RESUMEN
Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the ßAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure's suitability for human clinical applications while affording the additional safety of conditional suicide.
Asunto(s)
Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Hemoglobinopatías/genética , Hemoglobinopatías/terapia , Transducción Genética , Globinas beta/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Genes Transgénicos Suicidas , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Ratones , Ratones Transgénicos , TransgenesRESUMEN
A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered ß-globin gene (ß(A-T87Q)-globin) in a subject with ß thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 ß-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with ß-thalassemia major and sickle cell disease.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Talasemia beta/terapia , Anemia de Células Falciformes/genética , Animales , Antígenos CD34/metabolismo , Biología Computacional , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ratones Endogámicos C57BL , Talasemia beta/genéticaRESUMEN
Uranium level in drinking water is usually in the range of microgram-per-liter, but this value may be as much as 100 to 1000 times higher in some areas, which may raise question about the health consequences for human populations living in these areas. Our purpose was to improve knowledge of chemical effects of uranium following chronic ingestion. Experiments were performed on rats contaminated for 9 months via drinking water containing depleted uranium (0.2, 2, 5, 10, 20, 40, or 120 mg/L). Blood biochemical and hematological indicators were measured and several different types of investigations (molecular, functional, and structural) were conducted in organs (intestine, liver, kidneys, hematopoietic cells, and brain). The specific sensitivity of the organs to uranium was deduced from nondeleterious biological effects, with the following thresholds (in mg/L): 0.2 for brain, >2 for liver, >10 for kidneys, and >20 for intestine, indicating a NOAEL (No-Observed-Adverse-Effect Level) threshold for uranium superior to 120 m g/L. Based on the chemical uranium toxicity, the tolerable daily intake calculation yields a guideline value for humans of 1350 µg/L. This value was higher than the WHO value of 30 µg/L, indicating that this WHO guideline for uranium content in drinking water is very protective and might be reconsidered.