title: GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells.
abstract: 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.
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.
authors.forename: Mobashar Hussain Urf Turabe
authors.initials: MH
authors.lastname: Fazil
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.
authors.forename: Seow Theng
authors.initials: ST
authors.lastname: Ong
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.
authors.forename: Madhavi Latha Somaraju
authors.initials: ML
authors.lastname: Chalasani
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.
authors.forename: Jian Hui
authors.initials: JH
authors.lastname: Low
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.
authors.forename: Atish
authors.initials: A
authors.lastname: Kizhakeyil
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.|School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459, Singapore.
authors.forename: Akshay
authors.initials: A
authors.lastname: Mamidi
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors.forename: Carey Fang Hui
authors.initials: CF
authors.lastname: Lim
authors.affiliation: Institute of Medical Biology, A*STAR, Immunos, Biopolis, 138648, Singapore.
authors.forename: Graham D
authors.initials: GD
authors.lastname: Wright
authors.affiliation: Singapore Eye Research Institute, The Academia, 20 College Road, 169856, Singapore.
authors.forename: Rajamani
authors.initials: R
authors.lastname: Lakshminarayanan
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.|Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V1Y 1T3, Canada.
authors.forename: Dermot
authors.initials: D
authors.lastname: Kelleher
authors.affiliation: Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore.|Singapore Eye Research Institute, The Academia, 20 College Road, 169856, Singapore.
authors.forename: Navin Kumar
authors.initials: NK
authors.lastname: Verma
mesh_terms: D001692:Biological Transport / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010873:Pinocytosis / Q000235:genetics / Q000502:physiology*; D058305:Sorting Nexins / Q000235:genetics; D050777:Stathmin / Q000235:genetics; D013601:T-Lymphocytes / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D003598:Cytoskeletal Proteins; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D058305:Sorting Nexins; D050777:Stathmin; C477371:locked nucleic acid