Dual AAV gene therapy in combination with rapamycin rescues muscle and liver impairment in a mouse model of GSDIII.

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation in liver, and in cardiac and skeletal muscles, responsible for impaired hepatic metabolism, heart function impairment, and muscle weakness. To date, there is no curative treatment for GSDIII. The large 4.6 kb coding sequence of GDE represents a major limitation toward the development of clinically relevant AAV gene transfer strategy for GSDIII. We previously reported that liver and heart/muscle correction of the GSDIII phenotype can be achieved in a mouse model of GSDIII by using two distinct overlapping AAV vectors encoding GDE. Here, results of a long-term stability of an approach based on a dual AAV vector encoding for GDE under the control of a recently developed tandem liver-muscle promoter suggests that stable but partial correction of the muscle phenotype can be achieved at very long-term (12 months). In liver, the dual AAV had an only transient efficacy supporting the need for an optimization of the approach. We then assessed the efficacy of rapamycin, an autophagy inducer used in the clinic as an immunosuppressive drug that has shown efficacy in GSDIII. Combination of rapamycin with the dual vector expressing GDE resulted in better correction of glycogen accumulation and muscle strength impairment than AAV vector alone thus supporting a synergic effect of the two treatments. The combined treatment synergic effect was also demonstrated at the molecular level by transcriptomic analysis that indicated a better rescue of the lysosomal pathway in muscle, possibly due to the induction of autophagy by rapamycin and the clearance of glycogen achieved with the combination therapy. In GSDIII mice liver, rapamycin was also able to counteract an unexpected immune reaction to the AAV vector that seems specific of this model. In conclusion, these results indicate that correction of both liver and muscle can be achieved in symptomatic GSDIII mice by an overlapping vector expressing GDE with a tandem promoter when combined with rapamycin treatment. These data also indicate that the effect of rapamycin on AAV gene transfer although disease- and tissue-specific has a net positive impact on dual AAV gene therapy for GSDIII thus opening the way to the clinical translation of this combination approach.