By Sahana Shankar, Ph.D. Candidate
CRISPR-based gene therapy has been in development for several diseases, especially congenital diseases caused by loss-of-function mutations. With continuous improvements to techniques and methodologies, CRISPR’s limitations can soon be addressed for therapies pertaining to humans. In a new study, published in Nature Biomedical Engineering, a team of scientists from the University of California, Irvine, has demonstrated the use of CRISPR gene-editing to cure inherited retinal disease in mice with a proof-of-concept study.
Inherited Retinal Disease (IRD)
IRDs are a spectrum of ocular diseases caused by pathogenic mutations in over 250 genes. Gene therapy has emerged as a potential cure for IRDs. A USFDA approved gene therapy, Zolgensma is available for Leber Congenital Amaurosis (LCA), a sub-type of IRD wherein patients have biallelic mutations in the RPE65 gene. LCA is a disease caused by a nonsense pathogenic mutation in the exon 3 of the RPE65, which renders the gene inactive due to a premature stop. This results in no expression of the RPE65 protein and loss of vision. The gene augmentation therapy delivers a functional copy of the RPE65 gene via a viral vector.
However, there may be continual retinal degeneration and loss of vision due to insufficient expression of the transgene over time. “As an alternative to gene augmentation therapy, we applied a new generation of CRISPR technology, referred to as ‘base editing’ as a treatment for inherited retinal diseases,” said first author Susie Suh, an assistant specialist in the UCI School of Medicine Department of Ophthalmology.
Restoring RPE65 Expression
The authors delivered a viral vector containing a single-guide RNA targeting the mutation in an LCA mouse model through a subretinal injection. Using this approach, they could validate the correction of the mutation by sequencing, expression of RPE65 protein, and other methods with ~29% editing efficiency. By analyzing visual and retinal functions in the edited mice after 5 weeks of injection, the team found that restoring RPE65 expression could rescue blindness, as evidenced by multiple tests to assess RPE65 localization, function, and electroretinograms of edited mice as well as the ability of the mice to perceive direction, distance, spatial and temporal frequency. The editing could also revive neuronal activity, especially in the V1 neurons in the primary visual cortex in response to visual stimuli.
Use of Adenine Base Editors
While addressing some of the issues with CRISPR-based gene editing like low efficiency and off-target effects, the authors used cytosine and adenine base editors (ABEs). Unlike Cas9 editing, ABEs do not induce double-stranded breaks and homology-repair, which improves the efficiency of base editing. These limit the unintended off-target mutations and enable more precise and predictable correction of disease-causing mutations. The improved adenine base editing approach, developed at MIT and Harvard in Dr. David Liu’s lab, corrected the mutation, which resulted in the expression of RPE65 protein and restoration of vision.
Treating eye diseases is particularly challenging due to limitations in sensitivity, accessibility, and the eye’s immune-sensitivity. Hence, a combination of gene therapy and CRISPR-based gene editing is the optimal treatment model to cure mutation-based vision disorders. The authors recognize the need to validate each sgRNA and delivery vector to optimize the therapy in the clinic. However, their work is a good starting framework to develop more preclinical editing therapies.
Editor: Rajaneesh K. Gopinath, Ph.D.
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