We are focusing on the development of complex retroviral vectors containing human beta-globin gene and beta-LCR for the gene therapy of sickle cell disease and beta-thalassemias. First generation vectors containing mutated splice-sites to insure stability of proviral transfer enabled long-term reconstitution in 10/12 transplanted mice for a least 8 months with high expression levels in 2 out of 3 mice analyzed (5% and 20% murine beta). Transfer and expression were also achieved in secondary recipients (range: 3-11% murine beta). Position independent expression was not observed. In an effort to increase the efficiency of gene transfer and obtain complete reconstitution of recipient mice with exclusively transduced cells while enriching for proviral integration into active chromatin regions, we have incorporated a cassette expressing CD24 or the green fluorescent protein (GFP). Stable transfer to murine bone marrow cells allowed efficient FACS-sorting of pure populations of transduced cells. A family of vectors based on these principles and containing segments of gamma- or delta-globin genes were also designed for systematic analysis of their anti-sickling properties.