Expression from the human parvovirus B 19p6 promoter fused to the firefly luciferase (' Luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B 19p6-Luc plasmid DNA, no expression from the B 19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV.They indicate that expression from the B 19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.

SllmmllL~We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human cx-globin gene product towards developing a treatment modality for B-thalassemia since accumulation of free c~-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human ~x-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human cx-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neo ~) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that expresses high levels of c~-globin mRNA. Clonal populations of neo p" cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and o~-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus-infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense cx-globin sequences showed no inhibition of expression of the endogenous ol-globin gene, the SV40 promoter and the oe-globin gene promoter-driven antisense ot-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of t3-thalassemia.