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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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