Brolin Camilla, Shiraishi Takehiko, Hojman Pernille, Krag Thomas O, Nielsen Peter E, Gehl Julie
Center for Experimental Drug and Gene Electrotransfer (CEDGE), Department of Oncology, Copenhagen University Hospital Herlev, Denmark.
Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Denmark.
Mol Ther Nucleic Acids. 2015 Dec 1;4(12):e267. doi: 10.1038/mtna.2015.41.
Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.
肽核酸（PNA）是一种合成的DNA模拟物，已显示出发现新型剪接转换反义药物的潜力。然而，体内细胞递送一直是其开发的限制因素，仅有少数成功的研究报道。作为提高体内细胞可用性的一种可能方式，我们研究了电转染对体内肌肉注射（i.m.）PNA给药的影响。将靶向小鼠肌营养不良蛋白基因外显子23的反义PNA通过i.m.注射给予正常NMRI小鼠和营养不良的mdx小鼠的胫前肌（TA），注射时伴有或不伴有电穿孔。在低剂量、单次PNA给药（1.5、3或10μg/TA）时，在优化电参数后，7天后测量发现，电转染使健康小鼠肌肉中未修饰PNA诱导的反义外显子跳跃增加了两倍至四倍。单剂量治疗后长达4周在RNA水平检测到PNA剪接转换。在mdx小鼠的营养不良肌肉中，与仅注射相比，单次PNA给药后2周，电转染使肌营养不良蛋白阳性纤维数量增加了约2.5倍。总之，我们发现电转染可增强肌肉组织中PNA的反义作用。
