Gene therapy restored vision to blind patients by strengthening visual pathways from the retina, through the brain’s white matter, into the brain’s visual cortex, says a report in Science Translational Medicine.

An historic University of Pennsylvania/Children's Hospital of Philadelphia gene therapy trial, launched in 2007, restored vision to many Leber's Congenital Amaurosis Type 2 (LCA2) patients who would normally go completely blind by their early 40s. Although reviving the retina was one key to restoring these patients' sight, the new imaging study showed the gene therapy also prompted the brain to rewire, strengthening the visual pathway from eye to brain.

As this occurred even in a patient in her 40s, it means the brain is more plastic than was known.

“For many years, most of the focus on brain properties has been on the cortex, activation in the grey matter, because that is what neuroscientists typically measure with their electrodes,” Brain Wandell, Ph.D., director of Stanford University’s Center for Cognitive and Neurobiological Imaging, told Drug Discovery & Development. “Finally, somebody has looked at white matter properties, at the critical axons, and specialized cells and glia wrapped around those axons. Finally, they have established a confluence between white matter and gene therapy.” Wandell was not involved in the new study.

National Institutes of Health Nervous System Development and Plasticity Section Chief R. Douglas Fields, Ph.D., was also uninvolved in the new study. He agrees it is critical to look at oft-overlooked white matter in human brain studies. Mice don’t cut it. Only 10 percent of the mouse brain is made of white matter, versus half of the human brain.

“This study is significant in many respects,” Fields told Drug Discovery & Development.

Senior author Jean Bennett, M.D., Ph.D., director of U Penn’s Center for Advanced Retinal and Ocular Therapeutics (CAROT), told Drug Discovery & Development: “It’s an exciting time.”

Gene therapy that works

LCA2 affects people inheriting a dysfunctional copy of the RPE65 gene from each parent. Retinas slowly erode, and patients with limited vision at birth go completely blind by mid-life. 

In 2007, Bennett's team injected the correct version of the RPE65 gene into retinas of a small group of LCA patients. Her team used an adeno-associated virus (AAV) as the vector to ferry the corrective gene into the eye. As AAV does not integrate into human DNA, it was unlikely to cause the problems seen by other gene researchers a decade ago (when integrative viral vectors landed near oncogenes, causing some cancers.)

Bennett's group encountered no such problems. Their patients' vision improved in many small, but dramatic ways. As other LCA2 patients received similar injections—and saw similar improvements—it became clear the gene therapy field was seeing one of its first successes.

Proving it

Two-plus years after the gene therapy, lead author Manzar Ashtari, Ph.D., director of U Penn’s CNS Imaging at CAROT, signed on to find out how the patients had improved.  Her team analyzed 10 LCA2 patients who had regained vision in one eye (the worse eye) due to gene therapy.   

Ashtari compared the LCA2 patients to age-matched controls with normal vision. Since the patients all initially received gene therapy in one eye, she also compared the “good” and “bad” eye.

Pioneering use of DTI in gene therapy

She did this in part by becoming the first to use diffusion tensor imaging (DTI), an advanced method of MRI that tracks movement of water molecules along axons, on gene therapy patients. Ashtari found the pathway from the treated eye in LCA2 patients was similar to that of sighted controls, while the pathway of the untreated eye was weaker.

The team believes the corrected gene did part of the work—the patient did the rest. Using the restored gene, the patients strengthen their own visual pathways by stimulating them with environmental interactions. For treated pathways were strongest in patients furthest away from the procedure.

All this, even though many patients were in their 20s, and one was 45—an age where it was thought brain plasticity in visual regions was essentially over. The study showed the brain is capable of repair at all ages, if children may show a better response.

To confirm the bolstered connectivity, Ashtari’s team used functional MRI (fMRI). There was a symmetrical brain response when controls were visually stimulated, while LCA2 patients showed far stronger responses when treated eyes were stimulated.

All patients in the Phase I trial later received the intervention in their other eyes. With the Children's Hospital, and sponsored by Spark Therapeutics, the U Penn group is carrying out a larger Phase III clinical trial, bringing the number of LCA2 patients in the study to 41. The FDA reviews the results next year. Clinical use may not be far behind.

Bennett told Drug Discovery & Development it was key that injections were made into a very specific part of the retina, the subretinal space, as that allowed the diseased cells to be targeted. The imaging showed the pathways from those cells were the very connections made.

Stimulating the left or right eye in normal sighted individuals results in a symmetrical brain response shown as yellow areas in the visual cortex located in the back of the brain. This is because each eye is connected to both sides of the brain roughly equally and the connectivity of the visual pathways is alike. (Credit: Illustrated by Elena Nikonova)

Bennett said many eye disorders are now in gene therapy trials, including Stargardts disease, Usher’s Syndrome, choroideremia, retinitis pigmentosa, and a neovascular complication of macular degeneration. A total of 100 patients have received LCA gene therapy. “Many more gene therapy clinical trials targeting retinal degenerative disease are planned.”

Ashtari told Drug Discovery & Development when she came to U Penn, she was interested in psychiatric and developmental diseases. But after meeting Bennett two years after the Phase I trial, she was intrigued. “I saw it was really lucky the individuals were only treated in one eye. What better control is there than the other eye in the same person? You still have to have controls to compare pathways in sighted and diseased individuals. But here we could also look at treated and untreated pathways in the same person.” 

Bennett added the disease is “bilaterally symmetrical, so she had good internal controls.”

Another reason the trial was attractive to both: simplicity. The gene lost in LCA controls the vitamin A cycle. A derivative of vitamin A, produced in the retinal pigmented epithelium, ensures photoreceptors respond to light. “It is a simple situation,” said Bennett. The photoreceptors can’t respond without vitamin A. “Our correction restores the biochemical circuit, and the ability to respond to light.”

The pathways had not fully degenerated pre-therapy. “There were residual pathways,” said Ashtari. It is possible they were co-opted by other parts of the brain, as blind people have enhanced other senses. “So that pathway is there. But it strengthens dramatically after gene therapy. When the eye opens, information from the environment needs to reach the brain. The axons of the neurons stimulated by information from the retina scream for oliogodendrocytes,” or cells responsible for insulation (myelination). “They scream, `Come and myelinate me!’ As in a house, to make a wire carry more electricity, you thicken insulation. The brain works the same way. When there is much information flow, the brain needs to develop existing pathways more; it needs more insulation around those axons.”

The DTI was “incredibly important,” Ashtari said. “Without it, we would not see the microstructure of the white matter fiber bundles connecting the eye to the brain."

Bennett said her patients had little vision up front. “They could see fairly well only if they were on a beach at high noon in mid-summer, they required that much light to see anything. Inside it was too dark for them to walk independently, or see in fine visual detail, or read. The patients ranged from ages 8 to 45 at time of intervention. Some could only see very, very bright light, no details. Others could see big shapes. After therapy, children could read, play sports, and ride bicycles to their friends’ houses. Adults could navigate independently, attend college....and see their kids’ faces.”

The technology will undoubtedly now be used in many gene therapy trials. “Absolutely. There was recently a new gene therapy breakthrough in the auditory cortex for hearing, I can tell you they will do this.” See Drug Discovery & Development’s story here.

Added Ashtari: “Even though we hypothesized this, and it makes sense because many animal studies have been done, it was amazing to see things fall into place, one after another. To detect remyelinated pathways after gene theray, and see exactly what was seen in animals after opening a sutured eye, was really amazing.”

And while DTI only enabled a view of existing pathways, a new method may let Ashtari see the temporal aspect of brain rebuilding. How fast does the brain start rebuilding after the intervention? “A new method of diffusion may let us see if dendritic populations change shortly after gene therapy. I believe they will, as that was seen in animals,” which are easier to see because they can be sacrificed and autopsied. “I believe we may see new dendrites. This is the exciting thing about the human brain. It was believed you were born with all your brain cells. Science shows us this is not true. Your brain regenerates.”

Said Bennett: “We get an incredible handle on plasticity using this paradigm.”

Recent NEJM study

A May New England Journal of Medicine (NEJM) study offered the news, discouraging to some, that three of 15 patients in another team’s LCA trial saw only temporary improvement. Bennett said that study “looked at light sensitivity in a particular region of the retina in three individuals who received different doses of a slightly different reagent. So it is not directly comparable to this one.”

She said her team “also are doing long-term follow-up of patients using standard clinical testing, and exploratory outcome measures. But our first set of patients had their second eyes injected after Dr. Ashtari carried out her study, so our studies are not directly comparable to the NEJM study. We should report soon on persistence data on the visual function, as well as the visual pathway fortification.”

Wonderful medical implications

Fields has done much work in the area of activity-dependent myelination. He told Drug Discovery & Development the new study is significant, first, due to “the wonderful medical implications of gene therapy in restoring some vision to the blind.  Even limited improvement in vision can be life changing.” 

The Ashtari study advances basic science knowledge on two fronts. “First, most of what we know about brain plasticity comes from animal studies. This research is extremely important, but clearly the human brain differs greatly from brains of experimental animals. The ability to rescue retinal function in LCA patients through gene therapy provides the opportunity to determine how the human brain changes structure and function in response to changes in functional input.  This process is critical not only for recovery from disease and injury. The ability of the brain to alter its structure according to sensory input is the basis of learning.  The wiring and rewiring of our brain according to our individual experience is what makes every human brain different, uniquely suited to success in the particular environment inhabited.”

Second, Fields said, studies of brain plasticity traditionally examine neurons, synapses, and dendrites in gray matter.  Human neural computation does occur via synaptic connection alterations in gray matter. “But there is increasing appreciation that, as in the internet, the nervous system depends not only on local information processing, but on how efficiently this information is transmitted through the brain’s expansive networks.  These networks comprise white matter.  White matter is made of tens of thousands of axons insulated with myelin to transmit electrical impulses rapidly.  This study finds that in humans, disuse—blindness—leads to loss of integrity of the white matter tracts carrying visual information from the retina to the brain. Restoring retinal function through gene therapy enhances that integrity.” 

He concluded researchers are limited to words like “enhances integrity,” as human MRI brain imaging does not fully unveil cellular structure change. “But from animal studies, and types of changes researchers see in these MRI studies, they strongly suspect restoring function in the retina stimulates formation of myelin in fiber pathways to the brain.  This would promote transmission of information from the retina.  This makes sense.  If gene therapy only restored photoreceptor function, but the connections to the brain had withered away, there would be no hope of restoring vision, because we ‘see’ with our brain” and our eyes, both. 

Spark Therapeutics was formed for the Bennett team gene therapy. She has waived rights to profit.