Potential RNA therapeutics for Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an incurable disorder with neuroand cardiodegenerative progression. This monogenic disease is caused by the expansion of naturally occurring GAA repeat in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters, in iron metabolism and in stress-induced apoptosis. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Low levels of the protein are responsible for all clinical and morphological manifestations of FRDA critically affecting survival of large primary neurons of the dorsal root ganglia, cardiomyocytes and pancreatic β-cells. Thus, current therapeutic strategies in FRDA are mostly aimed to restore physiological frataxin expression. In this scenario, RNA therapeutics could represent an appealing approach to induce a gene-specific activation. Among regulatory RNAs, a new functional class of antisense long non-coding RNAs (lncRNAs) acting as enhancers of target mRNA translation was recently discovered. The activity of this lncRNAs requires an inverted SINEB2 sequence to increase translation (Effector Domain; ED) and an overlapping region to target its sense mRNA (Binding Domain; BD): this class of RNAs was designated as SINEUPs. Here we describe the development of a synthetic SINEUP targeting the FXN gene (SINEUP-FXN) able to increase protein synthesis at a post-transcriptional level. By swapping a natural BD, the synthetic SINEUP-FXN was designed to act through the binding to FXN mRNA. SINEUP-FXN was first tested by cotransfection with FXN cDNA and then redesigned to target endogenous FXN sequence, reporting in both cases a consistent accumulation of frataxin levels in human HEK-293 cells. Subsequently, a second generation of optimized RNAs, with a 6 shorten structure and named miniSINEUP-FXNs, was directly tested on endogenous mRNA in FRDA patient-derived cells. Some miniSINEUP-FXNs variants demonstrated to be the best candidates for subsequent investigations and intriguingly achieved frataxin protein restore to physiological levels. More importantly, our results indicated a consistent rescue of the disease-associated phenotype. Collectively, our study shows the first gene-specific therapeutic approach to activate frataxin translation in FRDA cells and, more broadly, the first potential therapy employing a specific translational activator for a human monogenic disease