A Field of Dreams for Vision Research

By Jason Socrates Bardi

"But from thine eyes my knowledge I derive,
And, constant stars, in them I read such art."

——William Shakespeare, from Sonnet XIV, circa 1594

"If you build it, they will come," was the oft-repeated line from the 1989 film Field of Dreams, which told the story of a farmer who had a vision to build a baseball diamond in the middle of his cornfield.

No single line more accurately describes the work of a handful of researchers at The Scripps Research Institute (TSRI) who for the last several years have been building their own diamond dream—collaborations and multidisciplinary research projects that aim to make an impact in the field of vision research and to touch lives by finding an approach to treating ocular diseases.

And now this work is beginning to attract attention. The National Eye Institute (NEI) and the rest of the vision research world is beginning to recognize TSRI, with its team of interdisciplinary researchers, as a major center for vision and eye research—research that is critically important, since vision loss does and will continue to affect the quality-of-life in our aging society.

"Virtually every American has one of the diseases or knows somebody who has one of the diseases that could be effectively treated if we develop a drug to prevent abnormal growth and function in the eye," says Martin Friedlander, associate professor in the Department of Cell Biology and chief of the Retina Service in the Division of Ophthalmology, Department of Surgery at Scripps Clinic.

Building Upon Past Successes

The latest recognition of the vision research at TSRI is a $9.6 million NEI grant, which was recently awarded to Friedlander and several other investigators at TSRI to take basic science observation as close to the clinic as possible.

The grant, titled Fragments of TrpRS to Treat Neovascular Eye Disease, also includes TSRI investigators Paul Schimmel, who is Ernest and Jean Hahn Professor of Molecular Biology and Chemistry and a member of the Skaggs Institute for Chemical Biology; Dale L. Boger, Richard and Alice Cramer Professor of Chemistry; Professor David Cheresh and Associate Professor Glen Nemerow, both of TSRI's Department of Immunology; and Gary Siuzdak, Adjunct Associate Professor of the Department of Molecular Biology.

For Friedlander, the grant is testament to the fact that TSRI is the perfect place for such a project, since it combines first-rate basic vision science with the nearby clinical resources of the Scripps Clinic. The program, which is already up and running, was favorably reviewed and funded by the NEI, and in part, this was because of the existing collaborations of the grant's investigators and the high potential this work has to translate bench work to the bedside.

For instance, Friedlander and Nemerow already participate in the NEI-funded Core Center for Vision Research at TSRI, which began operations last summer, providing shared support resources for 11 TSRI researchers and six researchers from the University of California, San Diego (UCSD) who have independent programs in vision science funded through the NEI.

The Core Grant for Vision Research supports several core facilities to provide additional resources for the independent programs of these researchers and others doing vision science research. A microarray core module produces DNA "chip" microarrays that can be used for observing changes in gene expression during the course of normal and pathological changes in the eye. Similarly, a proteomics core module provides global analysis of all the proteins expressed. A microscopy and imaging core module allows the phenotypic state of eye tissue to be studied in conjunction with the expression data that comes from the microarray and proteomics cores, taking advantage of a new state-of-the-art multiphoton scanning laser confocal microscope.

In May, a $500,000 supplement for technology enhancement was awarded to Friedlander for one of his grants from the National Eye Institute. These funds were used to purchase a new Maldi-TOF spectrometer and additional imaging equipment to further expand resources available to TSRI and UCSD vision researchers already working together through the Core Grant for Vision Research.

"We are very excited about this [new] interdisciplinary project to study a very exciting new anti-angiogenic and delivery systems that will enable us to bring it to sites of abnormal ocular angiogenesis," says Friedlander. "We are particularly grateful to the NEI for providing the resources to establish this program."

Angiogenesis and Vision Loss

The vast majority of diseases that cause catastrophic vision loss do so as a result of abnormal vessel growth in the back of the eye. In fact, the leading cause of vision loss in patients who are above the age of 65 is macular degeneration.

"Twelve to fifteen million people in this country alone have macular degeneration," says Friedlander. "And 10 to 15 percent of them will suffer acute loss of central, or 'reading,' vision."

In patients under the age of 65, the leading cause of vision loss is due to a complication of diabetes known as diabetic retinopathy. Some 16 to 18 percent of the U.S. population has diabetes. Virtually every one of those patients will eventually have a form of diabetic retinopathy after 20 years, says Friedlander, and every year 40,000 of them lose vision.

Both macular degeneration and diabetic retinopathy are characterized by angiogenesis, or the development of abnormal blood vessel growth in the eye. In the case of macular degeneration, new blood vessels grow under the retina. In diabetic retinopathy, abnormal vessels grow on top of the retina. The effect is much the same; the vessels interfere with normal structures or the transmission of light to the back of the eye, impeding vision.

There is currently no effective treatment for the vast majority of these patients. For several years, researchers have sought compounds that inhibit angiogenesis and curtail the diseases.

There are several anti-angiogenic compounds in clinical trials. But one of the more promising, says Friedlander, is TrpRS, the one the TSRI researchers will be developing with the NEI funding.

"People typically talk about 20, 30, 40 percent inhibition [of new vessel formation] for the compounds that are in clinical trials," says Friedlander. "What we have seen in our pre-clinical studies is that in 70 percent of cases, you get 100 percent inhibition."

"Our hope is that TrpRS may be something we someday use to treat patients with neovascular eye disease."

TrpRS and the Autoregulation of Angiogenesis

The original work started with Schimmel, who had been studying RNA synthetases for a number of years.

After a gene is transcribed from double-stranded DNA into a single-stranded form of RNA called messenger RNA (mRNA), a large molecule called the ribosome translates the mRNA into a protein. The ribosome recognizes another type of molecule, transfer RNA (tRNA), which brings the ribosome the amino acids from which it constructs proteins.

One of the first steps of protein synthesis involves "charging" the tRNA molecules with the amino acids, and this step is carried out by a set of molecules known as tRNA synthetases. TrpRS, for instance, charges tRNA molecules with the amino acid tryptophan. Since protein synthesis provides the raw material during angiogenesis, tRNA synthetases play a big role. And, indeed, several years ago, Schimmel showed that a full length tyrosine tRNA synthetase served as a pro-angiogenic molecule.

Noticing that another similar enzyme, the tryptophanyl tRNA synthetase "TrpRS", had similar motifs as the tyrosine enzyme, Schimmel and his laboratory reasoned that like the tyrosine tRNA synthetase, the TrpRS would promote angiogenesis. Much to his amazement, however, TrpRS not only was not a promoter of angiogenesis—it actually inhibited the process.

This was a surprising result, since one would not expect a molecule involved in protein synthesis and cell proliferation to be involved in shutting down that same proliferation.

"Then," says Schimmel, "a talented postdoctoral student in our laboratory—Kei Wakasugi—had the original idea that a fragment of human TrpRS could be active in angiogenic pathways."

Interestingly, two naturally occurring, shortened forms of the molecule proved to be even more powerful inhibitors of angiogenesis. These truncated forms are either made after one end of the full-size TrpRS is chopped off by proteolysis or they are synthesized from an "alternatively spliced" mRNA, which has been rearranged by the cell before the ribosome uses it to make a protein.

Wakasugi and others in the laboratory did many tests and established that the TrpRS fragments were, indeed, inhibitors of angiogenesis in cell culture. But Schimmel and his laboratory wanted to test the TrpRS fragments using more powerful models, such as those that Friedlander had already developed over the course of studying angiogenesis for several years. In this model system normal vessel formation in the eye resembles, in many ways, the type of angiogenesis observed in human neovascular eye disease.

"We were then fortunate to have wonderful collaborators here at The Scripps—the laboratories of Marty Friedlander and David Cheresh—who gave us the opportunity to then extend the work by testing two of the fragments in animal models that they had specifically developed for angiogenesis," says Schimmel.

In the subsequent experiments, they confirmed the earlier findings and extended them by demonstrating the TrpRS fragments were potent anti-angiogenics.

The fact that TrpRS is a naturally occurring protein may make it an even more effective treatment because it will not have the same problems of toxicity and immunogenicity that plague some other potential drugs.

"Moreover," says Friedlander, "this is something that we can teach the cell how to make." One clinical approach to treating angiogenic vision loss, he says, could be to deliver the TrpRS molecules directly into the eye through gene- and cell-based vectors.

The purpose of the grant is twofold. On the one hand, they are interested in simply understanding how TrpRS works. This includes determining what the TrpRS is binding to, elucidating the specific mechanism whereby it is inhibiting the angiogenesis, and perhaps in the process, learning more about angiogenesis and the action of other anti-angiogenics.

In nature, TrpRS could be controlling the direction and perhaps the termination of blood vessels, and organisms may have evolved to use the shortened form of TrpRS to regulate angiogenesis because the full-size protein was already at the site of proliferation.

"We're trying hard to figure out what role [the alternatively-spliced fragment] plays in nature," says Schimmel. "The key thing that we have to do now is identify its receptor."

"We still have no idea what the receptor is," says Friedlander. "That's a major focus of our current research efforts."

Helping in this effort will be Gary Suizdak, who will apply his expertise in mass spectrometry towards identifying putative receptors of TrpRS.

"This effort represents one of the true strengths of TSRI, in that individuals from very different areas of research can combine their expertise to tackle scientifically fundamental, yet medically important, problems," says Suizdak.

The other major focus of the grant is directed towards developing an effective way to deliver physiologically and pharmacologically meaningful doses of the TrpRS fragments into the back of the eye by means other than direct intraocular injection. The goal is to have some sort of alternative cell-, viral- or particle-based delivery vehicle.

One approach will involve combining the gene that encodes the TrpRS with a delivery system that Cheresh has been developing for several years and which he has already shown to be effective at delivering reporter molecules to the back of the eye in model systems.

Cheresh's delivery system is a 50- to 100-nanometer-sized particle that selectively targets the cells that form new blood vessels in angiogenesis without influencing the normal blood vessels or any other tissue.

These nanoparticles are like smart bombs that deliver their genetic payloads into endothelial cells that proliferate during angiogenesis. Unlike other, "systemic" angiogenesis blockers, which become diffused throughout the blood steam upon injection, the nanoparticle-targeting vehicle directs itself to areas of the body where the tumors exist and where local vascular cells are expanding to form new blood vessels. The nanoparticle, when combined with the TrpRS fragment genes, should home in on these cells and drop off multiple copies of the genes that will effectively block angiogenesis.

The delivery system looks like it's going to work," says Cheresh, "so we're off and running.

Another Possible Delivery Vehicle

Another, separate approach will involve using adenovirus vectors as delivery vehicles.

Nemerow plans to generate adenoviral vectors that have increased capacity to target blood vessels by using modified vectors that have increased tropism (binding) for endothelial cells via the fiber protein and by incorporating in the TrpRS gene a "promoter" sequence of DNA that has enhanced activity in these cell types and will drive its expression.

In preliminary studies, Nemerow and his colleagues have also had success delivering a reporter gene to retinal cells using modified adenovirus vectors that target photoreceptors on these cells. And they are planning to look at the efficacy of the vectors to deliver a normal gene (peripherin) to correct macular degeneration in murine models of ocular disease.

"Also," says Nemerow, "it may be that we don't actually need TrpRS fragments to be expressed exclusively in endothelial cells. Cells in the immediate vicinity of blood vessels and actively secreting it might also represent a therapeutic approach."

Adult Bone Marrow-Derived Stem Cell "Smart Bombs"

Cells that specifically target and actively participate in new blood vessel formation may be an even better way to directly deliver TrpRS fragments to sites of unwanted angiogenesis. Adult bone marrow derived stem cells that selectively target to areas of vascular injury and regeneration can do precisely this in a mouse model. In fact, when these cells are pre-loaded with a gene encoding the T2-TrpRS, they target to sites of blood vessel formation in the eye and selectively kill the new vessels. This work, recently published in Nature Medicine, will be actively pursued as part of the new NEI-sponsored program and may lead to yet another way to deliver drugs to the back of the eye.

It isn't clear which form of delivery vector will ultimately work the best in any given tissue, so the team is exploring several avenues and looking to choose the best approach to pursue further.

"The collaborative nature of this project is extremely important," says Nemerow. "Having a relatively large number of collaborators with expertise in different areas allows us to explore a wide range of options and bring our combined knowledge to bear on a complex problem."

The multiple backgrounds mean multiple approaches. Specific to the grant, in fact, is support not only for the development of TrpRS and its potential delivery systems, but for the development of TrpRS alternatives as well.

Dale Boger, a synthetic chemist, is developing a novel screen involving competition of small molecules with TrpRS for its biological target.

"We have a library of [around] 40,000 compounds that we have prepared to compete with such protein-protein interactions," says Boger. "We are confident we will find leads in our existing library that we can then optimize for potency and selectivity for this target."

"This work could not be conducted by a single group," he adds. "It is only through the coordinated efforts of several superb groups that a problem of such a magnitude could be attempted."

Obviously, the National Eye Institute agrees that such multidisciplinary approaches to treating disorders of the visual system are important, and they have awarded funding for a period of five years. And everyone hopes that by then, Friedlander and his colleagues might have a lead compound and delivery system heading to, or already in, the clinics.


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