Inherited long QT syndrome (LQTS) is an electrical heart disorder that is characterized by the abnormal prolongation of cardiac repolarization, which corresponds to the QT interval on an electrocardiogram. It has been observed that the prevalence of LQTS is ~1:2,500 among Caucasians (1). Clinically, patients with LQTS are at an increased risk of torsades de pointes, which manifests as palpitations, syncope, seizures and sudden cardiac death. To date, hundreds of mutations have been identified that contribute toward the development of LQTS, and these are present in at least 13 genes (Table I) (2–4). It has been determined that >90% of positive-genotype cases of LQTS exhibit one of three particular genotypes: LQT1, LQT2 and LQT3 (3,5–8).

Current treatment options for LQTS include the administration of β-adrenoceptor antagonists, the implantation of pacemakers or implantable cardioverter defibrillators (ICDs), and left cardiac sympathetic denervation (7). However, there are limitations associated with each of the current treatment options for LQTS (7–9). Firstly, ICDs are usually expensive and available only to a relatively small proportion of patients with LQTS. Drug-based therapies are widely available, but exhibit limited efficacy in patients with advanced LQTS. Furthermore, in blood relatives, LQTS symptoms may be severe, and generate complicated clinical situations. Although genomic studies have contributed significantly to the improvement of diagnostics and a limited number of treatments, the human genome is more complex than was initially expected. For example, individuals with identical genetic defects in the same pedigree may present with different characteristics. These differences may be partially due to the polygenic nature of genetic diseases, as there are multiple genetic factors that contribute toward the pathology of a disease, thus complicating the etiology of LQTS (10). Finally, the primary limitation of current therapeutic approaches is that their targets are relatively independent of the specific origin of inherited LQTS. To date, many mutations in various genes have been well-characterized and their toxic mutant protein products widely recognized as being responsible for the dominant-negative processes underlying the development of LQTS (11–14). However, for many of these culprit targets, there are currently no treatments available that successfully act on them. None of the current therapeutic approaches are able to differentiate a target consisting of a single-nucleotide polymorphism mutation. The inadequacies of current therapies have prompted the investigation and screening of novel alternative treatments for LQTS, including gene therapies. The process of RNA interference (RNAi) is increasingly recognized as serving a crucial function in regulating direct gene expression (15–19), and studies of the use of RNAi for modulating LQTS-associated gene function have been initiated (8,20).

Mutations have been identified to cause ion channel dysfunction by various mechanisms. In this section, the human ether-à-go-go-related gene (hERG), whose frequency in mutation-identified LQTS patients is the highest in Chinese patients, is discussed as an example. hERG is in chromosome 7 and encodes the α-subunit of the rapidly activating delayed rectifier K⁺ current (I_Kr) in the heart, a current that contributes toward the repolarization of the cardiac action potential (21,22). hERG potassium channels form homotetramers of identical six α-helical transmembrane-spanning domains (11,21,23,24). Delisle et al (12) and Gong et al (24) have reviewed a proposed classification system for LQT2 mutations, which organizes these mutations according to their underlying mechanisms. Class 1 mutations produce abnormal protein synthesis by defective transcription or translation. Class 2 mutations lead to defective protein trafficking. Class 3 mutations result in abnormal gating and/or kinetics. Class 4 mutations result in altered or absent channel selectivity or permeability. Class 5 mutations produce a reduction in mutant mRNAs through nonsense mediated decay, thereby altering the quantity of mRNA available for subsequent hERG protein generation (11–14,24). The majority of hERG mutations exhibit loss-of-function phenotypes, due to haploinsufficiency or suppression of wild-type (WT) function by a dominant-negative mechanism. Dominant-negative current suppression is a mechanism by which mutant hERG alleles may lead to a reduction of the number of WT hERG channels in heterozygous patients, with the result that during the co-assembly of hERG WT and mutant hERG channel subunits, the tetrameric hERG channel complexes are rendered non-conducting. The dominant-negative suppressive effects of hERG mutations on WT function are the result of a number of defects (13,14). In the majority of human LQT2 mutations, the dominant-negative suppression of WT function is a result of the trafficking-deficient mechanism (12–14,25). The dominant-negative current suppression is explained most accurately as a conditional trafficking block imposed on WT channels by co-assembly with trafficking-deficient mutant subunits, with increased degradation as a consequence of impaired trafficking.

RNAi may be generated by molecules other than mRNA and it may be possible to design and utilize RNAi-based techniques for numerous applications in molecular biology and medicine (15,17,18,26–29). RNAi is an evolutionary conserved mechanism for silencing sequence-specific post-transcriptional genes. RNAi was initially recognized by Fire et al (30) in 1998, who determined that double-stranded RNA was substantially more effective than a single strand at generating interference. Following injection into adult animals, purified single strands of RNA exhibited a modest effect, whereas double-stranded mixtures resulted in marked and specific interference. The effects of this interference could be observed in the injected animals and their progeny. Only a small number of double-stranded RNA molecules were required per affected cell, indicating that stoichiometric interference with endogenous mRNA is unlikely and suggesting that there may be a catalytic or amplification component in the interference process (30). Currently, RNAi exhibits the potential to be a novel therapeutic strategy in various areas of medicine. The capability of RNAi to reduce the expression of pathological proteins is potentially applicable to virtually all classes of molecular target, including those that are difficult to selectively modulate with traditional pharmaceutical approaches involving small molecules or proteins (17,26–29,31–34). The exploitation of small RNA biogenesis and silencing gene pathways for heart disease, using small interfering (si)RNA against single targets or inhibiting the action of misregulated micro (mi)RNA, has yielded promising results and such agents are approaching clinical trials (17,26). Fitzgerald et al investigated the safety and efficacy of ALN-PCS, an siRNA that inhibits PCSK9 synthesis, in healthy volunteers with elevated levels of cholesterol who had received no lipid-lowering treatment (29). Administration of ALN-PCS led to a rapid, dose-dependent reduction in plasma PCSK9 levels, with higher doses exhibiting an extended duration of effect. The maximum reduction in low-density lipoprotein (LDL) cholesterol noted in an individual participant was 57%. These results suggested that the inhibition of PCSK9 synthesis by RNAi provides a potential alternative approach for reducing LDL cholesterol levels in healthy individuals with raised cholesterol. The study by Fitzgerald et al was the first to demonstrate the effective clinical application of RNAi-based therapeutic strategies against a validated endpoint (LDL cholesterol) in humans (29).

There are numerous problems to be solved prior to the use of RNAi therapy in clinical practice. To date, multiple barriers to achieving efficient and safe RNAi-based treatment have become evident, including specificity for the target gene, delivery to the correct cell or tissues, the duration of RNAi activity and the stability of the target mRNA and encoded protein (8,15–17,27,33,45,46). Efficient delivery to target cells and tissues is the primary challenge for RNAi-based therapeutics. A key issue is the physical guiding of the vector to the target tissue (33). Cardiac targeting has emerged as an extremely difficult task that has not been achievable in a simple and efficient manner using earlier gene transfer systems. A second key issue is the stability of the therapeutic vector. Small regulatory RNAs are inherently unstable, and viral vectors are currently the only available tools for non-topical in vivo therapy using these molecules. However, multiple proteins are also inherently unstable and/or intracellular, which similarly suggests the use of long-term stable vectors for long-term therapy (33). Third, the safety issues associated with RNAi-based therapies require consideration. Even assuming that the delivery problems can be solved, toxicity of small hairpin (sh)RNA may result from competition with the normal cellular miRNA processing system. shRNAs exert their therapeutic effects by mimicking this endogenous process and achieve silencing; however, they may disturb cellular miRNA pathways and thereby result in adverse effects. For other target diseases experimentally treated with cardiotropic RNAi vectors with low affinity to the liver, no hepatic or other toxicity has been observed (17), however toxicity remains a possibility. The off-target effects of regulatory RNAs are another possible source of toxicity and are difficult to assess (46). As a consequence, side-effects at the cellular level and in vivo require further investigation to assess the possible toxicity of the RNAi-based therapeutics.

Mutations associated with LQTS typically lead to trafficking deficiencies, which introduce dominant-negative effects and thus result in ion channel dysfunction. Recently, RNAi has emerged as a robust technology for improving disease phenotypes in vitro and in in vivo models of human disease. Early studies supported the potential new class of RNAi-based therapy in the treatment of numerous genetic diseases, as selective and potent gene expression inhibitors. The present review discusses examples of mutations that induce dominant-negative effects, which are able to destabilize mRNA transcripts via a mechanism known as RNAi in inherited LQTS. This mechanism helps to reduce the quantity of mutant mRNA transcripts, thus reducing the production of toxic proteins. Although a number of studies are in progress, numerous challenges require solutions prior to the application of RNAi in clinical practice. Further studies are required to clarify the key obstacles, and to investigate strategies that may lead to safe and effective delivery technologies as RNAi-based therapies are adopted in clinical practice. Additionally, the combination of RNAi and certain emerging techniques, such as genetically-modified iPSCs to treat heart disease, are also promising. However, the field must progress further to enable the necessary development of this therapeutic approach for the treatment of patients with otherwise untreatable genetic diseases. Due to the pace of investigation, RNAi may emerge as a valuable therapy and has the potential to be used clinically for the treatment of LQTS.