Poster Presentation 39th Annual Lorne Genome Conference 2018

Development of Molecular Therapies for PRPF31-associated Retinitis Pigmentosa Using Splice-switching Antisense Oligonucleotides (#156)

Janya Grainok 1 2 3 4 , Ianthe Pitout 1 2 , May T. Aung-Htut 1 2 , Fred K. Chen 5 6 7 , Steve Wilton 1 2 , Sue Fletcher 1 2
  1. Centre for Comparative Genomics, Murdoch University, Western Australia, Australia
  2. Perron Institute for Neurological and Translational Science, Nedlands, Western Australia, Australia
  3. Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
  4. Murdoch University, Murdoch, Western Australia, Australia
  5. Centre for Ophthalmology and Visual Science, , University of Western Australia, Western Australia, Australia
  6. Lions Eye Institute, Nedlands, Western Australia, Australia
  7. Department of Ophthalmology, Royal Perth Hospital, Western Australia, Australia

Background: Pre-mRNA processing factor 31 (PRPF31) is essential for pre-mRNA splicing. Heterozygous loss of function mutations in PRPF31 cause autosomal dominant retinitis pigmentosa 11 (RP11), characterized by photoreceptor degeneration, with potential for blindness. Increasing the PRPF31 transcript level in the retina may rescue splicing function and therefore prevent or delay degeneration of photoreceptor cells. This study aims to upregulate PRPF31 expression, sufficient to support retinal function in an RP11 patient carrying c.1205 C>A nonsense mutation in exon 12 of PRPF31. Splice-switching antisense oligonucleotides (AOs) were designed to exclude exon 12 and restore the reading frame of the PRPF31 transcript, facilitating the production of potentially functional PRPF31 in RP11 patient cells.

Methods: AOs targeting splicing enhancer motifs in PRPF31 exon 12 were designed to promote exclusion of exon 12 during pre-mRNA processing, and transfected into RP11 fibroblasts. RNA was isolated 24 hours after transfection and assessed for PRPF31 exon 12 exclusion using end-point RT-PCR and total PRPF31 transcript using quantitative RT-PCR.

Results: An AO targeting splicing enhancer sequences at the position 70-94 on exon 12 excluded exon 12 from 80% of the PRPF31 transcripts, as determined by RT-PCR and Sanger sequencing. Exclusion of exon 12 resulted in approximately two-fold increase in total PRPF31 transcript at the mRNA level, compared to control.

Conclusion: Exclusion of the exon bearing the nonsense mutation restored the reading frame of PRPF31 and increased total PRPF31 transcript, which would otherwise have been degraded through nonsense mediated decay. Deletion of exon 12 does not disrupt any predicted functional domain and therefore the truncated protein is expected to retain some function. PRPF31 protein expression and the consequences on the photoreceptors will be further evaluated in retinal organoids derived from patient induced-pluripotent stem cells. This study will yield a potential candidate for PRPF31-associated RP.