Researchers find potential new gene therapy for blinding disease

August 20, 2018, University of Pennsylvania
Gene therapy adeno-associated viral (AAV) vector includes sequences that encode both the shRNA knockdown element and the replacement rhodopsin protein. Credit: Lesley Earl, National Eye Institute

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.

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," says William A. Beltran, professor of ophthalmology and director of the Division of Experimental Retinal Therapies at Penn Vet and co-lead author of the study, which appears in this week's Proceedings of the National Academy of Sciences. "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."

"We've known for decades that this specific molecule causes a specific form of retinitis pigmentosa, but developing a treatment has not been straightforward," says Artur V. Cideciyan, 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 work is the latest result in a long-standing partnership among Penn vision scientists. In addition to Beltran and Cideciyan, the team included Penn Vet's Raghavi Sudharsan, a research associate in the Beltran lab, and Gustavo D. Aguirre, professor of medical genetics and ophthalmology, and Penn Medicine's Samuel G. Jacobson, professor of ophthalmology and director of the Center for Inherited Retinal Degenerations.

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.

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," Jacobson says. "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," says Aguirre, "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.

Because mutant rhodopsin dogs are very sensitive to ambient light, a short exposure equivalent to midday luminance was used to accelerate the degeneration of photoreceptors. This sensitivity allowed the researchers to control which areas of the retina were most affected and when, and thus evaluate treatment success in a matter of weeks rather than the years it would take to develop degeneration without this trigger.

In the lab, the team confirmed that they could disrupt the mutant rhodopsin gene using a knockdown reagent called short-hairpin RNA (shRNA) engineered by Alfred Lewin and colleagues at the University of Florida to target an area away from the mutated sections of the gene. Adding back a healthy copy of the rhodopsin gene that is resistant to this shRNA compensated for the knockdown.

In dogs with the rhodopsin mutation, they used the same strategy, finding the best success when both the shRNA and the healthy copy of rhodopsin were co-delivered in the same vector, as opposed to using two different vectors. The team restored roughly 30 percent of the normal level of rhodopsin, enough to prevent deterioration of rod cells in the retina.

"What we showed was that if you just did the knockdown alone, you preserve the outer nuclear layer of rods, which is where the cell bodies are located," Beltran says. "But without another critical layer, the outer segments, where rhodopsin plays the essential role of capturing light and initiating vision, then the rods become useless. However, if you combine the knockdown with the replacement reagents, then the drastic difference is that you now have perfectly formed and aligned outer segments and functional photoreceptor cells."

The Penn researchers were able to confirm the beneficial effects on both structure and function of rod cells (as well as cones, responsible for color vision) by using specialized imaging techniques that can be used in human patients along with electroretinography, which provides a measure of rod and cone function.

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," Cideciyan says.

Explore further: Genome surgery for eye disease moves closer to reality

More information: Artur V. Cideciyan el al., "Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector," PNAS (2018). www.pnas.org/cgi/doi/10.1073/pnas.1805055115

Related Stories

Genome surgery for eye disease moves closer to reality

May 11, 2018
Researchers from Columbia University have developed a new technique for the powerful gene editing tool CRISPR to restore retinal function in mice afflicted by a degenerative retinal disease, retinitis pigmentosa. This is ...

Gene therapy research cures retinitis pigmentosa in dogs

January 23, 2012
Members of a University of Pennsylvania research team have shown that they can prevent, or even reverse, a blinding retinal disease, X-linked Retinitis Pigmentosa, or XLRP, in dogs.

New gene therapy corrects a form of inherited macular degeneration in canine model

March 5, 2018
Researchers from the University of Pennsylvania have developed a gene therapy that successfully treats a form of macular degeneration in a canine model. The work sets the stage for translating the findings into a human therapy ...

New advances in treating inherited retinal diseases highlighted in Human Gene Therapy

May 8, 2012
Gene therapy strategies to prevent and treat inherited diseases of the retina that can cause blindness have progressed rapidly. Positive results in animal models of human retinal disease continue to emerge, as reported in ...

Study stops vision loss in late-stage canine X-linked retinitis pigmentosa

October 12, 2015
Three years ago, a team from the University of Pennsylvania announced that they had cured X-linked retinitis pigmentosa, a blinding retinal disease, in dogs. Now they've shown that they can cure the canine disease over the ...

Visual pigment rhodopsin forms two-molecule complexes in vivo

July 25, 2016
The study of rhodopsin—the molecule that allows the eye to detect dim light—has a long and well-recognized history of more than 100 years. Nevertheless, there is still controversy about the structure in which the molecule ...

Recommended for you

Why some human genes are more popular with researchers than others

September 18, 2018
Historical bias is a key reason why biomedical researchers continue to study the same 10 percent of all human genes while ignoring many genes known to play roles in disease, according to a study publishing September 18 in ...

Class of neurological disorders share 3-D genome folding pattern, study finds

September 18, 2018
In a class of roughly 30 neurological disorders that includes ALS, Huntington's Disease and Fragile X Syndrome, the relevant mutant gene features sections of repeating base pair sequences known as short tandem repeats, or ...

Researchers resolve decades-old mystery about the most commonly mutated gene in cancer

September 18, 2018
The most commonly mutated gene in cancer has tantalized scientists for decades about the message of its mutations. Although mutations can occur at more than 1,100 sites within the TP53 gene, they arise with greatest frequency ...

Study of one million people leads to world's biggest advance in blood pressure genetics

September 17, 2018
Over 500 new gene regions that influence people's blood pressure have been discovered in the largest global genetic study of blood pressure to date, led by Queen Mary University of London and Imperial College London.

Genetic mutations thwart scientific efforts to fully predict our future

September 17, 2018
Ever since the decoding of the human genome in 2003, genetic research has been focused heavily on understanding genes so that they could be read like tea leaves to predict an individual's future and, perhaps, help them stave ...

Gene therapy via skin protects mice from lethal cocaine doses

September 17, 2018
There are no approved medications to treat either cocaine addiction or overdose. Frequent users tend to become less and less sensitive to the drug, leading to stronger or more frequent doses. The typical result is addiction. ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.