NOVEL MUSCLE SPECIFIC VECTOR FOR GENE THERAPY OF ACID MALTASE DEFICIENCY

溶酶体酸性麦芽糖酶（GAA）的遗传缺陷导致糖原 贮藏病II型（GSD-II）或酸性麦芽糖酶缺乏。婴儿- 发病患者（庞贝氏症）表现为大量积聚 糖原在心脏和骨骼肌，导致死亡的第一个 年的生活。相比之下，成人发病的患者有表现 仅限于骨骼肌。目前，没有治疗或治愈方法 治疗疾病基因治疗影响心脏和 骨骼肌或器官（如GSD-II）可能非常困难 因为大小、位置和转染效率。纳入不断 在正常小鼠和受影响的牛模型中的研究，我们利用 携带巨细胞病毒（CMV）启动子和人GAA的载体 cDNA与脂质体复合的DNA结合，试图纠正 缺损我们证明了质粒被组织吸收， 包括骨骼肌和心肌，在一次 输液然而，没有控制或调节过度表达， 这个系统可能。因此我们建议发展肌肉- 将控制过表达特异性调节载体系统 和组织特异性。 我们将构建一系列向量 含有巨细胞病毒或肌酸激酶或重链肌球蛋白 启动子-增强子，加上一个原核调控元件， 在多西环素（Dox）存在下的“关闭”转录。我们将 将人GAA cDNA亚克隆到每个载体中。我们也会检查 将小基因导入人GSD-II成纤维细胞和肌肉细胞，加上 鼠和人组织培养细胞，包括肌肉、皮肤和 淋巴样细胞细胞将在有和没有各种水平的情况下培养 关于Dox人GAA的转录、翻译和表达将是 通过GAA酶活性、GAA蛋白质（火箭） 免疫电泳（RIE）和Western分析和人GAA mRNA， 北方分析。实验室将用于寡核苷酸 合成、DNA测序、DNA分离、RNA分离、PCR、北方 Southern分析、超电泳和重组DNA 技术.

Inherited deficiency of lysosomal acid maltase (GAA) results in glycogen storage disease type II (GSD-II) or acid maltase deficiency. Infantile- onset patients (Pompe's disease) present with massive accumulation of glycogen in cardiac and skeletal muscle, resulting in death in the first year of life. In contrast, adult-onset patients have manifestations limited to skeletal muscle. Currently, there is no treatment or cure for the disease. Gene therapy for diseases that affect cardiac and skeletal muscles or organs (such as GSD-II) may be very difficult because of size, location and transfection efficiency. In ongoing studies in normal mice and in an affected bovine model, we have utilized a vector carrying the cytomegalovirus (CMV) promoter and the human GAA cDNA combined with liposome-complexed DNA in attempts to correct the defect. We demonstrated that the plasmid is taken up by tissues, including skeletal and heart muscle, for three months after a single infusion. However, no control or regulation for over-expression is possible with this system. We therefore propose to develop a muscle- specific regulatory vector system that will control for over-expression and tissue specificity. We will construct a series of vectors containing the cytomegalovirus or creatine kinase or heavy chain myosin promoters-enhancers, plus a prokaryotic regulatory element that "turns off" transcription in the presence of doxycycline (Dox). We will subclone the human GAA cDNA into each vector. We will also transfect the minigene(s) into human GSD-II fibroblast and muscle cells, plus murine and human tissue culture cells, including muscle, skin and lymphoid cells. Cells will be cultured with and without various levels of Dox. Human GAA transcription, translation and expression will be assessed by GAA enzyme activity, GAA protein by rocket immunoelectrophoresis (RIE) and Western analysis and human GAA mRNA by Northern analysis. The laboratory will be used for oligonucleotide synthesis, DNA sequencing, DNA isolation, RNA isolation, PCR, Northern and Southern analysis, ultracentrifugation, and recombinant DNA techniques.