Physicists devise gene therapy platform for macular degeneration patients

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Millions of adults over age 50 struggle each year with vision loss caused by damage to the retina or common macular degeneration.

Physics researchers at The University of Texas at Arlington have developed a new platform that uses ultrafast near-infrared lasers to deliver gene therapy to damaged areas of the retina to enable vision restoration in patients with photo-degenerative diseases.

“Most therapies focus on slowing down or halting degeneration but cannot target already-damaged areas of the retina,” said Samarenda Mohanty, assistant professor of physics and head of UTA’s Biophysics and Physiology Group, who led the research. “Our capacity to specifically target these damaged areas cell by cell opens up a new world of possibilities for vision restoration.”

Mohanty demonstrated the effectiveness of the new method in a recent article published by the Nature journal Light: Science & Applications. In his study, Mohanty and his team compared their ultrafast near-infrared laser-based method of delivering genes with the popular non-viral chemical gene delivery system known as lipofection.

The laser-based method creates a transient sub-mircometer hole that allows the gene for light-sensitive proteins, or opsins, to permeate into the damaged retinal cell. The genes are then activated to produce the opsins, which attach to the cell membrane and convert external light into the photocurrent signals that are basis of sight.

In Mohanty’s experiments, the laser-based method gave better results than chemical gene delivery in terms of the amount of opsins produced and the number expressed on the membrane of the cell. It was also able to target cells one by one where the chemical gene delivery system cannot be that specific.

Furthermore, the laser-based method was also able to effectively deliver large packages of genes encoding a wide spectrum of colors to damaged retinal cells, which could enable broadband vision restoration in patients with photo-degenerative diseases.

With aging populations in many countries, the number of macular degeneration sufferers is expected to reach 196 million worldwide by 2020 and increase to 288 million by 2040, according to The Lancet.

Mohanty is the principal investigator for the research detailed in the article, ‘Optical delivery of multiple opsin-encoding genes to targeted expression and white-light activation.’ The research team included Kamal Dhakal and Subrata Batabyal of the UTA biophysics and physiology laboratory, Weldon Wright of NanoScope Technologies and Young-Tae Kim of UTA’s Bioengineering Department. A National Institute of Health grant supported the initiative.

Earlier this year, Mohanty and UTA Psychology Professor Perry Fuchs published a study in the journal PLOS One that showed how to inhibit pain perception in the anterior cingulate cortex region of the brain. In their optogenetic stimulation method, genes for light-sensitive proteins are delivered to neurons and then activated by a laser.

That study demonstrated that optogenetic stimulation could be more accurate and effective than current methods of delivering stimulation for pain relief. It also enabled the researchers to see how different types of pain activated neurons in the brain’s thalamus.

Alex Weiss, UTA chair of Physics, said “Dr. Mohanty’s team has applied its expertise in the use of light to develop a new technique for effectively introducing genes into living cells. The research could lead to revolutionary new therapies for the restoration of sight in cases that are currently irreparable, but also has applications for the remediation of pain. ”

Mohanty joined UTA in 2009 from the Beckman Laser Institute of the University of California, where he did post-doctorate research in in biophotonics. He earned his doctorate in physics from the Indian Institute of Science.

His recent and varied investigations have included mapping neural circuits in the brain, looking at how neuron growth can be controlled in the laboratory and new methods to pinpoint cancer treatment.

Researchers discover way to improve image sharpness for blind people with retinal implants

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Retinal implants that deliver longer pulses of electrical current may noticeably improve image sharpness for individuals who have lost their sight due to retinitis pigmentosa, according to a new study by researchers from the USC Eye Institute and USC Viterbi School of Engineering.

The research will be published in the peer-reviewed journal Science Translational Medicine online on Dec. 16, 2015.

Retinitis pigmentosa (RP) is an inherited disease of the eye that causes blindness through gradual degeneration of photoreceptors, the light-sensing cells in the retina. The disease affects about one in 4,000 people.

Retinal implants (artificial retinas) give people with RP the ability to perceive light, using a system that includes a video camera mounted on a pair of eyeglasses, a video processing unit that transforms images from the camera into wirelessly transmitted electronic signals, and an implanted array of electrodes to stimulate visual neurons.

Retinal implants have enabled blind individuals to detect motion and locate large objects. However, because the implants may unintentionally stimulate axons in the retina, patients sometimes see large oblong shapes of light that reduce the quality of their vision. In order for patients to see more clearly, the images created by the implant should be composed of focal spots of light.

Current implant technology stimulates the retina with brief pulses of electrical current roughly 0.5 millisecond (ms) in duration. The researchers found that increasing the duration of the stimulus pulses allows visualization of distinct focal spots of light.

“This is a huge step forward in helping restore sight for people with retinitis pigmentosa,” said Andrew Weitz, PhD, assistant professor of research ophthalmology. “Being able to create focused spots of light is important. Think of each light spot as a pixel in an image. By arranging many light spots into the shape of an object, we can generate sharp images of that object. For those of us who wear glasses, imagine the difference between trying to read a distant neon sign with and without your glasses on. For people with retinal implants, being able to see more clearly should have a big impact on their ability to recognize objects and navigate their environments. These improvements in vision can really boost a person’s sense of independence and confidence.”

The researchers tested various stimulus pulse durations in an animal model and validated their findings in a patient with an early version of the Argus retinal implant (Second Sight Medical Products, Inc.). The results indicated that longer pulse durations allowed the retina to be stimulated more precisely. In the animal model, all pulses 8 ms and shorter activated axons, obscuring the ability to generate a focal spot of light. Sixteen-millisecond pulses also stimulated axons but to a much lesser extent. Pulses 25 ms and longer produced no evidence of axonal stimulation, instead resulting in focal spots of light.

“Our findings further support that it is possible for patients with RP to see forms using artificial vision,” said James Weiland, PhD, professor of ophthalmology and biomedical engineering. “This makes a strong case for developing high-resolution retinal implants.”

Retinal cells work with little reserve energy; may explain vulnerability to eye diseases

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Our eyes are especially demanding when it comes to energy: Along with our brain, they require a substantial amount of power to keep them functioning and healthy. Now a new study by the National Eye Institute suggests that because of their high-energy demands, our eyes function at high efficiency and with little reserve capacity, which scientists say may explain why they become vulnerable to degenerative diseases.

Better understanding of how cells in the eye become susceptible to degenerative diseases may point to biomarkers that could be used to identify people at risk, and also to develop potential therapies, said the study’s lead author, Anand Swaroop, Ph.D., chief of NEI’s Neurobiology-Neurodegeneration and Repair Laboratory.

In degenerative diseases such as retinitis pigmentosa and age-related macular degeneration, for example, photoreceptors are among the first cells to die. These are the cells in the retina that convert light into electrical signals that are sent to the brain. To get a handle on how energy requirements may factor into photoreceptor death, Dr. Swaroop and his colleagues focused on mitochondria, power plant structures within each cell that produce the energy needed for the cell to function. They studied mitochondrial function in the photoreceptors of mice both with and without retinal degenerative diseases. Their results were published in Investigative Ophthalmology & Visual Science (IOVS).

The team analyzed samples of mouse retina using a novel approach that involved directly measuring the mitochondrial oxygen consumption rate, an indicator of how efficiently mitochondria use oxygen and energy from nutrients to make energy that can be used by the cell. The novel technique involved mounting several retinal samples onto mesh and placing them onto a microplate for analysis. Previous methods involved inserting an electrode into a suspension of retinal cells, an approach that allowed for evaluation of only one retinal sample at a time and did not capture how the cells functioned within the context of tissue.

“We show for the first time that mitochondria within photoreceptors operate at 70 to 80 percent of their maximum capacity, with very little reserve, which suggests that the cells are in a perpetual state of high metabolic stress,” Dr. Swaroop said. “It’s like when a rubber band gets stretched all the way to the point where adding just a little more force breaks it.” The findings suggest that photoreceptors would be particularly vulnerable should mitochondria encounter stress that disrupts energy production.

“Our data strongly support the use of mitochondrial oxygen consumption rate as an early biomarker of retinal disease, before the onset of overt degeneration,” he said. However, this would also require a method to assess mitochondrial function in the intact eye, without having to even touch the retina.

Methods of detecting changes to mitochondrial health may already be in the pipeline.

Raul Covian, Ph.D., a study coauthor and a staff scientist at the National Heart, Lung and Blood Institute, said that his lab has been developing techniques that help determine the structure and connectivity of mitochondria in living cells of the heart. “Although the technical details would need to be worked out to apply these methods in an intact eye, in principle we could use these experimental approaches to study mitochondrial function in intact retinas.”

Taking a different approach, Bruce Berkowitz, Ph.D., an NEI-funded researcher and professor of anatomy and cell biology, and ophthalmology at Wayne State University in Detroit, recently published a study, also in IOVS, in which he and his colleagues used a noninvasive MRI-based method to measure oxidative stress in the photoreceptors of mice with diabetic retinopathy. Oxidative stress is a harmful reaction that can occur as a byproduct of oxygen consumption in cells. “With this new approach we were able to confirm that photoreceptors are a major contributor to oxidative stress in diabetic retinopathy,” Dr. Berkowitz said.

Damage in retinal periphery closely matches loss of blood flow in people with diabetes

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Research from the Joslin Diabetes Center’s Beetham Eye Institute demonstrated earlier this year that in people with diabetic retinopathy, the presence of lesions in the periphery of their retina substantially increases the risk that the disease will progress more rapidly. A follow-up study has shown that these peripheral lesions, which are not detected by traditional eye imaging, correlate very closely with the loss of retinal blood flow called retinal “non-perfusion” caused by loss of small blood vessels or capillaries.

In research reported this week in Ophthalmology, the Joslin scientists used ultra-wide-field (UWF) imaging to examine the eyes of 37 patients with diabetes and varying levels of retinopathy ranging from no disease to very advanced disease. UWF retinal imaging can view more than 80 percent of the retina in a single image. In comparison, traditional clinical retinal imaging combines seven smaller photos to cover about a third of the retina, says Paolo Silva, M.D., staff ophthalmologist and assistant chief of telemedicine at the Beetham Eye Institute.

Areas of non-perfusion were identified by UWF retinal angiography, which detects blood flow after patients are injected with a fluorescent dye. “With the UWF angiograms, we can more accurately measure the extent of non-perfusion in the peripheral and examine how this relates to the increased risk for retinopathy progression over time,” says Silva, who is lead author on the paper.

The areas of non-perfusion matched up very closely with the peripheral lesions detected when these patients’ eyes were scanned by normal UWF imaging, he emphasizes.

“The most surprising result was how very closely associated these two areas seemed to be,” says Lloyd Paul Aiello, M.D., Ph.D., director of the Beetham Institute, professor of ophthalmology at Harvard Medical School and senior author on the paper. He says that discovery raises the possibility that clinicians might be able to use the peripheral lesions to estimate the extent and location of the non-perfusion damage and the risk of disease progression without necessarily resorting to UWF angiography in every case.

A related trial by the Diabetic Retinopathy Clinical Research Network, now underway, will follow more than 350 diabetes patients across the United States with UWF imaging for at least four years.

If this national study confirms the Joslin findings about the association of peripheral lesions with risks of disease progression, and the close match of these lesions with non-perfusion, the grading system for diabetic eye disease probably will be revised to incorporate these risk factors and imaging approaches, Aiello says.

The Joslin team is now analyzing another alternative approach to assess levels of retinal perfusion–detecting levels of oxygen in blood cells non-invasively by measuring their absorption of a certain wavelength of light.

Aiello notes that the last decade has seen many major success stories for the treatment of another form of diabetic eye disease, diabetic macular edema, with drugs that target a protein called vascular endothelial growth factor (VEGF). It’s possible that such anti-VEGF drugs might also help to treat peripheral lesions, he speculates.

In any case, better understanding of these lesions and their causes represents “another step forward in the evaluation, risk identification and the treatment of patients with retinopathy.” Aiello says. “We have so many more effective treatment options than we had even three to four years ago.”

Retinal nerve cells grown in the lab – Work could eventually lead to cell transplants for people blinded by glaucoma

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Johns Hopkins researchers have developed a method to efficiently turn human stem cells into retinal ganglion cells, the type of nerve cells located within the retina that transmit visual signals from the eye to the brain. Death and dysfunction of these cells cause vision loss in conditions like glaucoma and multiple sclerosis.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” says study leader Donald Zack, M.D., Ph.D., the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

The laboratory process, described in the journal Scientific Reports, entails genetically modifying a line of human embryonic stem cells to become fluorescent upon their differentiation to retinal ganglion cells, and then using that cell line for development of new differentiation methods and characterization of the resulting cells.

Using a genome editing laboratory tool called CRISPR-Cas9, investigators inserted a fluorescent protein gene into the stem cells’ DNA. This red fluorescent protein would be expressed only if another gene was also expressed, a gene named BRN3B (POU4F2). BRN3B is expressed by mature retinal ganglion cells, so once a cell differentiated into a retinal ganglion cell, it would appear red under a microscope.

Next, they used a technique called fluorescence-activated cell sorting to separate out the newly differentiated retinal ganglion cells from a mixture of different cells into a highly purified cell population for study. The cells showed biological and physical properties seen in retinal ganglion cells produced naturally, says Zack.

Researchers also found that adding a naturally occurring plant chemical called forskolin on the first day of the process helped improve the cells’ efficiency of becoming retinal ganglion cells. The researchers caution that forskolin, which is also widely available as a weight loss and muscle building supplement and is touted as an herbal treatment for a variety of disorders, is not scientifically proven safe or effective for treatment or prevention of blindness or any other disorder.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” says lead author Valentin Sluch, Ph.D., a former Johns Hopkins biochemistry, cellular and molecular biology student and now a postdoctoral scholar working at Novartis, a pharmaceutical company. Sluch completed this research at Johns Hopkins before transitioning to Novartis.

“I was very excited when it first worked,” Sluch says. “I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for MS, Zack is working with Peter Calabresi, M.D., professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

How the retina marches to the beat of its own drum

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Researchers at Johns Hopkins and the University of Washington report new research that sheds light on how the retina sets its own biological rhythm using a novel light-sensitive pigment, called neuropsin, found in nerve cells at the back of the eye.

“No one knew what neuropsin actually did,” says King-Wai Yau, Ph.D., a professor of neuroscience at Johns Hopkins School of Medicine. “We only knew it existed in the mammalian genome and may help set the timing of reproduction for some birds. Now we think we know what it does in mammals.” The new study, described in a report online on Sept. 21 of the journal Proceedings of the National Academy of Sciences, ushers in a more complex view of the retina, according to Zheng Jiang, Ph.D., a postdoctoral fellow and one of the authors of the paper.

Neuropsin is one of seven related “opsin” proteins in mammals. Four enable the rod and cone cells of the retina to absorb light of different wavelengths and transmit that information to the brain so that the eye can see images. Another opsin, melanopsin, also absorbs light but uses it to guide processes like pupil constriction and circadian rhythms. It is found in nerve cells that connect the retina to the body’s master clock, the suprachiasmatic nuclei of the brain. On its own, this master clock tends to run slower than 24 hours in humans, and faster than 24 hours in mice, so it needs to be constantly reset to the light/dark environment by signals from the retina.

Yau explains that nearly every tissue in the body also has a local molecular “clock” for regulating patterns of activity, but most of them cannot be reset to light on their own. Instead, all but one of these molecular clocks are synchronized by the master clock within the brain — the exception being the retina, which maintains its own rhythms while sending the master clock the signals it needs to set the light-dark activity tempo for the rest of the body.

In previous work, Yau’s collaborator, Russell van Gelder, M.D., Ph.D., a professor of ophthalmology at the University of Washington, studied the circadian rhythms of genetically tweaked mice that were missing rod and cone cells and melanopsin. As expected, the circadian rhythms of these mice continued cycling but could no longer adapt to changes in light exposure. Surprisingly, though, the activity patterns of the retinas were still responsive to light changes, suggesting that there was another pigment in play.

“The retina is the only tissue known to ignore the master clock, but it does keep itself on a schedule, so we wanted to know how,” says Wendy Yue, a graduate student in Yau’s laboratory who worked on the project.

Suspecting one of the two opsin proteins with unknown functions, the teams created two more types of mice, each one missing one of the opsin genes. The retinas of mice without opsin 3 continued to be light-sensitive but their activity patterns were less robust. However, even with intact rods, cones and melanopsin, the retinas of the mice without neuropsin lost their ability to adapt to new patterns of light and darkness.

By repeating their experiments with different wavelengths of light, the team found that neuropsin responds to UV-A and violet light. “That means that the retina uses separate light signals to set its own clock and that of the body’s master clock, the latter of which is set based on the blue/green light absorbed by melanopsin as well as blue through red light absorbed by the rods and cones,” says Yau.

Next the team used a specialized “locator gene,” called a reporter gene, to figure out where neuropsin does its work. They found that, like melanopsin, it is located in neural cells that connect the retina to the brain, though where these particular cells go within the brain remains uncertain. Mysteriously, the researchers say, they also found it in the cornea, which was not thought to contain any pigments, since its job is to let light through to the rest of the eye.

“There is a lot of work left to do,” says Xiaozhi Ren, Ph.D., a postdoctoral fellow in Yau’s laboratory. “For example, we don’t know what kind of signals — chemical or electrical — neuropsin uses to set the retina’s clock.” Yau says they also want to investigate what role opsin 3 plays.

January is Glaucoma Awareness Month!

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Posted on 18th January 2016 by Pacific ClearVision Institute in General |Retina

Glaucoma is a leading cause of irreversible blindness in the United States. Glaucoma has no noticeable symptoms in its early stages, and vision loss progresses at such a gradual rate that people affected by the condition are often unaware of it until their sight has already been compromised.

During Glaucoma Awareness Month in January, the American Academy of Ophthalmology advises the public that the best defense against developing glaucoma-related blindness is by having routine, comprehensive eye exams.

Pacific Clearvision Institute in Eugene, Cottage Grove, and Oakridge, Oregon is supporting that encouragement, and is happy to schedule appointments for those with serious eye care needs.

Our three locations can be reached at the following numbers:

Eugene: 541-343-5000
Cottage Grove: 541-942-5000
Oakridge: 541-782-5000