Spinocerebellar ataxia type 3 (SCA3, Machado Joseph Disease) is a progressive neurological disorder (Paulson, 2007a
; Paulson, 2007b
). SCA3 is caused by heterozygous mutations within one allele of the ataxin 3 (ATX3
) gene. The ATX3
gene contains a tract with multiple copies of the trinucleotide CAG. In unaffected individuals, this tract is typically less than 31 repeats. Individuals with 45 to 51 repeats sometimes show symptoms but disease penetrance is incomplete. When greater than 52 repeats are present, there is full penetrance. Patients can have as many as 86 CAG repeats within the mutant allele. Clinical symptoms are affected by repeat size and mean repeat length can vary from 73 to 80 repeats in different patient populations (Sasaki, 1995
The symptoms of SCA3 are severe (Paulson 2007)
. Typically these symptoms begin to be observed in patients over fifty, but are also noted in younger individuals and age of onset correlates with the number of mutant repeats. Patients can have problems with walking, speech, and blurred vision. Symptoms worsen over ten to fifteen years and patients may require wheelchairs or other devices to maintain mobility. The patient’s conditions deteriorate over time and death from pulmonary complications can occur.
One approach to treating SCA3 would be to inhibit ATX3 protein expression, removing the cause of the disease and slowing or preventing its progression. Supporting this conclusion, in a conditional mouse model of SCA3 turning off ATX3 expression early in the disease state yielded a phenotype that was indistinguishable from wild-type mice (Boy et al., 2009
One approach to reducing levels of ATX3 protein is to use duplex RNAs or antisense oligonucleotides complementary to ATX3 mRNA. Researchers have identified antisense oligonucleotides and duplex RNAs targeting mRNAs for huntingtin (HTT) (Huntington’s Disease), ATX3, and other triplet repeat-containing genes (Gonzalez-Alegre and Paulson, 2007
; Denovan-Wright and Davidson 2006
; Scholefield and Wood, 2010
). Other elegant studies using antisense oligonucleotides have shown that blocking the long (>500 repeat) CUG repeat in the DMPK (Myotonic Dystrophy) gene can limit aberrant muscleblind protein binding to the expanded repeat region (Mulders et al., 2009
; Wheeler et al., 2009
Most trinucleotide repeat expansion diseases are autosomal dominant conditions caused by expression of a mutant allele. A key consideration for nucleic acid-based therapy is whether inhibition of both alleles can be achieved without undue toxicity due to reduced expression of wild-type protein. For ATX3, one recent report suggests that inhibiting expression of both the mutant and wild-type alleles did not cause observable toxicity, suggesting that approaches for therapy that reduce expression of both alleles might be feasible (Alves et al., 2010
There is no guarantee, however, that successfully inhibiting both alleles in mice will translate into successful treatments for humans. Preferential inhibition of the mutant allele may be beneficial and allele-selective strategies have the potential for fewer side effects in patients. To achieve allele-selective inhibition, Paulson targeted a duplex RNA to a single-nucleotide polymorphism linked to SCA3 (Miller et al., 2003
). Subsequently, Pereira de Almeida (Alves et al., 2008
) observed that targeting siRNAs to a SNP found in 70 % of patients with SCA3 led to allele-selective inhibition.
A fundamental difference between the wild-type and mutant alleles of all patients is the number of CAG repeats. CAG repeats are known to form hairpin structures when analyzed in cell free systems (Sobczak et al. 2003
; Kiliszek et al., 2010
). In the context of a complete cellular mRNA, these hairpins might differ significantly in structure depending on the number of CAG repeats present. We reasoned that short single-stranded oligomers complementary to CAG repeats might take advantage of differences in RNA structure between wild-type and mutant CAG repeat tracts, selectively recognize the mutant repeat region, and block expression of the mutant protein while leaving expression of the wild-type protein relatively unchanged.
We tested this hypothesis by targeting peptide nucleic acids (PNAs) to CAG repeat tracts in fibroblast cell lines derived from patients with SCA3 and a related triplet expansion disease, Huntington’s Disease (HD) caused by expanded CAG repeats within the huntingtin (HTT
) gene (Hu et al., 2009a
; Hu et al. 2009b
; Hu et al., 2009c
). PNA is a DNA/RNA mimic in which nucleotide bases are linked by an amide backbone (Nielsen et al., 1991
). PNA is known to be able to recognize complementary sequences within structured RNAs (Marin and Armitage, 2005
), making it a promising starting point for studies investigating recognition of CAG hairpins. Anti-CAG PNAs inhibited expression of both mutant genes and did not cause toxicity or affect expression of other cellular genes containing triplet repeats. We subsequently observed that single-stranded oligonucleotides (Hu et al., 2009a
; Gagnon et al., 2010
) and mismatch- containing duplex RNAs (Hu et al., 2010
) could also be allele-selective inhibitors of HTT expression. We now report inhibition of ATX-3 by additional PNA derivatives and by double-stranded RNAs designed to achieve allele-selective inhibition by shifting the mechanism used during RNAi.