Recombinant adeno-associated virus (rAAV)-mediated expression of a human γ-globin gene in human progenitor-derived erythroid cells

Abstract

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle- cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human γ-globin gene (pJM24/vHS432(A)γ*). Its 4725- nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the β-globin gene cluster, linked to a mutationally marked (A)γ-globin gene ((A)γ*) 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 medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human γ-globin genes. Twenty to 40% of human erythroid burst- forming unit-derived colonies expressed the rAAV-transduced (A)γ*-globin gene at levels 4-71% that of the endogenous γ-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a γ-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.