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速率限制问题：对转基因产物的免疫力和载体递送，来改善治疗性基因转移在杜氏肌营养不良症中的前景。采用一种新描述的杜氏肌营养不良犬动物模型——德国短毛犬，实验设计利用了肌营养不良蛋白基因的缺失来评估肌营养不良蛋白和肌营养不良蛋白的比较免疫原性。我们只使用rAAV载体。实验设计测试了在缺失的情况下，重组（犬）微型肌营养不良蛋白会引起有害的细胞免疫反应的假设。它进一步验证了一种类似设计的犬微型营养因子转基因的替代将绕过这种免疫反应的假设。基于广泛的初步数据，该建议还提出了一个假设，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，从孤立的肢体灌注开始，最终达到全身基因传递。这些研究还将广泛使用另一种自然发生的动物模型，即四肢带状肌肉萎缩症的仓鼠模型。实验计划的成功完成将提供有关体细胞基因传递的免疫反应和在内皮完整性发生深刻但迅速可逆的改变时器官功能的保存的一般信息。它还将提供关于合理设计系统性基因治疗策略的具体信息，以治疗人类最常见的单基因致死性疾病之一杜氏肌营养不良症。

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.