Recombinant adeno-associated virus-mediated expression of O-6-alkylguanine-DNA-alkyltransferase protects human epithelial and hematopoietic cells against chloroethylating agent toxicity

Recombinant adeno-associated virus (rAAV) encoding the human O-6-alkylguani ne-DNA-alkyltransferase (hAT) protein and a selectable marker (Neo(r)) was used to transduce human cervical carcinoma (HeLa) cells and erythroleukemic (K562) cells and clones were selected using G418 (0.4 mg/ml). Thirteen HeL a clones were isolated, 9 of which survived for 2-3 months before cell deat h ensued, presumably owing to the loss of G418 resistance. Northern blot an alysis of the remaining four clones, using a neo probe, showed high levels of RNA equivalent in size to the bicistronic RNA expected to be produced fr om this construct. Analysis of hAT activity showed that 2000-5000 fmol/mg p rotein was expressed relative to untransduced cells (800-900 fmol/mg protei n). Cell survival analysis following exposure to the chloroethylating agent mitozolomide revealed that expression of hAT at levels two- to fourfold hi gher than background conferred significant resistance (p < 0.001) to the to xic effects of this drug. Two days following infection of K562 cells with t he rAAV vector, immunoblot analysis showed that hAT protein was being produ ced. Three K562 clones, isolated using G418 selection, were studied in deta il and were shown to express hAT activities of 1500, 1010, and 890 fmol/mg protein, respectively, at 40 days posttransduction (mock-transduced K562 ce lls contain <2 fmol of hAT/mg protein). As with HeLa cells, Northern blot a nalysis showed the production of an appropriately sized transcript and immu noblot analysis indicated that hAT protein was being produced. These clones were assayed for cell survival following exposure to mitozolomide. Express ion of hAT at levels 800- to 1500-fold higher than background conferred sig nificant resistance (p < 0.001) to the toxic effects of mitozolomide. We ha ve therefore successfully conferred a protective advantage against mitozolo mide toxicity to cells by rAAV-mediated hAT expression.