27 Development of Precision Virotherapies for Diffuse Midline Gliomas

Abstract Introduction Diffuse Midline Gliomas (DMGs) are highly aggressive paediatric brainstem tumours with a median survival of 9-11 months - a figure unchanged for over four decades. This indicates a critical need for targeted therapies. The VITAL lab has developed Ad5NULL-RGD, an engineered adenovirus modified to ablate binding to native adenovirus receptors minimising off-target effects. The vector was redirected to αvβ3/5 integrins highly expressed in several brain tumours, via an RGD motif, enhancing tumour-specific uptake. The overarching aim of this project is to characterise the receptor profile of these tumours and evaluate Ad5NULL-RGD as a therapeutic vector for DMG. Methods Flow cytometry of DMG cell lines was performed to assess expression of αvβ3, αvβ5 integrins, and the Coxsackievirus and Adenovirus Receptor (CAR). Transduction efficiency was quantified using luciferase-based assays following infection with Ad5 and Ad5NULL-RGD vectors. Cytotoxic activity was evaluated using CellTiter-Glo viability assays post-infection with the oncolytic Ad5 and Ad5NULL-RGD vectors. Results DMG cell lines consistently overexpressed αvβ3 and/or αvβ5 integrins, validating them as entry receptors for the modified vector. Ad5NULL-RGD demonstrated enhanced transduction efficiency and preferential uptake compared to unmodified Ad5. Both oncolytic Ad5 and Ad5NULL-RGD induced significant cytotoxic effects, resulting in a marked reduction in DMG cell viability compared to uninfected controls, with Ad5NULL-RGD offering enhanced tumour selectivity. Conclusions Ad5NULL-RGD effectively targets DMG cells via αvβ3/αvβ5 integrins, offering improved transduction over native Ad5 and on-target cytotoxicity. These results highlight its potential as a safe and effective virotherapy for DMG, with broader applications for targeted gene therapy in brain tumours.