Optimization of recombinant adeno-associated viral vectors for human β-globin gene transfer and transgene expression

Therapeutic levels of expression of the β-globin gene have been difficult to achieve with conventional retroviral vectors without the inclusion of DNase I-hypersensitive site (HS2, HS3, and HS4) enhancer elements. We generated recombinant adeno-associated viral (AAV) vectors carrying an antisickling human β-globin gene under the control of either the β-globin gene promoter/enhancer or the erythroid cell-specific human parvovirus B19 promoter at map unit 6 (B19p6) without any enhancer, and tested their efficacy in a human erythroid cell line (K-562) and in primary murine hematopoietic progenitor cells (c-kit⁺lin^-). We report here that (1) self-complementary AAV serotype 2 (scAAV2)-β-globin vectors containing only the HS2 enhancer are more efficient than single-stranded AAV (ssAAV2)-β-globin vectors containing the HS2+HS3+HS4 enhancers; (2) scAAV2-β-globin vectors recombine with scAAV2-HS2+HS3+HS4 vectors after dual-vector transduction, leading to transgene expression; (3) scAAV2-β-globin as well as scAAV1-β-globin vectors containing the B19p6 promoter without the HS2 enhancer element are more efficient than their counterparts containing the HS2 enhancer/β-globin promoter; and (4) scAAV2-B19p6-β-globin vectors in K-562 cells, and scAAV1-B19p6-β- globin vectors in murine c-kit⁺lin^- cells, yield efficient expression of the β-globin protein. Thus, the combined use of scAAV vectors and the parvovirus B19 promoter may lead to expression of therapeutic levels the β-globin gene in human erythroid cells, which has implications in the use of these vectors in gene therapy of β-thalassemia and sickle cell disease.