Takeda Shin'ichi
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo.
No To Hattatsu. 2004 Mar;36(2):117-23.
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene. Although an adeno-associated virus (AAV) vector-mediated gene transfer provides an attractive approach to the treatment of DMD, limitation in insertion size up to 4.9 kb excludes incorporation of a full-length dystrophin cDNA (14 kb) into an AAV vector. We previously generated micro-dystrophin transgenic dystrophin-deficient mdx mice. In 4.9 kb rod-truncated micro-dystrophin CS1 transgenic mdx mice, dystrophic phenotypes were ameliorated almost completely (Biochem Biophys Res Commun 2002; 293:1265-72). We therefore constructed an AAV vector expressing micro-dystrophin CS1 driven by a skeletal muscle-specific MCK promoter, as the expression of the LacZ gene driven by the MCK promoter is longer in an AAV vector than in the CMV promoter in the skeletal muscle (Gene Ther 2002; 9:1576-88). We injected the AAV-MCK delta CS1 into the anterior tibial (TA) muscles of 5-week-old mdx mice, which exhibit active cycles of muscle degeneration/regeneration. At 8 weeks after the AAV vector injection, a large percentage of fibers were dystrophic-positive (10 to 50%). Even 24 weeks after injection, 15 to 75% of myofibers expressed micro-dystrophin. Dystrophin-positive fibers often had centrally located nuclei in the mice, however, the ratio was significantly reduced compared with that of dystrophin-negative fibers. We also measured tetanic force of AAV-MCK delta CS1-treated and non-treated mdx TA to evaluate functional amelioration. Non-treated mdx TA muscles showed marked reduction of specific tetanic force, while AAV-injected muscles showed moderate improvement. In conclusion, our study demonstrated that introduction of delta CS1 micro-dystrophin with an AAV vector successfully protected mdx muscles from progressive dystrophic degeneration.
杜兴氏肌营养不良症（DMD）是一种由肌营养不良蛋白基因突变引起的X连锁致死性肌肉疾病。尽管腺相关病毒（AAV）载体介导的基因转移为DMD的治疗提供了一种有吸引力的方法，但插入大小限制在4.9 kb以内排除了将全长肌营养不良蛋白cDNA（14 kb）整合到AAV载体中的可能性。我们之前构建了微肌营养不良蛋白转基因肌营养不良蛋白缺陷型mdx小鼠。在4.9 kb杆状截短的微肌营养不良蛋白CS1转基因mdx小鼠中，营养不良表型几乎完全得到改善（《生物化学与生物物理研究通讯》2002年；293:1265 - 72）。因此，我们构建了一种由骨骼肌特异性MCK启动子驱动表达微肌营养不良蛋白CS1的AAV载体，因为由MCK启动子驱动的LacZ基因在AAV载体中的表达在骨骼肌中比在CMV启动子中持续时间更长（《基因治疗》2002年；9:1576 - 88）。我们将AAV - MCKδCS1注射到5周龄mdx小鼠的胫前（TA）肌肉中，这些小鼠表现出活跃的肌肉退化/再生周期。在注射AAV载体8周后，很大比例的纤维呈肌营养不良蛋白阳性（10%至50%）。即使在注射24周后，15%至75%的肌纤维表达微肌营养不良蛋白。然而，在小鼠中，肌营养不良蛋白阳性纤维的细胞核通常位于中央，但其比例与肌营养不良蛋白阴性纤维相比显著降低。我们还测量了经AAV - MCKδCS1处理和未处理的mdx TA的强直力，以评估功能改善情况。未处理的mdx TA肌肉显示出特定强直力的显著降低，而注射AAV的肌肉则显示出适度改善。总之，我们的研究表明，用AAV载体导入δCS1微肌营养不良蛋白成功地保护了mdx肌肉免受进行性营养不良性退化。
