ADENOASSOCIATED VIRUS 2-MEDIATED TRANSDUCTION AND ERYTHROID CELL-SPECIFIC EXPRESSION OF A HUMAN BETA-GLOBIN GENE

Recombinant adeno-associated virus 2 (AAV) virions were constructed th at contained the genomic copy of a normal human beta-globin gene marke d with a 4-bp Clal linker, and the herpesvirus thymidine kinase (TK) p romoter-driven bacterial gene for resistance to neomycin (v beta(m)-gl obin), as well as those containing the DNase 1-hypersensitive site 2 ( HS-2) from the locus control region (LCR) of the human beta-globin gen e cluster (vHS2-beta(m)-globin). These recombinant virions were used t o infect a human erythroleukemia cell line which normally does not exp ress the beta-globin gene (K562), or a human nasopharyngeal carcinoma cell line (KB). Cell populations resistant to G418, a neomycin analogu e, were obtained following infections with the recombinant virions, in dicating high-efficiency transduction of the chimeric gene as well as functional activity of the transduced neo gene in both cell types. Sou thern blot analysis using a human beta-globin DNA probe substantiated stable integration of the exogenous beta-globin allele in these cells. There was no expression of the transduced beta-globin gene in K562 or KB cells infected with the v beta(m)-globin virus. High-level express ion of the transduced beta-globin gene occurred only in the vHS2-beta( m)-globin virus-infected K562 cells, but not in KB cells, as determine d by Northern blot as well as RNase protection analyses. Expression of the human beta-globin protein could also be detected in approximately 10-20% of the vHS2-beta(m)-globin virus-infected K562 cells. These st udies suggest that the AAV-based vector system may prove useful for hi gh-efficiency globin gene transfer in human hematopoietic cells.