Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks <i>in vitro</i>

Hunt, M.A.; Li, Dan; Hay, Michael P.; Currie, Margaret J.; Robinson, Bridget A.; Patterson, Adam V.; Dachs, Gabi U.

doi:10.1002/jgm.1635

Cited by 9 publications

(12 citation statements)

References 72 publications

(94 reference statements)

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“…22 as a candidate for bacterial GDEPT, we show that it also renders host cells more sensitive to metronidazole, a nitroheterocyclic antibiotic in clinical use for the treatment of anaerobic bacterial infections (Lö fmark et al, 2010). Critically, metronidazole exhibits no bystander effect (Hunt et al, 2012), allowing the same nitroreductase that acts as a therapeutic gene in the presence of a high-bystander prodrug such as PR-104A to act as a suicide gene for vector elimination. Enhancing the sensitivity of a pathogenic bacterial vector to a clinical agent that can safely be used to eliminate it (e.g., in the event of adverse events or at the cessation of treatment) is highly desirable from a safety and regulatory perspective.…”

Section: Discussionmentioning

confidence: 90%

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

Copp

Mowday

Williams

et al. 2017

Cell Chemical Biology

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115

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Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.

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Section: Discussionmentioning

confidence: 90%

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

Copp

Mowday

Williams

et al. 2017

Cell Chemical Biology

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115

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“…They demonstrated the bystander cell killing mediated by endothelial cells transfected with a suicide gene nitroreductase (NTR) on unmodified tumor cells by using a 3D multicellular nodule model. More recently, Hunt et al presented coated spheroids as the 3D spatially distinct model to represent tissue-like cell density [21]. However, they observed absence of cytotoxic response in the 3D conditions in contrast to the monolayer cultures with the enzyme/prodrug NTR/CB1954 combination expressed in a human bladder carcinoma cells T24.…”

Section: Discussionmentioning

confidence: 99%

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment

Bohovic¹,

Demkova²,

Cihova³

et al. 2015

neo

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Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

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“…2A) and on the basis of their bystander effects having previously been evaluated in mixed multilayer cell culture models. In order of ascending bystander effect in these models these were: metronidazole, negligible bystander effect [2,16]; CB1954, low bystander effect [30,26,31]; PR-104A and its more lipophilic di-bromo mustard analogue SN27686, medium to high bystander effects [26,10,21]; and nitro-CBI DEI, maximal bystander effect [32,13]. (Note: as the different studies that we refer to employed substantially different proportions of activator cells in their mixed multilayer cell culture models, it is not possible to directly compare the calculated bystander effect efficiencies of the different prodrug metabolites, and hence we have employed qualitative terms to describe their relative bystander efficiencies.…”

Section: Assay Designmentioning

confidence: 99%

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy

Chan-Hyams

Copp

Smaill

et al. 2018

Biochemical Pharmacology

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Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2-versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

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Characterisation of enzyme prodrug gene therapy combinations in coated spheroids and vascular networks in vitro

Cited by 9 publications

References 72 publications

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy

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