Abstract
Purpose :
RNA editing with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins is becoming an increasingly popular strategy for the development of safe therapies against genetic diseases. While DNA editing causes permanent changes in the genome, RNA editing can achieve therapeutic effects in a safer, reversible, and flexible manner, and be delivered from a single adeno-associated virus (AAV) vector. We hypothesised that RNA silencing of vascular endothelial growth factor A (VEGFA) using CRISPR-Cas13e RNA editing would control ocular neovascularisation.
Methods :
In vitro, HEK293FT and ARPE19 cells were transfected with Cas13e carrying either a non-targeting (NT) guide RNA (gRNA), VEGFA targeting gRNA or an array of three VEGFA targeting gRNAs. VEGFA mRNA expression was quantified using RT-qPCR, and off-target effects were identified from RNA sequencing. In vivo, the three constructs were packaged into AAV2Max vectors, and intravitreally injected into Kimba mice, expressing human VEGFA (hVEGFA+/+). Two months post injection, mice eyes were imaged using fundus fluorescence angiography and VEGFA mRNA expression was similarly quantified using RT-qPCR. Images were analysed using AngioTool for vessel density and lacunarity.
Results :
In both HEK293FT and ARPE19 cells, with >70% transfection efficiency, up to 90% of VEGFA mRNA knockdown was observed with both single and array of VEGFA targeting gRNAs, compared to NT sgRNA controls. In addition, this knockdown corresponded with no off-target effects with single guide RNA in HEK293FT cells. With an array of gRNAs, one other gene, IFIT2, was significantly downregulated, however, this was not found to be guide-dependent. In vivo, moderate knockdown (≤20%) of VEGFA mRNA was observed with Cas13e carrying an array of gRNAs, while single gRNA did not show an observable knockdown. With a gRNA array, there was also significant reduction in vessel density and improvement in lacunarity, suggesting control of neovascularisation from the RNA editing therapy.
Conclusions :
Efficient knockdown of VEGFA mRNA can be achieved in mammalian cells using single-AAV compatible plasmids, demonstrating proof-of-concept for anti-VEGF therapy using CRISPR-Cas13 RNA editing strategy. Phenotypic improvement in Kimba mice also show translational potential of CRISPR-Cas13 for treatment of neovascular ocular diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.