Novel gene therapy platform speeds search for ways to cure blindness

A collaboration among scientists from the University of Pennsylvania, University of Pittsburgh School of Medicine, University of California, Berkeley, and Carnegie Mellon University has developed a platform to identify top-performing viral vectors that could deliver gene therapies to the retina with maximum efficiency and precision.

The technology, described in a paper published today in the journal eLife, streamlines development of gene therapy approaches for the treatment of genetic blinding disorders, saving time and resources.

“This novel approach accelerates identification of the most efficient viral vectors that can be used to develop gene therapies to treat blinding conditions in people and in animals,” says William Beltran, a professor of ophthalmology at Penn’s School of Veterinary Medicine and an author on the new paper who led the experiments at Penn.

“Vision loss has a huge impact on quality of life. It has long been near the top of the greatest fears of people, alongside cancer and Alzheimer’s disease,” adds senior author Leah Byrne, assistant professor of ophthalmology at the University of Pittsburgh. “But the field of vision restoration has entered a new era, where many patients have received effective treatment for the very first time. Because of that, the potential of our new platform is thrilling—it will allow us to translate emergent therapies that are already working for some patients into the clinic much more rapidly.”

Even though blinding genetic disorders that affect the retina are considered rare, approximately 1 in every 3,000 people worldwide carries one or more copies of genes that cause retinal degeneration and vision loss. For centuries, many people with inherited blindness were all but guaranteed to spend a portion of their lives with vision loss.

Now, with several gene therapies already on the market in Europe and the United States, and dozens more entering clinical trials, hope for people with inherited blindness is within reach. But a key obstacle remains: ensuring that vectors, or inactivated viruses carrying the therapeutic genetic code, enter the particular cells that scientists are targeting. The retina is composed of hundreds of millions of cells that are arranged into a series of layers, so precisely targeting the vector to a specific location is not a trivial task.

To approach the problem, researchers developed a computational platform called scAAVengr, which uses single-cell RNA sequencing to quickly and quantitatively evaluate—among dozens of options—which adeno-associated virus vector, or AAV, is best suited for the task of delivering a gene therapy to a specific part of the retina.

The traditional approach of evaluating AAVs is painstakingly slow, requiring several years and many experimental animals. It is also not very precise, since it doesn’t directly measure if AAVs not only entered the cells but also delivered their gene therapy cargo.

In contrast, scAAVengr uses single-cell RNA sequencing, which detects if the cargo arrives at its destination safely. And with scAAVengr, that screening process takes months, not years.

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Penn Vet researchers from the Division of Experimental Retinal Therapies helped accumulate candidate vectors using a directed evolution approach in dogs, which Beltran and colleagues have long studied en route to establishing experimental gene therapies for inherited vision disorders. Dogs naturally develop blinding disorders that recapitulate many features of human disease, and the canine eye anatomy closely resembles that of human eyes. The directed evolution method enabled the team to identify variants that were particularly good at targeting the outer retina, where photoreceptors—rods and cones—and retinal pigment epithelial (RPE) cells are found.

Pitt researchers validated these candidates using non-human primates, leading to the identification of variants that precisely target cells of the outer retina, in particular, the light-sensing photoreceptor cells and RPE, two cell types crucially important to a successful vision restoring gene therapy. These candidate AAVs were also efficiently expressed in these cells following an intravitreal injection.

“An intravitreal injection is a much simpler and safer surgical route of delivery than the most commonly used subretinal approach,” says Beltran.

The platform’s uses aren’t just limited to the retina—the researchers showed that it works just as well for the identification of AAVs that target other tissues, including the brain, heart, and liver.

“Rapidly developing fields of gene editing and optogenetics all rely on efficient gene delivery, so the ability to quickly and strategically choose the delivery vectors would be an exciting leap forward,” says Byrne.

Byrne and Beltran’s coauthors are Penn’s Valérie Dufour, Simone Iwabe, Felipe Pompeo Marinho and Gustavo Aguirre; Bilge Öztürk, Molly Johnson, Serhan Turunç, Jing He, Sara Jabalameli, Zhouhuan Xi, William R. Stauffer, and José-Alain Sahel, all of Pitt; Michael Kleyman and Andreas Pfenning, both of Carnegie Mellon University; Meike Visel, David Schaffer, and John Flannery, all of the University of California Berkeley. Öztürk was first author and Byrne was senior author on the paper.

This research was supported by the National Institutes of Health (grants F32EY023891, R24EY-022012, R01EY017549, P30EY001583, UG3MH120094, DP2MH113095), the UPMC Immune Transplant and Therapy Center, Foundation Fighting Blindness, Ford Foundation, Research to Prevent Blindness, and the Van Sloun Fund for Canine Genetic Research.

Adapted from a release written by Anastasia Gorelova of the University of Pittsburgh.