Investigating CRB1 Mutations in Retinitis Pigmentosa 12

CRB1 mutations are associated with various inherited retinal diseases, including retinitis pigmentosa 12 (RP12) .Researchers from Lions Eye Institute in Australia investigatedthe effects of CRB1 variants on mRNA expression using CRISPR activation (CRISPRa) of CRB1 gene expression in human fibroblast cultures. Their research was presented at the Association for Research in Vision and Ophthalmology (ARVO) 2023 Annual Meeting, held April 23-27, 2023, in New Orleans, Louisiana, by Sang Yoon Moon, PhD, postdoctoral Research Associate, Lions Eye Institute.

Moon and colleagues found that the CRISPRa system was effective in screening the effects of CRB1 variants on gene expression. The platform should be able to be generally applied to screen novel variants of other genes of interest across inherited retinal diseases (IRD). CGTLive spoke with Moon to learn more about the research and its potential applications.

CGTLive: Can you tell me about the research you conducted and the rationale behind it?

Sang Yoon Moon, PhD: Inherited retina diseases are the most common cause of blindness in children and working-aged adults. Through the Western Australian Retinal Disease (WARD) Study, the Lions Eye Institute has established one of the largest retinal disease biobanks in Australia and is providing Western Australian patients with the genetic diagnoses and regular clinical monitoring that is critical for access clinical trials. However, a full genetic diagnosis remains elusive for a subset of patients who receive inconclusive results.

Thousands of retinal disease-causing mutations have been identified in several hundred genes, making the process of genetic diagnosis like looking for a ‘needle in a haystack'. Next generation sequencing technologies have greatly enhanced our ability to find the mutations causing inherited retinal diseases, as well as uncovering new disease-causing mutations. When these new mutations are discovered in a patient, it is not always clear if they are the cause of disease, or whether they are benign natural variation in the human genetic code. Thus, for some patients with newly discovered mutations, genetic diagnosis remains uncertain, leaving them ineligible for inclusion in future clinical trials.

To determine whether new mutations pathogenic, functional studies must be conducted to determine how the mutation affects the expression of the gene it is located in. To perform these studies, scientists are increasingly turning to cells that can be easily harvested from patients carrying these mutations, such as skin and blood, providing a rapid method for functional studies of gene expression. However, since many retinal disease genes are not expressed in skin or blood, these cells often require conversion into retinal cells in the laboratory, causing the retinal genes to be expressed and providing material for mutation classification studies. This process, which involves reprogramming cells into stem cells then differentiating them into retinal tissues can be a costly and time-consuming process.

This project, funded by the National Health and Medical Research Council of Australia, sought to develop a faster and more accessible method for performing functional studies on newly-discovered retinal disease-causing mutations. The availability of patient skin and blood cells in the WARD biobank provides a valuable resource for functional classification of mutations. To unlock the potential of this resource for the study of genes normally expressed only in the retina, I utilized recently developed CRISPR gene activation technology to ‘turn on’ retinal gene expression in human skin cells. The expression of retinal genes bearing disease-causing mutations in WARD patient skin cells provided a rapid method for assessing the effect of the mutation on gene expression. In parallel, I reprogrammed the same WARD patient skin cells into retinal cells, enabling the comparison of gene expression results in patient retinal cells and CRISPR-activated skin cells.

Can you give an overview of your findings? Were there any particularly notable or surprising findings?

Moon: The activation of CRB1 in the 3 RP12 patient fibroblasts provided an accessible method for screening human IRD mutations for splicing defects. The expression of CRB1 is not detectable in human skin cells and would usually require cellular reprogramming and retinal differentiation for it to be expressed. Using the CRISPRa system, I screened 5 known pathogenic CRB1 mutations and compared results with those obtained from retinal organoids from the same WARD patients. Four of 5 mutations showed expression of the same defect in both skin and retinal cells, with the fifth mutation showing a more severe defect in gene-activated skin cells compared with retinal cells. This demonstrated the efficacy of the CRISPRa system in accurate transcriptional activation in a fraction of the time taken through the retinal organoid study. These results provide a proof of principle for a new, rapid method for confirming newly discovered mutations are pathogenic.

How do you think your research could potentially help inform clinical trials and practice in the future?

Moon: For patients with variants of unknown significance, pathogenicity assessment usually entails full cellular reprogramming by retinal organoid differentiation. Some patients may be eligible for gene therapy or clinical trial enrolment but require a confirmed pathogenicity assessment, leaving them without an answer for an extended period. The establishment of a rapid method for screening retinal disease-causing genes in patient skin cells unlocks the potential of the WARD biobank for the classification of new pathogenic mutations. We will now turn our attention to characterizing the many gene mutations of uncertain significance that have been identified in WARD Study patients, ensuring these patients receive the confirmed diagnoses necessary for inclusion in future clinical trials. By using CRISPRa to directly activate IRD gene expression, we are able profoundly expedite this process. We hope that the established protocol can be used to screen patients in the near future.

The CRISPRa method established here provides medical researchers with a rapid method for classifying pathogenic mutations. Although our focus is inherited retinal diseases, this new technique could be easily applied to mutation discovery across a wide range of different inherited diseases.

Transcript edited for clarity.

REFERENCE

Moon SY, Zhang D, Chen SC, et al. CRISPR activation of endogenous CRB1 expression in fibroblasts from retinitis pigmentosa 12 patients. Presented at: ARVO 2023 Annual Meeting; April 23-27, 2023, New Orleans, Louisiana. Abstract #753 - C0354