Translational optimization of AAV vectors to cure GSDIII – TRACeGSDIII

Glycogen storage disease type III (GSDIII) is a rare (incidence: 1:100000) autosomal recessive disorder resulting from the lack of glycogen debranching enzyme (GDE) deputed to glycogen linearization in different tissues. Although GDE is expressed in all the tissues, the disease manifestation is primarily due to its absence in liver and muscle. In particular, during childhood, the disease is mainly metabolic with frequent sever hypoglycemia. During the adolescence the hypoglycemic episodes becomes less severe and a generalized muscle weakness and cardiomyopathy appear. Cirrhosis and hepatocellular carcinoma (HCC) have been reported in adult GSDIII patients. At present, the only curative treatment for GSDIII is based on the dietary administration of complex carbohydrates during childhood to prevent hypoglycemia and a high protein/high fat diet to reduce cardiomyopathy. Patients affected show a progressive impairment in the muscle function associated with exercise intolerance. The absence of curative treatment for GSDIII and the mono-genic character of the disease provide a strong rationale for the development of a gene replacement strategy for GSDIII proposed in TRACeGSDIII. Extensive experience in clinical trials and in preclinical model of muscle diseases indicates adeno-associated virus (AAV) as the vector of choice for in vivo gene therapy for GSDIII. These vectors efficiently transduce liver and muscle, their production is scalable and compared to other gene therapy vectors they have a relatively low immunogenicity profile. In the last years, animal models of the disease became available. In particular, the mouse model recapitulates the disease manifestation in humans with decreased glycemia and progressive muscle weakness. Molecular analysis performed in different tissues demonstrated the complete absence of GDE and an extensive accumulation of glycogen in different tissues. The mouse model is therefore suitable to develop a gene therapy approach for GSDIII. The major obstacle to the development of an AAV vector-based strategy for GSDIII is that the size of GDE transgene is bigger than the AAV packaging size limit. Dual AAV vectors have been proposed to overcome this size limitation. By using dual AAV vectors expressing GDE, we provided proof of concept of the feasibility of GDE gene replacement in GSDIII mice. In particular, by using dual AAV vectors specific for muscle targeting, we demonstrated a complete rescue of the muscle strength impairment. In addition to that, dual AAV vectors specific for the liver expressed GDE in this organ and rescued the hypoglycemic phenotype. Although promising, data obtained indicated several limitations of this approach. In particular the use of two different dual vector approaches to rescue a single disease is definitely not translationally viable. Additionally a high dose of vector was needed to rescue the disease phenotype in muscle and liver in GSDIII mice. Such a dose, corresponding to 5E13 vg/kg has never been tested in humans and could be possibly associated to uncontrolled immune response against the vector. Finally, the production of AAV vectors for human experimentation at this dose is not sustainable for both economics and production reasons. The optimization of the transgene expression cassette and the capsid to reduce the dose of vector needed to rescue the GSDIII phenotype in muscle and liver are the main aims of this grant proposal. At the same time we aim at the characterization of the metabolic pathways that are impaired in GSDIII to develop novel strategies to enhance therapeutic efficacy of gene transfer for GDE.