Phd in INNOVATIVE BIOMEDICAL TECHNOLOGIES IN CLINICAL MEDICINE

Thesis title: CRISPR/Cas9 technology for the deletion of (CTG)n pathological expansion in Myotonic Dystrophy type 1: in vitro characterization and in vivo application in a mouse model of the disease

Myotonic Dystrophy Type 1 (DM1), the most common form of muscular dystrophy worldwide, is an autosomal dominantly inherited disease caused by the abnormal expansion of the CTG repeat tract in the 3’ UTR of the DMPK gene. The molecular effector of the disease is the DMPK mutant transcript that accumulates into nuclear foci and sequesters RNA-binding proteins involved in the regulation of RNA splicing. Currently, different therapeutic approaches for DM1 are under investigation in preclinical studies, exploiting new strategies based on the administration of different molecules such as drugs, newly designed or repurposed, antisense oligonucleotides and short interfering RNAs. Nevertheless, these approaches yield only temporary effects and require repeated administration of the therapeutic molecules. At present, there is no long-term curative treatment for DM1. CRISPR/Cas9 technology has been shown to act directly on the genome to definitively correct the genetic defects in some inherited diseases. In this project we explored the possibility to apply this gene editing strategy for the treatment of DM1, employing the enhanced specificity Cas9 nuclease from Streptococcus pyogenes (eSpCas9) and two guide RNAs (sgRNAs) targeting sequences upstream and downstream the CTG repeats to elicit their permanent excision from the genome. First, we characterized the outcome of gene editing in vitro in DM1 patient-derived myogenic cells using a doxycycline-inducible CRISPR/Cas9 system to temporally control the activity of Cas9 with the aim of limiting undesired effects, such as the occurrence of off-target events. Once assessed the efficiency and precision of the treatment in vitro, we tested our inducible system in vivo in the DM1 mouse model DMSXL using a combination of two different adeno-associated viruses (AAVs) for the delivery of eSpCas9 and the two inducible sgRNAs, injected either locally or systemically. We found that our inducible system was effective in eliciting the removal of CTG repeats in different tissues but very inefficient in promoting the molecular rescue, evaluated as reduction of nuclear foci and amelioration of alternative splicing defects. Hence, we considered new strategies to improve editing efficiency and attain the recovery of the molecular alterations, focusing on cardiac and skeletal muscle tissues. To this aim, we selected a new couple of sgRNAs, utilized self-complementary AAV genomes for sgRNAs expression and employed the recently developed myotropic AAVs with enhanced tissue tropism for striated muscles. DMSXL mice were injected intraperitoneally at two different ages (P5 and P24) and the molecular analysis revealed that the treatment at P24 with the new sgC3/G4 couple was more effective in eliciting foci number reduction and splicing amelioration. Moreover, the treatment resulted effective in leading to a long-lasting increment of body weight in sgC3/G4-treated mice at both ages of injection, while partial results were obtained in recovery of muscular strength. Overall, these data show the feasibility of the gene editing approach in DM1 therapy and its effectiveness in eliciting both molecular and phenotypic rescue. However, in view of a future application in DM1 patients, new methods of delivery of the CRISPR-Cas9 system have to be evaluated to enhance editing efficiency in vivo and a deeper insight into DNA-repair mechanisms is needed to precisely control gene editing outcomes.