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Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.

Applied molecular evolution of O 6 -benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells

Applied molecular evolution of O ⁶ -benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells