Menezes Kareena M, Mok Hoyin S, Barry Michael A
Center for Cell and Gene Therapy, Baylor College of Medicine, Methodist Hospital, and Texas Children's Hospital, Houston, TX 77030, USA.
Hum Gene Ther. 2006 Mar;17(3):314-20. doi: 10.1089/hum.2006.17.314.
Gene therapy for Duchenne muscular dystrophy will likely require that the corrective dystrophin gene be delivered to a high fraction of muscle fibers in vivo. Because of the large size of the dystrophin cDNA, adenoviral (Ad) vectors have been developed for this application. However, Ad vectors transduce mature muscle inefficiently in part due to downregulation of Ad receptors on these cells. To circumvent this problem, we have tested fibroblast growth factor-2 (FGF) and insulin-like growth factor (IGF) as ligands for their ability to enhance Ad transduction of muscle cells. In this work, we demonstrate that covalent conjugation of FGF, but not IGF, to Ad5 vectors mediates substantial increases in transduction of skeletal muscle cells in vitro and dystrophic in vivo. Ad5 vectors expressing reporter genes were cross-linked to the ligands, using bifunctional polyethylene glycol (PEG) molecules. Ad-PEG-FGF mediated 1000- and 200-fold increases in transduction on C2C12 myoblasts and myotubes in vitro when compared with Ad5, Ad-PEG, or Ad-PEG-IGF. When tested in vivo in mdx mice, Ad-PEG-FGF mediated 6-fold higher transduction in skeletal muscle than unmodified Ad5. Similar results were seen when using lacZ as a reporter gene to observe transduction qualitatively. These data suggest that FGF may be a useful cell-binding ligand to enhance gene delivery by Ad and other vectors into skeletal muscle for the gene therapy of Duchenne muscular dystrophy and other muscle-related diseases.
杜氏肌营养不良症的基因治疗可能需要将矫正性肌营养不良蛋白基因在体内传递至大部分肌纤维。由于肌营养不良蛋白cDNA的尺寸较大，已开发出腺病毒（Ad）载体用于此应用。然而，Ad载体在成熟肌肉中的转导效率低下，部分原因是这些细胞上Ad受体的下调。为了解决这个问题，我们测试了成纤维细胞生长因子-2（FGF）和胰岛素样生长因子（IGF）作为配体增强肌肉细胞Ad转导的能力。在这项研究中，我们证明FGF而非IGF与Ad5载体的共价偶联介导了体外骨骼肌细胞和体内营养不良模型中转导的显著增加。表达报告基因的Ad5载体使用双功能聚乙二醇（PEG）分子与配体交联。与Ad5、Ad-PEG或Ad-PEG-IGF相比，Ad-PEG-FGF在体外C2C12成肌细胞和肌管上的转导增加了1000倍和200倍。在mdx小鼠体内进行测试时，Ad-PEG-FGF在骨骼肌中的转导比未修饰的Ad5高6倍。当使用lacZ作为报告基因定性观察转导时，也得到了类似的结果。这些数据表明，FGF可能是一种有用的细胞结合配体，可增强Ad和其他载体将基因传递到骨骼肌中，用于杜氏肌营养不良症和其他肌肉相关疾病的基因治疗。
