Evaluation of RNA trans-splicing as a therapeutic strategy for spinocerebellar ataxia type 1

Background and objective Spinocerebellar ataxia type 1 (SCA1) is caused by an expanded polyglutamine (polyQ) tract in the protein ataxin-1 encoded by the ATXN1 gene. 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 an RNA reprogramming technology. Methods Spliceosome-mediated RNA trans-splicing (SMaRT) creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and an exogenously delivered pre-trans-splicing molecule (PTM). We constructed and tested, in-vitro, several PTMs designed to substitute S776 or S752 (the mouse homologue for S776) for alanine. PTMs were constructed in pcDNA3.1 and used to generate lentiviral constructs containing a GFP expression cassette. Trans-splicing in transfected, or transduced cells was analysed by RT-PCR and sequencing. Endogenous and ataxin-1 minigene transcripts were analysed, minigenes were constructed using the pSPL3 exon trapping vector. Results Human (SH-SY5Y) and mouse (N2a) cell lines were transfected with PTMs with and without minigenes. SMaRT successfully edited, in-vitro, mouse and human ATXN1 transcripts to substitute S752 or S776 for alanine, with efficiencies of approximately 30% for endogenous human transcripts. We additionally observed trans-splicing of endogenous ataxin-1 in cultured primary cortical neurons from wild-type mice. The most efficient PTM design hybridises with the 3’ end of intron 8, upstream of the branch point. Discussion and conclusion SCA1 is an excellent prototypic system to demonstrate that a SCA-causing protein can be converted into a non-toxic form by SMaRT. SMaRT can theoretically repair any mutation downstream of the PTM binding site and is particularly suited for dominant gain of function mutations characteristic of SCAs. Our work demonstrates the potential of SMaRT to prevent a pathogenic phosphorylation event and provides proof-of-concept for in-vivo pre-clinical development.