Development of antisense oligonucleotides : a cellular model for enhancing receptor-mediated uptake
Iyer, Vaishnavi Srinivasan
Date of Issue2017
School of Biological Sciences
Antisense oligonucleotides (ASO) have been used to knockdown the expression of their target genes via ribonuclease (RNase) H-mediated mRNA degradation. One of the key issues faced with this promising method of gene targeting is delivery into the target cell. Current methods of delivery are focused on active free uptake. An emerging approach to increase uptake involves conjugation of the ASO with a ligand, corresponding to a highly expressed receptor on the target cell. The aim of this project is to establish a cellular model which allows for the regulation of receptor level. I manipulated the expression of receptors in the cellular model using tetracycline to induce the expression of the receptor gene in a highly controlled manner. I have achieved the aims of regulation of the level of asialoglycoprotein receptor-1 (ASGR1), shown physical uptake of the ligand-conjugated ASO and tested the knockdown potential of the ASO conjugate. I extended this system to another receptor, glucagon-like peptide-1 receptor (GLP1R). Future works will be dedicated to enhancing the knockdown further. In these experiments, the ASO used targets STAT3, the cell line is HEK293 Flp-In, which has an FLP Recombination Target (FRT) site into which ASGR1 and GLP1R needs to be integrated.
Final Year Project (FYP)
Nanyang Technological University