Davis H L, Whalen R G, Demeneix B A
Physiotherapy Program, University of Ottawa, Canada.
Hum Gene Ther. 1993 Apr;4(2):151-9. doi: 10.1089/hum.1993.4.2-151.
Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.
横纹肌是唯一被发现能够摄取并表达以质粒DNA形式转入的报告基因的组织。因此，直接基因转移是一种针对原发性遗传性肌病的潜在基因治疗方法。然而，迄今为止的结果显示，其表达不足且变化太大，无法考虑在人体试验中使用直接基因转移。我们已经确定，表达的大部分变异性是由于体内注入骨骼肌的物质分布不均匀所致，并已开发出一种模型来改善这一情况。预先向肌肉注射相对大量的高渗蔗糖可改善注入物质的分布，并使荧光素酶或β-半乳糖苷酶报告基因的表达变异性显著降低；平均荧光素酶活性的变异系数从约120%降至25%。表达与剂量并非直接成正比，但如果预先用蔗糖注射肌肉，则比例关系更明显。如果以较大体积而非较小体积注射DNA，表达会更高且变异性更小。启动子的选择似乎尤为重要。来自SV40启动子的荧光素酶报告基因表达短暂且水平低，而由劳氏肉瘤病毒（RSV）启动子驱动的表达则高且持续，以至于可以观察到1000倍的表达差异。基因摄取的机制仍然未知，但我们的研究结果表明，因注射过程受损的纤维不会摄取并表达质粒DNA。
