Fighting against inherited blindness: “Restoring Function in iPSC-Based Models of Late-Onset Retinal Degeneration (L-ORD) Using CRISPR-Mediated Gene Editing”

The Problem

Late-onset retinal degeneration (L-ORD) is a rare inherited eye disease that leads to vision loss.

A vital part of our vision is supported by a thin layer of cells called the retinal pigment epithelium (RPE) which sits between the light-sensing retina, and the choroid, a layer of blood vessels that supply blood to the retina. Its job is to supply nutrients and remove waste products from the light-sensing cells in your eyes.

As late-onset retinal degeneration progresses, it causes the retinal pigment epithelium to break down. This means they can’t support the light-sensing cells of the retina, which then die, leading to progressive and irreversible vision loss.

Currently, there is no cure for late-onset retinal degeneration.

Mice are often used in gene therapy for genetic disorders of the retina. However, their short lifespan means scientists often turn to larger, longer-lived animals. This means animals like cats, dogs, pigs, and even monkeys are used to study gradual vision loss.

This is where Casey’s project steps in. She aims to test a gene-editing therapy. Using CRISPR, a powerful gene-editing tool, she hopes to fix the faulty gene that causes vision loss in L-ORD. This will be done using a lab-based system, derived from patient samples.

The Project

Casey will be using skin cells donated from patients with late-onset retinal degeneration. In the lab, these ordinary skin cells are transformed into induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells act as a blank canvas and are capable of becoming almost any other type of cell in the human body.

Because these stem cells are so versatile, Casey will be able to transform them into the cells affected by late-onset retinal degeneration. This allows her to create a human-relevant disease model right in the petri dish. It’s like a miniature human eye tissue that accurately reflects the condition.

Casey intends to grow and study three different types of these RPE cells:

Healthy retinal pigment epithelium cells: These will serve as her gold standard, showing how normal, healthy cells function.

Late-onset retinal degeneration mutant cells: These are the cells carrying the faulty gene, allowing her to see the effects of the disease.

CRISPR-edited cells: These are the exciting ones! They are the late-onset retinal degeneration patient cells that have been ‘fixed’ using the CRISPR gene-editing tool.

She will then compare how these different cells are expressing their genes. Essentially, what instructions they’re following, and how well they’re functioning.

Casey’s ultimate goal is to understand if her CRISPR correction can restore the faulty retinal pigment epithelium cells to a healthy state. It’s like taking a broken piece of machinery and repairing the faulty part, hoping to get it working again.

The Potential

If all goes to plan, Casey’s work will help produce a truly human-relevant model for studying retinal diseases. This means we could significantly reduce our reliance on animals in research, especially for conditions like late-onset retinal degeneration. It’s a huge step towards more ethical and efficient scientific practices.

Beyond that, her system offers a much more precise way to test and assess new therapies for all inherited retinal diseases. Imagine a future where scientists can rapidly test potential cures in a dish, getting accurate results that directly apply to human patients, before even considering animal studies. This innovative approach could bring us much closer to finding effective treatments and, potentially, a cure for vision loss.

The update

Casey successfully grew all three types of cells in the lab. The healthy ‘control’ cells, the late-onset retinal degeneration mutant cells, and the CRISPR-edited cells, which were incubated and matured, all developed as she had hoped.

When she examined under the microscope, Casey found that all the cell lines appeared as expected, and her molecular analysis showed that all cell lines had active genetic markers associated with retinal pigment epithelium.

To find out whether the cell lines behaved as though they were retinal pigment epithelium, Casey monitored the live cells in real-time at different stages of their growth. She measured how they responded to stress by incubating them with three different drugs that inactivate different components of the cell’s metabolism. She also measured the electrical resistance across the cells as how they behave as a barrier is critical for maintaining the retina’s health.

Using microscopy, molecular, biochemical and electrical techniques, Casey was able to show that the induced pluripotent stem cells not only behaved like real retinal pigment epithelium when they were differentiated, but CRISPR-gene editing also partially restores the mutant cells’ metabolic activity to healthy levels.

The gene expression data from the control, mutant, and gene-edited cell lines adds to the growing body of evidence supporting the value of stem cell-based disease models in the development of new therapies. Casey suggested that future efforts could focus on increasing their complexity, for example by incorporating iPSC-derived 3D blood vessels, which could further help replace animal ‘models’.

Altogether, this would provide a more robust understanding of the how CRISPR-based gene editing could help restore retinal pigment epithelieum function in late-onset retinal degeneration, and guide the development of new therapies.