Cas-13 mediated DMPK-knockdown as a novel treatment approach for myotonic dystrophy type 1

• Applicant: Dr. Peter Meinke
• Subject Area: Human Genetics ; Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 470092532

Myotonic dystrophy (DM) is a multisystemic disorder with predominant myotonia and muscular dystrophy, which brands DM as the most frequent muscular dystrophy in adulthood. DM1 is caused by CTG-repeat expansions in the 3’ UTR of the DMPK gene. Mutant RNA (containing the extended CUG-repeats) forms hairpin structures which sequester several RNA binding proteins into so-called foci. This results in several molecular pathologies including alternative splicing and alternative polyadenylation, altered transcriptional regulation, inhibited translation, miRNA misregulation, and repeat associated non-ATG (RAN) translation (resulting in the presence of toxic peptides). Several therapeutic approaches have been tried but were not developed into therapies yet due to several reasons like inadequate biodistribution, reduced degradation of toxic RNA, or off-target effects. To overcome these problems I am planning to use Cas-13 for post-transcriptional silencing of DMPK. The recently described Cas-13 enzyme has the ability to specifically bind and cut RNA sequences and has been shown to have a high knockdown efficiency. The approach to use Cas-13 for DMPK knockdown promises a higher specificity of knockdown than antisense-oligonucleotides paired with high efficiency. My preliminary data do show indeed a very effective DMPK knockdown and a loss of foci in cell culture. To overcome the problem of inadequate biodistribution I will use adeno-associated virus (AAV)-based delivery. AAVs are largely non-integrative, low immunogenic, and non-pathogenic and are already successful in use for the treatment of different diseases. I plan to screen a set of different guide RNAs for the Cas-13 enzyme to achieve (i) the optimal knockdown and (ii) an isoform specific knockdown, which would still allow the expression of functional DMPK but eliminate all CUG-repeat containing transcripts. This will be of interest as the expendability of human DMPK protein hasn’t been proven yet. The effect of the knockdown on the molecular pathology will be determined using established readouts in cell culture. I will further test for any off-target effects in cell culture. The best performing guide RNAs will be used to test the efficacy in an established mouse model for DM1. This approach has the potential to be directly developed into a therapy for DM1.

• DFG Programme: Research Grants