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Cheryl Y Gregory-Evans, Naif S Sannan, Xianghong Shan, Kevin Gregory-Evans; RNA editing via spliceosome-mediated mRNA trans-splicing repairs a mouse Pde6b mutation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4931.
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© ARVO (1962-2015); The Authors (2016-present)
Although somatic gene augmentation therapy has been applied successfully to retinal diseases there are still limitations including packaging capacity of viral vectors and the appropriate control of transgene expression. Contrastingly, RNA therapies have the advantage of using the cell’s own machinery to regulate the natural spatiotemporal gene expression. Here we evaluate the potential of pre-mRNA trans-splicing to edit Pde6b mutations in cell culture and in a new mouse model of recessive retinitis pigmentosa (Pde6batrd3/1H).
Two 3’-pre-mRNA trans-splicing molecules (PTM1 and PTM2) were designed that bind to intron 11 of the mouse Pde6b gene, as this would target the compound heterozygous mutations (IVS18+1G>A and Asn606Ser) carried by a new Pde6batrd3/1H mouse model that we developed. These were tested for efficacy in mouse embryonic fibroblasts (MEFs). For in vivo testing, 1-2 µg of PTMs complexed with in vivo-JetPEI® were delivered by sub-retinal injection into P14 mouse eyes and then analyzed by histology, western blotting and electroretinography.
Two different recessive Pde6b strains of mice were crossed to produce a compound heterozygote Pde6batrd3/1H mouse. This mutant had a slower retinal degeneration (8 rows of photoreceptor nuclei at P14) than the Pdeb6rd1 mouse with a homozygous recessive Pde6b mutation (only 1 row of photoreceptor nuclei at P14). Photoreceptor cell death was detected by TUNEL staining at P14 and no Pde6b protein was made by either mutant allele. MEFs were transfected with PTM1 or PTM2 cloned into the sfGFP-N1 mammalian expression vector. After 48 hrs, MEFs produced a 4-fold and 2.5-fold increase in Pde6b protein, respectively. At 72 hours after sub-retinal injection, the localization of PTM1 was detected by wholemount immunohistochemistry and in retinal sections. Western blot detected retinal expression of a 90 kDa Pde6b protein. Electroretinography at P35 did not however show an improvement of visual function.
In conclusion, although trans-splicing and protein production was detected in vitro and in vivo, phenotypic rescue was not apparent. Trans-splicing efficiency using a non-viral vector approach needs to be improved and most likely administered before retinal degeneration begins at P14. This type of RNA therapy could be relevant to many different Pde6b genotypes as it is not a mutation-specific approach.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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