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发表于 2004-9-22 00:04
The Sound of RNA Silencing
by JR Minkel | 八月 01 '04
Drug developers have long pinned hopes on a series of technologies derived from the properties of short ribonucleic acid (RNA) polymers, or oligonucleotides. Antisense technology, one of the best known approaches, is intended to prevent the messenger RNA of an undesired gene from becoming a protein by gumming it up with a short, complementary RNA molecule. Aside from the still incompletely solved challenges of making such molecules stable and tissue-specific, antisense and other RNA technologies often have the added burden of being foreign to the cell. "You're trying to force something onto a cell that it doesn't know what to do with," says Nassim Usman, PhD, chief operating officer, Sirna Therapeutics Inc., Boulder, Colo.
The situation changed dramatically in 1998 when researchers discovered a natural and widespread system for taking out messenger RNAs, called RNA interference (RNAi). A slew of companies sprang up or, like Sirna (formerly Ribozyme Pharmaceuticals, which focused on a technology for cleaving one RNA with another, called a ribozyme), reinvented themselves to turn RNAi into a therapeutic tool. "Here comes something that's 100 to 1,000 times more potent [than previous technologies] based on RNA," says Usman. "It was a pretty easy choice" to switch to RNAi. Now Sirna is vying with those other companies (see table on page 30), hoping to leverage 12 years of experience in synthesizing and chemically modifying RNA to produce part of the first wave of RNAi therapeutics.
Taking a lead role in Sirna's scientific effort is Barry Polisky, PhD, senior vice president of research. Polisky began designing RNA-based drugs in the early 1990s with aptamers, highly structured and modified single-stranded RNA molecules that function like antibodies, which specifically latches onto proteins and inactivates them. The same fundamental issues apply to both aptamers and RNAi, he says. Both could have very broad applicability, so the basic challenge becomes how to use them intelligently and selectively.
Originally identified in Caenorhabditis elegans , RNAi starts with double-stranded RNA molecules, either transcribed, synthetic, or viral. A highly specific class of RNA nucleases (nucleic acid-digesting enzymes) called dicers chew these molecules into 21- to 25-nucleotide-long RNA molecules known as small interfering RNAs (siRNAs). The oligonucleotides join a protein-RNA complex, lose one strand, and bind messenger RNAs (mRNAs) with a complementary sequence. Binding results in degradation of the mRNA and suppression of its translation into protein. In 2001 researchers observed mammalian cells using RNAi to regulate gene expression, opening up the prospect of a natural, highly potent tool for gene silencing in humans. Unlike small molecules or other therapeutic approaches that inactivate a single target protein per molecule, RNAi has a more catalytic effect because a silenced messenger RNA would have produced numerous proteins.
The breadth of RNAi, like other potential RNA therapeutics, is both a blessing and a curse, says Polisky. Interference can target any transcribed gene whose product causes or contributes to disease through overactivity, but the technology is essentially untested as a therapeutic tool. Sirna has programs in age-related macular degeneration, hepatitis C virus, and oncology, and could expand into obesity, central nervous system (CNS), and HIV. "You need an initial proof of principle in the clinic," Polisky says. "Show the technology is an advance and can do something existing therapies can't, then expand the applications in a careful and thoughtful way. Some companies focus on a disease or a limited technology. Our job is to become focused, but with a much broader technology."
From a therapeutic standpoint, RNAi has some attractive features but is far from being an effective clinical tool, says Cy Stein, MD, PhD, Albert Einstein College of Medicine, New York. It produces good knockouts in tissue culture at low concentrations, "but there's a ways between getting that in tissue culture and [getting it] therapeutically." The main challenge to harnessing RNAi is the same as for antisense technology, he says, "It's delivery, delivery and delivery—get enough in the right cells at the right time. The delivery question is one that the antisense field has never answered effectively. For me, there's a lot of déjà vu all over again." (See antisense sidebar on p. 32.)
The lessons from antisense, Usman says, are to make sure an oligonuecleotide is potent and understand its pharmacokinetic challenges. Bulky, highly charged molecules such as RNA do not easily penetrate the oily cell membrane. The cell never evolved receptors to let them inside. There are also endo- and exonucleases that rapidly degrade RNA molecules. That degradation makes unmodified siRNAs unstable in serum, and the presence of nucleases inside the cell makes intracellular stability an obstacle to RNAi therapeutics too.
Sirna takes the view that chemistry can make siRNAs stable and enhance their selectivity for tissues and cell compartments. The company's researchers use cell culture and animal models to screen modified oligonucleotides, which may have a ligand or sugar attached, or an altered nitrogen base or sugar-phosphate backbone. Usman says numerous modifications can enhance intracellular and serum stability and keep the kidneys from eliminating the molecules from the blood, while others enhance the efficiency and potency of silencing.
One potential change is to reverse the "polarity" of the nucleotides capping the siRNA. In natural nucleic acids, each nucleotide binds the next one down the line between specific atoms in their sugar components. Flipping that bonding order on each end makes the whole molecule harder for so-called RNA exonucleases to recognize. (Exonucleases degrade the ends of nucleic acid chains.) Other chemical modifications protect olignucleotides from endonucleases, which sever them in the middle. Sirna researchers have found that the caps also make siRNAs more tissue selective in several animal models, Usman says.
Although chemistry is key to Sirna's approach, intelligent target selection is the first step. The company's target selection approach is conservative in that it pursues those that are already clinically validated, says Polisky. For the lead program, age-related macular degeneration (AMD), researchers chose to target a receptor for vascular endothelial growth factor (VEGF). In so-called wet AMD, which is responsible for most of the blindness caused by the disease, the retina overgrows with fresh, abnormal blood vessels in response to a lack of oxygen. Clinical research shows that inactivating VEGF, a promoter of blood vessel growth, helps counteract the disease. Similarly, knocking out genes the hepatitis C virus (HCV) needs for replication has a direct effect on the spread of the virus. "There's no question it will have an effect if we can knock down VEGF or HCV," says Usman.
Once a target is chosen, chemists have to select the optimal siRNA sequence. Sequences differ in potency and may overlap with and silence untargeted genes, potentially causing toxicities. Researchers can build bioinformatics algorithms to pick a sequence that is most specific to and potent against a target gene. Dharmacon Research Inc., Lafayette, Colo., which offers tools for chemical synthesis of RNA molecules, uses an algorithm to identify "hyperfunctional" siRNAs capable of silencing effectively at concentrations below one nanomolar, says William Marshall, PhD, executive vice president of research and operations and site manager at Dharmacon. One algorithm step ranks a candidate sequence based on 66 parameters, including the number of hydrogen bonds it will form with its complementary RNA, the differential between each strand's affinity for the silencing complex, and whether the siRNAs would tend to form their own structures, which would hinder their silencing ability. Another step checks every possible 21-nucleotide siRNA sequence, searching each one for matches to other genes.
A siRNA chemically modified for stability is useless if it doesn't home in on the right tissue and get into the cytoplasm where the silencing complex resides. One solution is to bypass systemic delivery altogether. For AMD, compounds can be designed for local administration, direct injection into the eye. Sirna is pursuing judicious formulation as a potential solution for hepatitis C. Polisky says Sirna chemists can modify the molecules to enhance their affinity for hepatocytes, in which the virus replicates. The company's researchers are now actively designing, assembling, and testing different formulations of oligonucleotides with lipophilic complexes, which are intended to transport them through the cell membrane and into the cytoplasm without causing toxicity. "Sequence analysis and modifications are the easy part," says Polisky. "Delivery is a formidable challenge [that's] never been adequately solved." And even if the formulations work in model systems, he says, researchers still won't know if the formulations will deliver the compound in people. "If we're successful at this, it will be a very big deal."
Some applications may not require chemically modified siRNAs. Unmodified oligonucleotides are actually preferred for a disease like AMD, says Samuel Reich, co-founder, senior director of research and development, Acuity Pharmaceuticals, Philadelphia. The eye is a much more inert space than the serum, so the natural molecules last longer. "We specifically don't want the molecule to be stable in serum" because it could be more likely to cause side effects. Acuity has selected a clinical siRNA candidate against VEGF in its AMD program and is preparing to file an IND later this year. Intradigm Corp., Rockville, Md., is also working on an unmodified siRNA candidate for cancer, ICS-283, which uses nanometer-sized, integrin ligand-coated organic particles to target new blood vessels in tumors.
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