DEVELOPMENT AND VALIDATION OF A NEW PTK7-TARGETED AAV-MEDIATED SUICIDE-GENE THERAPY FOR GLIOBLASTOMA

Abstract AIMS Glioblastoma (GBM, IDH WT, grade 4) is a dreadful primary brain tumor. Despite the current standard-of-care, patient survival remains dramatically poor. This highlighting the urgent need for new therapeutic strategies. In this context, we developed a novel therapy targeting Protein Tyrosine Kinase 7 (PTK7), highly expressed in GBM compared to non-tumoral brain tissue. By combining an anti-PTK7 Nanobody® (VHH) with a ‘suicide-gene therapy’ (SGT), we aim to design a highly-specific targeted therapy that holds promising therapeutic potential. METHODS PTK7 expression was assessed by immunofluorescence and flow cytometry on patient tissue samples and patient-derived GBM stem-like cell (GSC) cultures. In parallel, anti-PTK7 VHH were produced and validated using bio-layer interferometry and flow cytometry. These sequences were then combined with a recombinant AAV, and a suicide-gene delivery. The resulting anti-PTK7 AAV-SGT was tested for specific GBM cell death in- duction, using live cell imaging. To further confirm the specificity of this targeted SGT, we generated a PTK7-KO GSC culture, using the CRISPR-Cas9 technique, and used it as a control in all experiments. RESULTS PTK7 was highly expressed in GBM samples compared to non-tumoral brain, and highly expressed in GSCs, even upon therapy-mimicking stress conditions. The combination of anti-PTK7 VHH with the AAV-SGT effec- tively and specifically targeted and killed GBM cells following drug-induced activation of the SGT, leading to a concentration-dependent cytotoxicity. Notably, this cytotoxic effect was PTK7-dependent, as PTK7-KO cells remained unaffected by the targeted therapy. Ongoing in vivo studies aim to validate this strategy. CONCLUSION PTK7 was identified as a GBM-specific cell surface protein that could serve as a new target for therapy. We successfully validated VHH antibodies against PTK7 that show good affinity and specificity for GBM cells. We also generated promising preliminary data showing that AAV-mediated suicide-gene therapy, using PTK7 as an entry signal, induces GBM cell death.