The overall objective of this project is to use animal models of neuropathic lysosomal storage disorders (SDs) to develop and evaluate ex vivo and in vivo approaches to central nervous system (CNS)-targeted gene delivery. To facilitate these studies, we have established breeding colonies of cats with mucopolysaccharidosis Type I (fMPS I) and GM2 gangliosidosis (fGM2) and constructed 'knock out' mouse models of Type A Niemann-Pick disease (mNPD) and Schindler disease (mSD).
The specific aims of this project are to: 1) Characterize the biochemical abnormalities, neural pathology and clinical course of the murine models. The natural history of each murine disease will be studied, the levels of residual enzymatic activity and substrate accumulation will be documented, and the neuropathology will be assessed and quantitated by morphometric analysis. These studies will provide essential baseline data by which one can evaluate the effects of therapeutic intervention. 2) Compare the entrance, migration and persistance of hematopoietically- derived cells in the CNS following hematopoietic stem cell transplantation (HSCT). Hematopoietic stem cells (HSC) will be obtained from normal mice and cats and marked with retroviral vectors expressing Beta-galactosidase (Betagal) activity. HSCT will be performed in utero, in neonates and in developmentally mature animals in order to evaluate whether there are age dependent 'windows of opportunity' during which HSC-derived cells enter the CNS. Transplanted animals will be sacrificed at various times post-engraftment and their brains analyzed for Betagal expression to assess the persistance and migration of the transplanted cells. 3) Evaluate the biochemical, pathological and clinical effectiveness of ex vivo gene therapy in the neuropathic LSD animal models. Lysosomal overexpression/secretion cassettes (developed in Projects 1 and 2) will be inserted into retroviral vectors and used to transduce HSCs from the neuropathic LSD animal models. Autologous HSCT will be performed in affected animals using optimal conditions for CNS entry and the biochemical, pathological and clinical effectiveness of this therapeutic approach will be monitored. We will also evaluate the ex vivo delivery of lysosomal overexpression/secretion vectors into the CNS of affected animals using the neural cell transplantation strategies developed in Project 3. 4) Evaluate the effectiveness of in vivo CNS- targeted gene delivery and therapy using the animal models systems. Using the vectors and delivery systems developed in Projects 1-3, lysosomal overexpression/secretion vectors will be delivered directly into the brains of affected animals by transient disruption of the blood- brain barrier via carotoid infusion of hyperosmolar mannitol or direct intraventricular or subarachnoid injections. The biochemical, pathologic and clinical effectiveness of these therapeutic approaches will be evaluated.
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