For about five years now, pharmaceutical companies have been interested in developing therapeutics based on the RNA interference (RNAi) pathway. Such drugs take the form of small interfering RNAs (siRNA), which perform the duty of interfering with expression of targeted messenger RNAs. And although the field has grown by leaps and bounds since its inception, there are still problems.
“The only real problem with therapeutic RNAi is getting the siRNA into the cell; every other problem is minutia in comparison,” says Steven Dowdy, Ph.D., investigator at The Howard Hughes Medical Institute, University of California at San Diego School of Medicine. A major reason for the challenges in RNAi delivery is that siRNA is a large (14,000 Daltons, on average), highly negatively charged molecule that will not enter cells unless they are artificially perturbed by chemical means.
Two of the most popular methods of siRNA delivery are nanoparticle- and liposome-based systems. The liposome-based systems have, up to now, been associated with liver-targeted pharmacokinetics, a phenomenon that restricts the therapeutic potential of RNAi to common diseases of the liver such as hepatitis, hepatocellular carcinoma, and hypercholesterolemia.
At CHI’s “RNA Interference Summit” to be held this month in San Francisco, various emerging and revised mechanisms of RNAi delivery will be presented. Dr. Dowdy will be one of the speakers at this meeting. “Our approach is an entirely different one than the rest in that it looks to have a different pharmacokinetic profile, so that we can potentially go after diseases in tissues other than the liver,” he says.
Dr. Dowdy’s approach involves the development of a fusion protein that contains a peptide transduction domain (PTD), an eight or ten amino acid, arginine-rich peptide that enables siRNA to be delivered to the entire population of cells in the human body. The delivery also occurs in a noncytotoxic manner, which already gives it a leg up on liposome- and nanoparticle-based delivery systems notorious for their cytotoxicity.
Essentially, because the PTD is arginine-rich the protein can interact with the highly negatively charged siRNA; when this occurs, they neutralize each other. The system is further enhanced with the binding of the siRNA by a double-stranded RNA-binding protein (DRBP), which further masks the highly negatively charged surface of the siRNA, thus allowing the entire complex to effectively interact with the target cell’s plasma membrane.
The complex is taken up into the cell by a specialized form of endocytosis called macropinocytosis that occurs in all cells. Once inside the macropinosome, the pH decreases from 7 to 5 and the DRBP, which is pH-sensitive, releases the siRNA into the lumen of the vesicle. There, the PTD causes a breach in the vesicle membrane, thereby releasing the naked siRNA into the cytoplasm, where it can cause a knockdown of its target through the RNAi pathway.
Dr. Dowdy will also present data on the effectiveness of this platform in over 30 cell types in culture, including many cancer cell types. “RNAi is the ultimate warhead to kill cancer cells because it has the ability to adapt as the genetics of the cancer adapt,” he says. “For personalized cancer treatment, the only putative drug on the table is RNA interference. Nothing will come close to this, if you could deliver it. If you can’t deliver it, then, obviously, there is no drug.”