用于评估杜氏肌营养不良症中外显子跳跃的体外分析

In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy.

作者信息

Boisguerin Prisca, O'Donovan Liz, Gait Michael J, Lebleu Bernard

机构信息

Centre de Recherche de Biochimie Macromoléculaire, UMR 5235 CNRS, Université Montpellier, Montpellier, France.

出版信息

Methods Mol Biol. 2015;1324:317-29. doi: 10.1007/978-1-4939-2806-4_20.

DOI:10.1007/978-1-4939-2806-4_20

PMID:26202278

Abstract

Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity. This chapter provides details of experimental procedures to monitor exon skipping efficiency and cellular trafficking of Pip6a-PMO, a recently developed and particularly efficient conjugate, in skeletal H2k cells and in primary cardiomyocytes from mdx mice. Similar procedures may be used in principle to evaluate any free or vector-associated oligonucleotide for exon skipping.

摘要

细胞穿透肽（CPP）介导的磷二酰胺吗啉代寡聚物（PMO）递送可实现高效的外显子跳跃，并且作为杜氏肌营养不良症（DMD）的潜在治疗方法已展现出巨大前景。然而，不同CPP之间以及在递送至不同组织时，效率存在很大差异。细胞运输似乎是活性的一个重要决定因素。本章详细介绍了监测Pip6a-PMO（一种最近开发且特别有效的缀合物）在骨骼肌H2k细胞和mdx小鼠原代心肌细胞中外显子跳跃效率和细胞运输的实验程序。原则上，类似的程序可用于评估任何游离或载体相关的寡核苷酸的外显子跳跃情况。