Hailed as the breakthrough of the year in 2002 by Science, RNAi still holds promise as a powerful, novel therapeutic for a wide variety of diseases and viruses. Its exquisite ability to selectively silence any gene taunts scientists still struggling with substantial hurdles, including clinical transition, delivery, and safety. Experts agree that delivery remains the main obstacle, which is where most companies are currently focusing their efforts.
A recent report by Research and Markets forecast RNAi therapeutics to generate sales of around $1 billion by 2015. Several companies operating within this potentially lucrative arena will be presenting their latest advances at Keystone Symposium’s “Therapeutic Modulation of RNA Using Oligonucleotides” to be held later this month in Lake Louise, Alberta, Canada.
Although only about 20 nucleotides in length, microRNAs are believed to regulate a large number of genes. In fact, approximately 700 microRNAs have been discovered in the human genome to date, regulating more than one-third of all genes. Regulus Therapeutics, created as a joint venture by Alnylam Pharmaceuticals and Isis Pharmaceuticals in 2007, has inherited almost 1,000 patents from its two parent companies to be used exclusively in microRNA applications.
“The big excitement and advantage of working with microRNA is that we can go after targets that nobody has identified before,” explains Kleanthis Xanthopoulos, Ph.D, president and CEO. MicroRNA has the ability to interfere with an entire pathway—rather than a single protein. It has been shown to be associated with certain diseases including metabolic and inflammatory diseases, cancer, and viral infections. “We think microRNAs developed as regulators over millions of years to regulate complex diseases. These are fine-tunings and may turn out to be enormously beneficial in terms of drug discovery,” he adds.
The company has two main programs under development. miR-122 is expressed in the liver and appears to be essential for the replication of hepatitis C virus. Anti-miR-122 reduces cholesterol levels in blood and reverses fatty liver in obese mice. Clinical trials are anticipated to begin within the next 12 to 18 months.
Recently published data in Nature on the role of miR-21 showed that it is over-expressed in a failing heart, contributing to this condition through regulation of a stress-response signaling pathway associated with changes in heart muscle structure and function. Targeting miR-21 with antisense oligonucleotides prevented heart failure in mice, and administration of anti-miR21 after heart failure showed significant treatment benefit in the animals. “We believe this is the first study to clearly demonstrate therapeutic efficacy for targeting microRNAs in an animal model of human disease,” states Dr. Xanthopoulos.
A Novel Delivery Vehicle
Steve Dowdy, Ph.D., investigator, Howard Hughes Medical Institute, and professor, department of cellular and molecular medicine at University of California, San Diego (UCSD) School of Medicine, and his lab at UCSD have been focused on a delivery vehicle for siRNAs called protein transduction domain (PTD)—a short peptide that can be covalently linked to a macromolecule cargo and delivered into a cell. Once in the cell, they can be separated by enzymatic cleavage and free to perform whatever function they are supposed to do.
The advantage of siRNAs is that they can target specific genetic changes present in cancer cells but not normal cells. The challenge with these molecules, however, is that they are large (about 14,000 Daltons) and have a negative charge with no bioavailability. “It’s a superb drug, but if it doesn’t get inside the cell, it doesn’t count,” says Dr. Dowdy.
His group realized that the PTD is positively charged and the siRNA is negatively charged. So, in order to neutralize this negative charge, his group coats it with a protein domain called double-stranded RNA binding domain (DRBD). They developed a fusion protein of PTD-DRBD, which works well, and in every cell tested, and provides a complete RNAi response very quickly with no cytotoxicity, Dr. Dowdy says.
“The beauty of the siRNA is that you can knock down multiple targets at the same time. So, it looks quite promising,” he adds. In addition, he believes that, in the next five years, there will be a much wider variety of delivery approaches because one delivery approach won’t solve the problem for every disease. “RNAi has great potential—more potential than any drug regimen we have come up with,” he states.