41. Ex Vivo Gene Therapy Provides a Unique Therapeutic Benefit and Corrects Metachromatic (MLD) and Globoid (GLD) Leukodystrophy in the Mouse Models

Abstract

MLD and GLD are inborn lipidosis caused by the deficiency of the lysosomal enzymes arylsulfatase A (ARSA) and galactocerebrosidase (GALC). They are characterized by storage of undegraded sulfatides in the CNS and PNS, leading to progressive demyelination and early death. Using Lentiviral Vectors (LV), we efficiently transduced Hematopoietic Stem Cells (HSC) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice, and correct MLD and GLD phenotypes in the mouse models. We proved extensive transgene expression in the CNS and PNS of transplanted mice due to progressive turnover of microglia and endoneurial macrophages by HSC-derived cells. Recruitment of these cells was consistently observed independently from the conditioning protocol, and it was faster and more robust in MLD and GLD mice. By transplanting HSC transduced with the ARSA gene, we reconstituted enzyme activity in the hematopoietic system of MLD mice at supranormal levels and prevented all mayor disease manifestations. Transplanted mice were completely protected from the development of motor conduction impairment (normalization of motor conduction velocity and F wave latency, and of central conduction time), of learning and coordination deficits (normalization of the performance at rotarod test), and of neuropathological abnormalities (prevention of sulfatide accumulation and demyelination in CNS and PNS) typical of the disease. Remarkably, ex vivo gene therapy had a significantly better therapeutic impact than wild-type HSC transplantation on all examined parameters, highlighting the crucial role of enzyme over-expression and indicating the likely occurrence of in vivo cross-correction. Confocal analysis of the CNS of treated mice showed the presence of tagged ARSA enzyme within the lysosomes both of the transduced HSC-derived microglia cells and of resident neurons, demonstrating in vivo enzyme transfer and, most likely, cross-correction. To further evaluate the therapeutic impact of this approach we challenged it with the acute and devastating evolution of GLD in the mouse model. Neonate GLD mice were transplanted with HSC transduced with the GALC gene, supplemented with accessory Sca1- cells to reduce short-term toxicity of conditioning. We observed a dramatic improvement in survival of transplanted mice, which reached an average of 135 days, as compared to 40 days of untreated GLD mice and 80 days of mice transplanted with wild type bone marrow, providing striking evidence, in another disease setting, of the therapeutic advantage of ex vivo gene therapy versus allogeneic BMT. We observed a significant improvement in the GLD phenotype (absence of leg palsy and delayed, minor twitching) and prevention of mayor histopathological signs of the disease (demyelination, axonal loss). Overall, these results demonstrate that transplantation of LV-transduced autologous HSC represents a new, potentially efficacious therapeutic strategy for MLD, GLD and possibly other neurodegenerative disorders.