533. In Vitro and In Vivo Expression of Modified Mouse Galactosylceramidase in the Twitcher Mouse Model of Krabbe Disease

Abstract

The neurodegenerative disease globoid cell leukodystrophy (GDL), commonly known as Krabbe disease, is caused by an autosomal recessive disruption of the lysosomal enzyme galactosylceramidas (galactocerebroside β-galactosidase, GALC). Enzyme disruption causes an accumulation of undegraded psychosine, resulting in demylination due to loss of oligodendrocytes. With advancing pathology, there is a progressive loss of nervous system function, invariably leading to death. As currently there is no effective treatment, we have worked on developing gene therapy strategies using the HIV-1 based lentivector system. In initial in vitro experiments, monkey COS-7, human 293T, and human bone marrow derived mesenchymal stem cells were transduced with lentiviral vectors encoding mouse GALC. After 4 days, GALC expression increased from 15 to 190 fold over baseline activity, depending on the cell type, with elevated GALC expression persisting for at least 2 weeks. Transduction of skin derived fibroblast cells taken from the Twitcher mouse (a murine GDL model) with the GALC lentivector produced an in vitro correction of the enzyme deficiency. To facilitate monitoring transgenic GALC, and to distinguish transgenic from endogenous GALC, a myc epitope and 6 HIS residues were added to the C terminus of GALC (GALC-MH). A Tat protein transduction domain (PTD) was also added to improve in vivo distribution (GALC-TMH). In vitro comparisons showed that the addition of myc-HIS did not impair GALC specific activity, while that of GALC-TMH was only slightly impaired. These tags allowed detection of GALC protein in cell extracts and culture medium. GALC was found to be secreted by transduced cells, and taken up by neighboring cells via mannose-6-phosphate receptor (M6PR) mediated endocytosis. The addition of Tat significantly stimulated M6PR-independent uptake of GALC. Ongoing studies are assessing the biodistribution of both GALC expressing cells and secreted extracellular GALC after direct CNS injection of GALC lentiviral vectors. Additionally, therapeutic effects of transgenic GALC are being evaluated in the Twitcher mouse.