Lab Opens New Front in War on Common Retinal Diseases

One of the cruelest blows of aging, for many, is the loss of sight caused by abnormal blood vessel growth at the back of the eyes in conditions such as age-related macular degeneration and diabetic retinopathy.

The good news is that scientists—prominent among them Scripps Research Institute Professor Martin Friedlander—are developing powerful new therapies to prevent this kind of vision loss. Friedlander, who is also an ophthalmologist/retina specialist at Scripps Clinic, manages several major projects aimed at delivering therapies for diabetic retinopathy, macular degeneration, and other retinal vascular diseases, as well as neurodegenerative disorders. The latest project is backed by a new, five-year, $10.2 million grant from the National Eye Institute (NEI) at the National Institutes of Health.

“We’re very excited about the most recent direction our research has taken with microRNAs and induced pluripotent stem cells and continue to be extremely pleased by the strong support we receive from the National Eye Institute and other funding sources,” Friedlander says. “Even with the advent of new therapies over the past few years, there is still a very large, unmet medical need in the area of retinal vasculo- and neurodegenerative diseases.”

Ordinarily in adults, new blood vessels do not grow in the back of the eye unless there is an ongoing disease process such as age-related macular degeneration—the leading cause of vision loss in the elderly, currently affecting 15 to 20 million Americans. In age-related macular degeneration, abnormal material accumulates under the retina. This interferes with oxygen delivery from the underlying vascular plexus to the rod and cone photoreceptors, causing abnormal growth of new blood vessels in response to oxygen “starvation.”  This material itself can also directly stimulate the abnormal growth of new blood vessels, which can rupture and bleed or leak fluid, causing swelling of the retina.

In diabetes, a disease affecting more than 400 million individuals worldwide, abnormalities in the small retinal blood vessels lead to decreased flow of blood (causing chronic lack of oxygen) or leakage of fluid from the blood vessels (causing swelling of the retina). In both diabetes and age-related macular degeneration, these vascular changes can lead to catastrophic retinal damage and profound loss of vision.

Harnessing the Potential of Micro-RNA

The newest weapon Friedlander hopes to deploy against these conditions is a strip of RNA called an “anti-miR.” The project originated from a meeting two years ago with former Scripps Research scientist David Cheresh, a professor of pathology at the University of California (UC) San Diego School of Medicine. Cheresh and his team had found a small strand of gene-regulating RNA, a “micro-RNA” known as miR-132, that acts as an on-switch for the hyper-growth of new blood vessels (angiogenesis) in tumors. They had found that they could turn this angiogenesis switch off in both cell and mouse models by adding a solution of complementary micro-RNA, anti-miR-132, which latches onto miR-132 and blocks its biological activity.

Friedlander and Cheresh recognized that anti-miR-132 might also work against the abnormal blood vessel blooms that cause common retinal diseases. When Friedlander applied Cheresh’s anti-miR-132s to his own lab’s rodent models of pathological angiogenesis, “we observed a very dramatic effect in halting new blood vessel growth,” he says.

In adult eyes, angiogenesis usually occurs only in pathological conditions, so stopping it shouldn’t harm normal processes. In contrast, recently introduced treatments for retinal vascular diseases affect more than just angiogenesis. The leading therapies block the action of VEGF-1, a growth factor upregulated by hypoxia and inflammation associated with age-related macular degeneration and diabetic retinopathy. Blocking VEGF-1 does help stabilize abnormal blood vessels, but it also blocks VEGF-1’s nourishing effects on normal blood vessels and retinal photoreceptors (e.g. rods and cones), leaving them more vulnerable to stress and degeneration. By contrast, the biological pathway activated by miR-132 in the eye appears to be much more tightly associated with angiogenesis.

“We’re hoping that the blocking of miR-132 in the eye will have few or no significant side effects,” Friedlander says.

Using Nanoparticles to Reduce Injections

During the grant period, Friedlander and Cheresh will be working closely with UC San Diego biochemistry professor Michael J. Sailor, an expert on nanotechnology-based drug delivery systems. Standard methods of delivering drugs to the back of the eye require frequent injections into the eye, which bring risks of damage and infection.

“We have some very exciting preliminary results suggesting that we can inject nano-particles that Mike’s lab has designed, and these slowly release a steady dose of anti-miRs over several weeks to months, which could mean a lot fewer injections than with standard treatments,” Friedlander says.

Under the new grant, Friedlander and his colleagues also will explore the activities of other miRs in human retinal vascular disorders, and the effects of blocking them. “We know that several other micro-RNAs help drive abnormal blood vessel proliferation and abnormal vascular leakage,” Friedlander says. “So we may end up with a combination therapy that delivers anti-miRs against miR-132 and these other relevant micro-RNAs.”

The team expects to have one or more anti-miRs ready for clinical trials by the end of the five-year grant period. Regulus Therapeutics, a San Diego-based biotech company, and a leader in microRNA based drug design, will be a collaborator to assist with early-stage clinical development and modification of anti-miRs to increase potency or extend their duration of action.

Illuminating Basic Mechanisms and Protecting the Retina

Under another newly released, five-year $3.4 million NEI grant, Friedlander’s lab will continue its studies of the basic mechanisms underlying normal and pathological angiogenesis in the eye. These will include mouse studies in which the team will switch off a variety of genes involved in the regulation of the retina’s response to hypoxia and other forms of stress. The scientists will also be studying how various progenitor and stem cells in the eye and the bone marrow can protect the retina. A decade ago, members of Friedlander’s lab found that when they injected myeloid progenitor cells from bone marrow—stem-like cells that will eventually mature into microglia and endothelial cells—into the eyes of mice that develop ischemic and neuroretinopathy, the progenitor cells migrated specifically to areas of retinal damage. There, they interacted with vessel-associated helper cells called astrocytes and promoted the health of retinal cells and vessels.

Those findings and subsequent ones have suggested the possibility of marrow- and, more recently, cord blood-derived cell therapies to treat retinal disorders—therapies that Friedlander and his colleagues are developing under another, ongoing, $18 million NEI grant. The cells used in such therapies could be therapeutic by their own youthfulness, but they also could be modified to produce proteins or micro-RNAs that specifically protect stressed retinal cells and/or shut off angiogenesis.

“These stem-like cells provide trophic rescue activity themselves, as well as the possibility of selectively targeting therapeutic molecules to the sites of disease,” says Friedlander.

Engineering Stem Cells for the Future

With an additional $6 million grant from the stem-cell-research-boosting California Institute for Regenerative Medicine, Friedlander and his lab also are developing therapies based on a relatively new, engineered type of stem cell. This is the “induced pluripotent stem cell” (iPSC), which many researchers now see as a basic tool in the stem cell medical armamentarium of the future.

The iPSC technique begins with the harvesting of a small number of ordinary mature cells from the body, such as skin cells. These cells are briefly cultured in a dish, and then their normal patterns of gene activity are reprogrammed with a cocktail of transcription factors, so that they enter an immature state, not dissimilar to that of an embryonic cell. The iPSC technique has the advantage that, theoretically, it can generate cells capable of differentiating into virtually any type of cell in the body, unlike the bone marrow-derived cells, which have limited potential in this regard.  The iPSCs also have advantages over embryonic stem cells, which raise ethical issues and can trigger a tissue rejection response from a host’s immune system. The iPSCs can be used to generate “autologous” grafts, in that they were derived from the patient’s own tissue.

But iPSCs are not without drawbacks. One is that the reprogramming process, as originally developed several years ago, requires the use of a retrovirus that inserts itself into the target cell genome and pumps out copies of the four cell-reprogramming transcription factor proteins. If the virus inserts in the wrong place, it could cause damage that puts the cell on the path to cancer. The transcription factor proteins themselves can drive cells towards cancer if their expression gets out of control. “Working with Sheng Ding at UC San Francisco and Yang Xu at UCSD,” says Friedlander, “we’ve developed alternative ways to reprogram. One involves eliminating all but one of these transcription factors, using small molecules. The other involves the use of an episomal vector to deliver the four transcription factors. The episomal vector, unlike the retrovirus, produces these reprogramming molecules without having to integrate itself into the cell genome.”

Friedlander and his colleagues can program these potentially safer iPSCs to mature into the retinal pigmented epithelial cells that line the back of the retina. Retinal pigmented epithelial cells die off in common conditions such as “dry” macular degeneration, and currently there is no good therapy to prevent that. The scientists have found that injecting new human-iPSC-derived retinal pigmented epithelial cells into rats’ eyes can serve to replace the degenerated cells with newly generated cells that are fully functional. So far, the team has been using cells from newborns.

“The next step is to make the same quality retinal pigmented epithelial cells, starting from the skin cells of a 40- or 50-year-old adult,” says Friedlander. “Those studies are ongoing now, and if they work out, then in principle we’ll be able to think about making patient-derived RPE cells that would be safe enough to test in a clinical trial.”

Many Opportunities to Contribute

In addition to heading several major collaborative research projects over the 19 years he has been at Scripps Research, Friedlander has been active in the broader scientific community. To name only a few of these activities, he has participated in numerous NIH study sections, review panels, and committees; acted as the Scripps Research faculty liaison to the Sanford Consortium for Regenerative Medicine; currently serves on the Executive and Alcon Award Review Committees for the Alcon Research Institute; and is the director of research laboratories for the MacTel Project, a large multi-national study sponsored by the Lowy Medical Foundation.

With three major translational projects under way, a bevy of basic eye-science experiments, and activities in the scientific community, Friedlander is extremely busy, but grateful for the opportunity to contribute.

“I am very fortunate to have a great group of individuals working in our laboratory who are not only highly productive and brilliant as individuals, but work extremely well together and share a passion for the work we do and the translational aspects of our basic science,” he says. “In addition, I’m thankful for the very generous support we have received from the National Eye Institute, the California Institute for Regenerative Medicine, and the Lowy Medical Research Foundation as well as various individual donors. Without this support, none of what we have accomplished would have been possible. Only a few years ago, I had very little to offer my patients who have these retinal degenerative diseases; today there many new therapeutic options, with the prospect of many more becoming available over the next decade.”

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