Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

Abstract

Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heatshock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allelespecific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

A 40‐year‐old woman with difficulty going down stairs in high‐heeled shoes

“…23 Therapies that reduce neurotoxic gene expression thus might slow, halt, or reverse disease progression. 25,26 The eyes are a promising target because of the ability to focally deliver small interfering RNAs by topical instillation or direct injection. 24 Therapeutic approaches using RNA interferencebased gene silencing are currently under investigation in SCA7 patient-derived fibroblasts.…”

CADASIL and CARASIL

“…Based on the assumption that the decisive pathogenesis is the toxic gain of function, probably associated with the unpaired cysteine in the affected EGFr/EGFrs, there are two possible gene therapeutic approaches: (i) silencing the mutated gene allele with inhibitory RNA molecules and (ii) antisense‐mediated skipping of the NOTCH3 exon harboring the defective DNA sequence. Allele‐specific siRNAs have already been designed for several diseases, including familial forms of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and spinocerebellar ataxia 7 . Tikka et al demonstrated efficient silencing of NOTCH3 in control VSMCs (from human umbilical artery) transfected with two different vectors (by 36% respectively 63%), which encode shRNAs targeting NOTCH3 gene.…”

Mutant CAG Repeats Effectively Targeted by RNA Interference in SCA7 Cells

“…We selected the UCHL1 gene, encoding the de-ubiquitinating enzyme, which was shown earlier to be silenced together with ATXN7 with a decreased expression level [14]. The UCHL1 mRNA level was decreased in SCA7 fibroblasts compared with the control cell lines (Figure 3A).…”

“…The targeting of a common SNP variant, which is also linked to an ATXN7 mutation, resulted in the high discrimination of silencing. In another study, synthetic small interfering RNA (siRNAs) were tested in SCA7 fibroblasts [14]. ATXN7 silencing was demonstrated at the transcript level, and there was a lack of selectivity in a broad range of siRNA concentrations.…”