Correcting the Mutation Behind a Genetic Eye Disease

Almost 200 million people worldwide suffer from macular degeneration, a condition that affects central vision and can result in near blindness.¹ Although most forms of macular degeneration affect older people, those caused by mutations in a single gene are usually more severe and affected people show signs at a young age.²

Among the forms of monogenic macular degeneration, the most common is Stargardt disease. This disease is caused by mutations in the ATP-binding cassette transporter alpha 4 subunit (ABCA4) gene, giving rise to a defective protein. This eventually leads to the accumulation of toxic forms of vitamin A, causing retinal cell death. As these cells die, people gradually lose the ability to read, drive, and recognize faces.

“Because we know the genetic cause [of Stargardt disease], we felt that a new technology, base editing, could potentially [provide] hope for these patients where we can correct the underlying mutation as a therapeutic aid,” said Bence György, a translational scientist at the Institute of Molecular and Clinical Ophthalmology Basel (IOB).

Recently, György and his colleagues designed a base editing therapy that achieved high levels of mutation correction in cells, organoids, mice, and non-human primates.³ Their results, published in Nature Medicine, highlight the promise of treating certain forms of inherited blindness by addressing the root cause of the disease.

“This is a well-conducted study, and very relevant,” said Indumathi Mariappan, a cell and molecular biologist at the LV Prasad Eye Institute, who was not involved in the study. “And they have nicely validated this editing system [in different models].”

Researchers have previously shown that patients with Stargardt disease commonly carry a mutation in ABCA4: A single nucleotide switched from guanine to adenine.⁴ To correct this point mutation, György and his team decided to use DNA base editors, which can repair single nucleotide mutations without breaking both DNA strands. This editing machinery uses guide RNAs (gRNAs) to direct the Cas9 enzyme—also called molecular scissors—to the target site on the DNA to make the appropriate cuts; then, another enzyme modifies the nucleotide base.

György and his team started out by designing and testing different gRNAs. They tested these in a human kidney cell line carrying mutant ABCA4 and sequenced the cells’ DNA to identify the gRNAs that resulted in the most efficient gene editing.

After selecting the most suitable gRNAs, the researchers set out to identify ways to deliver these efficiently into target cells. They used modified viral vectors, which other researchers have previously used to successfully deliver gene-editing tools into the eye.⁵

The researchers used various systems to model the retina in vitro for optimizing vector components that would enable the highest editing efficiency. They injected their editing tools into the eyes of mutation-carrying mice and analyzed gene editing via sequencing. They found that the mutation was corrected in both light-sensing photoreceptor cells and retinal pigment epithelial (RPE) cells in the outermost retinal layer.

Encouraged by these results, György and his team tested this therapy in macaques. Because the monkeys did not carry the Stargardt disease mutation, the researchers used the editing tool they had developed to alter a nucleotide near the usual mutation site as a proxy. Sequencing the DNA isolated from the eyes of treated primates revealed high editing efficiencies; on average, editing occurred in 75 percent of the photoreceptor cells and 87 percent of the RPE cells.

Finally, the researchers analyzed genome-wide off-target effects of their editing machinery using computational tools to identify the sites at which their gRNAs could bind. They did not find any off-target sites that showed significant editing, suggesting that their base-editing strategy is precise.

The results, said György, “[highlight] the immense potential of gene correction as a medical technology.”

“This is one of the first papers that used all [of the available] human models to correct the mutation,” said study coauthor Botond Roska, a neuroscientist at the IOB. This approach, the authors noted, maximizes the chances of clinical translation. Roska believes that with a few more experiments to optimize the delivery system and evaluate its safety, the team will have a gene therapy vehicle that is ready for clinical trials.

While the results have important clinical implications, Mariappan noted that the treatment may not restore vision if the majority of a patient’s retinal cells have already degenerated. However, if the disease is caught early enough and people have retained a sufficient number of retinal cells, the therapy could prevent further degeneration. This makes it important to consider the right choice of patients and vision assessment parameters to define a successful outcome, she cautioned.

György noted that the organoids and mice carrying ABCA4 mutations do not contain the fovea, which is the central region of the retina that degenerates in Stargardt disease. So, the researchers could not assess whether their gene therapy would stop cell degeneration in this part of the retina. Testing the editors in humans would shed light on this aspect, he said. “Once we are confident with the safety, it makes sense to try this in humans.”

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