Guanine Analogues Enhance Antisense Oligonucleotide-induced Exon Skipping in Dystrophin Gene In Vitro and In Vivo

Abstract

Exon skipping has demonstrated great potential for treating Duchenne muscular dystrophy (DMD) and other diseases. We have developed a drug-screening system using C2C12 myoblasts expressing a reporter green fluorescent phosphate (GFP), with its reading frame disrupted by the insertion of a targeted dystrophin exon. A library of 2,000 compounds (Spectrum collection; Microsource Discovery System) was screened to identify drugs capable of skipping targeted dystrophin exons or enhancing the exon-skipping effect by specific antisense oligomers. The 6-thioguanine (6TG) was effective for inducing skipping of both human dystrophin exon 50 (hDysE50) and mouse dystrophin exon 23 (mDysE23) in the cell culture systems and increased exon skipping efficiency (more than threefolds) when used in combination with phosphorodiamidate morpholino oligomers (PMO) in both myoblasts and myotubes. Guanine and its analogues were unable to induce detectable skipping of exon 23 when used alone but enhanced PMO-induced exon skipping significantly (approximately two times) in the muscles of dystrophic mdx mouse in vivo. Our results demonstrate that small-molecule compounds could enhance specific exon skipping synergistically with antisense oligomers for experimental therapy to human diseases.

Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice

“…All injections were performed under isoflurane anesthesia, and all efforts were made to minimize suffering. 3,5,6,18 animals and intramuscular injections Dystrophic mdx mice aged 4-5 weeks were used for in vivo testing (five mice each in the test and control groups) unless otherwise stated. The PMOE23 (5′-GGCCAAACCTCGGC TTACCTGAAAT-3′) targeting the boundary sequences of exon and intron 23 of the mouse dystrophin gene (GeneTools) was used.…”

“…Protein extraction and Western blot were performed as described previously. 3,10,18 Briefly, the membrane was probed with NCL-DYS1 monoclonal antibody against dystrophin rod domain (1:200 dilution, Vector Laboratories, Burlingame, CA, USA) followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (1:3,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL™ Western blotting analysis system (Perkin-Elmer, Waltham, MA, USA). The intensity of the bands with appropriate size was measured and compared with that from normal muscles of C57BL mice using ImageJ software.…”

“…Expression of GFP was controlled by effective skipping of the inserted human dystrophin exon 50 sequence (hDysE50) or mouse dystrophin exon 23 sequence (mDysE23). 18 The C2C12E50 cell line was maintained in 10% fetal bovine serum-DMEM in a humidified 10% CO 2 incubator at 37°C. Approximately 5×10 4 C2C12E50 cells per well in 500 µL of 10% fetal bovine serum-DMEM medium were seeded and allowed to grow until confluence of 70%.…”

“…3,6 Furthermore, the complicated synthesis and purification in modification increases the costs significantly, and potential immune responses to the targeting peptide could prevent repeated administration. The nonvirus-mediated delivery strategy remains attractive because of the vector's structural flexibility chosen from synthetic or natural compounds, capacity for delivery of larger therapeutic agents, ease of handling, greater safety, and less expense than viral vectors, [18][19][20] especially for those commercial available compounds that have been widely applied in the field of environmental biology and biotechnology.…”

Duchenne Muscular Dystrophy: Therapeutic Approaches to Restore Dystrophin

“…Many approaches have been tested. Cell penetrating peptides (Jearawiriyapaisarn et al, 2008;Jearawiriyapaisarn et al, 2010; www.intechopen.com al., 2008; Yin et al, 2008), muscle targeting peptides connected to cell penetrating peptides (Yin et al, 2009) and guanidine analogs (Hu et al, 2010;Wu et al, 2009) showed the most promising results in the mdx mouse and in the hDMD mouse model (Wu et al, 2011a). Notably, pPMOs (containing arginine-rich peptides covalently bound to the morpholino AONs) have shown great potential in the mdx mouse, inducing high levels of exon skipping in skeletal muscles and heart and high levels of dystrophin rescue.…”