RNA interference (RNAi) has been called “one of the most has exciting discoveries in
biology in the last couple decades,” and since it was first recognized by Andrew Fire et
al. in 1998, it has quickly become one of the most powerful and indispensable tools in the
molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can
selectively silence essentially any gene in the genome. It is an ancient mechanism of gene
regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a
central role in controlling gene expression in all eukaryotes. In the lab, RNAi is
routinely used to reveal the genetic secrets of development, intracellular signaling,
cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by
the journal
Science as the “Breakthrough of the Year” in 2002 break out of the lab
and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several
biotech companies have bet their futures on it, but not everyone is sanguine about the
future of RNAi therapy.
At the heart of its promise as a powerful therapeutic drug lies the exquisite
selectivity of RNAi: like the fabled “magic bullet,” an RNAi sequence seeks out and
destroys its target without affecting other genes. The clinical applications appear
endless: any gene whose expression contributes to disease is a potential target, from viral
genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes,
and more.
But for all its promise, RNAi therapy is a long way from entering the clinic. While it
is a proven wunderkind in the lab, to date no tests have been done in humans, and only the
most modest and circumscribed successes have been demonstrated in animals. The road to
clinical success is littered with great ideas that have come a cropper along the way,
including two other RNA-based therapies, antisense and ribozymes, both of which showed
promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also
likely to fall short? Or is it different enough to make this third try the charm?
Clinical Naïveté, Mysterious Mechanisms
To be a successful drug, a molecule must overcome a long set of hurdles. First, it must
be able to be manufactured at reasonable cost and administered safely and conveniently.
Then, and even more importantly, it must be stable enough to reach its target cells before
it is degraded or excreted; it must get into those cells, link up with its intracellular
target, and exert its effect; and it must exert enough of an effect to improve the health
of the person taking it. And, finally, it must do all this without causing significant
toxic effects in either target or nontarget tissues. No matter how good a compound looks in
the lab, if it fails to clear any one of these hurdles, it is useless as a drug.
For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery,
stability, and potency have been the most significant obstacles. “There was a lot of
clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman,
Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado.
“Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup
Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions,
including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it
to cleave specific target RNA molecules (see Figure 1). “The initial results with hepatitis
C were not that inspiring,” says Usman, because the molecule they used had low potency and
a short half-life once in the body. Despite “enormous doses,” the viral load was not
significantly affected. “It just didn't have the characteristics to be a drug,” he says. No
ribozyme has yet been approved for use by the United States Food and Drug Administration
(FDA).
Similarly, despite much initial enthusiasm, attempts to develop antisense drugs have
been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a
target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents
translation of the mRNA (see Figure 2). At least that was the theory, and early clinical
results seemed to support the theory: antisense drugs effectively reduced tumor sizes in
anticancer trials and viral loads in antiviral trials. But closer inspection revealed these
results were largely due to an increase in production of interferons by the immune system
in response to high doses of the foreign RNA, rather than to specific silencing of any
target genes. (The relatively high proportion of C–G sequences in antisense mimics
bacterial and viral genes, thus triggering the immune response.) When the antisense dose
was lowered to prevent the interferon response, the clinical benefit largely disappeared as
well. Thus, rather than being a highly specific therapy, antisense seemed to be a general
immune system booster.
But as long as patients were getting better, does it matter what the mechanism was? “It
doesn't matter if you are a patient, but it does matter if you are trying to develop the
next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia
University College of Physicians and Surgeons in New York City. Stein has researched
antisense for more than a decade. “If you get the mechanism wrong, you're not going to be
able to do it.”
To date, only one antisense drug has received FDA approval. Vitravene, from Isis
Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the
eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral
DNA, though, says Usman, “it's unclear whether it actually works by an antisense
mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense
drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed
in high levels in cancer cells, which is thought to protect them from standard
chemotherapy. The FDA is currently reviewing an application for Genasense, based on
promising results in the treatment of malignant melanoma.
RNAi: A Natural Alternative
Growing disillusionment with antisense and ribozymes coincided with the discovery of
RNAi and the realization that it was a far more potent way to silence gene expression. RNAi
uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a
cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called
short interfering RNAs (siRNAs) (see Figure 3). siRNAs become bound to the RNA-induced
silencing complex (RISC), which then also binds any matching mRNA sequence. Once this
occurs, the mRNA is degraded, effectively silencing the gene from which it came. (Details
of the structure and function of the RISC are still largely unknown, but it is thought to
act as a true enzyme complex, requiring only one or several siRNA molecules to degrade many
times that number of matching mRNAs.)
The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few
dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics
(determining what a gene product does and with what other products it interacts) and for
drug target discovery and validation. By “knocking down” a gene with RNAi and determining
how a cell responds, a researcher can, in the course of only a few days, develop
significant insight into the function of the gene and determine whether reducing its
expression is likely to be therapeutically useful. But does RNAi have a better chance to
succeed as a drug than antisense or ribozymes?
“The fundamental difference favoring RNAi is that we're harnessing an endogenous,
natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam
Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company
developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is
the main reason RNAi is orders of magnitude more potent than either of the other two types
of RNA strategies.
Delivery, Delivery, Delivery
More potent in the test tube, at least. But stability and delivery are also the major
obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical
nature of RNA itself, as well as the body's defenses against infection with foreign
nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The
problem is that a charged oligonucleotide will not pass through a lipid layer,” which it
must do in order to enter a cell. John Rossi, Director of the Department of Molecular
Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based
therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which
makes evolutionary sense, since extracellular RNA usually signifies a viral infection.
Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or
degraded by enzymes.
To solve the problem of cell penetration, most researchers have either complexed the RNA
with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa
thinks the complexing approach is unlikely to be a simple solution, since drug approval
would require independent testing of the lipid delivery system as well. Instead, Alnylam is
pursuing backbone modification. “Some minimal modification is going to be necessary” to
increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What
we have learned from the antisense field is that even without other delivery strategies,
when you administer RNA at sufficient doses, if it's stable, it gets taken up by
cells.”
“Anything that can be done to increase half-life in circulation would improve delivery,”
says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate
Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may
not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of
RNAi to silence expression of the Fas gene in mice, protecting them from fulminant
hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of
cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection
into the tail. The RNA got to the liver, silenced Fas, and protected the mice from
hepatitis. However, a significant fraction of animals died of heart failure, brought on
because the injection volume was about 20% of the mouse blood volume. Such a delivery
scheme simply will not work in humans. “Delivery to the cell is still an obstacle,”
Lieberman explains. “Unless you really focus on how to solve that problem, you're not going
to get very far.”
Unanswered Questions
Even assuming delivery problems can be solved, other questions remain, including that of
whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer
is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi
isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he
says. “That's where you get toxicity.”
But if small RNAs can be delivered to target cells efficiently and without significant
toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If
you can get it there, and if it's in one piece, there no doubt in our minds that it will
work,” he says. To date, numerous experiments in animal models suggest RNAi can
downregulate a variety of target genes effectively. However, there are still two unanswered
questions about whether that will translate into effective therapy.
The first is whether RNAi's exquisite specificity is really an advantage beyond the
bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says
Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms
of action. The more specific you make it, the less robust the therapeutic activity is
likely to be.” Rossi agrees: “Overspecificity has never worked,” he says.
The second question is what effect an excess of RNA from outside the cell will have on
the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number
of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a
therapeutic effect may occupy all the available complexes. “We are usurping a natural cell
system that is there for some other purpose, for knocking out endogenous gene function,”
says Rossi. With the introduction of foreign RNA, will the system continue to perform its
normal function as well, or will it become saturated? “That's the big black box in the
field,” he says.
Looking Ahead to the Clinic
Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies
are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably
only likely to be answered in the process of therapeutic development. The first
applications are likely to be in cancer (targeting out-of-control oncogenes) or viral
infection (targeting viral genes). To avoid some of the problems of delivery, initial
trials may deliver the RNA by direct injection into the target tissue (for a tumor, for
instance) or ex vivo, treating white blood cells infected with HIV, for example.
Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the
rough road it faces. “We haven't solved all the problems, but we know how to proceed to
work through them. It's no surprise to us—we've seen this movie before.” Usman expects
Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and
to have a human clinical trial in progress in 2005. “Without a doubt, there will be an
RNAi-based drug within ten years,” Usman predicts.
Stein isn't so sure and thinks that too much is still to be learned about RNAi and the
body's reaction to it to be confident that RNA-based therapies will ultimately be
successful. “The whole field was founded on the belief it was rational, but there are huge
gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think
people are surprised at how complicated it is, but why should it be any other way? It's an
intellectual conceit to think that nature is simple.”
