Ribonuclease Dicer functions in cells to excise microRNAs from their precursors and process long double-stranded RNAs into short interfering RNAs. We show that transcripts containing long hairpin structures composed of CNG repeats are another class of Dicer targets. The cellular levels of transcripts from mutant genes involved in triplet repeat expansion diseases such as myotonic dystrophy type 1, Huntington's disease, and spinocerebellar ataxia type 1 are under Dicer control. The Dicer-induced downregulation of the mutant transcript in myotonic dystrophy cells is accompanied by the downregulation of transcripts containing long complementary repeats. Short CUG repeats generated from long repeat hairpins act as siRNAs and use the RNA interference pathway to trigger the downstream silencing effects. We demonstrate that synthetic oligonucleotides composed of repeats are highly specific in the silencing of mutant transcripts containing complementary repeats and may be considered as potential therapeutic agents.

This review presents detailed information about the structure of triplet repeat RNA and addresses the simple sequence repeats of normal and expanded lengths in the context of the physiological and pathogenic roles played in human cells. First, we discuss the occurrence and frequency of various trinucleotide repeats in transcripts and classify them according to the propensity to form RNA structures of different architectures and stabilities. We show that repeats capable of forming hairpin structures are overrepresented in exons, which implies that they may have important functions. We further describe long triplet repeat RNA as a pathogenic agent by presenting human neurological diseases caused by triplet repeat expansions in which mutant RNA gains a toxic function. Prominent examples of these diseases include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome, which are triggered by mutant CUG and CGG repeats, respectively. In addition, we discuss RNA-mediated pathogenesis in polyglutamine disorders such as Huntington's disease and spinocerebellar ataxia type 3, in which expanded CAG repeats may act as an auxiliary toxic agent. Finally, triplet repeat RNA is presented as a therapeutic target. We describe various concepts and approaches aimed at the selective inhibition of mutant transcript activity in experimental therapies developed for repeat-associated diseases.

The specific silencing of the gene of interest is the major objective of RNA interference technology; therefore, unique sequences but not abundant sequence repeats are targeted by silencing reagents. Here, we describe the targeting of expanded CAG repeats that occur in transcripts derived from the mutant allele of the gene implicated in Huntington’s disease (HD) in the presence of the normal allele and other human mRNAs containing CAG and CUG repeat tracts. We show that a high degree of silencing selectivity may be achieved between the repeated sequences. We demonstrate preferential suppression of the mutant huntingtin allele and concomitant activation of the normal huntingtin allele in cell lines derived from HD patients that were transfected with short RNA duplexes composed of CAG and CUG repeats containing mutations at specific positions. These effects may lead to promising therapeutic modalities for HD, a condition for which no therapy presently exists.

MicroRNAs comprise a large family of short, non-coding RNAs that are present in most eukaryotic organisms and are typically involved in downregulating the expression of protein-coding genes. The detailed mechanisms of miRNA functioning in animals and plants have been under investigation for more than decade. In mammalian cells, miRNA guides the effector complex miRISC to bind with partially complementary sequences, usually within the 3′UTR of mRNAs, and inhibit protein synthesis with or without transcript degradation. In addition to these main mechanisms, several other modes of miRNA-mediated gene expression regulation have been described, but their scale and importance remain a matter of debate. In this review, we briefly summarize the pathway of miRNA precursor processing during miRNA biogenesis and continue with the description of the miRISC assembly process. Then, we present the miRNA-mediated mechanisms of gene expression regulation in detail, and we gather information concerning the proteins involved in these processes. In addition, we briefly refer to the current applications of miRNA mechanisms in therapeutic strategies. Finally, we highlight some of the remaining controversies surrounding the regulation of mammalian gene expression by miRNAs.

Huntington’s disease (HD) is a neurodegenerative genetic disorder caused by the expansion of the CAG repeat in the translated sequence of the HTT gene. This expansion generates a mutant huntingtin protein that contains an abnormally elongated polyglutamine tract, which, together with mutant transcript, causes cellular dysfunction. Currently, there is no curative treatment available to patients suffering from HD; however, the selective inhibition of the mutant allele expression is a promising therapeutic option. In this study, we developed a new class of CAG repeat-targeting silencing reagents that consist of self-duplexing CUG repeats. Self-duplex formation was induced through one or several U-base substitutions. A number of self-duplexing guide-strand-only short interfering RNAs have been tested through transfection into cells derived from HD patients, showing distinct activity profiles. The best reagents were highly discriminatory between the normal and mutant HTT alleles (allele selectivity) and the HTT transcript and other transcripts containing shorter CAG repeats (gene selectivity). We also demonstrated that the self-duplexing CUG repeat short interfering RNAs use the RNA interference pathway to elicit silencing, and repeat-targeting reagents showed similar activity and selectivity when expressed from short hairpin RNA vectors to achieve more durable silencing effects.

Considerable advances have been recently made in understanding the molecular aspects of pathogenesis and in developing therapeutic approaches for polyglutamine (polyQ) diseases. Studies on pathogenic mechanisms have extended our knowledge of mutant protein toxicity, confirmed the toxicity of mutant transcript and identified other toxic RNA and protein entities. One very promising therapeutic strategy is targeting the causative gene expression with oligonucleotide (ON) based tools. This straightforward approach aimed at halting the early steps in the cascade of pathogenic events has been widely tested for Huntington's disease and spinocerebellar ataxia type 3. In this review, we gather information on the use of antisense oligonucleotides and RNA interference triggers for the experimental treatment of polyQ diseases in cellular and animal models. We present studies testing non-allele-selective and allele-selective gene silencing strategies. The latter include targeting SNP variants associated with mutations or targeting the pathologically expanded CAG repeat directly. We compare gene silencing effectors of various types in a number of aspects, including their design, efficiency in cell culture experiments and pre-clinical testing. We discuss advantages, current limitations and perspectives of various ON-based strategies used to treat polyQ diseases.

In Huntington's disease and other polyglutamine (polyQ) disorders, mutant proteins containing a long polyQ stretch are well documented as the trigger of numerous aberrant cellular processes that primarily lead to degeneration and, ultimately, the death of neuronal cells. However, mutant transcripts containing expanded CAG repeats may also be toxic and contribute to cellular dysfunction. The exact nature and importance of RNA toxicity in polyQ diseases are only beginning to be recognized, and the first insights have mainly resulted from studies using simple model systems. In this review, we briefly present the basic mechanisms of protein toxicity in polyQ disorders and RNA toxicity in myotonic dystrophy type 1 and discuss recent results suggesting that the pathogenesis of polyQ diseases may also be mediated by mutant transcripts. This review is focused on the experimental systems used thus far to demonstrate RNA toxicity in polyQ disorders and the design of new systems that will be more relevant to the human disease situation and capable of separating RNA toxicity from protein toxicity.

Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-010-8137-3) contains supplementary material, which is available to authorized users.