RECOMBINANT ADENOASSOCIATED VIRUS-MEDIATED GENE-TRANSFER INTO HUMAN LEUKEMIA-CELL LINES

Adeno-associated virus (AAV)-based vector is a promising gene transfer vehicle by virtue of the characteristics of wild-type AAV:tropism to a wide range of human tissues and locus-specific integration at chromo some 19q13.3. To elucidate the nature of the recombinant AAV (rAAV), t ransduction of neomycin phosphotransferase enzyme gene (NeoR gene) int o seven human leukemia cell lines was performed. Transduction efficien cies were assessed by colony formation assay and limiting dilution ass ay. The results suggested that both assays are comparable. Transductio n efficiencies of the NeoR gene into K-562, MEG-OI, Raji, MOLT-3, HL-6 0, U937 and NKM-1 at a multiplicity of infection (MOI) of 0.1 were 0.2 7, 0.25, 0.015, 0.009, 0.009, < 0.0025 and < 0.0025%, respectively. Af ter purification and concentration of rAAV, 27% efficiency was observe d in K562 at an MOI of 7 and a linear relationship between MOI and eff iciency was confirmed, suggesting that this system may be useful for g ene transduction into leukemia cells. Integration of the NeoR gene int o the host genome was detected by Southern blotting analysis, which sh owed various sizes of digested fragments. A fluorescent in situ hybrid ization (FISH) study was carried out on 11 clones, in all of which the NeoR gene was integrated out of chromosome 19q13.3. In five of the cl ones, whole chromosome painting probes revealed that the integration s ites were chromosomes 1q, 2q, 2q, 11p, 12p and 13q. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.