Prime Editing Can Become the Future of Vision Loss Treatment

Gene therapy research is full steam ahead trying to provide novel treatments and cures for diseases caused by harmful genetic alterations in humans. The advent of CRISPR and other related gene-editing tools, such as base editing and the recently emerged prime-editing method, collectively offer vast options to directly edit human DNA ex vivo and in vivo.

Congenital retinal diseases comprise a heterogenous group of conditions that result in progressive loss of sight. To date, treatment options including recently developed gene supplementation therapies are only available for a subset of these diseases, leaving a large number of patients without a treatment. After winning a grant of just over 3 million Danish kroner (400,000 EUR) from the Velux Foundation (a philanthropic funding body in Denmark), PhD student Silja Hansen MD, who is based at Aarhus University in Denmark, aims to develop a new treatment based on a gene-editing method called prime editing, to slow the progression of inherited retinal diseases.

Here, Silja Hansen speaks with CRISPR Medicine News to share an inside view on where this new project may lead.

A way to tackle heterogeneous genetic diseases

- Hi Silja, it’s nice to meet you. Before we get into your project, can you tell us about the causes of inherited retinal diseases and how impactful these conditions are?

Inherited retinal diseases are a large group of diseases, which all affect the retina in the eye. The diseases are characterised by the dysfunction and degeneration of photoreceptors and/or the retinal pigment epithelium, resulting in progressive vision loss and blindness.

Each condition is different in relation to the severity of vision loss and time of onset, i.e., whether symptoms start in childhood or later in life. Inherited retinal diseases affect 1 in 2,000 people worldwide and represent a leading cause of vision impairment.

- What was the motivation to investigate gene editing to ameliorate vision impairment?

To date, various variants in more than 250 genes have been directly linked with an inherited retinal disease. Although gene replacement therapies have recently shown to be successful for the treatment of autosomal recessive variants, this treatment option is not feasible for the large number of diseases that are either caused by variants in genes that are too large to be supplemented or that are inherited in an autosomal dominant manner. This means that there are still thousands of people out there that will suffer from severe vision loss or even become blind from a retinal disease for which there is no treatment.

In many cases, the only thing a patient can do to manage their disease is go to an ophthalmologist and have their case followed and monitored, but without any effective treatment. A versatile gene-editing platform that could treat many of these diseases would completely transform the lives of these patients. The motivation behind this project is to develop a gene-editing approach that may have the potential to treat more of these diseases, and thereby improve the current treatment landscape and the outcome for the individual patient in the future.

- What are the main goals of the project?

The aim of the project is to establish prime editing as a possible treatment option for inherited retinal diseases that cannot be treated by gene replacement therapy or other gene-editing approaches, e.g., base editing, and could therefore uniquely benefit from prime editing. In a first step, we want to design and validate prime-editing guide RNAs (pegRNAs) to target some of the main disease-causing variants. pegRNAs function both as a guide to the desired target and as a template for the desired edit, and are therefore essential for prime editing efficiency. Then, we will use pre-clinical cell models and reporter cell lines to test those pegRNAs to get a good idea about the best performing options for each of the variants.

Prime editing: the new kid on the block

- What exactly can prime editing do here that the other gene-editing modalities cannot?

Prime editing is a new CRISPR-Cas9-based gene-editing tool that is very versatile compared to other gene-editing methods. Let’s start with CRISPR-Cas9. This method causes the formation of a double-stranded break at the target site followed by non-homologous end joining (NHEJ) or homology-directed repair (HDR). While the NHEJ pathway is error prone, HDR requires a donor template and is inefficient in non-dividing cells, which accounts for the majority of retinal cells. In addition to that, the creation of a double-strand break has shown to increase the risk of chromothripsis (chromosome scattering).

Base editing does not generate a double-stranded break, but it is limited to transition mutations, and depending on the target site, its use may be limited by the target window and the risk of bystander edits.

Prime editing, although still very new, has the potential to correct all kinds of point mutations as well as smaller deletions and insertions that cover a large amount of the variants underlying inherited retinal diseases. It functions as an all-in-one system – you have the pegRNA that directs the enzyme complex to the target site and it also functions as its own template, and it does not require a double-strand break.

- Are there any challenges associated with prime editing?

Yes, the prime editing method is still in its infancy. One of the challenges in prime editing is finding a pegRNA that can create the desired edit and achieve high targeting efficiency. Right now, we have a number of candidate pegRNAs to validate and once we find the most effective ones, we will go forward with those for further testing in pre-clinical models.

Another major challenge, which is not only relevant for prime editing but any large gene-editing tool, is the delivery of the gene-editing cargo. The most established delivery vector for the receptor cells in the retina is the adeno-associated virus (AAV), but the prime-editing construct is too large to fit in one AAV particle. This is also something we would like to address in our project. Previous studies have looked at the possibility of using a dual-AAV to deliver a split prime editor system. We would like to test this strategy further, but we may also investigate different delivery options, including non-viral delivery methods.