Extempore preparations

Oncolytic virotherapy (OVT) uses viruses to selectively destroy tumor cells and stimulate antitumor immunity, independently of specific tumor antigens. Despite promising results in preclinical and clinical studies, OVT is not yet widely accepted as a therapy, partly due to manufacturing challenges and the need for high standards of purity in viral preparations. The aim of this research is to develop ex tempore (prepared immediately before use) oncolytic RNA virus preparations, with a particular focus on viruses such as mumps virus, measles virus, Newcastle disease virus, and vesicular stomatitis virus. The project plans to standardize small-scale production processes, optimize purification and characterization methods, and develop procedures that ensure the stability and reproducibility of the preparations. The methods include virus propagation in cell cultures, collection of supernatants, and purification through centrifugation, filtration, ultrafiltration, and chromatography. The quality of the preparations will be assessed by analysing viral infectivity, particle quantity, cellular contaminants (DNA and proteins), and the content of fetal bovine serum. Attention will be given to distinguishing viral particles from extracellular vesicles (EVs), which can interfere with measurement accuracy and therapeutic efficacy. In addition, the genetic diversity of viral populations will be investigated using deep sequencing to identify acceptable variations within the quasispecies composition of RNA viruses. Sensitive bioinformatic tools will be developed to differentiate true genetic variations from technical sequencing errors, enabling detailed quality and safety control of viral preparations intended for future clinical applications.

The Extempore project was financed as an institutional project of the University of Zagreb with grants from European funds from the Recovery and Resilience Mechanism (RRM) in the amount of EUR 202,681.37 for a period of 4 years (2025/12 – 2029/09).