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.”