Targeting the hematopoietic stem cell to correct osteopetrosis

This thesis focuses on developing stem cell targeted gene therapy for the severe hereditary disorder Infantile Malignant Osteopetrosis (IMO) as well as increasing the understanding of how the genetic defect present in IMO affects the hematopoietic stem cells and hematopoiesis in general. IMO is a rare congenital disorder associated with an increased number of non-functional osteoclasts and the absence of bone resorption results in accumulation of sclerotic bone leading to abnormal bone marrow cavity formation insufficient to support hematopoiesis. The most common mutation is in the TCIRG1 gene seen in over 50% of the patients. The children suffer from bone marrow failure, anemia, thrombocytopenia, hepatosplenomegaly, immune dysfunction and recurrent infections. Furthermore, the impaired remodeling of the developing bone results in compression of cranial nerves which leads to neurological impairment, affecting especially vision and hearing which may progress to blindness and deafness. The only curative treatment available today for IMO is bone marrow transplantation. However, this can only be performed if the patient has a suitable stem cell donor. If untreated, the children die around the age of five. Currently no alternative treatment exists, thus IMO is a candidate disease for development of gene therapy. The first two papers are based on studies conducted with the oc/oc mouse model that has a mutation in the homologous gene and shares the same osteopetrotic phenotype as seen in humans. Paper one focuses on developing a clinically relevant model for cell therapy in the oc/oc mouse by replacing radiation conditioning with low-dose busulfan conditioning prior to neonatal bone marrow transplantation and optimizing the administration procedure. In paper two, neonatal oc/oc mice were treated with bone marrow transplantation without any prior conditioning, which showed a rapid reversal of the severe osteopetrotic phenotype despite low level of engraftment and lack of selective expansion of the osteoclastic lineage. In manuscript three human stem cells from patients with IMO were studied and the paper provides the first proof-of-principle of lentiviral-mediated correction restoring the resorptive function of osteoclasts derived from peripheral blood CD34+ cells of IMO patients. In manuscript four the osteoclast significance in hematopoietic stem cell maintenance was studied by generating two adult osteopetrotic mouse models and subsequently analyzing the hematopoiesis of the mice. The results from this study indicate that osteoclast function is not essential for hematopoietic stem cell maintenance in adult mice. Results obtained from the oc/oc mouse model and from IMO patient cells are encouraging for further development of hematopoietic stem cell targeted gene therapy, and will serve as basis for upcoming development of clinical gene therapy of osteopetrosis