Suppression of Single and Double Nonsense Mutations Introduced into the Diphtheria Toxin A-Chain Gene: A Potential Binary System for Toxin Gene Therapy

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Abstract
We have previously shown that ablation of specific cells can be achieved through the transcriptionally regulated expression of the diphtheria toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cancer and acquired immunodeficiency syndrome (AIDS) therapy. The use of mutants of DT-A with attenuated toxicity may allow targeting of cells for which only moderately selective gene regulatory elements are available. Alternatively, conditional mutants might be used to target cells in which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons to amber (TAG) nonsense codons in the DT-A coding sequence. In transient transfection of HeLa cells, DT-A activity was markedly reduced by the introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from both single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyrosine codon 28 in DT-A. Thus, nonsense suppression can be used to control the expression of DT-A in mammalian cells, potentially allowing binary control over the targeting of tissues for selective ablation. Intracellular expression of the diphtheria toxin A-chain (DT-A) causes cell suicide and potentially represents a novel approach to cancer therapy, provided expression can be controlled tightly enough. Stringent control over DT-A expression might be achieved using a binary system involving controlled suppression of nonsense mutations in a transcriptionally regulated DT-A gene. Robinson and Maxwell introduce amber codons at selected positions in the DT-A coding sequence. Using transiently transfected HeLa cells, they show that both single and double nonsense mutations can be suppressed by a cotransfected suppressor tRNA expression plasmid, allowing substantial expression of DT-A activity. Importantly, although single nonsense mutants retained detectable activity in the absence of added suppressor, the double mutants were essentially inactive under these conditions. Such DT-A mutants might be of use for gene therapy, provided appropriate vehicles can be developed for delivery and/or expression of suppressor tRNAs.