Retinitis Pigmentosa CRISPR Knockout Models for Drug Discovery

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive photoreceptor degeneration, leading to vision loss. According to the World Health Organization (WHO), RP affects approximately 1 in 4,000 people globally, with an estimated 1.5 million individuals affected worldwide. It is a leading cause of inherited blindness in working-age adults. The disease typically presents with night blindness in adolescence, followed by progressive visual field constriction and eventual central vision loss. There is no cure, and current treatments are limited to vitamin A supplementation and gene therapy for specific subtypes (e.g., RPE65 mutations). The 5-year survival for RP patients is not applicable as it is not a life-threatening condition, but quality of life is severely impacted. Key risk factors include family history and specific genetic mutations. Data from the National Eye Institute (NEI) and ClinVar highlight over 80 genes associated with RP, with autosomal dominant, autosomal recessive, and X-linked inheritance patterns.

RP is an ideal model for studying photoreceptor biology, ciliary function, and protein trafficking. The genetic heterogeneity of RP provides a rich landscape for functional genomics, allowing researchers to dissect the role of individual genes in retinal health. Public datasets from the NCBI Gene database and the RetNet network provide comprehensive lists of RP-associated genes. Open questions include the mechanisms of cone death in rod-cone dystrophy, the role of oxidative stress, and the potential for neuroprotective strategies. Gene-edited cell models are critical for validating candidate genes and testing therapies in a human photoreceptor context.

The pathogenesis of RP involves several key pathways:

1. Phototransduction cascade disruption:

• • Mutations in RHO (rhodopsin) lead to misfolding and ER stress.
• • Impaired visual cycle due to RPE65 or LRAT mutations.

2. Ciliary transport defects:

• • RPGR and RP2 mutations disrupt protein trafficking to the outer segment.
• • Defective intraflagellar transport (IFT) leads to photoreceptor degeneration.

3. Oxidative stress and apoptosis:

• • Accumulation of reactive oxygen species (ROS) triggers cell death.
• • Activation of caspase-dependent and independent pathways.

4. RNA splicing defects:

• • Mutations in PRPF31, PRPF8, and other spliceosome genes cause aberrant splicing.
• • Leads to loss of photoreceptor-specific transcripts.

Data from ClinVar, NCBI Gene, and RetNet.

Key deregulated networks in RP include:

• • Phototransduction cascade: RHO, GNB1, GNGT1, PDE6A/B, CNGA1, CNGB1.
• • Ciliary transport network: RPGR, RP2, IFT88, IFT172, KIF3A.
• • ER stress and unfolded protein response (UPR): ATF6, XBP1, CHOP.
• • Oxidative stress response: NRF2, KEAP1, SOD2, catalase.
• • Apoptosis pathways: BAX, BAK, cytochrome c, caspases 3/9.
• • Spliceosome complex: PRPF31, PRPF8, SNRNP200.

Organoids derived from induced pluripotent stem cells (iPSCs) offer 3D retinal structures with photoreceptor layers, enabling study of cell-cell interactions and drug testing. Patient-derived iPSCs can be differentiated into retinal organoids to model specific RP mutations.

• • Royal College of Surgeons (RCS) rat: Spontaneous mutation in Mertk, models RPE phagocytosis defect.
• • rd1 mouse: Mutation in Pde6b, rapid photoreceptor degeneration.
• • rd10 mouse: Mutation in Pde6b, slower degeneration.
• • RHO P23H mouse: Transgenic model of autosomal dominant RP.
• • RPGR knockout mouse: Models X-linked RP.
• • Zebrafish models: Used for high-throughput drug screening due to rapid development and transparency.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with precise RP-associated mutations. For example, RHO P23H knock-in in ARPE-19 or hTERT-RPE1 cells recapitulates rhodopsin misfolding. RPGR knockout in RPE1 cells models ciliary transport defects. These models allow for controlled experiments comparing mutant vs. isogenic wild-type controls, eliminating genetic background noise. Commercially available, sequence-verified isogenic cell lines accelerate drug screening and target validation. Examples include RHO knockout, RPGR knockout, and USH2A knockout lines. These models are essential for studying disease mechanisms and testing gene therapies, small molecules, or CRISPR-based corrections.

CRISPR knockout and knock-in lines allow functional validation of candidate RP genes. For example, knocking out RPGR in RPE1 cells leads to defective ciliogenesis, confirming its role in ciliary transport. Knock-in of RHO P23H in ARPE-19 cells induces ER stress and apoptosis, providing a platform to test UPR modulators. Genome-wide CRISPR screens in isogenic backgrounds can identify genetic modifiers of RP phenotypes.

Isogenic pairs (mutant vs. wild-type) are used for high-content screening of small molecules that rescue photoreceptor survival. For example, screening for compounds that reduce ER stress in RHO P23H cells. Resistance modeling: chronic treatment with neuroprotective agents can select for resistant clones, revealing compensatory pathways. Gene-edited cells are also used to test AAV-based gene therapy vectors.

CRISPR-based synthetic lethality screens identify genes that, when knocked out, selectively kill RP mutant cells. For example, targeting oxidative stress pathways in RPGR-deficient cells. Proteomic and transcriptomic analysis of isogenic lines reveals biomarkers of disease progression, such as secreted factors or cell surface markers, which can be used for non-invasive monitoring.