573. Enhanced Level of Gene Correction Mediated by Oligonucleotides Containing CpG Modification in the mdx Mouse Model for Duchenne Muscular Dystrophy

Abstract

Single point mutations and large deletions of the dystrophin gene are responsible for Duchenne muscular dystrophy (DMD) a severe neuromuscular disorders characterized by complete absence of dystrophin expression in skeletal muscles. Gene editing mediated by single stranded oligonucleotides (ssODNs) represents an appealing option to treat the disease since it has the potential to treat both single point mutations as well as deletions that cause frame shift of the dystrophin mRNA. The major limitation of the technology has been so far the low level of genomic correction detected in muscle cells. We have focused on the development of new vectors capable to activate specific repair mechanisms and capable of directing the repair process specifically on the sequence of the genomic DNA targeted for correction. The methyl binding protein 4 (MBD4) takes an active role in the base excision repair mechanism and is highly expressed in muscles. In addition to containing a binding site for the methylcytosine, MBD4 contains also a specific glycosilase capable of recognizing a T to G transversion at CpG sites and direct the conversion of the thymine into methylcytosine. CpG modifications were introduced on the mutating base of the targeting oligonucleotide in the attempt to mimic a deamination of methylcytosine which results in the activation of MBD4. All the studies were conducted using the mdx mouse as a model for DMD. This strain contains a stop codon in exon 23 of the dystrophin gene that is responsible for the absence of dystrophin protein in skeletal muscles. As a target for the single base substitution we have chosen the splice boundary of exon 23 of the mouse dystrophin gene in order to induced exon skipping to bypass the nonsense mutation and induce expression of internally deleted but functional dystrophin proteins. We have designed ssODNs complimentary to the coding or the non-coding strand of the donor site of exon 23. CpG modifications were introduced at the targeted base. The ability of these modified ssODNs to increase gene repair was studied in muscle cells both in vitro and in vivo. The level of dystrophin protein expression was significantly increased by the use of ssODNs containing CpG modifications on the targeting base. Studies conducted on muscle cells in culture demonstrate up- regulation of MBD4 mRNA and the activation of the base excision repair mechanism through which MBD4 acts, demonstrating the specificity of the repair process recruited by the ssODNs. Correction of the dystrophin gene was shown to occur at the genomic level and was stable over prolonged periods of time. In muscle cells in culture, restoration of dystrophin expression was analyzed at the protein level by western blot and immunostaining analysis and at the mRNA level by RT-PCR. In vivo analysis also showed restoration of dystrophin in skeletal muscles of injected mice by immunostaining. These studies demonstrate that the efficacy of oligonucleotide- mediated gene correction can be increased by improving oligonucleotide design.