AVR Funded Grant – 2022
Project Title:
RNA base editing for treatment of inherited retinal diseases
Chief Investigator:
A/Prof Liu Guei-Sheung
Co-Investigators:
Dr Livia Carvalho, Prof Bang V. Bui, Dr Lewis Fry, Dr David Cordeiro Sousa
Aim
This project aims to demonstrate the feasibility of using a genetic technology known as “CRISPR RNA base editing” to correct genetic mutations and treat inherited retinal diseases.
Methods
RNA base editing is designed to correct single-base mutations in RNA transcripts. In this proof-of-concept study, we employ our in-house developed CRISPR-Cas13bt3 RNA base editor to – for the first time – directly correct single base mutations found in two IRDs (Leber congenital amaurosis and Usher syndrome type 2A).
Key results
In this project, we developed an adeno-associated virus (AAV)-compatible RNA base editor (dCas13bt3-ADAR2) to target a nonsense mutation found in Rpe65 gene, causative for Leber congenital amaurosis. To establish the potential of our RNA base editor, we investigated its capacity to correct the Rpe65 mutation efficiently and specifically against the only other established AAV-compatible RNA base editor, known as CRISPR-Cas-inspired RNA targeting system (CIRTS)-ADAR2. Firstly, we screened various guide (g)RNAs to identify the optimal gRNA for each system using a dual-luciferase assay. Both dCas13bt3-ADAR2 and CIRTS-ADAR2 were then delivered with their optimal gRNAs and a mutant Rpe65 gene into human retinal pigment epithelium cells. We have thereby shown that the dCas13bt3-ADAR2 proves far superior to CIRTS-ADAR in correcting the Rpe65 mutation. It can also achieve up to 36% editing in human cell lines, as well as recovery of Rpe65 protein in human retinal pigment epithelium cells.
We then further employed dCas13bt3-ADAR2 RNA base editor against a nonsense mutation found in Ush2a gene, causative for Usher syndrome type 2A.
Our work demonstrated the feasibility of targeting Ush2aW3947X variants with dCas13bt3-ADAR2 base editor. Our screening revealed the 28th position mismatch (50-28) produced the most efficient editing with dCas13bt3-ADAR2. The 50-28 sgRNA also resulted in editing efficiencies of up to 70%.
Implications for Clinical Practice/Science and Future Research
In this project, we demonstrated the feasibility of using the CRISPR-Cas13bt3 RNA base editor to correct the genetic mutations to treat IRDs. The outcome of the project will help develop a better therapeutic approach. This study will also greatly impact the management of IRDs, as it will introduce a novel and versatile strategy for other genetic diseases. We are now expanding this study to investigate the feasibility of using this RNA base editor to correct the mutations in ex vivo (retinal organoids) and in vivo (IRD mouse) models of IRDs.
Conclusion
Through this study, we have shown proof-of-concept for a novel gene editing treatment strategy that offers a widely applicable treatment option for IRDs, one that can adequately address the heterogeneous nature of the disease.
Lay Summary of Outcomes
Inherited eye diseases are significant contributors to global blindness, and there is no cure to date. This project demonstrated that RNA gene editing technology allows precise correction of the underlying genetic mutations for gene therapies. This unprecedented approach would revolutionise current gene therapy approaches.