Abstracts

Rationale: Lafora Disease (LD) is an autosomal recessive, progressive myoclonuic epilepsy caused by mutations in the EPM2A (laforin) or EPM2B (malin) genes. The onset is typically in adolescence followed by rapid decline and death within 10 years. LD is characterized by increased glycogen levels with the accumulation of Lafora bodies (LB) which are poorly branched and insoluble glycogen in different tissues. LD mouse models exhibit similar pathologies including LBs and neurodegeneration. Glycogen Synthase 1 (Gys1) gene knockout in LD mouse results in an absence of LB formation, glycogen levels are significantly decreased, and neurodegeneration is prevented. Our objective is to evaluate the therapeutic potential of Gys1 inhibition using antisense oligonucleotides (ASO) to suppress Gys1 activity in LD mouse models. Methods: ASOs targeting mouse Gys1 were identified, characterized, and used to treat LD mice. 1, 3 or 8 months old LD mice (Epm2a-/- and Epm2b-/-) for 2, 3 and 6 months, respectively. The Gys1 and control mismatch ASOs were administered by intracerebroventricular (ICV) injection. Brain tissues from the treated mice were analyzed for Gys1 mRNA and protein levels, glycogen, LB content and histology. Results: We designed 300 anti-Gys1 ASOs, which were screened for efficacy in B16-F10 cells. The 12 most effective ASOs were tested in wild-type mice by intracerebroventricular (ICV) injection. Two ASOs, ION-648327 and ION-648402, were well-tolerated and highly effective. Their dose-dependent efficacies in the cell culture experiments had IC₅₀’s of 1.4 and 0.9 μM, respectively, and in the murine experiments they reduced brain Gys1 mRNA to 38+2% and 47+4% of PBS-injected controls, respectively. LD mice (n=6/group) treated with the Gys1 ASOs at age 1, 3 or 8 months showed a dramatic reduction in brain Gys1 mRNA (75%-90%) and protein (90%) leading to a dramatic reduction in glycogen levels in the brain. Remarkably, this attenuation of glycogen synthesis resulted in a rubust reduction of  LB formation.  Conclusions: We have demonstrated that glycogen accumulation and LB formation can be halted with anti-Gys1 therapy. Our results provide positive proof-of-concept that supports development of ASO therapy for Lafora disease that has the potential to substantially modify the course of this catastrophic, universally fatal, adolescent-onset epilepsy. Funding: This study was funded by the families of patients with LD through Chelsea's Hope (USA), Associazione Italiana Lafora, France-Lafora, Tatjana and Milana Gajic Foundation (Bosnia-Herzegovina), Genome Canada, The Ontario Brain Institute, and Ionis Pharmaceuticals. SA was supported by the Sigrid Juselius Foundation, Maire Taponen Foundation, Pikki and Sakari Sohlberg Foundation, and the Foundation for Pediatric Research.