Search

Glycogenosis type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by a deficiency of lysosomal acid a-glucosidase (GAA), responsible for a glycogen accumulation mainly in skeletal muscle, heart and diaphragm. Two main clinical presentations can be distinguished: the infantile form presenting with severe cardiomyopathy and hypotonia and the adult form exhibiting progressive proximal muscle weakness and impairment of respiratory function. Different therapeutic approaches are currently under development for GSDII patients such as enzyme replacement therapy (ERT) and gene therapy. In this study we aimed to develop a new gene transfer approach based on lentiviral vectors. We first designed SIN-lentiviral vectors expressing GAA or EGFP genes under the control of the ubiquitous retroviral MND-promoter. Batches of high-titer recombinant lentiviral vectors were obtained after concentration reaching 5.109 IU/ml. Fibroblasts from 4 GSDII patients (2 infantile and 2 adult forms of the disease) were transduced at various multiplicity of infection (MOI 1, 10 and 100). Transduction efficiency based on EGFP-expression analysis ranged from 3 to 93% EGFP+ cells. After GAA gene transfer a strong increase of GAA activity was observed (up to 85 fold vs normal) with a complete restoration obtained at MOI 10 (24 to 34% EGFP+ cells). A slight decrease of glycogen accumulation was observed after GAA gene transfer but only at MOI 100. We then developed lentiviral vectors expressing EGFP or GAA genes under the control of muscle-specific (MS) enhancer/promoters. We compared both the mouse muscle creatin kinase (MCK) minimal promoter (Mp) with/without MCK enhancer (Me) or the human desmin promoter (Dp) with/without desmin enhancer (De). We analysed EGFP expression level of the MS-vectors with that of the MNDp in C2C12 myoblasts and myotubes after induction of cell fusion. The highest expression level was obtained with the MCKp-lentiviral vector reaching up to 25% of the MNDp level. Myoblasts from GSDII mice were transduced with either MND-GFP/GAA or Mp-GFP/GAA lentiviral vectors at MOI 5 or 50. Transduction efficiency ranged from 25 to 69% for MNDp versus 13 to 40% for Mp-lentivectors. In primary myoblasts, a 14-fold higher EGFP expression level was observed for MNDp vs Mp promoter (1234 |[plusmn]| 243 vs 90 |[plusmn]| 5 MFI). A complete restoration of GAA activity was observed after transduction with MNDp-based vectors (5 to 25-fold increase vs normal cells). We demonstrated also that GAA was efficiently secreted and uptaken by GSDII myoblasts leading to an almost complete enzymatic restoration (74% activity vs normal). Only a modest increase enzymatic activity was obtained after GAA gene transfer using the Mp-based vector (11% GAA activity vs normal cells). We are currently testing a new MS lentiviral vector based on the synthetic C5-12 promoter which results will be presented at the meeting. Finally our data demonstrate that SIN-lentiviral vectors expressing human GAA can efficiently transduce GSDII myoblasts leading to a metabolic correction. These vectors form the basis of new therapeutical strategies for Pompe disease using genetically-modified autologous muscle stem cell transplantation.

IntroductionThis study aims to define the phenotypic and molecular spectrum of the two clinical forms of β-galactosidase (β-GAL) deficiency, GM1-gangliosidosis and mucopolysaccharidosis IVB (Morquio disease type B, MPSIVB).MethodsClinical and genetic data of 52 probands, 47 patients with GM1-gangliosidosis and 5 patients with MPSIVB were analysed.ResultsThe clinical presentations in patients with GM1-gangliosidosis are consistent with a phenotypic continuum ranging from a severe antenatal form with hydrops fetalis to an adult form with an extrapyramidal syndrome. Molecular studies evidenced 47 variants located throughout the sequence of the GLB1 gene, in all exons except 7, 11 and 12. Eighteen novel variants (15 substitutions and 3 deletions) were identified. Several variants were linked specifically to early-onset GM1-gangliosidosis, late-onset GM1-gangliosidosis or MPSIVB phenotypes. This integrative molecular and clinical stratification suggests a variant-driven patient assignment to a given clinical and severity group.ConclusionThis study reports one of the largest series of b-GAL deficiency with an integrative patient stratification combining molecular and clinical features. This work contributes to expand the community knowledge regarding the molecular and clinical landscapes of b-GAL deficiency for a better patient management.