Characterization of the nitroreductase/metronidazole suicide gene system as a safeguard for cell based therapies

Miranda Li

Abstract


Cell-based therapies are promising treatment strategies for a variety of disorders ranging from cancer to spinal cord injuries. However, there is a risk of the transplanted cells becoming malignant. As a safeguard against this, suicide gene systems can be implemented so that transplanted cells can be eliminated if necessary by administering a pro-drug. Herpes simplex virus thymidine kinase (HSV-tk) paired with the pro-drug ganciclovir (GCV) is one of the most studied suicide gene systems. However, it can only kill cells that are actively dividing. Here we characterize another suicide gene system, nitroreductase (NTR) with its pro-drug metronidazole (MNZ), to investigate where in the cell cycle the killing occurs, hypothesizing that it could become an ideal candidate for eliminating transplanted cells irrespective of their proliferative status. Murine embryonic stem cells were transfected with vectors expressing
either HSV-tk or NTR and treated with the corresponding pro-drug. Confocal imaging and FUCCI (fluorescent ubiquitination-based cell cycle indicator) were used to identify where in the cell cycle the drug was active. MNZ was found to kill both dividing and non-dividing cells whereas GCV killed only the dividing cells. These results
suggest that the NTR system may be a valuable addition or complement to HSV-tk

Les thérapies cellulaires sont des stratégies promettantes en tant que traitements pour une variété de maladies. Celles-ci incluent le cancer et les traumatismes médullaires. Cependant, il y a un risque que les cellules implantées puissent devenir malignes. Afin de prévenir cela, des systèmes de gènes suicides peuvent être utilisés afin d’éliminer les cellules implantées si nécessaires par l’administration d’une prodrogue. La thymidine kinase, une enzyme
trouvée chez les patients atteint du virus de l’herpès simplex (HSV tk), utilisée en conjonction avec la prodrogue ganciclovir (GCV), est un des systèmes de gènes suicides les plus étudiés. Cependant, il peut seulement tuer les cellules qui se divisent activement. Ici, nous caractérisons un autre système de gènes suicidaires, nitroréductase (NTR) avec sa prodrogue metronidazole (MNZ), afin d’étudier à quel point dans le cycle cellulaire la tuerie se déroule. L’hypothèse est que ce système pourrait être un candidat idéal afin d’éliminer les cellules transplantées, peu importe leur statut prolifératif. Des cellules de souche embryonnaires murines ont été transfectées avec
des vecteurs qui exprimaient soit HSV- tk ou NTR et traitées avec la prodrogue correspondante. La microscopie confocale et le système FUCCI (pour fluorescent ubiquitination-based cell cycle indicator) ont été utilisés afin d’identifier le point du cycle pendant lequel la drogue était active. Il a été trouvé que MNZ tuait les cellules qui se
divisaient et qui ne se divisaient pas, alors que GCV tuait uniquement les cellules qui se divisent. Ces résultats suggèrent que le système NTR pourrait être une addition ou un complément utile à HSV-tk.


Keywords


suicide gene; nitroreductase; metronidazole; stem cells; cell therapy

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References


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DOI: https://doi.org/10.13034/jsst.v10i2.226

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