)将优化后的TAT-APOBEC嵌合体基因转染到rAAV2载体中，用于体外和体内感染肝细胞；(3)在实验动物中验证其分泌转导策略及其安全性和对血清脂蛋白的影响。

DESCRIPTION (provided by applicant): The proposed research will develop a novel therapeutic using as proof of principle the reduction of the atherogenic risk factor, LDL. Approximately 10% of the population at risk for developing cardiovascular and peripheral vascular diseases is unable to take therapeutic doses of current lipid lowering therapies due to undesirable side effects or limited response. Clinical management of obesity and type II diabetes faces complications due to hepatosteatosis and the atherogenic risk associated with lipid mobilization from the liver into the circulation. The theoretical value of editing enzyme (APOBEC-1) gene therapy for the induction of apolipoprotein B (apoB) mRNA editing in human liver and reduction of serum LDL through the expression and secretion of apoB48-containing very low-density lipoproteins (VLDL) has long been recognized. The challenge has been how to deliver APOBEC-1 therapeutically while minimizing the risk of side effects due to unregulated enzyme activity. Controlled editing activity has been achieved in primary hepatocytes through protein transduction using TAT on APOBEC-1. A novel approach for enzyme replacement therapy has been devised involving production of APOBEC-1 chimeras in situ that are secreted from producer cells and activate apoB mRNA editing only after they transduce into neighboring hepatocytes. Secretion of APOBEC by infected hepatocytes limits its accumulation in the producing cells, eliminating the risk of off-target effects from excess editing. Chimeric APOBEC-1 constructs will be optimized in vitro for secretion-transduction and their therapeutic benefit assessed in acute animal studies. The Specific Aims are: (1) construction of a APOBEC chimera optimized for secretion, transduction and induction of activity in recipient cells; (2) transfer of the gene for the optimized TAT-APOBEC chimera into an rAAV2 vector for infection of hepatocytes in vitro and in vivo, and (3) demonstration of the secretio transduction strategy in experimental animals and its safety and affect on serum lipoproteins. Public Health Relevance: The biomedical impact of the proposed research is the development of a novel gene therapeutic approach for the safe and effective replacement of enzyme activity. The proposed research uses as proof- of-principle the induction of apolipoprotein B mRNA-editing in liver by the delivery of the enzyme APOBEC-1 in order to produce a lipid carrying protein that will reduce the atherogenic risk factor in serum known as LDL cholesterol. In addition, this therapeutic approach will satisfy the current need for interventions that can reduce fatty liver disease without resulting in elevated serum LDL and atherogenic risk.