GENETIC CONTROL OF PROTEIN STRUCTURE AND SYNTHESIS

Although allogeneic hematopoietic stem cell transplantation has been successful in correcting some of the clinical features of Hurler's syndrome such as hepatosplenomegaly and obstructive sleep apnea, the skeletal complications are refractory to this treatment. In addition, graft versus host disease (GVHD) and infections are serious, possible life threatening complications of this treatment, We hypothesize that transplantation of autologous mesenchymal stem cells (MSCs) in which the missing/dysfunctional a-L-iduronidase gene has been corrected may overcome the enzyme delivery limitations inherent to hematopoietic stem cell transplantation and circumvent allogeneic transplant associated complications. To accomplish this we will isolate and characterized mesenchymal stem cells in human and murine bone marrow. We will define conditions for ex vivo expansion of such cells in an undifferentiated state and conditions that will allow successful retroviral transduction. We will also define conditions required for successful transplantation of MSCs in a murine model including optimal age of the recipient animal, need for irradiation, MSC cell dose and number of infusions. MSCs used in such transplant model will be transduced with an eGFP retroviral gene which should allow analysis of engraftment in mesenchymal tissues by PCR and FACS. Finally we will perform syngeneic transplant of M-Idua-eGFP MSCs from Idua-/-animals in Idua -/- recipient animals. Successful replacement of the defective enzyme will be determined by detection of a-L-iduronidase in mesenchymal tissues (RT-PCR and fluorometric assay), decrease of GAG accumulation in target organs (in situ histochemical staining for GAGs and transmission electron microscopy) and normalization of the clinical features (GAG level in urine, morphometric and radiographic studies).