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Researchers Develop New Method for Silencing Specific Genes in Human T-Cells

An international team of researchers led by Nanyang Technological University, Singapore, has developed a novel technique for gene silencing in human T-lymphocytes (T-cells), a type of immune cells.

Left: an illustration of GapmeR design, cellular internalization leading to specific gene silencing. Right: super-resolution microscopy of GapmeR co-localization with SNX5 protein -- a member of the sorting nexin family of proteins -- in human T-cells. Image credit: Fazil, M. H. U. T. et al, doi: 10.1038/srep37721.

Left: an illustration of GapmeR design, cellular internalization leading to specific gene silencing. Right: super-resolution microscopy of GapmeR co-localization with SNX5 protein — a member of the sorting nexin family of proteins — in human T-cells. Image credit: Fazil, M. H. U. T. et al, doi: 10.1038/srep37721.

In human body, information for synthesizing proteins that make up living cells is stored in genes. Genetic code is transcribed into messenger RNA (mRNA), which is translated into specific proteins.

An antisense oligonucleotide that binds to the target mRNA can interfere the process of protein synthesis.

Harnessing this RNA interference (RNAi) mechanism for modulating cellular responses is an area of ongoing intensive research.

While various gene silencing approaches, such as siRNA and CRISPR-Cas9, are available, these cannot be effectively applied to ‘hard-to-transfect’ primary T-lymphocytes.

“The main challenge to the success of RNAi approach is cellular delivery, stability and in vivo applicability of inhibitory oligonucleotides,” explained Dr. Navin Verma, a researcher at the Singapore Eye Research Institute and the Nanyang Technological University’s Lymphocyte Signalling Research Laboratory.

“Even more challenging is to silence individual gene with high sensitivity while maintaining specificity and avoiding potential off-target effects.”

“GapmeR is an emerging new class of gene silencing molecule, which is created by adding chemically modified Locked Nucleic Acid at both the ends of a central stretch or ‘gap’ of 5-10 base single strand antisense DNA,” said Dr. Verma, who is senior author of a paper on the findings published in the journal Scientific Reports.

“These chemical and structural modifications provide the chimeric antisense GapmeR with high target affinity, sequence specificity, biological stability, favorable pharmacokinetic and tissue-penetrating properties.”

In the current study, Dr. Verma and co-authors have developed and validated specific GapmeR molecules targeting a panel of genes in human primary T-cells.

In doing so, the researchers discovered two new proteins CG-NAP/AKAP450 and Stathmin as critical regulators of T-cell motility.

They detected that GapmeR internalizes into cells through macropinocytosis — a process used by cells to take-up nutrients.

“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 leukocytes and other cell types,” said co-author Prof. Dermot Kelleher, from the University of British Columbia.

“If we manage to fine-tune immune cells in a specific tissue, we may be able to devise selective therapies to better treat autoimmune diseases where drugs targeting T-cells have had unacceptable and serious side effects, such as progressive multi-focal leukoencephalopathy in the brain.”

“In addition to addressing intricate biological questions, our study offers a general framework for future research for developing novel therapeutics and we are beginning to explore the opportunities,” Prof. Kelleher said.

“The next step is to tweak the design and scale-up the synthesis for testing in animal models for various diseases,” Dr. Verma added.

“The approach may be combined with small molecules, therapeutic peptides or antibodies that can be incorporated into purpose-made nanodevices capable of controlled release for next-generation therapeutics.”

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Fazil, M. H. U. T. et al. 2016. GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells. Sci. Rep. 6, 37721; doi: 10.1038/srep37721

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