Abstract
Purpose :
Over two million people worldwide suffer from inherited retinal diseases (IRDs) with most classified clinically as retinitis pigmentosa (RP) or macular dystrophies. Although significant progress has been made in the application of gene therapy for the treatment of some IRDs, there is a need for the development of new and improved therapeutic strategies based on emerging technologies that overcome the limitations of previous gene therapy. The purpose of this study is to explore the use of genome base editing in combination with lipid nanoparticle (LNP) delivery system to correct a nonsense mutation in the rhodopsin (Rho) gene linked to RP and a nonsense mutation in the ABCA4 gene linked to Stargardt disease.
Methods :
HEK293T cells stably expressing C-terminus EGFP-tagged rhodopsin with or without a Q344Ter mutation were treated with pNG-ABE8e (addgene #138491) and sgRNA that place the target base in position A3 or A6 using either polyethylenimine (PEI) or LNPs as transfection agents. LNPs were synthesized by rapid mixing of ionizable lipids, phospholipid, cholesterol, PEG lipid, pNG-Abe8e, and sgRNA. Editing efficiency was determined 72 hours post-transfection by restoration of EGFP signal as quantified by flow cytometry with non-transfected cells used as negative control. Base editing was also evaluated by immunofluorescence (IF), Western blotting (WB), and DNA Sanger sequencing.
Results :
The Rho gene was successfully edited using pNG-ABE8e and sgRNA in position A6. PEI transfection of pNG-Abe8e resulted in 18% editing for A6-sgRNA and 3% editing for A3-sgRNA (n=5). In contrast, the delivery of pNG-ABE8e and A6 sgRNA using LNP resulted in 81% editing efficiency (n=3). WB and IF confirmed the restoration of EGFP signal after treatment with pNG-ABE8e and A6 sgRNA. No editing was detected when a scrambled control sgRNA was used. Sanger sequencing confirmed editing at the target nucleotide and the absence of off-target edits within the editing window.
Conclusions :
This data demonstrates the efficiency of LNP transfection and the ability to successfully edit the Rho c.1030C>T (p.Q344Ter) mutation. This easily customizable technology is now being applied to repair nonsense mutations in ABCA4. These findings provide the foundation for the pairing of LNP and base editing technology for potential in vivo rescue and survival of photoreceptors.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.