This grant was funded by the Australian and New Zealand Society of Retinal Specialists (ANZRS)
Investigator: A/Prof Fred K. Chen
Co-investigator: Prof Sue Fletcher
Aim: In this project the aimed was to:
1) generate personalised retinal cell models for patients with CRB1 mutations;
2) design and test antisense oligonucleotide drugs for treatment of CRB1 mutations; and 3) develop a new method for inducing CRB1 expression in skin cells.
Methods: Fibroblasts derived from patients with CRB1-associated retinopathy were reprogrammed
into induced pluripotent stem cells, differentiated into retinal organoids and screened for splicing defects by RT-PCR. Patient fibroblasts were transfected with Cas9- VPR and guide RNAs targeting the CRB1 promoter and CRB1 expression was analysed by quantitative PCR.
Key Results: Analysis of patient-derived retinal organoids indicated CRB1 splicing defects occurred in 2/4 patients analysed (Aim 1). Antisense oligonucleotidedrugs for treating CRB1 splice mutations have been designed and are being screened in patient retinal organoids (Aim 2). Furthermore, we demonstrated induction of CRB1 expression in patient skin fibroblasts by CRISPR activation (Aim 3).
Conclusion: We have identified two Western Australian patients with splice-altering mutations in CRB1 that may be amenable to treatment with antisense oligonucleotide drugs. Additionally, we demonstrated the feasibility of inducing CRB1 expression in patient- derived skin cells through CRISPR activation.
Implications for Clinical Practice/Science and Future Research: We have successfully optimised our pipeline for developing treatments for patients with CRB1- associated retinopathy. These pilot studies provided preliminary data for a successful application to the NHMRC for funding to continue our work in developing therapies for CRB1-associated retinopathy and other inherited retinal diseases.
- Moon SY, et al. ‘Generation of two induced pluripotent stem cell lines from a retinitis pigmentosa patient with compound heterozygous mutations in CRB1.’ Stem Cell Res. 2021 54:102403.
- Zhang X, et al. ‘Characterization of CRB1 splicing in retinal organoids derived from a patient with adult- onset rod-cone dystrophy caused by the c.1892A>G and c.2548G>A variants.’ Mol Genet Genomic Med. 2020 8(11):e1489