Glycogen storage disease III (GSDIII) is a rare (1:100,000) autosomal recessive disorder resulting from the deficiency of glycogen debranching enzyme (GDE) the enzyme that hydrolyzes glycogen branches in the cytosol. The disease manifestation occurs in two phases. Early symptoms of the disease includes hepatomegaly, hypoglycemia, failure to thrive and recurrent illness. After adolescence, the metabolic impairment becomes less evident and a generalized muscle weakness appears. A dietary treatment with frequent meals high in carbohydrates, slows the progression of the pathology that is however inevitable and severely affecting the quality of life of affected individuals.
At present, no cure is available for GSDIII and the disease represents an unmet medical need.
GLYCODIS3 had the overall aim to obtain a proof-of-concept of an adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of GSDIII. AAV gene therapy has been successfully used for the correction of several genetic diseases in animal models and humans. GLYCODIS3 had also, as an accessory aim, the development of human induced pluripotent stem cells (iPSc) derived from GSDIII patients.
AAV are the vectors of choice for in vivo genetic correction of monogenic disorders. Human trials of liver gene transfer for hemophilia A and B and eye gene transfer for congenital blindness have unveiled the therapeutic potential of this viral vector platform. Follow-up data of subjects treated with AAV vectors is showing sustained correction of the disease phenotype for several years after gene transfer, and recent data confirmed that AAV vectors can drive expression of a transgene in humans for >10 years. Despite these advantages, one limitation of AAV vectors is that they cannot package vector genomes significantly larger than 5kb inside their capsid. Due to the length of the sequence of the GDE enzyme (4.6 Kb), a transgene expression cassette for the efficient expression of GDE in both muscle and liver, hardly fit in a single AAV vector. To overcome this limitation, we engineered a dual-vector system with a recombinogenic bridging DNA sequence to drive reconstitution of the full-length GDE sequence within a cell. GDE-knockout mice treated with dual vectors showed an efficient expression of the protein in both muscle and liver and the rescue of the muscle weakness and the metabolic impairment associated with the disease.
In conclusion, data obtained in the frame of the GLYCODIS3 project, constitute a fundamental step in the translation of this approach to patients and represent an important advance over the state of the art as:
1) No gene therapy approach has been tested in vivo in models of GSDIII, so far only symptomatic treatments were attempted based on diet or conventional drugs administration. The paper showed that gene therapy can be used to express GDE and correct the actual disease.
2) Low levels of GDE expression are needed to revert the disease. This is a very important point when devising strategies based on gene therapy to treat the disease.
3) Concomitant correction of both liver and muscle phenotype has been achieved with gene transfer for GDE
4) Previous reports showed that recombinant GAA, a lysosomal enzyme, has the potential to reduce glycogen accumulation in human myoblasts from GSDIII patients. This approach has been proposed to treat patients. Here, we tested the approach in vivo and we demonstrated that the approach is efficient in clearing glycogen from liver (thus confirming that a lysosomal enzyme can clear cytosolic glycogen). However, no long-lasting effect is seen in muscle.
