Knockdown of DMPK in myotonic dystrophy type 1 using Cas13

Genetically, myotonic dystrophy type 1 (DM1) is caused by a pathological CTG-repeat expansion in the 3′UTR of the DMPK gene. The mRNA containing the extended repeat is described to be toxic and forms foci with RNA-binding proteins like MBNL1-3. Here, our objective is to employ Cas13b-mediated cleavage to knock down the DMPK in DM1 patient muscle cells. Primary myoblasts from controls and DM1 patients were immortalized using lentiviral transduction of CDK4 and hTERT to generate comparable cell lines. Immortalized myoblasts were transduced with adeno-associated virus (AAV) vectors containing Cas13b and DMPK specific gRNAs against (i) total DMPK and (ii) specific isoforms containing the repeat expansion. The latter approach aims to allow expression of DMPK isoforms not containing the repeat expansion. RT-qPCR was employed to assess knockdown efficiency. Following this, RNA sequencing results are analyzed using bioinformatic tools to capture and interpret transcriptomic changes. Here we present preliminary results of the Cas13 approach to target DMPK mRNA showing knockdown efficiencies and RNA-sequencing results. The RNA-sequencing results show that the Cas13b-mediated DMPK knockdown leads to a partial rescue of the DM1-relevant transcriptomic changes. The Cas13-mediated knockdown of DMPK has the potential to achieve target-specific and highly efficient results. Unlike lentivirus-mediated delivery, combining Cas13b treatment with AAV ensures safer delivery, potentially minimizing off-target effects by avoiding permanent genomic alterations. Multiple Cas13b gRNAs are under testing to enhance knockdown efficiency, with subsequent in vivo experiments planned in mouse models.