New discovery could help overcome a key challenge in gene editing approaches

IRD Overview:

Inherited retinal dystrophies (IRDs), which include retinitis pigmentosa (RP) and stargardt disease, are the most common cause of blindness in children and working aged people in the UK. Yet the conditions are rare, with only around 25,000 people affected in the UK. More than 300 genes have been linked to inherited sight loss conditions and many more are yet to be identified, making the search for treatments complex. There is only one treatment, called Luxturna, available for a tiny proportion of the community who live specifically with RPE65-associated RP and Leber Congenital Amaurosis (LCA). However, for the majority of people living with IRDs, there is currently no available treatment.

Advances in genetic medicine are raising hopes that the underlying causes of inherited sight loss could eventually be tackled with various therapeutic approaches in development, from gene therapies to optogenetics. However, the exciting update in this article focuses on an approach known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).

What is CRISPR?

To put it simply, CRISPR is a genetic technology that can precisely target and edit sections of DNA. It is often described as a molecular ‘cut and paste’ tool.

There are different types of CRISPR-based technologies that work in different ways. For example, traditional CRISPR-Cas9 works by using an enzyme to make a cut in both strands of DNA at a targeted location and then relies on the cell’s own machinery to repair it.

The CRISPR approach mentioned in this article, is CRISPR-base editing. Unlike CRISPR-Cas9, CRISPR-base editing uses an enzyme to chemically change just one DNA letter into another at a specific location without making a cut in both strands of DNA. It is often described as a ‘spellcheck’ tool. This approach is preferable for some IRDs because many disease-causing variants involve single-letter changes to DNA. However, base editing isn’t appropriate for every mutation or gene.

The problem:

One of the difficulties with CRISPR is getting the machinery to the right place in the cell in the first place. The cell itself presents numerous obstacles for the delivery of CRISPR to the cell’s nucleus, such as entry to the cell and navigating around biological structures. These obstacles can interfere with the delivery of CRISPR to the nucleus affecting its overall ability to make the desired genetic change.

What did this study find:

As part of an effort to identify such obstacles to CRISPR delivery and improve the efficacy of genetic editing technologies, researchers at the University of Wisconsin-Madison have conducted promising early-stage research into IRD treatment approaches and their roadblocks. The team found that specific genes appear to make CRISPR less efficient, and temporarily manipulating these factors may substantially improve gene editing in retinal cells.

The researchers developed a platform to screen more than 19,000 genes to find out which genes were linked to the disruption of CRISPR delivery. This means that the researchers were looking for genes that, once switched off, led to an increase in the efficiency of CRISPR genetic edits.

Over six years, the team found 26 genes that impacted the efficacy of CRISPR out of the 19,144 that were screened. Once this list of genes was refined, the top two (GJB2 and BET1L) were tested in human model cell systems. It was found that depleting either of these two genes increased the editing efficiency by more than six times using CRISPR-base editing.

More importantly, when this approach was tested in retinal cells derived from individuals with Leber congenital amaurosis, the inhibition of these two genes increased editing efficiency by more than three-and-a-half times, suggesting that this lab-based discovery has meaningful practical applications for patients.

Why does this matter?

With genetic editing approaches such as CRISPR, getting enough retinal cells successfully edited is a major challenge. We need to ensure that the editing machinery gets into the retinal cell and that enough of it reaches the nucleus to make the intended genetic change whilst avoiding unwanted changes. By identifying and potentially overcoming the barriers that impede CRISPR efficiency, researchers may eventually be able to make gene editing more effective and potentially more practical for inherited retinal diseases.

However, it is important to note that this is not a treatment for vision loss. Turning off genes such as GJB2 and BET1L will not restore vision, but may lead to genetic editing techniques becoming more efficient. This is also early-stage laboratory research and the team at University Wisconsin-Madison do not yet know how these genes are barriers to genetic editing and what effect dampening these genes would have in humans. Further research is needed to identify whether this approach works inside an eye, rather than only in cultured cells, whether it improves actual retinal function and vision in animal models and whether the approach can be delivered safely alongside the CRISPR machinery.

This is an exciting research update, not only for the IRD field but also for other tissue or disease areas, where different cell types may have different obstacles.

“My hope is that the field picks up the methodology and helps understand what cellular mechanisms, pathways, are important for the delivery of really anything that could be packaged into lipid nanoparticles. And in that way, I believe the impacts could be beyond just genome editing”. – Krishanu Saha, Associate Professor, University of Wisconsin-Madison