Cell Culture Models for Testing Dystrophobic Muscle Gene Therapy

该项目提出了一系列的体内实验，以测试和提高肌肉特异性表达的病毒载体交付的微型，迷你，和全长肌营养不良蛋白，以及各种补偿蛋白，将病毒交付到mdx 4cv小鼠的营养不良的肌肉。已经设计了不同大小和表达能力的调节盒，以便最佳地与不同大小的治疗性cDNA一起包装到AAV、慢病毒和腺病毒载体中。该提案中描述的体内研究对于建立调控基因盒的组织特异性、改善其表达水平、提高其生物学活性和生物学活性是至关重要的。 慢肌纤维中的转录活性，用于确保免疫系统抗原递呈细胞不表达调控盒，并确定调控盒是否长时间维持高表达水平。在确定了表达微小、微小和全长肌营养不良蛋白的盒功能的充分性之后，将使用最有活性和组织特异性的调节盒来过表达单个或多个补偿蛋白，例如肌营养不良蛋白、α 7-整联蛋白、GalNac转移酶-2、 α-肌营养不良短蛋白和肌营养不良蛋白-糖蛋白复合物的其他成员，以及测试dysferlin和其他候选蛋白的有益功能，其补偿特性尚未被检查。这些研究的总体假设是，虽然没有单一的治疗性蛋白质-特别是如果载体限制要求其以微形式递送-可以治愈DMD，但部分替代或规避肌营养不良蛋白功能方面的蛋白质的组合过表达，以及其缺失的修复后果，可以很好地改善肌肉变性的进展，并且可以改变DMD的病程，使得疾病表型类似于轻度形式的贝克尔肌营养不良症。这 这对患有严重DMD的人来说是一个重大的改善。

This project proposes a series of in vivo experiments to test and improve the muscle-specific expression of viral vector-delivered micro-, mini-, and full-length dystrophin, as well as a variety of compensatory proteins which will be virally delivered to the dystrophic muscles of mdx4cv mice. Regulatory cassettes of different sizes and expression capabilities have been designed so as to be optimal for packaging into AAV, Lentiviral, and Adenoviral vectors together with therapeutic cDNAs of different sizes. The in vivo studies described in this proposal are critical for establishing the tissue specificity of regulatory gene cassettes, for improving their transcriptional activity in slow muscle fibers, for assuring that the cassettes are not expressed by immune system antigen presenting cells, and for determining whether regulatory cassettes maintain high expression levels for prolonged periods of time. After establishing the adequacy of cassette function in the expression of micro-, mini-, and fuIl-length dystrophin, the most active and tissue-specific regulatory cassettes will be used to over-express single or multiple compensatory proteins such as utrophin, alpha7-integrin, GalNac transferase-2, alpha-dystrobrevin and other members of the dystrophin-glycoprotein complex, as well as to test the beneficial function of dysferlin and other candidate proteins whose compensatory properties have yet to be examined. The overall hypothesis of these studies is that while no single therapeutic protein- especially if vector constraints require its delivery in a micro-form -- may cure DMD, the combinatorial over-expression of proteins that partially substitute for or circumvent aspects of dystrophin's function, and that repair consequences of it absence, may well ameliorate the progression of muscle degeneration, and may change the course of DMD such that the disease phenotype resembles mild forms of Becker muscular dystrophy. This would represent a major improvement for persons with severe DMD.