Rucker Mary, Fraites Thomas J, Porvasnik Stacy L, Lewis Melissa A, Zolotukhin Irene, Cloutier Denise A, Byrne Barry J
Powell Gene Therapy Center, and Departments of Molecular Genetics and Microbiology and Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Development. 2004 Jun;131(12):3007-19. doi: 10.1242/dev.01169.
Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid alpha-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.
通过创建基因敲除小鼠品系，已经对几种影响横纹肌的人类遗传疾病进行了建模。然而，其中许多是围产期致死性突变，会导致出生后数小时内因呼吸窘迫而死亡。由于膈肌直到出生时才收缩，膈肌活动的突然增加会对肌肉造成永久性损伤，导致其无法满足呼吸需求。因此，这些突变的影响在整个胚胎发育过程中一直被隐藏，早期死亡使研究人员无法对新生儿期之后的其他横纹肌群进行详细研究。II型糖原贮积病（GSDII）由酸性α-葡萄糖苷酶（GAA）缺乏引起，会导致所有细胞类型中糖原在溶酶体中积累，并在横纹肌中出现异常的肌原纤维生成。婴儿期发病和晚期发病的个体中，膈肌的收缩功能均受到严重影响，常因呼吸衰竭而死亡。尽管所有横纹肌群逐渐衰弱，但GSDII的基因敲除小鼠模型能存活至成年。利用该模型，我们研究了编码人GAA cDNA的重组腺相关病毒（rAAV）载体向发育中的胚胎的递送情况。结果表明，膈肌组织有特异性的高水平转导，导致活性水平比正常水平高10倍，并恢复了正常的收缩功能。在处理过的膈肌中，每个二倍体基因组中估计有多达50个载体拷贝被定量。与未处理的对照组相比，处理过的膈肌的组织学糖原染色显示，几乎所有纤维中溶酶体糖原积累均得到预防。该方法可用于特定挽救膈肌能提高存活率的疾病模型，从而进一步研究基因缺失对其他横纹肌群的影响。
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