Self-deliverable GapmeR gene silencers designed to hit molecular targets in human cells

A team of researchers led by Assistant Professor Navin Kumar Verma at Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore (LKCMedicine) has developed a new method of gene silencing in human T-lymphocytes, a type of immune cell. The immune system plays a key role in the protection of our body from inflammation and infections. The ground-breaking research findings have just been published in the internationally renowned Nature group’s journal Scientific Reports.

In the 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 on-going intensive investigation worldwide.

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 main challenge to the success of RNAi approach is cellular delivery, stability and in vivo applicability of inhibitory oligonucleotides. Even more challenging is to silence individual gene with high sensitivity while maintaining specificity and avoiding potential off-target effects”, says Dr Verma.

“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. These chemical and structural modifications provide the chimeric antisense GapmeR with high target affinity, sequence specificity, biological stability, favourable pharmacokinetic and tissue-penetrating properties – Dr Verma explains.

In the current study, Dr Verma and his colleagues at LKCMedicine Dr MHUT Fazil, Dr Seow Theng Ong, Dr Madhavi Latha Chalasani and Mr Jian Hui Low have developed and validated specific GapmeR molecules targeting a panel of genes in human primary T-cells. In doing so, they 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.

Professor Dermot Kelleher, co-author from the University of British Columbia (UBC), who is also a Visiting Professor at LKCMedicine says: “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”.

“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 (PML) in the brain” – according to Professor Kelleher.

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

Article: GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells, Fazil MHUT, Ong ST, Chalasani MLS, Low JH, Kizhakeyil A, Mamidi A, Lim CFH, Wright GD, Lakshminarayanan R, Kelleher D, Verma NK, Scientific Reports, doi: 10.1038/srep37721, published online 24 November 2016.

Be the first to comment

Leave a Reply

Your email address will not be published.


*