Targeting Artificial Transcription Factors to the Utrophin A Promoter EFFECTS ON DYSTROPHIC PATHOLOGY AND MUSCLE FUNCTION

Duchenne muscular dystrophy is caused by a genetic defect in the dystrophin gene. The absence of dystrophin results in muscle fiber necrosis and regeneration, leading to progressive muscle fiber loss. Utrophin is a close analogue of dystrophin. A substantial, ectopic expression of utrophin in the extrasynaptic sarcolemma of dystrophin-deficient muscle fibers can prevent deleterious effects of dystrophin deficiency. An alternative approach for the extrasynaptic up-regulation of utrophin involves the augmentation of utrophin transcription via the endogenous utrophin A promoter using custom-designed transcriptional activator proteins with zinc finger (ZFP) motifs. We tested a panel of custom-designed ZFP for their ability to activate the utrophin A promoter. Expression of one such ZFP efficiently increased, in a time-dependent manner, utrophin transcript and protein levels both in vitro and in vivo. In dystrophic mouse (mdx) muscles, administration of adenoviral vectors expressing this ZFP led to significant enhancement of muscle function with decreased necrosis, restoration of the dystrophin-associated proteins, and improved resistance to eccentric contractions. These studies provide evidence that specifically designed ZFPs can act as strong transcriptional activators of the utrophin A promoter. These may thus serve as attractive therapeutic agents for dystrophin deficiency states such as Duchenne muscular dystrophy.

杜氏肌营养不良是由抗肌萎缩蛋白基因的遗传缺陷引起的。抗肌萎缩蛋白的缺失导致肌纤维坏死和再生，进而造成进行性肌纤维丧失。肌聚多糖蛋白是抗肌萎缩蛋白的一种紧密类似物。在抗肌萎缩蛋白缺陷的肌纤维的突触外肌膜中大量异位表达肌聚多糖蛋白能够防止抗肌萎缩蛋白缺陷的有害影响。一种用于肌聚多糖蛋白突触外上调的替代方法涉及使用具有锌指（ZFP）基序的定制设计的转录激活蛋白，通过内源性肌聚多糖蛋白A启动子增强肌聚多糖蛋白转录。我们测试了一组定制设计的ZFP激活肌聚多糖蛋白A启动子的能力。其中一种ZFP的表达以时间依赖性方式有效地提高了体外和体内的肌聚多糖蛋白转录本和蛋白质水平。在肌营养不良小鼠（mdx）肌肉中，给予表达这种ZFP的腺病毒载体导致肌肉功能显著增强，坏死减少，抗肌萎缩蛋白相关蛋白恢复，以及对离心收缩的抵抗力提高。这些研究提供的证据表明，专门设计的ZFP能够作为肌聚多糖蛋白A启动子的强转录激活剂。因此，它们可能作为治疗抗肌萎缩蛋白缺乏状态（如杜氏肌营养不良）的有吸引力的治疗剂。