The application will test the hypothesis that caprine fetal liver hematopoietic stem cells (HSC) transplanted in utero, during the period of immunotolerance, will engraft and modify the severe phenotype, including neuro- degeneration, in caprine b-mannosidosis. Factors which enhance HSC engraftment and the fate of HSC after transplantation will be evaluated. In utero transplantation of HSC transduced with an overexpressed copy of the b-mannosidase gene is proposed as a novel therapeutic approach.
Specific aims for this proposal are: 1) to optimize the efficiency of HSC engraftment and define the tissue targets: an HSC pretreatment regimen with phytohemagglutinin stimulated leukocyte condition medium (PHA-LCM) will be evaluated. To assess effects of HSC pretreatment of engraftment, male HSC will be administered to normal female recipient caprine fetuses at 60 days gestation. Engraftment will be assessed in bone marrow and peripheral blood at postnatal intervals up to 6 months of age. Donor HSC will be identified in tissue at 4 weeks posttransplantation and at 6 months of age using in situ hybridization with a Y-chromosome specific probe. 2) To overexpress b-mannosidase by introducing an overexpressed copy of the b-mannosidase gene into normal caprine fibroblasts and HSC in vitro. Optimal factors to obtain high level b-mannosidase expression in HSC using a retroviral gene transfer system will be investigated first utilizing a model system with neo(R) or b-galactosidase recombinant retrovirus to introduce a marker gene into normal caprine fibroblasts and HSC. Optimal conditions will be sued for b-mannosidase recombinant retroviral gene transfer into these same cells. 3) To compare modifications of the caprine b-mannosidosis phenotype by treatment of mutant caprine fetuses with normal, marker-transduced HSC or HSC transduced to overexpress b- mannosidase. Normal, marker-transduced HSC or HSC transduced to overexpress the b-mannosidase gene will be used for in utero therapy in caprine b-mannosidosis fetuses. The extent of engraftment in recipient blood, bone marrow and tissue samples will be determined by several methods including in situ detection of donor cells. Expected modifications of the b-mannosidosis phenotype will be assessed by measurement of tissue b- mannosidase activity, immunocytochemical localization of b-mannosidase, determination of urinary and tissue oligosaccharide concentrations, assessment of neurological and physiological functions and analysis of the histology and ultrastructure of lesions. In addition to expected modifications of CNS disease by prenatal therapy, new insights into the pathogenesis of this storage disease and HSC biology are expected outcomes.
| Jones, Margaret Z; Alroy, Joseph; Downs-Kelly, Erinn et al. (2004) Caprine mucopolysaccharidosis IIID: fetal and neonatal brain and liver glycosaminoglycan and morphological perturbations. J Mol Neurosci 24:277-91 |
| Lovell, K L; Kraemer, S A; Leipprandt, J R et al. (2001) In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases. Fetal Diagn Ther 16:13-7 |
| Downs-Kelly, E; Jones, M Z; Alroy, J et al. (2000) Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy. J Mol Neurosci 15:251-62 |
