Title

Engineering bacterial nitroreductases for anticancer gene therapy and targeted cell ablation

Conference Dates

September 24-28, 2017

Abstract

Specific tumour ablation through gene therapy holds great promise as an anti-cancer strategy. Gene therapy has potential to achieve specificity and ablation through a mechanism unlike that of chemo and radio-therapies, and to be used in combination with these therapies without overlapping toxicities. In one promising therapy, a bacterial or viral tumour-tropic vector can be ‘armed’ with genes encoding enzymes that convert prodrugs, non‑toxic precursor molecules, into a highly cytotoxic form. Current cancer treatments are disadvantaged by their non-specificity resulting in undesirable side-effects for patients, and historically gene therapy has been hindered by the inability of the vector to infect all cancer cells necessary to eradicate the tumour and prevent recurrence. Arming a vector with a nitroreductase (NTR) enzyme which activates a prodrug to a metabolite with a high bystander effect can address these limitations. We are using directed evolution to expand our repertoire of candidate NTRs for use in cancer gene therapy and identify NTRs with an improved ability to activate a nitroaromatic prodrug. The NTR NfsB from Vibrio vulnificus (NfsB_Vv) was found to have good activity with the prodrug and as such was selected as a scaffold for directed evolution. We have crystallised and solved the structure of NfsB_Vv to 2.65 Å and a double mutant, NfsB_Vv F70A/F108Y, to 1.98 Å. These structures were used to direct the selection of five additional residues to be targeted in the creation of a site-saturation mutagenesis library. Through library screening strategies involving rounds of both positive and negative selection pressures, several mutants that displayed improved prodrug activity in vivo were identified. Several engineered nitroreductases from this library were also serendipitously improved in activation of the prodrug antibiotic metronidazole, which has been used for targeted cell ablation in transgenic zebrafish. In ongoing work, we are investigating the possible applications of these engineered nitroreductases for targeted cell ablation in zebrafish models, both to provide more efficient nitroreductases that will resolve limitations in the existing system, and to enable selective targeting of multiple tissues within a single transgenic fish line.

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