Nucleic acid delivery using poly(ethylenimine)-based polymers for programmed death-ligand 1 (PD-L1) knockdown in ovarian cancer to enhance immunotherapy

摘要

Ovarian cancer remains the most lethal gynaecological cancer mainly due to the lack of reliable biomarkers and eventual development of chemo-resistance. This emphasizes the need for better therapies. Ovarian cancer is considered as an immunogenic tumour and adoptive immunotherapy is a promising treatment strategy. However, co-inhibitory molecules such as programmed death-ligand 1 (PD-L1), highly expressed on ovarian cancer cells interacts with its receptor, programmed death-1 (PD-1), expressed on T cells, causing immunosuppression. udThe aim of this Ph.D. was to 1) develop more efficient and targeted gene delivery agents by functionalizing poly(ethylenimine) (PEI) with various hydrophobic groups and folic acid (FA) targeting ligand, 2) deliver PD-L1 small interfering RNA (siRNA) or short hairpin RNA (shRNA) into ovarian cancer cells to block PD-1/PD-L1 interactions and 3) to study how T cell function and anti-tumour activity are affected as a consequence of PD-L1 knockdown. 4) In addition, detection of soluble PD-L1 (sPD-L1), using an enzyme linked immunosorbent assay (ELISA) and an Acoustic Membrane MicroParticle (AMMP) technology was also studied for its potential use as a biomarker for ovarian cancer diagnosis.udWhen PEI was functionalized with various hydrophobic functional groups (ethyl, octyl, deodectyl, benzl, phenyl urea) using the established methyl-carboxytrimethylene carbonate (MTC) platform, cytotoxicity was greatly reduced (for PEI, Mn=10 kDa) and gene transfection efficiency was substantially enhanced (Teo et al., 2013, Yang et al., 2013). Hydrophobic modification, however, did not improve siRNA delivery and PEI polymers functionalized with FA targeting groups (FA, poly(ethylene glycol) (PEG), PEG-FA), were investigated for PD-L1 delivery. FA-functionalized PEI displayed a higher specificity of uptake into ovarian cancer cells. PD-L1 knockdown in ovarian cancer cells using FA-functionalized PEI/PD-L1 siRNA polyplexes rendered tumour cells more susceptible to killing by chimeric antigen receptor (CAR) expressing T cells (Teo et al., 2015). In addition, an in-house ELISA, but not the AMMP platform, successfully detected sPD-L1 in patient plasma which differentiated patients with benign or malignant disease.udIn conclusion, PD-L1 has tremendous potential as a therapeutic and diagnostic target for ovarian cancer. Furthermore, modified PEI gene delivery agents has been shown to be an effective therapeutic tool in ovarian cancer and its use may be extended to a wide range of diseases which arise due to errant genes.