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.

Project Start
1998-12-01
Project End
2000-11-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Miranda, S R; Erlich, S; Friedrich Jr, V L et al. (2000) Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of Niemann-Pick disease. Gene Ther 7:1768-76
Schuchman, E H; Erlich, S; Miranda, S R et al. (2000) Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells based on acid sphingomyelinase expression. Methods Enzymol 312:330-8
Miranda, S R; He, X; Simonaro, C M et al. (2000) Infusion of recombinant human acid sphingomyelinase into niemann-pick disease mice leads to visceral, but not neurological, correction of the pathophysiology. FASEB J 14:1988-95
Erlich, S; Miranda, S R; Visser, J W et al. (1999) Fluorescence-based selection of gene-corrected hematopoietic stem and progenitor cells from acid sphingomyelinase-deficient mice: implications for Niemann-Pick disease gene therapy and the development of improved stem cell gene transfer procedures. Blood 93:80-6
Chen, F W; Davies, J P; Ioannou, Y A (1998) Differential gene expression in apoptosis: identification of ribosomal protein 23K, a cell proliferation inhibitor. Mol Genet Metab 64:271-82
Miranda, S R; Erlich, S; Friedrich Jr, V L et al. (1998) Biochemical, pathological, and clinical response to transplantation of normal bone marrow cells into acid sphingomyelinase-deficient mice. Transplantation 65:884-92
Schuchman, E H; Miranda, S R (1997) Niemann-Pick disease: mutation update, genotype/phenotype correlations, and prospects for genetic testing. Genet Test 1:13-9
Davies, J P; Cotter, P D; Ioannou, Y A (1997) Cloning and mapping of human Rab7 and Rab9 cDNA sequences and identification of a Rab9 pseudogene. Genomics 41:131-4
Miranda, S R; Erlich, S; Visser, J W et al. (1997) Bone marrow transplantation in acid sphingomyelinase-deficient mice: engraftment and cell migration into the brain as a function of radiation, age, and phenotype. Blood 90:444-52
Ioannou, Y A; Chen, F W (1996) Quantitation of DNA fragmentation in apoptosis. Nucleic Acids Res 24:992-3

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