Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Abstract

Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3 0 untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels. Gene Therapy (2003) 10, 795-802.

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

“…21 In DM1, a strategy to remove or correct mutant mRNA is appropriate in view of evidence that the accumulation of mutant mRNA in the nucleus is the primary basis of the disease. 3,22 Approaches targeted at the mRNA level to remove mutant mRNA, namely the use of antisense, 23 nuclear-retained hammerhead ribozyme 4 and siRNA, 5 have been shown to be able to remove mutant mRNA in in vitro DM1 cells. However, the removal of wild-type mRNA together with mutant mRNA that occurs with all these techniques may have potential untoward consequences.…”

Myotonic Dystrophy: Therapeutic Strategies for the Future

“…Puymirat and colleagues 61 have tested the functional effects of CUG exp RNA degradation by antisense gene therapy. In that study, human DM1 muscle cells in culture were transfected with a retrovirus expressing antisense RNA that was designed to bind and degrade the mutant DMPK allele.…”

Myotonic Dystrophy Type 1 (DM1): From the Genetics to Molecular Mechanisms

“…Antisense RNA complementary to (CUG)13 repeat-sequence AON was employed in human DM1 myoblasts carrying ~750 CTG repeats (Furling et al, 2003), resulting in preferential decay of mutant over wild-type DMPK transcripts and consequent normalization of myoblast fusion and glucose uptake via restoration of the expression and binding activity of CELF1. Further studies with a 2'-O-Methyl-phosphorothioate-modified AON (2'-MePS-AON) that targets CUG repeats were performed in immortal mouse myoblasts expressing the human DMPK gene with 500 CTG repeats (DM500 cell model) (Mulders et al, 2009) and in two following DM1 mouse models: the first carrying the Human skeletal alpha-actin (HSA) gene modified by the insertion of 250 CTG repeats in the 3'UTR (HSA LR mouse model) and the second, bearing the human DM1 locus with 500 CTG repeats (DM500 mouse model) (Mankodi et al, 2000;Seznec et al, 2000).…”