February 1, 2009 (Vol. 29, No. 3)

Abundance of Vehicles Are in Development to Help Translate the Technology into Therapeutics

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.

Regulus Therapeutics believes that microRNA has the ability to interfere with an entire pathway, rather than just a single protein.

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.

Using Polymers

It’s this great potential that has many companies working diligently to develop novel techniques to overcome the challenges of RNAi. Xiaodong Yang, M.D., Ph.D., vp of research and preclinical development at Intradigm, says these challenges include: excretion from the kidneys due to its small size, susceptibility to degradation by RNAses, a short half-life, difficulty entering the cell, as well as the RISC (RNA-induced silencing complex).

The company’s core delivery technology contains multiple components. One is a cationic polymer, a branched lysine and histadine polypeptide that acts like a siRNA condenser and protects the molecule from degradation. When this binds to siRNA, it forms a nano-sized complex particle, hence its name: RNAi Nanoplex.

In order to protect it from nonspecific binding and to improve pharmacokinetics, the scientists attached polyethylene glycol (PEG) to it. In addition, the Nanoplex is modular, allowing for the addition of ligands or antibody components to direct the particles toward specific tissue or cell types. “What we are trying to achieve with our technology is to address all these potential delivery hurdles,” explains Dr. Yang.

The company has libraries of synthetic polymer vectors, (PolyTran™), and can select any defined structure or composition to fit a specific application. This technology enables loading of more than 95% siRNA to the Nanoplex and the ability to load more than one siRNA to target the same genes and/or to load multiple siRNAs to target multiple genes, shutting down several disease targets. His group is currently focused on developing RNAi Nanoplex primarily for treatment of solid tumors. They are also working with other partners to explore various other tissue deliveries. “I believe our technology can have broad applications.”

Researchers at the David H. Koch Institute for Integrative Cancer Research at MIT have developed a large library of lipid-based molecules called lipidoids, which form novel nanoparticle formulations for the systemic delivery of RNAi therapeutics.

“When we first started thinking about RNAi, we looked to a long history of DNA delivery, and one of the key materials was lipids. We decided to develop a combinatorial approach to allow us to synthesize and test literally thousands of materials with broader diversity relative to the kinds of things tried with DNA,” explains Daniel Anderson, Ph.D., scientist.

Initially, the goal was to create lipid-like features. “But as we made more, they started to look less like the traditional two-tailed cationic lipids that people had used for gene therapy.” The advantage of the large library approach allows for rapid screening and for a variety of materials that work. Dr. Anderson says it also enables the discovery of unexpected solutions.

These molecules have shown successful delivery of lipidoid formulations of siRNAs in several animal models including mice, rats, and nonhuman primates. Data also demonstrated potent, specific, and durable effects on gene expression in multiple tissues, including liver, lung, and peritoneal macrophages.

The lipidoids were also successfully used for delivery of microRNA antagonists, and for the delivery of two different siRNAs simultaneously with no apparent competition—demonstrating the potential for multitargeting strategy for formulations of RNAi therapeutics, Dr. Anderson reports. Alnylam Pharmaceuticals has secured all rights to the lipidoid technology for delivery of RNAi therapeutics for all uses via an exclusive licensing agreement with MIT.

One Delivery Method Does Not Fit All

As research efforts progress, it has become clear that there is no universal vector for therapeutic RNAi applications in humans. Viral-based RNAi vectors present a potential alternative to siRNA delivery. Mark Kay, M.D., Ph.D., director, program in human gene therapy, and professor, department of pediatrics and genetics at Stanford University School of Medicine, has been developing viral gene therapy vectors for shRNA administration.

“We got interested in siRNA a few years ago to treat viral hepatitis.” The group engineered double-stranded AAV vectors (adeno-associated viruses) to express shRNAs against hepatic targets, including hepatitis B virus genes and various reporters expressed in mouse livers. Systemic delivery of a single low-dose of anti-HBV vector resulted in a five-month suppression of HBV expression and replication. The advantage of the AAV vectors for RNAi expression is the ability to use any of the viral capsids for specific tissue targeting.

“In general, the issue is that siRNAs aren’t quite as efficient, and they don’t last because the double-stranded RNA has a finite half-life and you have to redeliver. The advantage of the DNA template is that you can use gene transfer vectors rather than using a gene product to express the shRNA. So the limitations are based on the gene-transfer vector. Depending on what you’re trying to treat, theoretically, with the vectors you can give a single treatment and get expression of the shRNA. This may be advantageous in certain genetic diseases, like Huntington’s disease.”

Dr. Kay’s group is also developing another vector for tissue-specific expression of an shRNA from a liver-derived polymerase (pol) II promoter. This induces target silencing in hepatoma cells in vitro and in vivo. In addition, the liver-specific pol II shRNA expression lasted for more than a year after injection.

Dr. Kay believes that the secret of success is the platform for delivery—whether it’s siRNA or shRNA. “Our lab is interested in using these to combat viral infections, and we’ve focused on liver infections. So with something like HCV, you can use three or four different targets at once—substantially decreasing the probability of developing a resistant strain.”

Chemical Modification Pattern

Thermo Scientific began to focus on siRNA design a few years ago and developed a design algorithm to identify siRNAs for target genes that include all splice variants, while eliminating any undesirable sequences (such as SNPs), which interfere with siRNA performance. “As we better understand microRNA effects or seed-mediated silencing, it has become more important to incorporate criteria into siRNA design for specificity,” says Devin Leake, Ph.D., director of R&D, genomic technologies, Thermo Fisher Scientific.

Thermo Scientific Dharmacon Accell™ siRNA incorporates a novel chemical modification pattern on the RNA duplex, allowing for cellular uptake without requiring transfection reagents or viral vectors. It has been shown to transfect common cell types like HeLA as well as difficult cell types like immune-derived cell lines.  “Once you start exploring chemical modifications on the siRNA design, you can compromise the other two desirable attributes: performance and specificity. The challenge we overcame is effectively addressing all three to have an siRNA that retains activity, specificity, and cellular uptake,” states Dr. Leake. This required a chemical modification pattern to help neutralize the negative charge of the phosphate backbone and to protect the duplex from degradation without any type of lipid formulation. The company is seeing success in animal models through academic collaborations.

There’s no doubt that RNAi has quickly advanced from its initial discovery in worms to a key genetic research tool, and now, perhaps one of the most promising novel therapeutics with the potential to change the future of medicine. Despite the somewhat major hurdles that exist, delivery being the key one, Big Pharma is betting on it.

GlaxoSmithKline recently signed a $600 million collaboration agreement with Regulus. Alnylam has milestone deals ranging from $700 million to more than $1 billion with Novartis and Roche, respectfully. Merck bought Sirna Therapeutics for $1.1 billion. That translates into a lot of faith in RNAi, which may take years to realize.

Accell siRNA reagents deliver effective knockdown without cytotoxicity, according to Thermo Scientific.

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