Courtesy of Antisense Therapeutics Ltd.
Antisense strategies use polymeric nucleic acids or nucleic acid analogs to bind to and silence specific messenger RNAs. The silencing can be caused either by physically blocking the translational machinery or by RNA degradation via RNAse H. cleotides for research applications.)
These days RNA interference seems to be everywhere. A bonafide hit in research labs worldwide, the sequence-specific gene-silencing approach is now making inroads in drug-development circles. But RNAi is not the first targeted nucleic acid-based approach to garner both accolades and pharmaceutical dollars.
For at least 30 years scientists and drug developers bent on a sorely needed new class of therapeutics have been studying antisense RNA. The concept is deceptively simple: Binding of an oligonucleotide complementary to a specific mRNA transcript – in other words, an antisense molecule – to that transcript either blocks translation of, or stimulates degradation of, the...
DELIVERY ISSUES
In many ways Vitravene is a natural choice to lead antisense drugs to market. For one thing, it is designed to combat a single gene. Other illnesses will surely prove more formidable. "Most applications are tough applications – like cancer," with multiple genetic lesions, says Charles M. Roth, assistant professor of chemistry and biochemical engineering at Rutgers University in New Jersey.
Additionally, in developing a therapy that is applied directly to the target tissue (in this case, the eye) Vitravene's makers largely circumvented the problem of ensuring that enough intact antisense oligonucleotides (ASOs) arrive at the target tissue. During their journey from storage vial to the targeted mRNA, ASOs face degradation from both intracellular and extracellular nucleases, as well as the problem of getting across the cell membrane. "Delivery is still definitely an issue," says Roth.
Despite its name, antisense RNAs often are not built of ribonucleotides. Gene Tools, in Philomath, Ore., builds antisense molecules for diagnostic applications from morpholino analogs. Containing six-membered, nitrogen-containing ring in place of the standard pentose sugar, morpholino sequences are highly stable, according to company literature.
Santaris Pharma, near Copenhagen, develops therapeutics based on the nucleic acid analogs: locked nucleic acids (LNA) and peptide nucleic acids. According to the company's Web site, Santaris plans to take its LNA-based anti-Bcl-2 (anticancer) candidate into clinical trials in the first quarter of 2005. (Santaris licensed its LNA technology from Exiqon, a Danish company that offers LNA oligonucleotides for research applications.)
Isis Pharmaceuticals builds its drug candidates on a DNA backbone. The company's first ASOs included modifications in which the molecule's phosphoryl oxygen was replaced by sulfur. Addition of a 2'-O-methoxyethyl group to the sulfur, in the second generation rendered the molecules more resistant to nuclease attack. These new ASOs blend the properties of both DNA and RNA. As RNA has a greater affinity for mRNA than does DNA, this enables the molecule to form a tighter bond with its target.
Mark Wedel, Isis' chief medical officer and vice president of clinical research, calls the new chemistry "extraordinarily promising." He says, "I've been here long enough to watch the evolution from first generation to second generation, which has already performed beyond our expectation in terms of effectiveness in duration of activity."
But refining the oligonucleotide backbone involves other considerations besides stability. With each modification, chemists must consider pharmacokinetics and binding affinity while also maintaining the oligonucleotide's specificity and functionality. They also must consider how to get the molecules to cross the cell membrane, which is difficult, given that ASOs are highly charged.
One approach, Jens Kurreck wrote recently, involves liposomes that are internalized by the cell via endocytosis.1 Once inside the cell, transfection reagents delivered with the ASO release the molecules from the liposome and prevent lysosomal degradation. Kurreck, a professor of chemistry and biochemistry at Freie University in Berlin, also points to other potential delivery systems, including dendrimers, gels, nanoparticles, and specialized polymers. Use of receptor-mediated endocytosis, with antibody-tethered ASOs, is another option.
ASOs can enter the cell without a delivery system. But, according to Kurreck, "the development of delivery systems that mediate efficient cellular uptake and sustained release of the drugs remains one of the major challenges in the antisense field."1
NUCLEOTIDE SCRABBLE
Once across the membrane, the hard work begins for the ASO, says Roth. "Once the oligos arrive in the cytosol, they must find the complementary target RNA in a sea of mRNAs before being degraded by nucleases." Success hinges upon finding the perfect target sequence within the target mRNA, as not all sequences will block translation or recruit RNAse H equally well.
Researchers must consider a potential target site's accessibility in light of the transcript's secondary structure and the binding locations of accessory proteins. To maximize safety and minimize toxicity, the sequence must be sufficiently specific to silence the target gene without affecting the expression of other genes.
While researchers have largely identified target sequences through a combination of educated guesswork and tedious trial-and-error, a computational option is available: in silico selection. Volker Patzel, professor of immunology at the Max Planck Institute for Infection Biology in Berlin, says basic algorithms and software are available free of charge and can be modified to suit specific needs. The market also offers software packages for purchase, as well as companies that perform the selection using their proprietary codes.
Using the mRNA sequence as a guide, the software generally predicts secondary structure, sequence accessibility, and the efficiency and strength of binding, among other factors. It discards sequences that might be shared by genes other than the targeted gene and generates a ranked list of candidate sequences.
"In silico selection is not used as frequently as it could be," says Patzel, who recently started a company called Steinbeis Transfer Center for Nucleic Acids Design, which provides target-prediction and ASO-design services. "You can't be sure to find the best sequence, but you find the targets with the highest probability of success."
STAYING THE COURSE
In light of these obstacles, several companies have abandoned their antisense efforts. Yet some are forging ahead. Recent clinical results, though largely preliminary, suggest their persistence could be paying off.
"I'm hopeful that there will be multiple antisense drugs on the market," says Frank Bennett, vice president of antisense research at Isis. Along with licensees including Eli Lilly & Co., Vancouver-based OncoGenex, and Australia-based Antisense Therapeutics, Isis has several ASOs in clinical trials. In August 2004 the company announced promising results from a Phase I trial using an ASO to silence expression of apoB-100, which plays a role in elevating cholesterol levels. The drug caused significant decreases in low-density lipoproteins, very-low-density lipoproteins, and total cholesterol levels, as well as lowered apoB-100 levels.2
Also conducting clinical trials is Genta of Berkeley Heights, NJ, a pharmaceutical company developing both RNAi and antisense therapies. At the American Society of Hematology meeting last December, Genta announced results from a Phase III trial of its antisense drug Genasense, which targets Bcl-2, in patients with advanced chronic lymphocytic leukemia. According to a company press release the drug "significantly increased the proportion of patients who achieved a major response, which was the primary end-point of the trial."3 The news came just one week after the company announced disappointing data from a Phase III trial of Genasense in patients with multiple myeloma.4
The news for Isis has likewise been mixed. The company announced December 2 that in two Phase III trials of patients with Crohn disease, alicaforsen, an antisense molecule against ICAM-1, "did not demonstrate statistically significant induction of clinical remissions compared to placebo."5 But the company intends to stick with the drug for ulcerative colitis, based on positive Phase II studies.
"What has largely been viewed [in the pharmaceutical industry] as a disappointment for antisense therapeutics is something that's going to turn around," says Scott Cormack, CEO of OncoGenex, whose ASO candidate to target the expression of clusterin, a protein that inhibits cells from responding to chemotherapy, recently completed a Phase I clinical trial.
Cormack compares the progress of antisense technology to that of other highly hyped technologies. After the excitement and fanfare fade, "the realization of implementing and making it a commercial reality" sets in, he says. "If you asked seven years ago if monoclonal antibodies would ever be a success, people would say that it would be a failure. It didn't deliver on being the [touted] magic bullet, but then there was an inflection point in the technology to make monoclonal antibodies a major opportunity for novel medicines."
ALTERNATE AVENUES
Silencing gene expression posttranscription is not the exclusive domain of ASOs and RNAi. Others are developing drugs based on catalytic RNAs called ribozymes. Like RNAi, these sequences target and destroy specific mRNAs. But where RNAi relies on a protein-RNA complex to do its work, the ribozyme cleaves the RNA itself.
Facing the same kind of difficulties as ASOs, ribozyme therapeutics have made progress toward the clinic. Boulder, Colo.-based Sirna Therapeutics (formerly Ribozyme Pharmaceuticals) has tested some of its ribozyme candidates in human clinical trials, including Angiozyme, an anticancer drug that targets vascular endothelial growth factor, and Herzyme, which targets human epidermal growth factor 2 in patients with breast cancer. Neither trial has released results yet. Johnson & Johnson is currently recruiting patients for a Phase II trial of its anti-HIV ribozyme OZ1.
Another strategy involves microRNAs, which operate in a fashion akin to RNAi. In October Isis Pharmaceuticals and RNAi therapeutics company Alnylam Pharmaceuticals of Cambridge, Mass., announced they had licensed key patents from the Max Planck Society to develop therapeutic applications of microRNAs.
Whether microRNAs or any other transcript-targeting strategy will ultimately prove successful in the clinic is uncertain. What is certain, however, is that all this R&D will surely advance future development efforts, by teaching researchers how to stabilize and deliver their drugs more effectively. Says Raymond P. Warrell, Jr, chairman and CEO at Genta: with "the richness of the antisense experience," the development timeline for RNAi will be "markedly accelerated."
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