Application of phagemid-AAV vector in gene therapy of paediatric brain cancer

Abstract

Medulloblastoma and Diffuse intrinsic pontine glioma (DIPG) are childhood brain tumours with high malignancy, the former being the most common and the latter is the most aggressive. Although medulloblastoma patients have a high likelihood of long-term survival after conventional treatment, the prognosis is unfavourable and the treatment options result in long-term neurologic side effects. On the other hand, DIPG patients have a poor survival rate of only 6-10% beyond two years with no effective therapies. Thus, the development of novel, safer and effective therapeutic strategies, or improvement of existing treatments are urgently needed. Gene therapy is an attractive field for the treatment of cancer and offers potential to overcome the limitations of current therapeutic strategies. Here we applied a targeted cytokine gene therapy by using an engineered hybrid adeno-associated phagemid-based vector (PAAV) to selectively target the tumour through cancer-selective integrin binding ligand. PAAV bacteriophage is genetically engineered to express the RGD4C peptide on the vector capsid to ensure targeting of tumour cells and tumour vasculature through binding to α_v β_3 integrin receptor that is overexpressed in tumours and absent or barely detectable in the healthy tissues. In this study, we investigated the efficacy of this targeted vector (RGD4C/PAAV) as a gene delivery vehicle for medulloblastoma and DIPG. The tumour necrosis factor (TNFα) was used as the cytokine therapeutic gene to be delivered and expressed in the tumour cells. This prototype (RGD4C/PAAV-TNFα) induced cell death and activated the caspase pathway in DIPG. This targeted delivery of TNFα was also investigated in medulloblastoma cells in vitro, using UW228 and Daoy medulloblastoma cell lines, and showed high efficacy in inducing cell death. This effect was further augmented by using low dose cisplatin. Moreover, we showed that cisplatin induced the activity of GRP78 and CMV promoters and boosted transgene expression by RGD4C/PAAV in both medulloblastoma and DIPG cells. Finally, the efficacy of our prototype was tested for the treatment of medulloblastoma in vivo through intravenous administration. RGD4C/PAAV- TNFα showed high a and selective tumour homing resulting in targeted TNFα transgene delivery to the tumours. Beside the induction of tumour cell death, this treatment strategy induced damage to the tumour-associated blood vessels, in vivo. TNFα cytokine has long been known to possess anti-tumour activity but also has high systemic toxicity limiting its use in the clinic. In this thesis, we demonstrated that the use of RGD4C/PAAV provides a solution to this problem by targeting TNFα expression selectively to the tumour environment, ensuring systemic safety and therapeutic efficacy for the treatment of childhood brain tumours.