STRATEGIES FOR OPTIMIZING THE EFFICIENCY OF LENTIVIRUSES IN APPROACHES OF GENE THERAPY FOR PATIENTS WITH BETA-THALASSEMIA MAJOR

Beta-thalassemia major is a severe congenital anemia for which there is presently no curative therapy other than allogenic hematopoietic stem cell transplantation. This therapeutic option, however, applies only to the minority of thalassemia patients who have an HLA-matched bone marrow donor. Gene therapy by the delivery of a regulated globin gene to autologous hematopoietic stem cells is an attractive alternative approach as it is in principle applicable to all thalassemic subjects. In the last years, a number of studies in thalassemic mice has shown rescue from thalassemia by randomly delivering into the chromosomal DNA of HSCs a regulated globin gene shuttled within a virus shell derived from HIV. Occasionally, however, the random DNA insertion can interrupt and thus inactivate tumor suppressor genes or activate oncogenes near the site of DNA insertion with the globin regulatory sequences of the vector. In both instances, gene therapy can lead to unwanted tumors. In this project we propose to minimize the risk of insertional mutagenesis, increasing the globin expression to allow equal therapeutic efficacy with 1or 2 instead of the usual higher number of copies of vector per cell. At the same times, to reduce the copy number required for therapeutic effect it is crucial to avoid silencing effect due to chromatin conformation at the site of integration. Therefore we propose to shield vector by flanking it with chromatin insulator. The use of insulator will also improve the safety of vectors. Therapeutic efficacy will be evaluated in cell lines and human erythroid progenitors in the Center of Cagliari, Palermo and Pavia and in murine models in the laboratory of Prof. Sadelain in New York.