Another strategy exploits a universal difference between mutant and wild-type alleles: the mutant allele has more trinucleotide repeats [35
] (). The existence of additional repeats does not create the type of sequence differences that, as demonstrated for SNPs or deletion polymorphisms, might be exploited for allele-selective recognition. An oligonucleotide that is complementary to the mutant allele will also be complementary to the wild-type allele.
The expanded mutant repeat will, however, offer more binding sites for complementary oligonucleotides. It is also known that CAG repeat sequences can form hairpin structures [36
]. The longer mutant repeat will form a longer secondary structure, and it is possible that these longer structures will be more conducive for binding to complementary oligonucleotides. It is impossible to predict whether these characteristics would yield selective recognition of a trinucleotide repeat gene inside cells because the intracellular structure of RNA cannot be determined; neither is it known with certainty which proteins associate with repeat RNAs. The hypothesis must be tested empirically by introducing oligomers into cells and assaying selectivity.
Myotonic dystrophy (dystrophia myotonica) type 1 (DM1) is caused by expansion of a CUG tract within the 3′-untranslated region (UTR) of dystrophia myotonica-protein kinase (DMPK) mRNA. In patients, expansions range from 50 to >2000 repeats [38
]. The expanded repeat binds to muscleblind-like protein 1 (MBNL1) and reduces its effective concentration. This blocks the normal function of MBNL1 in modulating splicing. The expanded repeat also causes aberrant activation of protein kinase C (PKC), leading to upregulation of CUG binding protein (CUGBP1), which regulates alternative splicing [39
]. Two laboratories found that either phosphorothioate 2′-O-methyl oligonucleotides [40
] or morpholino oligomers [41
] () complementary to the CUG repeat reduced formation of toxic RNA–protein aggregates (). MBNL1 function was restored and splicing defects were corrected in vivo
Chemical structures of unmodified or modified nucleic acids used for antisense oligonucleotides (ASOs) and/or double-stranded (ds)RNAs targeting trinucleotide repeat genes.
Our laboratory has been testing the repeat-targeting strategy for inhibition of HTT expression. For HTT, the mutation is an expansion of a CAG repeat that exists near the 5′ end of the protein-coding region of HTT mRNA. We first assayed peptide nucleic acid (PNA) () oligomers complementary to CAG repeats [42
]. PNAs are amide-linked oligonucleotide mimics known for their ability to invade nucleic acid structure.
To improve entry into cells, the PNAs were conjugated to the peptide d-Lys8. Because the amino acids are in the unnatural D configuration, it is probable that they will survive inside the cells and participate in target recognition through electrostatic interactions with the phosphodiester backbone of RNA. We observed that PNAs with d-Lys8 at the C-terminus showed 3.5-fold selectivity for inhibition of mutant HTT expression in a HD patient-derived cell line (69/17 repeats). Attaching the peptide to the N rather than C terminus of the PNA improved selectivity to >eightfold, demonstrating that selectivity can be increased by simple chemical modifications.
PNAs are not currently being developed for clinical use, so to facilitate development, we tested analogous oligonucleotides containing locked nucleic acids (LNAs) () [42
]. Introduction of LNA bases into oligonucleotides enhances affinity and allows binding strength to be tailored for a particular application [43
]. LNAs are being used in phases I and II clinical trials [15
]. Methods for their large-scale synthesis have been optimized and initial trial data suggest that they are well tolerated by patients. We observed potent and allele-selective (>sixfold) inhibition of HTT expression by LNAs and subsequently observed similar promising results with oligonucleotides containing (S)-cEt bridged nucleic acid and carba-LNA () [44
dsRNAs are another widely used approach to silencing gene expression. We observed that duplex RNAs that were fully complementary to the CAG repeat showed little or no allele selectivity [42
]. We reasoned that this failure might have been because the RNAi was too powerful, overwhelming the differences in repeat length and secondary structure between the mutant and wild-type mRNA targets. To test this hypothesis, we weakened recognition by introducing mismatched bases within the central region of the RNA duplex. These mismatches would be predicted to weaken binding, disrupt cleavage of mRNA by argonaute 2 (AGO2) and shift the mechanism towards that used by miRNAs () [45
The mismatch-containing RNAs were potent and selective (up to >40-fold) inhibitors of gene expression [46
]. As many as four mismatches were tolerated without sacrificing potency and several different mismatch designs functioned equally well. The existence of several excellent inhibitors with different sequences provides molecular options for future development. Having diverse options might be important if a sequence being tested in vivo
is found to have unacceptable sequence-specific adverse effects. Although most experiments were performed in patient-derived fibroblast cells with 69 repeats in the mutant allele (much higher that the 45 repeat mean), good selectivity was also observed in cell lines with 41 or 44 repeats.
The mechanistic reasons for this high selectivity have yet to be fully investigated, but it is possible that binding of multiple RNAs to the longer mutant repeat without cleavage leads to cooperative effects for translational inhibition. Similar allele selectivity was observed by Krzyzosiak and colleagues using RNA duplexes with mismatches at positions 13 and 16 [47
]. Mismatched RNA duplexes targeting the expanded repeat of ataxin-3 were tested in cells derived from patients with Machado Joseph Disease [MJD; spinocerebellar ataxia type 3 (SCA3)] patients and showed >16-fold selectivity, suggesting that the approach can be applied to multiple expanded repeat genes [48
Targeting expanded trinucleotide repeats has the potential to benefit entire patient populations for multiple diseases. However, there are also several disadvantages. Many genes contain CAG or CUG repeat regions [1
] and some of these genes are known to be crucial for normal function. These other genes have relatively small numbers of CAG or CUG repeats and examination of the expression of repeat-containing genes, such as TATA box binding protein (TBP
) and forkhead box P2 (FOXP2
), has not revealed inhibition [42
]. Nevertheless, specificity must be observed carefully during development.
As noted above for targeting SNPs, the number of compounds that are candidates for development is more limited than for oligomers that can target any sequence within the mRNA of a disease gene. Another issue is that the degree of allele selectivity of repeat-targeting oligomers depends on differences in repeat length between wild-type and mutant alleles [42
It has also been noted that repeat-targeted RNAs have been reported to have lower potency than duplex RNAs that do not target repeats when comparisons are made between results from different laboratories [33
]. This observation raises an important point. Strategies should be compared against one another and, ideally, this should be done directly. However, when values are taken from the literature and compared, the difference in protocols and assay systems should be carefully evaluated. Different transfection methods are used for different types of oligomer, such as PNA, LNA and siRNA, giving different potential for gene silencing. The use of different cell types might also affect potencies. Therefore, relative values should be evaluated with caution unless obtained through side-by-side assays.