NOVEL MUSCLE SPECIFIC VECTOR FOR GENE THERAPY OF ACID MALTASE DEFICIENCY

遗传性溶酶体酸性麦芽糖酶（GAA）缺乏导致糖原累积病II型（GSD-II）或酸性麦芽糖酶缺乏。婴儿型患者（庞贝氏病）表现为心肌和骨骼肌中糖原的大量积累，导致出生后第一年死亡。相比之下，成人发病患者的表现仅限于骨骼肌。目前，没有治疗或治愈这种疾病的方法。影响心脏和骨骼肌或器官（如GSD-II）的疾病的基因治疗可能是非常困难的，因为大小，位置和转染效率。在正常小鼠和受影响的牛模型中正在进行的研究中，我们利用携带巨细胞病毒（CMV）启动子和人GAA cDNA与脂质体复合DNA相结合的载体，试图纠正缺陷。我们证明了质粒被组织吸收，包括骨骼肌和心肌，在单次输注后持续三个月。然而，用该系统不可能控制或调节过表达。因此，我们建议开发一种肌肉特异性调控载体系统，将控制过表达和组织特异性。 我们将构建一系列载体，其中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，以及在强力霉素（Dox）存在下关闭转录的原核调控元件。我们将人GAA cDNA亚克隆到每个载体中。我们还将小基因转染入人GSD-II成纤维细胞和肌肉细胞，以及鼠和人组织培养细胞，包括肌肉、皮肤和淋巴细胞。将在有和没有各种水平的Dox的情况下培养细胞。通过GAA酶活性评估人GAA转录、翻译和表达，通过火箭免疫电泳（RIE）和Western分析评估GAA蛋白，通过北方分析评估人GAA mRNA。该实验室用于寡核苷酸合成、DNA测序、Southern分析、ELISA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 rocketimmunoelectrophoresis (RIE) and Western analysis and human GAA mRNA by Northern analysis. The laboratory was used for oligonucleotide synthesis, DNA sequencing, Southern analysis, ELISA, and recombinant DNA techniques.