Use of positive selection methods for discovery and improvement of nitroreductase enzymes for cancer gene therapy

Bacterial nitroreductases are members of a diverse family of oxidoreductase enzymes that are capable of activating nitroaromatic compounds, including anticancer prodrugs such as CB 1954 and PR-104A. This capability is useful in the anti-cancer gene therapy strategy known as gene-directed enzyme prodrug therapy (GDEPT), which involves the killing of tumour cells through activation of an inert prodrug to its cytotoxic form, following selective delivery of a genetically encoded prodrug-converting enzyme to cancerous tissues. A key limitation in nitroreductase-based GDEPT has been the inability to rapidly and non-invasively determine vector localisation and gene delivery prior to systemic administration of prodrug. To address this we have developed dual-purpose nitroreductases that exhibit the ability to efficiently activate both GDEPT prodrugs and next-generation radioisotope-labelled PET imaging probes, in a manner that renders the probes temporarily cell-entrapped for detection using a PET scanner. This capability places greater control of the therapy in the hands of the clinician, and will facilitate clinical development of this treatment. One key focus has been the engineering of more efficient enzymes using both random and targeted mutagenesis strategies. A complementary strategy has been the discovery of novel nitroreductases through the screening of metagenomic fragments of DNA from the unculturable bacteria present in New Zealand soil. To enable efficient screening of these libraries, we have developed an array of genetic and biochemical tools for the rapid selection of active nitroreductases. Here we have investigated the effectiveness of these different approaches for improving nitroreductase activity, and demonstrate their utility in improving activity with specific target substrates including next- generation prodrugs and PET imaging probes

Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

The Australian Coral Reef Fin-Fish Fishery ITQ Market

The Australian coral reef fin-fish fishery (CRFFF) on the Great Barrier Reef has been managed under ITQs since 2004. A large number of different reef species (>155) are covered by the management plan, but the primary species in terms of value and volume landed are coral trout and red throat emperor. All remaining species are classified as other species for management purposes. The operational side of the fishery is relatively heterogeneous and distinct sub-divisions exist between participants in terms of individual vessel characteristics and the species and product forms landed. The ITQ system has introduced an additional dimension for heterogeneity between fishers whilst also allowing investors not participating in the fishery to hold and trade quota. This analysis uses data on individual level quota holdings and trades to assess the CRFFF quota market and its evolution through time. Fishery level trends in ownership and trade are determined, and market participants are identified as belonging to one out of a set of seven generalized types. The emergence of groups such as investors and lease dependent fishers is clear. In 2010-11 42% of coral trout quota was owned by participants that did not fish it and 69% of total coral trout landings were made by fishers that owned only 11% of the quota. The incentives faced by the groups within the fishery differ, as do their vulnerabilities and resilience to natural or management related changes in the fishery's situation

Assessing the Impact of Stakeholder Engagement in Management Strategy Evaluation

After completing a large, regional, multi-use Management Strategy Evaluation, we attempt to assess the impact of stakeholder engagement on the project. We do so by comparing the original project plan to the actual project development and highlight the changes which can be more directly related to stakeholder engagement aided by the application of a logic model for program evaluation. The impact can be summarised into four broad classes: a) change in the actual project development; b) a measurable change in the network of interactions both stakeholders (which includes researchers); c) changes in how the computer model was developed and run; and d) changes in attitudes among stakeholders (including researchers). We discuss these changes, the way they have been detected and some lessons we learnt which may benefit future Management Strategy Evaluation projects

The Complementarity of No-Take Marine Reserves and Catch Shares for Managing The Coral Reef Line Fishery of The Great Barrier Reef

Management changes in the coral reef fin fish fishery of the Great Barrier Reef provide a natural experiment of the efficacy of combining no-take areas and dedicated catch shares. A spatially-explicit bio-economic model of the fishery is used to analyze the tradeoffs between biomass and the net returns from fishing under different management regimes. Results for the scenarios examined show that: (1) the more the fishery is depleted, the greater are the payoffs from combining catch shares with no-take marine reserves; (2) a lower harvest while at high rates of exploitation only lowers net returns slightly for a wide range of reserve sizes; and (3) an increase in the reserve area from when it is zero or small- sized leaves net returns virtually unchanged at any catch level. Thus, catch shares and reserves are complementary and, when used jointly, promote lowering of TACs if rates of exploitation are high and increasing reserve sizes when no-take areas are small

Modeling the economic and ecological co-viability of the Northern Prawn Fishery in Australia

With the development of the ecosystem approach to f isheries (EAF), fisheries management is increasingly required to deal with multiple, often conflicting objectives. In this context, the stochastic co-viability approach has been proposed as a useful modeling framework as it allows for the combined representation of complex fisherie s dynamics, uncertainty and the need to include multiple dimensions in the evaluation of su stainability. The present paper focuses on the application of this approach to the case of the Nor thern Prawn Fishery (NPF) in Australia. The fishery is based on several high valued prawn speci es each with different biology: an uncertain resource, the white banana prawn (Penaeus merguiens is), a more predictable resource comprising two target tiger prawns (Penaeus semisul catus and P. Esculentus) and the by-product blue Endeavour prawn (Metapenaeus endeavouri). A bi o-economic multi-species and multi- fishing strategies model is developed to examine ho w the fishery can operate within a set of constraints relating to spawning stock size of each prawn species, impacts on benthos and habitats, and economic profitability of the fleet