RECOMBINANT ADENOASSOCIATED VIRUS (RAAV)-MEDIATED EXPRESSION OF A HUMAN GAMMA-GLOBIN GENE IN HUMAN PROGENITOR-DERIVED ERYTHROID-CELLS

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickl e-cell anemia and thalassemia, will require an efficient method to tra nsfer, integrate, and express a globin gene in primary erythroid cells . To evaluate recombinant adeno-associated virus (rAAV) for this purpo se, we constructed a rAAV vector encoding a human gamma-globin gene (p JM24/vHS432(A) gamma). Its 4725-nucleotide genome consists of two 180 -bp AAV inverted terminal repeats flanking the core elements of hypers ensitive sites 2, 3, and 4 from the locus control region of the beta-g lobin gene cluster, linked to a mutationally marked (A) gamma-globin g ene ((A) gamma) containing native promoter and RNA processing signals . CD34(+) human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medi um containing several cytokines. A reverse transcriptase polymerase ch ain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythr oid burst-forming unit-derived colonies expressed the rAAV-transduced (A) gamma-globin gene at levels 4-71% that of the endogenous gamma-gl obin genes. The HbF content of pooled control colonies was 26%, wherea s HbF was 40% of the total in pooled colonies derived from rAAV transd uced progenitors. These data establish that rAAV containing elements f rom the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoiet ic cells resulting in a substantial increase in HbF content.