Spliceosome‐mediated RNA trans-splicing prevents the pathogenic phosphorylation of ataxin-1

Background and objectives
Spinocerebellar ataxia type 1 (SCA1) is caused by an expanded CAG repeat in the ATXN1 gene, resulting in an elongated polyglutamine (PolyQ) tract in the ataxin-1 protein. The exact pathogenic mechanism is not understood but phosphorylation of ataxin-1 at S776 is critical for the stabilisation and neurotoxicity of polyQ-expanded ataxin-1. Our objective is to evaluate the therapeutic potential of preventing pathogenic phosphorylation of ataxin-1 using spliceosome-mediated RNA trans-splicing (SMaRT).

Methods
SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and an exogenously delivered RNA trans-splicing molecule (RTM). We designed and constructed a FLAG-tagged RTM to substitute S776 for alanine. SMaRT was performed via lentiviral transduction in SH-SY5Y cells stably expressing mutant YFP-ataxin-1(82Q) and patient iPSC-derived neuronal cultures. Trans-splicing and S776 phosphorylation were analysed by a combination of RT-PCR, qPCR, sanger sequencing, immunoprecipitation and western blotting.

Results
SMaRT successfully edited the substitution from S776 to A776 in SH-SY5Y cells and patient-derived neuronal cultures at both the RNA and protein level. A dose dependent effect was observed with increasing lentiviral concentrations. Importantly, a significant reduction (37%) in S776 phosphorylation was observed as well as a significant reduction in the intensity of nuclear ataxin-1 aggregates in YFP-ataxin-1(82Q) SH-SY5Y cells.

Discussion and conclusion
These results demonstrate the potential of SMaRT to prevent a pathogenic phosphorylation event and provides proof-of-concept for in-vivo pre-clinical development. Here, we demonstrate utility of SMaRT for a 3’ exon replacement. However, SMaRT strategies can be designed to replace 5’, internal or 3’ regions of an mRNA making this is an attractive versatile approach for genetic diseases.