Surgical Approaches to Systemic Gene Transfer

描述(由申请人提供)：拟议研究的总体目标是通过解决两个基本的限速问题来改善Duchenne肌营养不良症的治疗基因转移的前景：对转基因产品的免疫力和载体传递。使用一种新描述的Duchenne肌营养不良症的犬动物模型-德国短毛指针，实验设计利用dystrophin基因的缺失来评估dystrophin和utroin的相对免疫原性。我们独家使用rAAV载体。实验设计验证了这样的假设，即在缺失的情况下，重组(犬)迷你肌营养不良蛋白将引发有害的细胞免疫反应。它进一步验证了这样一种假设，即用类似设计的犬类微量促性腺激素转基因替代将绕过这种免疫反应。基于大量的初步数据，该建议还提出了这样一种假设，即在机械循环支持期间暂时注入组胺，可以绕过系统基因传递的内皮屏障。我们提出了一系列分级的实验来解决后一种假设，从孤立的肢体灌流开始，最终到全身基因输送。这些研究还将广泛使用另一种自然发生的动物模型，即四肢带状肌营养不良的仓鼠模型。实验计划的成功完成将提供与躯体基因传递的免疫学反应相关的一般信息，并在内皮完整性发生深刻但快速可逆的变化期间保护器官功能。它还将提供关于对人类最常见的单基因致命疾病之一--杜氏肌营养不良症--的系统基因治疗策略的合理设计的具体信息。

DESCRIPTION (provided by applicant): The overall aim of the proposed research is to improve the prospects for therapeutic gene transfer in Duchenne muscular dystrophy by addressing two essential rate-limiting issues: immunity to the transgene product and vector delivery. Using a newly described canine animal model for Duchenne muscular dystrophy, the German Short Haired Pointer, the experimental design takes advantage of a deletion of the dystrophin gene to evaluate the comparative immunogenicity of dystrophin and utrophin. We make exclusive use of rAAV vectors. The experimental design tests the hypothesis that in the context of the deletion, recombinant (canine) mini-dystrophin will elicit a deleterious cellular immune response. It further tests the hypothesis that substitution of a similarly designed canine mini-utrophin transgene will circumvent this immune response. Based on extensive preliminary data, the proposal also addresses the hypothesis that the endothelial barrier to systemic gene delivery can be bypassed by temporarily infusing histamine during a period of mechanical circulatory support. We propose a graded series of experiments to address the latter hypothesis, starting with isolated limb perfusion and culminating in systemic gene delivery. These studies will also make extensive use of another naturally occurring animal model, the hamster model for limb-girdle muscular dystrophy. Successful completion of the experimental plan will provide general information relevant to the immunological response to somatic gene delivery and the preservation of organ function during profound but rapidly reversible alterations in endothelial integrity. It will also provide specific information about the rational design of strategies for systemic gene therapy in one of the most common single-gene lethal diseases in man, Duchenne Muscular Dystrophy.