Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development

Bo Wu,Peijuan Lu,Allen Zillmer,Ehsan Benrashid,Caryn Cloer,Qi Long Lu,Mona Shaban,Gisela Nogales-Gadea

doi:10.1371/journal.pone.0019906

Abstract

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/mdx mouse, a transgenic model carrying the full-length human dystrophin gene with mdx background, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD/mdx mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD/mdx mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

Highlights

Antisense therapy has been shown promising for the treatment of Duchenne muscular dystrophy (DMD) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
The efficiency of human dystrophin exon 50 (hE50) skipping with the Phosphorodiamidate morpholinos (PMO) chemistries was consistent to the 2’O methyl phosphorothioate (2OMePS) oligomers of the same sequences and the highest efficiency was obtained in the cells treated with hE50AO12PMO, hE50AO5PMO, hE50AO23PMO, hE50AO13PMO, hE50AO 6PMO and hE50AO24PMO
No increases in muscles damage, inflammatory cellular infiltrations, or necrotic fibers were observed in the muscles treated by any of the Vivo-PMOs when compared to the PMO (Figure 4C). These results suggest that Vivo-PMOs can achieve high levels of human dystrophin exon skipping in the muscle of the hDMD/mdx mice without notexin pre-treatment which has been required for inducing human dystrophin exon skipping with untagged PMO and 2OMePS [26]

Summary

Introduction

Antisense therapy has been shown promising for the treatment of Duchenne muscular dystrophy (DMD) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Phosphorodiamidate morpholinos (PMO) have been reported with higher efficiency and functional levels of dystrophin production in body-wide skeletal muscles in mdx mice and dystrophic dog [6,10]. We and others demonstrated that arginine-rich peptides and dendrimeric octaguanidine conjugated morpholino (PPMO and Vivo-PMO) are able to restore the reading frame and produce almost normal levels of dystrophin protein in body-wide skeletal and cardiac muscles in the mdx mice [7,8]. The applicability of AO therapy has been indicated in Phase I/II clinical trials with both 2OMePS AOs and PMO targeting human dystrophin exon 51 in DMD patients [13,16]. Patients after systemic administration of 2OMePS AO (PRO051) targeting human dystrophin exon 51 have been reported with improvement in muscle function [16]

Methods

Results

Conclusion