Knockdown and Replace: A Gene Therapy Twofer to Treat Blindness

PHILADELPHIA—The last year has seen milestones in the gene therapy field, with FDA approvals to treat cancer and an inherited blinding disorder. New findings from a team led by University of Pennsylvania vision scientists, who have in the past taken gene therapies into clinical trials, are proving successful, this time treating a form of retinitis pigmentosa, a disease that progressively robs people of their night and peripheral vision before blindness develops.

The researchers, from Penn’s School of Veterinary Medicine and Perelman School of Medicine, in collaboration with University of Florida scientists, developed a therapy that effectively eliminates the abnormal copy of rhodopsin, a light-sensing molecule, and then restores it with a healthy copy of the protein. This knockdown and replacement approach preserved the retina’s light-sensing photoreceptor cells in affected dogs, which can develop a very similar disease to affected humans. The team published their findings in this week’s Proceedings of the National Academy of Sciences.

What’s more, they accomplished this using a single viral vector to co-deliver the genetic material needed to achieve both the knockdown and replacement. Though more than 150 different mutations in rhodopsin have been identified to cause retinitis pigmentosa, this approach is intended to work regardless of the mutation or the mechanism by which rod photoreceptor cells, those responsible for vision in dim light, die. That means that a large percentage of patients with rhodopsin autosomal dominant retinitis pigmentosa could benefit if the therapy is found to be safe and effective in people.

“It’s a one treatment fits all,” said William A. Beltran, DVM, PhD, a professor of ophthalmology and director of the Division of Experimental Retinal Therapies at Penn Vet and co-lead author of the study. “The treatment targets a region of the rhodopsin gene that is homologous in humans and dogs and is separate from where the mutations are located. That gives us great hope about making this a translational treatment.”

Retinitis pigmentosa refers to a set of progressive hereditary retinal disorders. Three decades ago, researchers identified mutations in the gene encoding rhodopsin as the first known genetic cause of the condition. Other genes have since been implicated as well, but rhodopsin mutations remain a major contributor, accounting for up to 30 percent of autosomal dominant retinitis pigmentosa.

“We’ve known for decades that this specific molecule causes a specific form of retinitis pigmentosa, but developing a treatment has not been straightforward,” said Artur V. Cideciyan, PhD, research professor of ophthalmology at Penn Medicine and co-lead author. “Now, with these elegant results based on years of study in dogs we can start working toward treating these mutations and prevent deterioration of photoreceptor cells in humans.”

The vast majority of rhodopsin mutations are passed through families in a dominant fashion, meaning that a parent need only pass one mutated copy on for their child to be affected.

“In our investigations, we’ve seen people in the 1990s with this genetic type of retinitis pigmentosa and now we’re seeing their grandchildren also affected,” said Samuel G. Jacobson, MD, PhD, professor of ophthalmology and director of the Center for Hereditary Retinal Degenerations at Penn Medicine. “It’s a multi-generational disease and it’s a serious disease.”

Several rhodopsin mutations that lead to retinitis pigmentosa result in what’s known as a toxic gain-of-function, thought to produce a protein that is harmful to the photoreceptor cells. Thus, to address the problems that arise in these patients, researchers have determined that the best strategy is to eliminate the mutant protein.

“We’ve developed in the past gene therapies for other conditions where the mutation causes a loss of function,” said Gustavo D. Aguirre, VMD, PhD, professor of medical genetics and ophthalmology at Penn Vet. “so in these cases we just needed to add back the normal copy of the gene in order for the photoreceptors to regain their normal structure and function. When you have a dominant disease like this one, where the gene product is really damaging to the cell, you have to get rid of it.”

Earlier efforts by the team had attempted to do just that: simply knockdown both the mutant and normal rhodopsin in dogs. This prevented the disease but caused a loss of the key cellular compartment of rod photoreceptors, the outer segment, which initiates vision. Beltran and colleagues determined that adding back a healthy copy would be the best and necessary strategy to achieve healthy functioning retinal cells.

In the current work, the researchers employed this strategy, which relied on both in vitro assays and, significantly, on a canine animal model with a naturally occurring mutation in the rhodopsin gene that faithfully recapitulates the form of human retinitis pigmentosa caused by rhodopsin class B mutations. While patients with class A mutations in rhodopsin lose functioning rod photoreceptor cells early in life, those with class B mutations may retain their rod cells for decades, making them candidates for a vision-preserving gene therapy.

Thus far, tracking the treatment effect more than eight months after delivery of the gene therapy, the effect seems stable and lasting. The research team is currently working to move the findings into clinical trials.

“The current work has strong implications for the treatment of patients with autosomal dominant retinitis pigmentosa due to Class B rhodopsin mutations,” said Cideciyan.

Additional authors on the study were Penn Vet’s Raghavi Sudharsan, Valérie Dufour, Simone Iwabe, Luis Felipe Marinho, and Tatyana Appelbaum; Penn Medicine’s Malgorzata Swider, Brianna Lisi, and Alexander Sumaroka; and University of Florida’s Michael Massengill and Brian Rossmiller.

The research was supported by the National Eye Institute (grants EY022012, EY06855, EY001583, and EY021721), Foundation Fighting Blindness, Research to Prevent Blindness, and the Shaler Richardson Professorship Endowment.

You can read more about this research on Penn Today.