Repair of Rhodopsin mRNA by Spliceosome-Mediated RNA Trans -Splicing: A New Approach for Autosomal Dominant Retinitis Pigmentosa

Abstract

The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa

“…As regards retinal dystrophies, most advanced therapeutic approaches that target splicing are aimed at correcting the splicing of individual genes using mutation-adapted U1 small nuclear RNA for the RPGR gene45 or spliceosome-mediated RNA trans-splicing in RHO46. Both these approaches are based on cellular and animal models and have provided encouraging results.…”

“…Both these approaches are based on cellular and animal models and have provided encouraging results. Once in the clinic, these promising approaches could be generalised and applied to other genes with splice donor site mutations45 and to all adRP genes rather than only to RPGR and RHO, respectively46.…”

mRNAtrans‐splicing in gene therapy for genetic diseases

“…Thus, the trans ‐splicing rate achieved in these studies, by the order of only a few percent, will likely not lead to a therapeutic threshold in patients affected by these dominant conditions to be reached. Our team has recently reported a rate of trans ‐splicing of about 40% in a cell‐line expressing a dominant mutation of rhodopsin, the gene responsible for retinitis pigmentosa . Although encouraging, this rate is probably still insufficient for the point mutations that cause the most aggressive cases of retinitis pigmentosa.…”

The mutation p.E113K in the Schiff base counterion of rhodopsin is associated with two distinct retinal phenotypes within the same family

“…A continuing reduction of mid- and peripheral visual fields often leads to tunnel vision. In the course of the disease, cone photoreceptor loss may result in photophobia and even complete blindness78. Initially, RP-affected patients suffer from night blindness, due to a functional defect in rod photoreceptors or associated retinal neurons.…”

“…Until now, more than 60 RP genes and more than 10 CSNB genes have been identified2 (RetNet, https://sph.uth.edu/retnet/). They show expression patterns in rod photoreceptors and/or retinal pigment epithelium, are involved in the phototransduction pathway and retinal metabolism, or play a role in structure or maintenance of retinal cells7. Rhodopsin ( RHO ) has been associated with more than 180 mutations (http://www.biobase-international.com/product/hgmd).…”