Targeted EDV™ Nanocells carrying small interfering RNA (siRNA) molecules to overcome drug resistance in Non-small cell lung cancer
- Publication Type:
- Thesis
- Issue Date:
- 2021
Open Access
Copyright Clearance Process
- Recently Added
- In Progress
- Open Access
This item is open access.
𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱. Over two million people worldwide suffer from lung cancer, the main sub-type (85%) being NSCLC and despite many chemotherapeutics approved for NSCLC, only 2% of patients with NSCLC metastatic disease survive 5 years post diagnosis with multidrug resistance being the major cause of mortality in NSCLC patients.
𝗔𝗶𝗺. The overall aim of this project was to evaluate targeted EnGeneIC Dream Vector™ (EDV™) nanocells for loading and delivering small interfering RNA (siRNA) molecules, Polo like kinase-1 (PLK1), Ribonucleoside reductase subunit M1 (RRM1) & Kinesin Spindle Protein (KSP), in order to silence proteins essential to tumour cell survival and proliferation, and to evaluate their therapeutic potential in overcoming the hitherto intractable multiple drug resistance in non- small cell lung cancer (NSCLC).
𝗠𝗲𝘁𝗵𝗼𝗱𝘀. The expression of cell cycle genes PLK1, RRM1 & KSP in NSCLC cell lines was measured using RT-qPCR and Western Blot. Efficacy of siRNAs targeting PLK1 (siPLK1), RRM1 (siRRM1) & KSP (siKSP) transfected into NSCLC cell lines was measured by the MTS proliferation assay and Western Blot. Flow cytometric analysis was used to measure apoptosis and cell cycle arrest in NSCLC cell lines transfected with the siRNAs targeting PLK1, RRM1 and KSP. EDV™ nanocells were targeted to the epithelial growth factor receptor (EGFR) and the copy number of siRNAs loaded into the nanocells was measured by staining with an RNA specific dye and measured on a fluorometer compared to known standards. EDV™-siRNAs were used to treat NSCLC cells lines grown as 3D spheroids using the hanging drop plates (Perfecta3D®:HDP1096) and cell proliferation inhibition was assessed using trypan blue cell viability assay. The EDV™s-siRNAs were then tested in vivo using the A549-Dox-R a xenograft mouse model, tumours were excised and assessed for gene knockdown by RT-qPCR.
𝗥𝗲𝘀𝘂𝗹𝘁𝘀 𝗮𝗻𝗱 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻. In this study we show that in vitro and in vivo, EDV™s can effectively deliver targeted-cell cycle-siRNAs to hanging drop 3D spheroids and into a mouse xenograft model to inhibit cell and tumour growth, and that EDV™s can encapsulate and deliver a significant siRNA payload directly inside the tumour cells without affecting non-target tissue. Overall, this study highlights the exciting possibility that siRNAs against mitotic regulators loaded into EDV™s will be safe alone or in combination with drug-loaded EDV™s, and may overcome drug resistance in NSCLC patients. This project has true translational potential for both delivering hitherto “undeliverable” functional nucleic acids, and for potentially addressing drug-resistance mechanisms in lung cancer.
Please use this identifier to cite or link to this item: