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The most frequent cause of retinitis pigmentosa (RP), a group of hereditary retinal dystrophies leading to blindness, is the occurrence of point mutations in the Rhodopsin (RHO) gene. Most of these mutations lead to gain of functions or dominant negative effects deleterious for photoreceptors, thus resulting in a dominant mode of transmission. Moreover, it has been shown that variations in RHO expression level are also deleterious for the retina. Gene transfer strategies modifying RHO mutations should therefore lead both to suppression of mutant protein expression and restoration of the normal one at a physiological level. Spliceosome-mediated RNA trans-splicing should in theory respect these constraints by repairing mutations at the pre-mRNA level. Using this technology, the expression level of the repaired mRNA will indeed depend solely on that of the gene endogenous promoter. To achieve this goal, this approach consists of introducing, by gene transfer, an exogenous RNA – called PTM, for Pre-Trans-splicing Molecule – able to bind the pre-mRNA and promote splicing in trans, leading to replacement of the mutant part of the RHO pre-mRNA.We engineered fourteen different RHO-PTMs able to repair any mutation in RHO exons 2, 3, 4 and 5, which differ only on their binding sequence to RHO pre-mRNA. To determine the efficiency of each PTM, we transiently co-transfected HEK293T cells with a plasmid encoding a PTM and another encoding the wild-type or mutant RHO. The maximum trans-splicing efficiency observed at the mRNA level was about 25%. We improved this efficiency to 40% by refinement of the PTM sequence, through reintroduction of an endogenous RHO intron in the cDNA part of the PTM. We then tested PTM at the protein level in a cellular model expressing RHO stably. While wild-type RHO was localized to the plasma membrane, mutant RHO was sequestrated in the cytoplasmic compartment. Using a flow-imaging methodology (ImageStreamX), we were able to precisely quantify this phenotype and showed that trans-splicing partially restored a correct localization of RHO in this cellular model. Thus, we constructed an AAV vector containing our best PTM to assess its activity in vivo in a humanized mouse model of rhodopsin mutation following subretinal injection. Using co-injection with an AAV-GFP and micro-dissection of the transduced part of the retina, we demonstrated that trans-splicing reached 30% at the RNA level, while it was not detectable in the untransduced part, or in animals that received a control PTM. This study demonstrates that trans-splicing may provide a therapeutic benefit in case of dominant mutations of the rhodopsin gene.