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Creation of Human Alkyltransferases Resistant to O⁶-Benzylguanine¹

The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington, Seattle, Washington 98195-7705

O⁶-benzylguanine (BG), an inhibitor of O⁶-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10⁶ human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 µM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.

¹ This work was supported by Outstanding Investigator Grant CA39903 (to L. A. L.) and NIH Fellowship IF32CA66291 (to F. C. C.).

² Present address: Maxygen, 3410 Central Expressway, Santa Clara, CA 95051.

³ To whom requests for reprints should be addressed.

Received 11/27/96. Accepted 3/25/97.

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