Wong Sharon H A, Lowes Kym N, Quigley Anita F, Marotta Rosetta, Kita Magdalena, Byrne Edward, Kornberg Andrew J, Cook Mark J, Kapsa Robert M I
National Muscular Dystrophy Research Centre, Howard Florey Institute, Parkville, Victoria 3010, Australia.
Neuromuscul Disord. 2005 Oct;15(9-10):630-41. doi: 10.1016/j.nmd.2005.04.008.
Non-viral gene transfer into skeletal muscle is enhanced by electroporation and myotoxin preconditioning of muscle following plasmid injection. We investigated in vivo delivery of naked DNA to mdx mouse muscle, utilising enhanced green fluorescent protein reporter vector (pEGFP) and a corrective nucleic acid to promote targeted corrective gene conversion at the mutant mdx mouse dystrophin (DMDmdx) locus. Electroporation, myoablation with bupivacaine and a combined protocol, were applied to mdx muscle. We report up to 90% EGFP expression in electroporated mdx tibialis anterior muscle. Muscles preconditioned with bupivacaine showed low transgene expression with or without EP. Single EGFP+ve muscle fibre explants showed EGFP expression in mature fibres in preference to satellite cells. We observed a two-fold increase (P<0.005; t) in dystrophin protein, accompanied by wild-type (wt) DMD transcript in muscles injected with corrective nucleic acid over contralateral saline-injected TAs. By targeting the muscle fibres in preference to the satellite cells, plasmid-bourne transgenes delivered to dystrophic muscle will not penetrate the regenerative component of muscle. Whether in the context of targeted corrective gene conversion or therapeutic non-viral transgenes, under these conditions periodic re-administration will be required to promote phenotypic benefits in dystrophic muscle.
质粒注射后，通过电穿孔和肌肉毒素预处理可增强非病毒基因向骨骼肌的转移。我们利用增强型绿色荧光蛋白报告载体（pEGFP）和一种矫正核酸，在mdx小鼠的肌营养不良蛋白（DMDmdx）突变位点促进靶向矫正基因转换，研究了裸DNA在体内向mdx小鼠肌肉的递送情况。对mdx肌肉应用了电穿孔、布比卡因诱导的肌消融以及联合方案。我们报告在电穿孔的mdx小鼠胫前肌中，EGFP表达高达90%。用布比卡因预处理的肌肉，无论有无电穿孔，转基因表达都很低。单个EGFP阳性肌纤维外植体显示，成熟纤维中的EGFP表达优于卫星细胞。我们观察到，与对侧注射生理盐水的胫前肌相比，注射矫正核酸的肌肉中肌营养不良蛋白水平增加了两倍（P<0.005；t检验），同时伴有野生型（wt）DMD转录本。通过优先靶向肌纤维而非卫星细胞，递送至营养不良肌肉的质粒携带转基因将不会穿透肌肉的再生成分。无论是在靶向矫正基因转换还是治疗性非病毒转基因的背景下，在这些条件下都需要定期重新给药，以促进营养不良肌肉的表型改善。
