Top of pageAbstract We have recently shown that in order to attain therapeutic efficacy by HSC gene therapy for Metachromatic Leukodystrophy (MLD), Arylsulfatase A (ARSA) over-expression in HSC and their progeny is required. The recent identification of Sulfatase Modifying Factor 1 (SUMF1) as a common activator of sulfatases and a rate-limiting factor in the biological activation of these enzymes, raises concerns for possible adverse effects of enzyme over-expression within certain cell type (Cosma M.P. et al., Cell 2003). In fact, over-expression of one type of sulfatase may lead to reduced activity of other sulfatases by a competitive interaction with their common activator. In order to assess the safety of ARSA over-expression in vivo we generated transgenic mice over-expressing the ARSA gene by lentiviral vectors (LV). Pups were genotyped for the presence of the LV backbone by PCR, and screened for vector content (expressed in LV copies per cell, CpC) and ARSA activity. Transgenic mice bearing multiple LV copies (up to 30CpC) and expressing ARSA at high levels were bred to generate a high expressor colony. In mice of the first 5 generations, we detected over-expression of ARSA up to 30 fold above wild type syngenic controls at the level of all tested tissues (liver, kidney, brain, bone marrow). Immunofluorescence and confocal microscopy showed the tagged enzyme widely expressed in these tissues, within different cell types, and correctly sorted to the lysosomal compartment. Despite these supra-physiological conditions, these mice, which have been monitored for survival, body weight, hematopoietic and neurological functions (behavioral and neurophysiological studies), showed no sign of toxicity in the 5 to 6 generations already bred. These data demonstrate the absence of major toxicity or detrimental effects related to ARSA over-expression. We then evaluated the activity of several other SUMF1-dependet sulfatases (Arylsulfatases C, D and E, of Sulfamidase and Iduronate 2-sulfatase) in the blood and tissue samples of ARSA transgenic mice. No significant differences were documented between transgenic mice and syngenic controls, suggesting that either SUMF1 could be inducible upon over-expression of one of the sulfatases, or that other molecules, such as the newly identified SUMF2, could play a compensatory role in sulfatases activation.
