Transgenic mice with pancellular enhanced green fluorescent protein expression in primitive hematopoietic cells and all blood cell progeny.

Transgenic mice homogeneously expressing enhanced green fluorescence protein (EGFP) in primitive hematopoietic cells and all blood cell progeny, including erythrocytes and platelets, have not been reported. Given previous data indicating H2Kb promoter activity in murine hematopoietic stem cells (HSCs), bone marrow (BM), and lymphocytes, an H2Kb enhancer/promoter EGFP construct was used to generate transgenic mice. These mice demonstrated pancellular EGFP expression in both primitive BM Sca-1+Lin-Kit+ cells and side population (SP) cells.

Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.

Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer.

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number.

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines.

Overexpression of human O6-alkylguanine DNA alkyltransferase (AGT) prevents MNU induced lymphomas in heterozygous p53 deficient mice.

O6-alkylguanine DNA alkyltransferase (AGT) is a key mechanism in the prevention against MNU induced malignant transformation by removal of O6 methyl guanine (O6mG) adducts. We asked whether heterozygous p53 deficient mice (p53+/-) would be more susceptible to MNU induced lymphomas than wild type mice, and whether O6mG adducts were responsible for this susceptibility. To determine whether MGMT overexpression would be protective, p53+/- mice were bred to human MGMT transgenic mice (MGMT+) and treated with 50 mg/kg MNU.

Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy.

As initial human gene therapy trials for beta-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve beta-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe beta-thalassemia. Generation of beta-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis.