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

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.