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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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