Abstract
Purpose :
Inherited retinal diseases (IRDs) encompass diverse genetic disorders resulting in progressive visual impairment and blindness. Gene therapy has emerged as an exciting therapeutic option to either treat or delay the progression of various IRDs. While clinical trials for viral vector-mediated gene therapy for numerous eye illnesses are ongoing, there is consensus that developing other alternative treatments is needed. In this context, the non-viral delivery of in vitro transcribed (IVT) mRNA offers many advantages, such as achieving high gene expression while eliminating the risk of genomic integration or the requirement to cross the nuclear barrier. Furthermore, new genome editing tools, such as prime editing, offer unparalleled precision and efficiency in targeted gene manipulation.
Methods :
In this study, we investigated the delivery of IVT mRNAs coding for green fluorescent protein (GFP), CHM Rab escort protein 1 (REP-1), or genome editing components in human induced pluripotent stem cells (hiPSCs)-derived Retinal pigment epithelium (RPE) by flow cytometry, immunofluorescence, and western blot. Moreover, we assessed the expression of the different mRNAs by intraocular injections in mouse eyes using immunofluorescence and RNAscope.
Results :
The assays conducted in hiPSCs-RPE revealed higher mRNA transfection efficiency compared to cells transduced with Adeno-associated virus (AAV2) vectors. Furthermore, assessments of mRNA cellular uptake capacity and transfection efficiency using GFP-mRNA demonstrated a peak at 6 hours post-transfection, with detectable GFP protein levels persisting for at least 30 days post-transfection. In CHM-mRNA assays conducted in hiPSCs-RPE derived from Choroideremia patients, the CHM augmentation therapy proved sufficient to rescue several disease features.
In adult mouse eyes, GFP and REP-1 were observed within most RPE cells for several days following intravitreal and subretinal injections. Lastly, preliminary results from IVT mRNA prime editing components delivered to CHM mutated cells revealed precise genomic correction of a point mutation.
Conclusions :
Our findings demonstrate that in vitro and in vivo models consistently exhibit highly efficient expression of IVT mRNA, predominantly in RPE cells. These results underscore the potential of non-viral mRNA technology in the retina, paving the way for developing cost-effective gene therapies for IRDs, such as Choroideremia.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.