Oligonucleotide-based strategies to combat polyglutamine diseases

Abstract

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.

Figure 1.

Toxic entities in the pathogenesis of polyQ diseases. The main products of the mutant gene are the mutant transcript containing the expanded CAG repeats and the mutant protein containing the expanded polyQ tract. The hallmarks of primary toxic events are nuclear aggregates containing mutant RNAs (CAG foci) or mutant proteins (full length or fragments). The interactions and events leading to the production of additional toxic entities from the mutant transcript and mutant protein are indicated. The antisense transcription of the mutant polyQ gene may result in transcripts containing expanded CUG repeats. siRNAs generated by the RNase Dicer from the expanded tracts may interact with complementary sequences in the transcriptome and cause the downregulation of the expression of numerous genes. Aberrant translation may lead to peptides containing polyA, polyS or polyQ tracts. See the text for more details.

Figure 2.

Strategies directed at the elimination of toxic entities in polyQ diseases. The main steps of mutant gene expression at which therapeutic intervention may be applied are indicated. The possible interventions include: 1. editing the CAG expansion in the mutant gene, 2. inhibiting mutant gene transcription, 3. interacting of potential drugs with mutant transcript leading to its degradation or inhibition of translation, 4. inactivating the mutant protein by its degradation or blockage and 5. targeting the main downstream pathways. See the text for more details.

Figure 3.

The most advanced ON-based tools for silencing the HTT gene tested in HD mouse models. The sequences with a schematic representation of chemical modification patterns are given for three selected ON-based silencing tools (A, B, C) tested as potential therapeutics for HD. (A) represents the non-allele-selective approach for HTT silencing, while (B) and (C) are designed to preferentially target the mutant allele by the SNP-targeting strategy (B) or by CAG-targeting (C) (nucleotides essential for the selective activity of these ONs are underlined). (A) and (B) are AONs that activate RNase H for transcript degradation, while (C) is ss-siRNA that activates an RNAi-based mechanism. In the experiments in HD mouse models, all ONs were delivered by intraventricular infusion. See the text for more details.

Figure 4.

The variety of CAG repeat-targeting ON-based tools showing preferential activity for the mutant allele. On the left side, various CAG repeat-targeting ONs and oligomers are presented. These molecules include a group of AONs, PNA and morpholino oligomers, as well as LNAs and RNAi-based tools including siRNAs, which are delivered as synthetic RNAs or expressed as shRNAs in cells. Specific chemical modifications, as well as the positions of base substitutions, are marked (see figure legend in the left upper corner). In some ONs, the CAG strand must have been included, while other ONs are composed of the CUG repeats only. On the right side, the interactions of ONs with CAG tracts in normal (marked in orange) and mutant alleles (marked in red) are presented. Only the binding of several ONs to the expanded CAG repeat tract in mRNA results in efficient gene silencing. The major mechanism is translational inhibition, which may occur by a steric blockade formed by AONs or by the RISC machinery recruited by siRNAs. See the text for more details.