Suppression of human alpha-globin gene expression mediated by the recombinant adeno-associated virus 2-based antisense vectors.

We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human alpha-globin gene product towards developing a treatment modality for beta-thalassemia since accumulation of free alpha-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human alpha-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human alpha-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neoR) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that express high levels of alpha-globin mRNA. Clonal populations of neoR 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 alpha-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 alpha-globin sequences showed no inhibition of expression of the endogenous alpha-globin gene, the SV40 promoter and the alpha-globin gene promoter-driven antisense alpha-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 beta-thalassemia.