(F-228) Utilization of EPM2A as a Target for Gene Replacement Therapy in Lafora Disease

Introduction:: Lafora Disease (LD) is a monogenic neurodegenerative disorder affecting adolescents and is associated with mutations in either EPM2A or EPM2B genes which encode for laforin and malin respectively. In the absence of either protein, there is an excessive accumulation of poorly branched glycogen which become aggregated and are called lafora bodies (LB). These lafora bodies are formed in neurons and glial cells and drive neurodegeneration and neuroinflammation. Following the recent success of gene therapy for treating spinal muscular atrophy (SMA), the objective of this project is to determine the feasibility of gene replacement therapy as a treatment strategy for Lafora Disease. We hypothesize that the replacement of either EPM2A or EPM2B genes in their respective knockout mouse model would reduce or alleviate lafora body formation.


Materials and Methods:: Recent studies have shown that malin, an E3 ubiquitin ligase, cannot localize on glycogen molecules and ubiquitinate glycogen metabolism enzymes in the absence of laforin. Hence, we decided to begin this project by analyzing the feasibility of laforin as a target for gene replacement therapy. To achieve this, we designed an AAV9 vector driven by the ubiquitous Chicken Beta hybrid intron promoter to deliver EPM2A into the Central nervous system (CNS) using intracerebroventricular injection in EPM2A wildtype and knockout mouse model. Mouse Models were injected at post natal day 1 and sacrificed at 3 months old, an age when lafora bodies are already present. Following sacrifice, western blots were used to analyze laforin expression, and Immunofluorescence imaging was used to determine laforin distribution in the brain. The reduction or alleviation of lafora bodies is the primary outcome of this study hence enzymatic determination of glycogen and periodic acid schiff diastase staining (PASD) was used to measure lafora bodies.



Results, Conclusions, and Discussions: Immunofluorescence imaging showed overexpression and widespread distribution of laforin across all brain regions, particularly in regions of the brains greatly affected by lafora bodies. Surprisingly enough, we observed a dominant negative effect when laforin is overexpressed in wild type mice; the brains of wild type mice form novel lafora bodies in regions of the brain which receive excessive amounts of laforin. Intrathecal and intramuscular injections in wildtype mice also show that tissues proximal to high doses of overexpressed laforin protein are more prone to forming lafora bodies, the driver of Lafora Disease. We can infer from these results that laforin is a highly regulated protein and is probably needed in little amounts in any cell. In Summary, we hypothesized that laforin might be a target for gene replacement therapy when driven by a low expressing promoter. In conclusion, gene replacement therapy for Lafora Disease would be evaluated by conducting toxicity and efficacy studies utilizing EPM2A gene as a target and delivered intrathecally using AAV serotype 9 driven under the low expressing promoter, JeT in Lafora Disease Mouse Model.