U1 (and U7) snRNA for Splicing Therapy

Abstract

The mainstream approach to splicing therapy today involves synthetic antisense oligonucleotides. ASOs interfere with spliceosomal ribozyme assembly by blocking splice sites or influence protein regulators by blocking or adding their binding sites. ASOs are not compatible with human biology, which limits their efficacy and they come with a burden of chemical toxicity. Spliceosomal snRNAs adapted for a specific target can either enhance or suppress exon inclusion. Recombinant snRNAs with their pre-mRNA binding sites changed still assemble into functional snRNPs and integrate into active spliceosomes. Native to human cells, they are versatile for in vivo delivery and can be either encoded and expressed from rAAVs or delivered as RNA molecules by exosomes, benefitting from excellent biodistribution. Imported snRNAs are taken up by cytoplasmic maturation factors and transferred into the nucleus. Adapted snRNAs can be used to target different stage spliceosomes: U1 and U2 for early complexes, U2/U6 for precatalytic or U5 for catalytic complexes. U1 is the only spliceosomal snRNA thoroughly examined in pre-clinical studies (~100 mutations in 24 genes) and it is necessary to review the past 20 years of experience with U1 before moving on to other snRNAs. U1 is involved in the initial splice site selection and usually binds the 5’ss, but it also promotes 5’ss usage ‘at a distance’ if bound in the vicinity. Adapted U1 suppresses 3’ss usage, as likely does WT U1 if 3’ss CAG|GU protosplice site repeat is followed by a sequence resembling the start of the intron. However, historically the non-spliceosomal U7-OPT is often used as a scaffold molecule for exon-skipping ASOs. This chimeric U7 snRNA cannot join histone bodies, its WT destination. While both U1 and U7 show promising safety profiles in mice, WT U7 expression is 1000 times lower, than U1. U7 snRNA modulates master transcription regulators by binding histone-fold domain protein NF-Y; the effects of U7-OPT overexpression remain underexplored. U1, a splicing molecule, naturally works better for promoting exon inclusion, than U7, but years of experiments also revealed U1 and U7 limitations. New adaptations of the core spliceosomal snRNAs are necessary to overcome these limitations.