Work package 4: Characterisation of the cells (safety, efficacy, aging)
Leader: Dr. Alexandra Stolzing, Loughborough University
As part of the expansion process for genetically corrected BOEC cells, Partner 4 UNILO will perform regular safety tests with particular regard to tumorigenic potential and chromosomal abnormalities of the cells, in order to ensure patient safety. The output of this work package will be a set of release criteria for functional cells. More precisely it will provide the maximal number of cell population doublings, release criteria based on ageing and functional markers for the cell production followed by cost analysis. All steps in endothelial manufacturing from starting material up to the final cell product will be highly standardised to assure reproducibility, consistency and safety.
Description of the tasks
Too many cell therapy products have only been described by detailing cell numbers and maximal cell doublings. There is also a lack of published data looking into testing cells with different doublings in vivo for their therapeutic activity. A set of markers forecasting in vivo cell activity would provide a bench mark for improving Advanced Therapeutic Medicinal Product (ATMP) production, minimize production costs and improve therapy outcome. This would improve our EU based capacity to develop ATMPs and help to optimise cell production conditions.
Task 1: Test tumorigenic potential of BOEC cells in vitro.
Cell batches from each individual donor will be tested for tumorigenic potential and will be karyotyped at the beginning and the end of the expansion process performed by Partner 4 UNILO. In addition, cells ready to be released will be tested for their tumorigenic potential in vivo in an appropriate immunodeficient mouse model (Partner 3 UPO). The mice that will be implanted with the Cell Pouch SystemTM containing the genetically corrected and expanded BOEC cells will also be monitored and verified for tumorigenic events as a final level of quality control.
Task 2: Functionality testing.
The supernatants from expanded BOEC cells (part of WP3) will be stored by UNILO, and sent for functionality testing to Partner 1 UKW. The functionality testing will verify the amount of factor VIII (FVIII) produced by the expanded cells as the main parameter. Feedback from UKW will provide UNILO with an amount of FVIII required to facilitate therapeutic success.
Task 3: Testing the expansion limit.
Cells age during expansion and therefore change their behavior. In order to quantify these changes, UNILO will test the epigenetic markers (ITGA2B, ASPA, PDE4C, GRM7, CASR, PRAMEF2, SELP, CASP14 and KRTAP13-3) of the expanded cells, using real time PCR. These results, once quantified, will be linked to the FVIII production with the help of Partner 1 UKW.