Ex Vivo Gene Therapy Approaches for the Treatment of Globoid Cell Leukodystrophy

Visigalli, Ilaria (2009). Ex Vivo Gene Therapy Approaches for the Treatment of Globoid Cell Leukodystrophy. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f210

Abstract

Globoid cell leukodystrophy (GLD) is a rare lysosomal storage disorder (LSD) due to the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The enzymatic deficiency results in intracellular storage of undegraded metabolites in the nervous system, leading to progressive dysmyelination. We are testing the feasibility and efficacy of a gene therapy strategy based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV) in the murine model of GLD. Differently from what observed with other lysosomal enzymes, GALC gene transfer and expression in HSPC causes apoptosis and functional impairment of the transduced cells due to an inbalance of the intracellular content in bioactive sphingolipids consequent to de novo enzyme expression. Differentiated cells of the myeloid and lymphoid lineages are not affected by GALC expression, suggesting a unique sensitivity of HSPC to enzyme toxicity.

To overcome this issue, we explored strategies aimed at de-targeting vector expression from HSPC, while permitting GALC over-expression in differentiated cells. The first approach aims at a transcriptional regulation of GALC expression by the microglia/macrophage specific promoter CD11b. The second strategy exploits endogenous microRNAs for post-transcriptional regulation of GALC expression. The use of LV containing the target sequence of miRNA126, expressed only in HSPC, allows expressing GALC only in the differentiated progeny of transduced HSPC. Although these two approaches proved to be very promising in protecting HSPC from enzyme toxicity both in vitro and in vivo in heterozygous GLD mice, gene-corrected HSPC failed to repopulate lethally irradiated homozygous GLD mice. This poor result might be explained by the existence of a niche defect in homozygous GLD mice, which might hamper the engraftment of HSPC.