Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212, USA.
Hum Gene Ther. 2012 Jan;23(1):98-103. doi: 10.1089/hum.2011.131. Epub 2011 Oct 24.
Six- to 8-kb mini-dystrophin genes are promising candidates for Duchenne muscular dystrophy (DMD) gene therapy. Several dual adeno-associated virus (AAV) mini-dystrophin vectors have been tested in dystrophin-deficient mice. Despite the encouraging preclinical results, none of the existing dual AAV vectors can restore sarcolemmal neuronal nitric oxide synthase (nNOS) expression. Localization of nNOS to the sarcolemma may greatly improve the therapeutic outcome in DMD (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635). In this study, we developed a series of dual AAV expression vectors to express a synthetic minigene that carries the nNOS localization domain. To help validate dual vector reconstitution, we also included a FLAG tag and a GFP reporter at different ends of the minigene. These dual AAV vectors were packaged in Y445F tyrosine mutant AAV-6 and tested in dystrophin-null mdx4cv mice by direct muscle injection. All dual vectors expressed GFP/FLAG-tagged mini-dystrophin and restored sarcolemmal nNOS. However, the reconstitution efficiency was significantly different among different sets. The dual vector set YZ27/YZ22 yielded the highest transduction efficiency (∼90%). Further development of this set dual vector may lead to more effective DMD gene therapy.
六到八 kb 的微小肌营养不良蛋白基因是治疗杜氏肌营养不良症 (DMD) 的有前途的基因治疗候选物。几种双腺相关病毒 (AAV) 微小肌营养不良蛋白载体已在肌营养不良蛋白缺陷型小鼠中进行了测试。尽管临床前结果令人鼓舞，但现有的双 AAV 载体都不能恢复肌膜神经元型一氧化氮合酶 (nNOS) 的表达。将 nNOS 定位于肌膜可能会极大地改善 DMD 的治疗效果 (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635)。在这项研究中，我们开发了一系列双 AAV 表达载体来表达携带 nNOS 定位结构域的合成微小基因。为了帮助验证双载体的重建，我们还在微小基因的不同末端包含了一个 FLAG 标签和一个 GFP 报告基因。这些双 AAV 载体被包装在 Y445F 酪氨酸突变型 AAV-6 中，并通过直接肌肉注射在肌营养不良蛋白缺陷型 mdx4cv 小鼠中进行了测试。所有双载体都表达 GFP/FALG 标记的微小肌营养不良蛋白并恢复了肌膜 nNOS。然而，不同组之间的重建效率有显著差异。双载体组 YZ27/YZ22 的转导效率最高（约 90%）。进一步开发这种双载体可能会导致更有效的 DMD 基因治疗。
