An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases

Abstract

BackgroundRNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.ResultsUsing the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.ConclusionsUsing three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.

Targeting CAG repeat RNAs reduces Huntington’s disease phenotype independently of huntingtin levels

“…LNA-ASOs formed by 6 or 7 GTC repeats with 3′ and 5′ terminal guanines and LNA nucleotides at every third thymine residue strongly inhibited protein expression (16,17). However, those consisting of CTG repeats were less efficient (17,18), which agrees with our finding of a lack of effect of LNA-CTG on HTT protein levels.…”

“…Our efforts have surprisingly shown that blocking the activity of expanded CAG repeats in HTT-e1 transcripts without modifying HTT RNA or protein levels is sufficient to reverse motor symptoms in an HD mouse model. Several types of LNA-ASOs complementary to the CAG repeats have been used for allele-selective inhibition in HD human fibroblasts at the protein level, with virtually no effect on HTT mRNA (16)(17)(18)29). These studies showed that the configuration of the LNA-ASO largely affects the efficiency of protein inhibition.…”

“…Locked nucleic acid-modified (LNA-modified) ASOs (LNA-ASOs) complementary to the CAG repeats have been found to block RNA translation to a variable extent, depending on the type of LNA-ASO (16)(17)(18). We have previously shown that a particular type of LNA-ASO with CTG repeats (LNA-CTG) abrogates the toxicity produced by CAG repeat small RNAs (sRNAs) generated from mutant HTT transcripts in a human neuronal cell model (9).…”

Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases

“…In addition, expanded CAG RNAs form more stable hairpin structures than their unexpanded counterparts, which may promote differential ASO interaction [48]. Significant allele-selectivity of CAG-targeted PMO, PNA, and LNA oligos has been demonstrated in HD patient fibroblasts [49][50][51][52]; however, these studies were performed using a cell line with 69 CAG repeats on the mutant allele, an unusually large repeat that would result in juvenile-onset HD. Juvenile-onset HD, which manifests before age 20, is a more severe form of the disease that accounts for only a small percentage of patients [34].…”

Oligonucleotide-based strategies to combat polyglutamine diseases

“…This gene-knockdown and replacement strategy was tested for SCA6 and SCA3 in cell culture models (178,179). In these studies, the expression of both alleles of the CACNA1A and ATXN3 endogenes was silenced in human cultured cells using siRNAs.…”