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Adeline Berger, Stéphanie Lorain, Melissa Desrosiers, Peggy Fabre, Cécile Peccate, Thomas Voit, Luis Garcia, José-Alain Sahel, Alexis-Pierre Bemelmans; Trans-splicing of Rhodopsin mRNA: Modeling and Therapeutic Strategy for Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1904.
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© ARVO (1962-2015); The Authors (2016-present)
To implement new gene therapy strategies for autosomal dominant Retinitis Pigmentosa, we applied Spliceosome-Mediated RNA Trans-splicing to correct mutations of the rhodopsin (RHO) gene. This technology promotes a splicing event in trans between the endogenous mutated rhodopsin pre-mRNA and an exogenous pre trans-splicing mRNA (PTM) containing a partial RHO cDNA devoid of mutation. Targeting the first intron of the RHO gene should thus allow the repair of any mutations present in exon 2 and the following exons. A major advantage of this approach is that the expression level of the "repaired" protein depends only on the endogenous gene expression regulation.
We constructed several vectors expressing wild-type or P347L alleles of the human RHO gene driven by an ubiquitous promoter. We introduced these vectors in HEK293 cells to create cellular models of RHO mutation. We then designed three different PTM targeting the first RHO intron. We evaluated these PTM in our cellular model, using a silent mutation inserted in the exogenous mRNA which allows us to precisely quantify trans-splicing efficiency by conventional molecular biology techniques (PCR and restriction digest).
After transient transfection of vector encoding whole rhodopsin gene, we observed by flow cytometry and immunocytofluorescence an expression of wild-type or P347L RHO in approximately 80% of the cells. Among the three PTM tested so far, one has no activity, one leads to a trans-splicing efficiency of 12% of RHO mRNA, whereas the most efficient one reached a trans-splicing efficiency of 34%. The level of trans-splicing was equivalent for wild-type and P347L alleles, confirming that trans-splicing occurred independently from the mutation. The phenotype of cells expressing wild-type or P347L alleles with or without trans-splicing is currently being characterized.
We have shown that Spliceosome-Mediated RNA Trans-splicing allows to partially repair RHO mutation at the transcriptional level in a cellular model. The PTM showing the best activity in vitro will be introduced into an AAV vector to be tested in vivo in an animal model expressing a mutated RHO allele. If efficient, this new tool will be directly applicable to patients.
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