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• W2022020971 abstract "With a prevalence of 1/2500, long QT Syndrome (LQTS) is one of the leading causes of sudden cardiac death in young and otherwise healthy individuals. LQTS is a proarrhythmic disorder hallmarked by prolongation of cardiac repolarization due to underlying ion channelopathies. This translates to the typical electrocardiographic (ECG) findings of prolonged QT interval, notched or biphasic T waves, and T wave alternans, and puts individuals at high risk of developing ventricular tachyarrhythmias and sudden cardiac death. To date, there are 13 subtypes of LQTS classified according to the 13 identified genetic defects, all encoding subunits of cardiac ion channels (K+, Na+, Ca2+) or ion channel regulatory proteins. Current treatment options for LQTS including beta-blockers, left cardiac sympathetic denervation, and implantable cardioverter-defibrillators are largely palliative [1]. Each of these existing modalities is associated with limitations such as adverse effects related to drug and surgical therapies and the limited lifespan of implantable devices. In the age of molecular genetics, a molecular diagnosis of the underlying ion channel defect not only establishes the disease state but paves the way for personalized medicine. Recently, Lu et al. adopted the use of RNA interference technology to target and rescue the channelopathy phenotype observed in LQTS type 2 due to KCNH2 mutation [2]. RNA interference therapy is based on the concept derived from the natural cellular mechanism where sequence-specific gene silencing can be achieved by creation of double-stranded RNA [3]. A synthetic effector small interfering RNA (siRNA) can be introduced into cells to silence cellular genes via an endogenous post-transcriptional gene silencing mechanism. This specific gene targeting approach is thus a new and attractive treatment option for genetic diseases such as LQTS which may avoid the disadvantages of conventional treatment modalities. Lu et al.'s study is based on the concept that LQTS type 2 is associated with mutations in the human ether-a-go-go-related gene, encoding for the alpha-subunit of the IKr channel. Such mutations lead to defective proteins that may either cause a dominant negative effect on wild-type subunits, happloinsufficiency, or defective trafficking. Blockage of IKr efflux from cells will lead to a delay in phase 3 rapid repolarization of the action potential and hence prolongation of QT interval. They hypothesized that by gene silencing of E637-hERG mutants (a dominant-negative mutation where a lysine was substituted with glutamic acid at position 637 in the pore-S6 loop transmembrane segment of hERG), would lead to relief of IKr inhibition and restore K+ current that is essential for cardiac repolarization, thereby preventing arrhythmias associated with IKr. They transfected plasmids containing wild-type or mutant hERG and siRNA in HEK293 cells which do not usually express the hERG protein, and then a systematic screening method via Western blot analysis was used to select the siRNA sequence with the highest knocking-down efficiency against E637-hERG mutant. Subsequently they characterized the cellular localization and electrophysiological properties of the hERG channel in order to determine the effect of siRNA on the molecular and electrophysiological function of IKr. For the validation of the rescuing effect of siRNA, a three-step approach was used. This was first done at a molecular level using Western blot analysis of hERG protein expression. Next, they examined the functional rescue of the defective electrophysiology in hERG mutants via patch clamp analysis. Several parameters were quantified including the voltage dependence of activation of the hERG channel, the steady-state inactivation kinetics, as well as the time course of deactivation. It was shown that siRNA treatment not only restored the maximal current and tail current amplitude of cells to a level comparable with their wild-type counterparts, they also reinstated the lost kinetic properties of the dominant negative hERG mutation. siRNA transformed both the activation and the steady-state inactivation curves of cells to closer to that observed in the wild-type controls, while it caused a reduction in the activation voltage to a level comparable to that of cells with wild-type hERG. siRNA also slowed down the rapid channel inactivation of mutants. The kinetics of recovery from inactivation was found to be unaffected by the addition of siRNA as shown by the unaffected deactivating tail currents. These observations occurred only in the cells with the presence of mutant hERG proteins, further validating the selectivity of the action of the designed siRNA. Finally, the authors postulated the mechanistic role of siRNA in the restoration of function of mutant hERG channels by showing that siRNA not only caused lower expression of the dominant negative mutant proteins (and thereby reducing its suppressive effect on wild-type channels), but it also prevented defective trafficking of the channel protein. Fractionation assay as well as confocal microscopy confirmed that siRNA treatment restored the mature hERG protein to the plasma membrane, where it serves its function gating the rapidly activating delayed rectifier potassium current. This study opens the possibility of novel gene therapies for the treatment of genetic cardiac disorders such as LQTS. The authors showed elegantly how a well-designed siRNA targeting a single genetic mutation was effective in restoration of the protein function in cells carrying a missense mutation. Their work can easily be expanded further to targeting other known mutations of the LQTS such as gain-of-function mutations in SCN5A encoding for cardiac sodium channels or G572R and A561V of hERG proteins which have also been shown to result in trafficking defective phenotypes and suppression of IKr current. However, arguably the main limitation of this study is that it was conducted using HEK293 cells, and will thus need to be validated in cardiomyocytes. While further work will be needed to show that siRNA treatment rescues the defective IKr current without affecting other ionic currents. To take the current study a step further would be to design and construct a lentiviral-mediated system of siRNA delivery in an animal model and to study the prevention of cardiac arrhythmias and sudden cardiac death. Another potential limitation to the use of RNA interference therapeutics as done by Lu et al. is the mode of delivery of siRNA. Currently, there are two main approaches to introduce siRNA into cells [4]: (1) an RNA-based approach where a preformed synthetic 21 bp siRNA effector is delivered via various carriers to the target cells (as used by Lu et al. in this study; or (2) a DNA-based strategy in which the siRNA effectors were produced by intracellular processing of longer RNA hairpin transcripts. The advantage of using direct introduction of synthetic siRNAs is that it allows room for enhancement of stability and efficacy and decreases unintended off-target effects via chemical modifications. The disadvantage of the approach adopted by Lu et al. is that the gene silencing effect of direct use of synthetic siRNA is short-lived, albeit potent. Furthermore, due to the size and their negative charge, siRNAs cannot readily cross cell membranes making delivery a challenging task [5], [6]. In contrast, promoter-expressed siRNA sequences potentially allow for a single treatment to achieve lasting effects of gene silencing. Finally, one must also critique the results of RNA interference mediated target gene knockdowns with due caution. This is because certain unwanted side effects have been observed from the myriad of studies done on RNAi based therapies in a wide range of diseases [7]. These include unintentional activation of Toll-like receptors and type 1 interferon responses, as well as competitive interference of the endogenous RNA interference pathway components. High level of exogenous siRNAs may sequester molecules in the endogenous RNA interference processing machinery, preventing their processing of cognate cellular microRNA. The ability of RNA interference to control expression of disease-associated genes makes siRNA-based intervention an exciting new therapeutic modality for personalized medicine for genetic diseases, such as LQTS. However, before taking a step further to translate bench research findings to the bedside, one will need to tackle several practical aspects including establishing a tissue-specific or even cell-specific delivery system of siRNA and prevention of unwanted side effects such as dysregulation of non-target genes. There remains further work to be done to bring this powerful tool from the laboratory into a clinical setting for LQTS, but the prospect of RNA interference therapeutics is promising. There is no conflict of interest related to this article." @default.
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• W2022020971 title "Personalized medicine for long QT syndrome: Restoration of ion channel function with RNA interference technology" @default.
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