Optimization of trans-Splicing for Huntington's Disease RNA Therapy

Hansjörg Rindt,Christian L Lorson,Colton M Tom,Virginia B Mattis

doi:10.3389/fnins.2017.00544

Hansjörg Rindt, Christian L Lorson + Show 2 more

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https://doi.org/10.3389/fnins.2017.00544

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Abstract

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin (HTT) gene. We have previously demonstrated that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here, the targeted tethering efficacy of the pre-mRNA trans-splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3′ end of HTT intron 1 or the intron 1 branch point were shown trans-splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the trans-splicing efficacy from 1–5 to 10–15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mtHTT allele in the context of HD.

Highlights

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in one allele of the Huntingtin (HTT) gene (The Huntington’s Disease Collaborative Research Group, 1993)
In this new series of pre-mRNA trans-splicing modules (PTM), a non-disease encoding HTT exon 1 with 21 CAG repeats was fused to a synthetic stretch of RNA that functioned as the targeting sequence or the “tether” (Figure 1A)
To enhance splicing to the endogenous HTT pre-mRNA, three tandem repeats of a previously identified intronic splice enhancer (ISE) element were incorporated into the PTM, as well as the HTT intron 1-derived branch point

Summary

Introduction

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat (polyQ) in one allele of the Huntingtin (HTT) gene (The Huntington’s Disease Collaborative Research Group, 1993). The disease affects ∼6 per 100,000 in Caucasian populations (Pringsheim et al, 2012), with devastating consequences including progressive motor dysfunction, chorea, cognitive impairment, psychiatric abnormalities (Vonsattel et al, 2008), sleep disturbances (Arnulf et al, 2008), eventually leading to death (DiFiglia et al, 1995). These symptoms are due to the dysfunction and eventual death of specific projection neurons within the brain (Ross and Tabrizi, 2011). The loss of these neurons can occur years before overt clinical symptoms (Rosas et al, 2008; Aylward et al, 2011)

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