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

作者：Fazil Mobashar Hussain Urf Turabe; Ong Seow Theng; Cha****ni Madhavi Latha Somaraju; Low Jian Hui; Kizhakeyil Atish; Mamidi Akshay; Lim Carey Fang Hui; Wright Graham D; Lakshminarayanan Rajamani; Kelleher Dermot; Verma Navin Kumar

来源：Scientific Reports, 2016, 6(1): 37721.

DOI：10.1038/srep37721

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 PKC epsilon, 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.

• 出版日期2016-11-24
• 单位河北医科大学; 南阳理工学院