An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies

A variety of gene therapy strategies are under development for the treatmen t of sickle cell anemia and other hemoglobinopathies. A number of alternati ve vectors have been developed to transfer and express the beta-globin gene and other therapeutic molecules, but none has resulted in efficient transd uction and stable long-term expression in primary hematopoietic cells. One reason for this problem is that most vectors are initially evaluated in imm ortalized cell lines which may not faithfully recapitulate the biology of p rimary erythroid cells. In order to provide a more relevant system for effi ciently evaluating alternative vector constructs for beta-globin disorders, we have developed (I) a simple method for generating primary human red blo od cell (RBC) precursors in liquid culture established with mononuclear cel ls obtained from normal donors as well as patients with Hb SC disease; (2) a high titer retroviral vector which can be easily modified to optimize gen e transfer and transgene expression, and (3) methods for transducing the RB C precursors at high efficiency. The development of simple and efficient me thods and reagents for generating and transducing primary human RBC precurs ors provides a facile and effective means for screening alternative gene th erapy strategies.