RNAi Therapeutics: How Likely, How Soon?

/Home/Biologics Delivery - Protein Synthesis/RNA - Protein Synthesis/RNAi - Protein Synthesis

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.?