Knockout punch: the promise of RNAi
RNA interference is a breakthrough in the lab,
but can it be turned into useful medicine?
Deep in your DNA, a gene has gone haywire and is driving
up the production of a protein that is messing with
your body. Wouldn’t it be great to sift through
all your 20,000-something genes, find the offender,
and swat it like a fly?
Fortunately, a new technique eventually could do just
that, targeting that gene and only that gene,
knocking it out of operation and relieving your distress
with zero side effects.
That’s the audacious theory, anyway, behind the
medical application of RNA interference (RNAi).
When RNAi emerged on the research scene five years
ago from experiments at Whitehead and other labs, it
was hailed as a critical breakthrough for science. The
approach involves delivering tiny strands of RNA into
target cells. These strands interfere with the messenger
RNA molecules that control protein production and hence
gene expression, giving scientists the power to knock
out individual genes at will.
Now a vital tool for genomic exploration, RNAi also
promises to create new drugs that would target the genetic
roots of disease.
Several classes of RNAi-based drugs are at advanced
stages of development. One—a treatment for age-related
macular degeneration (AMD) of the eye—is in Phase
1 clinical trials. Other RNAi-based drugs still in pre-clinical
development target HIV, hepatitis C, Huntington disease,
and various neurodegenerative disorders.
Systemic delivery to target cells in the body
remains a huge obstacle. Researchers are focusing
on ways to bypass cell membranes and evade immune
responses that might degrade the drugs too soon.
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In the race toward the clinic, RNA is at a turning
point. While scientists generally remain upbeat about
its clinical potential, they also are still cautious
in their views. Getting the tiny strands of RNA (known
as short interfering RNAs, or siRNAs) into target cells
is no easy task.
Delivering RNAi compounds and ensuring that they last
long enough to be useful pose continuing challenges.
Some researchers doubt that RNAi will ever make it out
of the lab, pointing to the decades of largely unsuccessful
struggle to commercialize antisense DNA, another approach
to selective gene silencing.
Preparing for trial
While working at Whitehead from 1999 to 2001, Whitehead
Member David
Bartel and colleagues who include Thomas Tuschl
(now at Rockefeller University), Phillip Zamore (now
at the University of Massachusetts Medical School),
and Phillip Sharp (still at MIT) generated early insights
into RNAi mechanisms in cell biology.
The group knew the research would be important for
both fundamental and clinical research. “None
of us had the resources or ability to develop this as
a clinical tool,” Bartel says. But as pioneers
in the field, with a series of influential papers behind
them, the scientists and their business colleagues had
little trouble securing capital for a startup. Raising
$17 million, in 2002 they founded Alnylam Pharmaceuticals,
which has since become a public company.
Headquartered a few blocks away from Whitehead, Alnylam
focuses on diseases that are well understood genetically
but have proved difficult to treat effectively with
more conventional drugs. That list currently includes
age-related macular degeneration (AMD, the leading cause
of vision loss in the U.S.), respiratory syncytial virus
(RSV), spinal cord injuries, Parkinson disease, and
cystic fibrosis. Half of Alnylam’s 70-plus employees
are MD or PhD researchers.
The company’s drug process kicks off by selecting
a suitable disease, says John Maraganore, president
and chief executive.
Second, researchers design and synthesize siRNAs that
are predicted to be effective against the target. There
might be 100 or 200 of these molecular candidates, with
sequences chosen to work preferentially against the
target gene’s messenger RNA. Another practical
consideration is to choose candidate sequences that
are identical between humans, mice and any other model
organisms.
“Then we take the synthetic siRNAs and screen
them in cell-based arrays to target reductions in messenger
RNAs and proteins,” Maraganore says. “We
can test a couple of hundred siRNAs within a week or
so. It’s pretty common that we find 10 to 20%
of them that are much more potent than the others.”
Next, the researchers screen out siRNAs that might
activate the interferon reaction in cells. (RNAi molecules
that look like viruses to the cell can risk being wiped
out by interferon proteins that cells produce and release
to the bloodstream in response to viral invasions.)
Then the researchers modify siRNAs chemically to enhance
their ability to pass through the cell membrane and
withstand attack by enzymes within the cell. Now the
survivors are ready for toxicology studies in animals,
the final step before human trials.
Alnylam expects that its AMD candidate will reach human
clinical trials by the end of this year, followed by
its RSV drug. The pre-clinical development cycle is
surprisingly fast compared to that of small-molecule
or protein drug discovery, since it starts with such
carefully honed targets, Maraganore says. And while
human trials will follow the same procedures and schedules
that they do with more conventional drugs, he hopes
that success rates will be higher since RNAi follows
natural pathways.
The vision thing
Here’s a similar startup story: During the late
1990s, Michael Tolentino, an ophthalmologist at the
University of Pennsylvania, and colleague Sam Reich
found that RNAi effectively silenced genes in the mammalian
eye. The scientists proposed that RNAi could offer new
treatments for AMD, a condition that occurs when the
vascular endothelial growth factor (VEGF) protein becomes
hyperactivated. This protein contributes to the growth
of abnormal blood vessels in the eye, which leak and
produce dim central vision in millions of aging patients.
Other treatments have offered only limited success.
Reich licensed the technology from the university, and
Acuity Pharmaceuticals of Philadelphia was created in
2002.
The company’s drug development efforts have been
based largely on the initial research by Reich and Tolentino.
Reich—who serves as Acuity’s vice president
for research and development—staffed the company
with experts in basic research, manufacturing and regulatory
affairs.
Acuity president Dale Pfost says that company researchers
built on the university experiments with a series of
in vitro and in vivo experiments designed to screen
for potential drug candidates. These efforts eventually
led to the isolation of Cand5, an RNAi compound that
is now the company’s chief product.
The firm’s scientists then moved on to a series
of pre-clinical studies designed to evaluate the drug’s
absorption, distribution, metabolism and excretion in
a range of mammalian species. These data, gathered in-house
and by contract laboratories, must be submitted with
an investigational new drug application filed with the
Food and Drug Administration.
Phase 1 clinical trials with Cand5 began last fall.
Pfost predicts the drug will be approved by the FDA
and on the market by 2009.
Special deliveries
AMD is likely to be the first human illness for which
RNAi yields approved treatments, says Irena Melnikova,
a senior research analyst with Life Science Insights,
a subsidiary of IDC Research. That’s because RNAi
compounds can be injected directly into the eye, avoiding
the systemic barriers that plague effective delivery
of the compounds elsewhere in the body. Several companies
are now working their own angles on the disease.
Acuity’s closest rival, Sirna Therapeutics of
Boulder, Colorado, also began clinical trials late last
year, with an RNA compound called Sirna-027 that targets
a VEGF receptor protein rather than VEGF itself.
Compound delivery methods differ. While Cand5 is unmodified
from its natural state, Sirna-027 is chemically modified
to enhance its stability.
Another class of RNAi-based drugs now on the verge
of clinical trials operates through a different approach
often described as gene therapy––or as “expressed
RNAi” because it harnesses the cell’s own
genetic machinery to produce a gene-silencing response.
Typically, a viral vector (viral DNA modified to carry
the desired DNA) transfers instructions for making RNAi
molecules directly into the cell’s genome. The
cell then produces the molecules as part of a natural
process.
One potential treatment based on the expressed approach
targets HIV. Benitec in Mountain View, California, is
creating a drug that targets a set of three genes involved
in HIV. Among them, a “master regulator”
called TAT controls other genes in the virus.
John Rossi, who chairs Benitec’s science advisory
board, is a professor at the Beckham Research Institute
at the City of Hope cancer center in Duarte, California.
“We feel that with an RNAi cocktail that targets
three genes, we’ll be able to keep HIV in check,”
he says. Rossi, who previously worked on antisense strategies
for gene silencing, is quite keen on the potential of
RNAi. Benitec plans to take its compound into Phase
1 clinical trials this November.
Yellow lights
Some researchers suggest that opportunities with RNAi
may never rise beyond basic research. At best, these
skeptics say, RNAi screening will speed up identification
of proteins that can be better targeted with standard
drugs.
Part of this skepticism comes from disappointments
with previous attempts at selective gene control. For
more than a decade, researchers have struggled to create
successful therapies with antisense drugs that also
bind to messenger RNA. Only one such drug is in use,
treating certain eye infections in AIDS patients.
One issue is that antisense drugs tend to degrade rapidly,
so their potency is low. RNAi proponents say that RNAi-based
preparations are up to 1,000 times more active than
their antisense counterparts, indicating a vastly greater
likelihood of therapeutic success.
For RNAi, systemic delivery to target cells in the
body remains a huge obstacle. Researchers are focusing
on ways to bypass cell membranes and evade immune responses
that might degrade the drugs too soon.
Alnylam made headlines with a paper, published in Nature
last November, showing that RNAi compounds administered
by injection could silence clinically relevant genes
if they were attached to cholesterol molecules. The
work demonstrated for the first time that gene silencing
could be achieved in live animals through a systemic
route of administration.
But dose levels were extremely high and yielded only
a partial effect. The company is working to design a
compound that goes directly to the target tissue and
is more easily taken up.
Into the pharm leagues
For RNAi companies to succeed, they must get the blessing
of major pharmaceutical companies. For the time being,
says Sara Cunningham, Benitec chief executive, the major
pharma companies are on the sidelines, waiting for safety
and efficacy data to emerge from pre-clinical and Phase
1 research.
In the meantime, RNAi researchers are running as fast
as they can.
“RNAi has enormous potential as a therapy,”
says Judy Lieberman, an RNAi researcher who teaches
pediatrics at Harvard Medical School.
“It’s hard to predict at this stage if
it’s going to be as promising as some people might
think,” sums up Lieberman. “But it’s
extremely active at very low concentrations with a high
degree of specificity. I think it can be used with almost
any gene, so the disease opportunities are pretty unlimited.
I’m very optimistic.”
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