SYNTHESIS OF HUMAN GLOBIN POLYPEPTIDES MEDIATED BY RECOMBINANT ADENOASSOCIATED VIRUS VECTORS

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were con structed. The cDNAs were spliced into an AAV plasmid, pAAV Delta K, do wnstream from the viral P40 promoter, substituting the capsid gene reg ion. The correctness of the insertion with regard to the transcription al polarity was ascertained by both restriction enzyme analysis and DN A sequencing. One of the constructs, pAAVcHBBLCR, contained the erythr oid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 10 4, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line ) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythr oleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical an d immunohistochemical means. The LCR made the construct respond to an inducer, N,N'-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-glo bin in MEL cells. Electrotransfection of mouse bone marrow hematopoiet ic stem/progenitor cells with the constructs dramatically increased th e number of benzidine-positive cells in liquid suspension culture, ind icating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobi nopathies.