426. Cure of Established Disease and Direct Evidence of Cellular Cross-Correction in the Nervous System of Metachromatic Leukodystrophy Mice after HSC-Based Gene Therapy
Authors: Alessandra Biffi, Alessia Capotondo, Angelo Quattrini, Ubaldo Del Carro, Riccardo Brambilla, Stefania Fasano, Sergio Marchesini, Claudio Bordignon, Luigi Naldini
Journal: Molecular Therapy (2005)

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Metachromatic Leukodystrophy (MLD) is an inborn lipidosis caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). It is characterized by storage of undegraded sulfatides in the CNS and PNS, leading to severe demyelination. We showed that transplantation of LV-transduced Hematopoietic Stem Cells (HSC) allowed: i) targeting therapeutic genes to CNS and PNS by turnover of microglia and endoneurial macrophages by HSC-derived cells; ii) preventing functional and neuropathological manifestations of MLD in the mouse model, when applied at pre-symptomatic stage (A. Biffi et al., J. Clin. Invest 2004, 113(8): 1118|[ndash]|29). Here we assessed whether this approach can be effective not only in preventing, but also in correcting already established MLD manifestations. By 5|[ndash]|6 months of age, MLD mice display motor conduction abnormalities in CNS and PNS, impaired motor coordination and learning, and sulfatide storage in the NS. We compared the outcome of transplant performed before disease onset with transplantation of ARSA-transduced HSC in 6|[ndash]|7 months old, already symptomatic, MLD recipients. We reached ARSA over-expression in the hematopoietic system of transplanted mice. Neurophysiological evaluations performed on 12 months old mice demonstrated normalization of motor stimuli conduction along CNS and PNS. Rotarod test showed, by comparison with age matched sham-treated MLD mice, protection from further worsening of motor coordination and learning abilities. Remarkably, 12 months old treated mice showed improved performance respect to 6 months old MLD controls and a similar performance to that of age-matched wild type mice. These data indicated that ex vivo gene therapy not only prevented disease progression, but also corrected already established functional deficits. In order to assess the mechanism responsible for the widespread correction of CNS pathology, and define the contribution of ARSA over-expression, we performed immunohistochemical studies of the brain of MLD mice transplanted with HSC expressing a HA-tagged ARSA. Confocal analysis showed the widespread occurrence of tagged enzyme within HSC-derived microglia and resident CNS neurons, oligodendrocytes and astrocytes. Importantly, ARSA-HA was correctly sorted to the lysosomal compartment in all cell types, proving the occurrence of in vivo enzyme transfer. Because these findings were associated to complete reversal of the neuronal degeneration which affects the hippocampus and cerebellum of aging MLD mice, in vivo enzyme transfer achieved robust metabolic cross-correction of the crucial CNS cell targets of the disease. Overall, these results demonstrate that transplantation of LV-transduced autologous HSC represents a promising therapeutic strategy for correcting MLD and possibly other neurodegenerative disorders in humans.