Abstract
Purpose :
Advances in CRISPR gene editing have enabled the modification of DNA, which can be applied to the treatment of congenital and multifactorial disorders. Increasing the cell-type specificity of delivery is a significant challenge to editing cells within the eye. To provide a safer and more targeted delivery, nonviral delivery is emerging as an alternative to viral delivery. Here, we evaluated engineered RNPs (eRNPs), a new class of modular and programmable biologics with the potential to be cell-selective, gene-targeting precision therapeutics.
Methods :
Six-week-old Ai14 mice of either sex were chosen to validate the CRISPR SpCas9 eRNP platform. First, we generated an eRNP comprising a sgRNA that excises the stop codons and depress the tdTomato gene within Ai14 mice, then delivered it to the mice via subretinal injection. The mice were euthanized after 14 days and retinal pigment epithelium (RPE) was flat mounted and imaged for the tdTomato fluorescent expression. Second, heterozygous mice for the Leber Congenital Amaurosis-16 disease (Kcnj13+/-) were used to test the eRNPs for the editing of an endogenous wildtype allele to create the humanized disease model. sgRNA targeting the Kcnj13 wildtype allele was complexed into a eRNPs and injected subretinally. Electroretinography (ERG) was performed on the mice before the injection and at multiple timepoints post disruption of wild-type allele using eRNPs.
Results :
The RPE flat mount images after the payload injection into the Ai14 mice showed tdTomato fluorescence, indicating successful delivery and editing of the gene and the ability to precisely delete the stop codons. The tdTomato positive area to total RPE floret area distribution ranged from 5% to 25%, with an average of 12.02 + 1.6% (n=15). Delivery of eRNPs into Kcnj13+/- mice disrupted the targeted allele and reduced the a- and b-waves amplitudes. c-waves originating from RPE cells also were reduced and did not recover after 6 weeks. When compared to the baseline ERG, the c-wave amplitude was reduced by 60%. eRNPs with non-targeting guide RNAs had no influence on the ERG waveforms.
Conclusions :
Robust editing of RPE cells via subretinal injection was observed with eRNP delivery. This strategy holds promise as a potentially safer and more effective alternative to packaged delivery systems, such as nanoparticle, viral and LNP, for CRISPR genome editors.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.