Future File is a Retinal Physician feature designed to highlight new and innovative early-stage and preclinical concepts that could one day help to advance the everyday practice of retina specialists.

New Gene Therapy Approach Restores Visual Function

■ A breakthrough study, led by researchers from the University of California, Irvine, results in the restoration of retinal and visual functions of mice models with inherited retinal disease. Published in Nature Biomedical Engineering, the study illustrates the use of a new generation of CRISPR technology and lays the foundation for the development of a new therapeutic modality for a wide range of inherited ocular diseases caused by different gene mutations.

“In this proof-of-concept study, we provide evidence of the clinical potential of base editors for the correction of mutations causing inherited retinal diseases and for restoring visual function,” said Krzysztof Palczewski, PhD, the Irving H. Leopold chair in the Gavin Herbert Eye Institute, Department of Ophthalmology at the UCI School of Medicine. “Our results demonstrate the most successful rescue of blindness to date using genome editing.”

“As an alternative to gene augmentation therapy, we applied a new generation of CRISPR technology, referred to as ‘base editing,’ as a treatment for inherited retinal diseases,” said first author Susie Suh, assistant specialist in the UCI School of Medicine Department of Ophthalmology.

“We overcame some of the barriers to the CRISPR-Cas9 system, such as unpredictable off-target mutations and low editing efficiency, by utilizing cytosine and adenine base editors (CBE and ABE),” said co-first author Elliot Choi, also an assistant specialist in the UCI Department of Ophthalmology. “Use of these editors enabled us to correct point mutations in a precise and predictable manner while minimizing unintended mutations that could potentially cause undesirable side effects.”

Converting Skin Cells to Retina-like Cells

■ Researchers have discovered a way to directly reprogram skin cells into light-sensing rod photoreceptors used for vision. The lab-made rods enabled blind mice to detect light after the cells were transplanted into the animals’ eyes. The work, funded by the National Eye Institute (NEI), was published in Nature. The NEI is part of the National Institutes of Health.

Until now, researchers have replaced dying photoreceptors in animal models by creating stem cells from skin or blood cells, programming those stem cells to become photoreceptors, which are then transplanted into the back of the eye. In the new study, scientists show that it is possible to skip the stem-cell intermediary step and directly reprogram skins cells into photoreceptors for transplantation into the retina.

“This is the first study to show that direct, chemical reprogramming can produce retinal-like cells, which gives us a new and faster strategy for developing therapies for age-related macular degeneration and other retinal disorders caused by the loss of photoreceptors,” said Anand Swaroop, PhD, senior investigator in the NEI Neurobiology, Neurodegeneration, and Repair Laboratory, which characterized the reprogrammed rod photoreceptor cells by gene expression analysis.

“Of immediate benefit will be the ability to quickly develop disease models so we can study mechanisms of disease. The new strategy will also help us design better cell replacement approaches,” he said.

Stem Cells May Rescue Dying Retina Tissue

INTERNATIONAL NEWS – Dying retinal cells send out a rescue signal to recruit stem cells and repair eye damage, according to the findings of a new study published in the journal Molecular Therapy. The findings open the door to restoring eyesight by modifying stem cells to follow the signal and transplanting them into the eye.

Martina Pesaresi, PhD, together with a group led by Pia Cosma at the Centre for Genomic Regulation in Italy identified 2 cell signals, known as Ccr5 and Cxcr6, using different models of retinal degeneration in humans and mice. They then genetically engineered the stem cells with an overabundance of Ccr5 and Cxcr6 cell receptors. When these modified stem cells were transplanted back into the models, they displayed a significantly higher rate of migration to degenerating retinal tissue, rescuing them from death and preserving their function.

“One of the main hurdles in using stem cells to treat damaged eyesight is low cell migration and integration in the retina,” says Pia Cosma, senior author of the study. “After the cells are transplanted they need to reach the retina and integrate through its layers. Here we have found a way to enhance this process using stem cells commonly found in the bone marrow, but in principle can be used with any transplanted cells.” RP

Protein Protects Against Diabetic Retinopathy

Researchers at Joslin Diabetes Center of Harvard University have shown that a protein found in the eye can protect against and potentially treat diabetic eye disease. At high enough levels, retinol binding protein 3 (RBP3) prevents the development of diabetic retinopathy. If introduced early enough in the development of the disease, RBP3 was shown to reverse the effects of the complication in rodent models of diabetes. These results are reported in Science Translational Medicine.

“The level of RBP3 in the eye’s vitreous and retina are higher in people who don't progress to diabetic eye disease than in those who do,” says George L. King, MD, chief scientific officer at Joslin Diabetes Center and senior author on the paper. “We saw that if you overexpress RBP3 by molecular methods [in animal models], you can prevent the onset of diabetic eye disease. And when we injected RBP3 itself into the vitreous of diabetic rats, we reversed some of the early changes of diabetic eye disease.”

A rare subset of people who have had insulin-dependent diabetes for more than 50 years have avoided complications. For 15 years, Joslin researchers have tracked these individuals as part of the Medalist Study. They noted that 35% of patients avoided retinopathy, even when they had elevated glucose levels.

Dr. King and his team deduced that something endogenous neutralizes the toxic effects of high glucose levels in these patients. This new study aimed to build on this observation and to determine which molecules could be responsible for the protection of the eye. They took biosamples from the eyes of Medalist Study patients — both from living patients during surgery and from people who had donated their eyes postmortem. They then characterized the many proteins that were present, to determine if any proteins were elevated more in the protected eyes than in eyes of people who developed retinopathy.

They recognized that RBP3, a protein only made in the retina/eye, was elevated. To determine if this was indeed the protective factor for which they were looking, they constructed experiments to compare normal vs increased expression of RBP3 in mouse models. Mice with increased RBP3 expression were protected from developing diabetic retinopathy.

Next, the researchers injected pure RBP3 into the vitreous of the eyes of mice in the early stages of retinopathy. The infusion of RBP3 reversed the damages done by early eye disease. They also discovered that diabetes seems to reduce the expression of RBP3 in eye in many subjects, which could explain why its protective effects are limited to only some patients.

“If we could find out what's causing the decrease of RBP3 in the retina in the first place, we could design some kind of treatment to maintain its production, allowing all diabetic patients to have an endogenous protection against eye disease,” says Dr. King.

NEI Uses Patients’ Own Retinal Cells for Leber Congenital Amaurosis Therapy

Scientists at the National Eye Institute (NEI) have developed a promising gene therapy strategy for a form of Leber congenital amaurosis (LCA), the disease caused by autosomal-dominant mutations in the CRX gene, which are challenging to treat with gene therapy. The scientists tested their approach using lab-made retinal tissues built from patient cells, called retinal organoids. This approach, which involved adding copies of the normal gene under its native control mechanism, partially restored CRX function. The study report appears in Stem Cell Reports.

“Our treatment approach, which adds more copies of the normal gene, could potentially treat autosomal-dominant LCA caused by a variety of mutations,” said Anand Swaroop, PhD, chief of the NEI Neurobiology, Neurodegeneration and Repair Laboratory and senior author of the report.

The FDA approved Luxturna (Spark Therapeutics) in 2017 for the treatment of LCA patients with mutations in a gene called RPE65. Although hailed as a major advance in gene therapy, Luxturna is ineffective against other forms of LCA, including those caused by autosomal-dominant mutations in CRX. The CRX gene encodes a protein (also called CRX) that binds to DNA and instructs the retina’s photoreceptors to make light-sensitive pigments called opsins. Without functional CRX protein, photoreceptors lose their ability to detect light and eventually die.

Study Identifies Possible Therapeutic Approach to Diabetic Retinopathy in Type 2 Diabetes

INTERNATIONAL NEWS — A new study in The American Journal of Pathology reports on the efficacy of a possible treatment candidate that showed anti-inflammatory and neuroprotective effects on the retina and optic nerve head in early type 2 diabetic retinopathy using a diabetic mouse model.

"Inflammation causes neurodegeneration as well as microvascular abnormalities in the retina," explained lead investigator Jin A. Choi, PhD, Department of Ophthalmology and Visual Science, St. Vincent's Hospital, The Catholic University of Korea. "Diabetic retinal neurodegeneration can occur before the onset of clinical diabetic retinal microvascular abnormalities. Therefore, therapeutics for neurodegeneration may provide a novel interventional strategy in the window period between the diagnosis of type 2 diabetes and the onset of clinically manifested diabetic retinopathy."

Investigators analyzed and compared the anti-inflammatory and neuroprotective effects of the glucagon-like peptide-1 receptor agonist (GLP-1RA) lixisenatide in the retina and the optic nerve head with those of insulin in a mouse model of type 2 diabetes. They divided diabetic mice into three groups; GLP-1RA (LIX); insulin (INS) with controlled hyperglycemia based on the glucose concentration of LIX; and a control group (D-CON). Nondiabetic control mice were also characterized for comparison.

After 8 weeks of treatment, neuroinflammation caused by type 2 diabetes was much lower in GLP-1RA-treated retinas and optic nerve heads than in untreated or even insulin-treated retinas of early type 2 diabetic mice, showing that the outcomes are independent of the glucose-lowering effect of GLP-1RA.

TLR2 Implicated in Dry AMD

INTERNATIONAL NEWS — Scientists from Trinity College Dublin have discovered that the molecule TLR2, which recognizes chemical patterns associated with infection in the body, also seems to play an important role in the development of retinal degeneration.

Dr. Sarah Doyle, assistant professor of immunology at Trinity, who led the study, which has just been published in the journal Cell Reports, said, "The lack of approved therapies for AMD is mainly because the factors involved in triggering the disease are not very well understood. Understanding and identifying early molecular events that may trigger dry AMD will allow us to develop a more targeted approach to therapy. In this case, we believe that regulating the activity of TLR2 may, over time, help to prevent the progression of dry AMD."

Two biological processes involved in AMD are the uncontrolled oxidative stress that results in the formation of bleach-like chemicals in the retina, and the laying down of a protein called complement, that "tags" whatever it touches for elimination.

In this study, the scientists implicated TLR2 as a critical bridge between oxidative damage and complement-mediated retinal degeneration. TLR2, which is found on the surface of cells, is part of the immune system because it is known to sense infection through recognizing chemical danger signals found on microorganisms like bacteria and yeast.

Once TLR2 is activated by a danger signal, it triggers a signal cascade, which is a bit like a cellular assembly line, with information about the cells immediate environment passed to our genes, which then respond with an inflammatory response.

"In the case of the eye, TLR2 appears to act as a sensor of oxidative-stress, recognizing a chemical pattern that is generated during oxidation, rather than infection, and triggering a signal cascade that ends in promoting the laying down of complement," said first author on the paper, Dr. Kelly Mulfaul, from Trinity.

Regrowing Nerve Cells by Removing a Membrane

Using mouse and human cells, researchers at Wilmer Eye Institute of the Johns Hopkins University School of Medicine in Baltimore, Maryland, say they have found that removing a membrane that lines the back of the eye may improve the success rate for regrowing nerve cells damaged by blinding diseases. The findings are specifically aimed at discovering new ways to reverse vision loss caused by glaucoma and other diseases that affect the optic nerve, the information highway from the eye to the brain.

“The idea of restoring vision to someone who has lost it from optic nerve disease has been considered science fiction for decades. But in the last five years, stem cell biology has reached a point where it is now feasible,” says Thomas Johnson, MD, PhD, assistant professor of ophthalmology at the Wilmer Eye Institute.

Researchers soon found that the barrier is the internal limiting membrane, a translucent connective tissue created by the retina’s cells to separate the fluid of the eye from the retina. After using an enzyme to loosen the connective fibers of the internal limiting membrane, the researchers then removed the membrane and applied the transplanted human cells to the retinas. They found that most of the transplanted retinal ganglion cells grew in a more normal pattern, integrating themselves more fully. The transplanted cells also showed signs of establishing new nerve connections to the rest of the retinal structure when compared with retinas that had intact membranes.

“These findings suggest that altering the internal limiting membrane may be a necessary step in our aim to regrow new cells in damaged retinas,” says Johnson. The research was published in the journal Stem Cell Reports.

Retinal Cells Transplanted From Cadaver Eyes

Retinal cells derived from a cadaver human eye survived when transplanted into the eyes of primate models, an advance in the development of cell therapy to treat blindness, according to a study published in Stem Cell Reports. The retinal pigment epithelium (RPE) functions as a barrier and regulator in the eye to maintain normal vision. Retinal pigment epithelium dysfunction can lead to eye disorders, including macular degeneration, and cause blindness.

To restore this population of cells, the researchers extracted retinal stem cells from donated cadaver adult eyes, which can enable donor compatibility matching and can serve as a recurring source of human RPE. The team then assessed the safety and feasibility of implanting adult retinal stem cells into non-human primates.

The study found that RPE patches transplanted under the macula, or the central part of the retina remained stable and integrated in vivo for at least 3 months without serious side effects, such as immune attack or light sensitivity. The researchers also found that the stem cell-derived RPE at least partially took over the function of the original RPE and was able to support the endogenous photoreceptor, which helps with light and water absorption, among other functions.

A Gene That Regulates the Aging of the Eye

A gene called Elongation of Very Long Chain Fatty Acids Protein 2, or ELOVL2, is an established biomarker of age. In a new paper, published online in the journal Aging Cell, researchers at University of California San Diego School of Medicine say the gene appears to play a key role in age-associated functional and anatomical aging in vivo in mouse retinas, a finding that has direct relevance to age-related eye diseases.

The research team found that an age-related decrease in ELOVL2 gene expression was associated with increased DNA methylation of its promoter. Methylation is a simple biochemical process in which groups of carbon and hydrogen atoms are transferred from one substance to another. In the case of DNA, methylation of regulatory regions negatively impacts expression of the gene. When researchers reversed hypermethylation in vivo, they boosted ELOVL2 expression and rescued age-related decline in visual function in mice.

“These findings indicate that ELOVL2 actively regulates aging in mouse retina, provides a molecular link between polyunsaturated fatty acids elongation and visual functions, and suggests novel therapeutic strategies for treatment of age-related eye diseases,” wrote the authors. ELOVL2 is involved in production of long-chain omega-3 and omega-6 polyunsaturated fatty acids, used in several crucial biological functions, such as energy production, inflammation response, and maintenance of cell membrane integrity. The gene is found in humans as well as mice.

NASA Awards Funding for Artificial Retina

LambdaVision, along with implementation partner, Space Tango, has been selected by NASA for an award of $5 million. This new funding will support LambdaVision’s development of the first protein-based artificial retina to restore meaningful vision for patients who are blind or have lost significant sight due to advanced retinitis pigmentosa (RP), with follow-on applications in AMD.

As part of this award, the company, together with Space Tango, will explore the benefits of microgravity for producing LambdaVision’s artificial retina on the International Space Station US National Laboratory located in low-earth orbit. The contract will cover a series of flights to the International Space Station over 3 years to evaluate and improve on-orbit production processes, and to produce artificial retinas that will then be evaluated on earth for the potential to restore vision to patients suffering from retinal degenerative diseases.

Mice Ward Off Retinal Disease Through Exercise

Exercise can slow or prevent the development of macular degeneration and may benefit other common causes of vision loss, such as glaucoma and diabetic retinopathy, new research suggests. The new study from the University of Virginia (UVA) School of Medicine found that exercise reduced the harmful overgrowth of blood vessels in the eyes of lab mice that exercised by up to 45%. This tangle of blood vessels is a key contributor to macular degeneration and several other eye diseases. The study represents the first experimental evidence showing that exercise can reduce the severity of macular degeneration.

"There has long been a question about whether maintaining a healthy lifestyle can delay or prevent the development of macular degeneration. The way that question has historically been answered has been by taking surveys of people, asking them what they are eating and how much exercise they are performing," said researcher Bradley Gelfand, PhD, of UVA's Center for Advanced Vision Science. "That is basically the most sophisticated study that has been done. The problem with that is that people are notoriously bad self-reporters ... and that can lead to conclusions that may or not be true. This [study] offers hard evidence from the lab for very first time."

Enticingly, the research found that the bar for receiving the benefits from exercise was relatively low — more exercise didn't mean more benefit. "Mice are kind of like people in that they will do a spectrum of exercise. As long as they had a wheel and ran on it, there was a benefit," Gelfand said. "The benefit that they obtained is saturated at low levels of exercise.

New Protein Restores Sight to Blind Mice

A newly developed light-sensing protein called the MCO1 opsin restores vision in blind mice when attached to retina bipolar cells using gene therapy. The National Eye Institute provided a Small Business Innovation Research grant to Nanoscope, LLC for development of MCO1. The company is planning a US clinical trial.

Nanoscope's findings, reported in Nature Gene Therapy, show that totally blind mice regain significant retinal function and vision after treatment. Studies described in the report showed that treated mice were significantly faster in standardized visual tests, such as navigating mazes and detecting changes in motion. Opsins are proteins that signal other cells as part of a cascade of signals essential to visual perception. In a normal eye, opsins are expressed by the rod and cone photoreceptors in the retina. When activated by light, the photoreceptors pulse and send a signal through other retinal neurons, the optic nerve, and on to neurons in the brain.