GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells

• Main Authors: Fazil, Mobashar Hussain Urf Turabe, Ong, Seow Theng, Chalasani, Madhavi Latha Somaraju, Low, Jian Hui, Kizhakeyil, Atish, Mamidi, Akshay, Lim, Carey Fang Hui, Wright, Graham D., Lakshminarayanan, Rajamani, Kelleher, Dermot, Verma, Navin Kumar
• Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
• Format: Article
• Language: English
• Published: 2016
• Subjects: T cells; Translational immunology
• Online Access: https://hdl.handle.net/10356/84347; http://hdl.handle.net/10220/41790
• Institution: Nanyang Technological University

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to “hard-to-transfect” primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called “GapmeR”, is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.