In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina

Abstract

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

Development of Engineered-U1 snRNA Therapies: Current Status

“…It may be worth mentioning that a significant part of the studies relying on the use of both modified U1 snRNAs and ExSpeU1s was developed in the labs of Mirko Pinotti (University of Ferrara, Italy) and of Franco Pagani (Centre for Genetic Engineering and Biotechnology in Trieste, Italy) [12,13,38,44,48,51,58,59], where the whole pipeline to address their therapeutic potential in vitro and in vivo, seems to be well-implemented. However, it is important to stress that many other teams, in different labs, have been able to replicate the same sort of methods, achieving similar results for other disease-causing mutations [11,16,41,61,66]. This adds up to the overall therapeutic potential of engineered U1s, by demonstrating the replicability of the method and the robust correction levels it may promote.…”

“…A similar approach combining the modified U1 snRNAs and U6 snRNAs was also very recently developed by the same group [66] for the correction of the previously referred 5 ss OPA1 mutation, c.1065+5G>A, which underlies a severe ocular disorder. These authors tested this combined therapy in a mouse model carrying the Opa1 c.1065+5G>A 5 ss mutation (Opa1 enu/+ ), which causes the same splicing defect observed in the patient's fibroblasts.…”