Myotonic dystrophy (DM) is the second most common muscular dystrophy occurring with a frequency of 1:8,000 individuals. DM is a progressive adult muscle disease marked by muscle stiffness (myotonia) and atrophy, intellectual decline, gastrointestinal dysfunction, cataracts and heart conduction defects. The molecular basis of myotonic dystrophy type 1 or 2 (DM1 or DM2) is a microsatellite repeat expansion of CTG or CCTG in the dystrophia myotonica protein kinase gene, DMPK, or the CCHC-type zinc finger nucleic acid binding protein gene (CNBP/ZNF9), respectively. Remarkably, this is a toxic RNA gain-of- function disease in which the repeat-expanded RNA (RER) is expressed from either the 3'UTR of DMPK or intron 1 of CNBP. The RER is trapped in the nucleus and sequesters the splicing factor muscleblind 1 (MBNL1). Significant defects in pre-mRNA splicing accumulate in over 200 different mRNAs that are the targets of MBNL1 processing activity due to MBNL1 sequestration. Dysregulation of splicing results in fetal isoform expression, leading to truncation or loss of proteins due to stop codon introduction. In this manner, seemingly unrelated phenotypes manifest from a common defect in the splicing machinery. To develop a therapy to treat DM1 we use an RNA interference (RNAi) approach to target the DMPK RER, so that the phenotypic changes downstream of the RER expression will be mitigated and potentially reversed in ongoing disease. In proof-of-principle studies we have shown in vivo efficacy in the HSALR mouse model of DM1 with adeno-associated virus (AAV) delivery of genes that carry out RNAi-mediated RER destruction. Since our initial studies, gene silencing efficiency was augmented through elimination of toxicity related to reporter gene expression. More recently we have constructed 37 RNAi expression cassettes to test target engagement of the disease-relevant DMPK mRNA in vitro. Our goal is to screen for DMPK-RNAi gene silencing of DMPK in DM1 myogenic cell lines with delivery of plasmids containing the 37 DMPK RNAi expression cassettes. In parallel we will test several tissue-specific expression cassettes to optimize activity in muscle and minimize expression in cell types that could cause immune clearance of the in vivo AAV- DMPK-RNAi vector. The most effective RNAi hairpins identified in vitro will be incorporated into AAV for local muscle injection in the DMSXL mouse model for target engagement and knockdown of the DMPK RER. Systemic injection of the DMSXL hemizygous mice will determine the therapeutic potential of AAV- DMPK-RNAi through striated muscle cell target engagement bodywide and assessment of safety in the DMPK mRNA expression context. Future therapy development would include screening a variety of AAV serotypes for more widespread delivery in vivo to optimize reversal of the multisystem disease phenotypic manifestations of DM1. If successful for treatment of DM1, this approach could be applied to reduction of the CNBP RER for development of a DM2 therapy and for treatment of other dominant muscle diseases.
The goals of this project will provide the basis for a treatment for myotonic dystrophy type I and establish a platform for treatment of additional dominant genetic diseases, where there is a need to reduce disease- causing mRNA or to block its translation into protein.
