In an effort to take siRNAs from the lab to the clinic, Howard Hughes Medical Institute researcher Steve Dowdy has created a way to improve the delivery of these silencing molecules into cells. His approach, which was published in May in Nature Biotechnology, showed that siRNA could be effectively delivered into three human primary cells by attaching a fusion protein to the molecule. The technique improves upon current methods, which have so far failed to make a successful therapeutic out of RNA interference.
For the past 12 years or so, Dowdy, who is also affiliated with the University of California, San Diego, has been working on ways to improve peptide and macromolecule delivery into cells using a small section of protein called a peptide transduction domain (PTD), which is ideal for passing through cell membranes.
They've made 50 of these fusion proteins — PTD to macromolecule — and guessed that the method might work for delivering siRNAs, too. "The problem is, when you attach the PTD to the siRNA, the PTD is positively charged, the siRNA is negatively charged, and they stick together like glue," Dowdy says. "So the ionic interaction neutralizes the PTD and you get no cellular delivery." To overcome this, the group made a fusion protein comprised of a PTD with a double-stranded RNA binding domain (DRBD), which binds the outside of the double-stranded siRNA. "So the DRBD binds the siRNA and essentially masks the negative charge, and that allows the PTD then to stick out in solution and productively interact with the cell membrane," Dowdy says.
To determine the ability of the fusion protein to deliver siRNA, Dowdy generated a human lung cancer reporter cell line. Using an engineered constitutively active GFP and flow cytometry to measure single cell RNAi response, they found that not only was there a stronger response compared to control methods, but that the entire cellular population was affected. They tried their method out on three primary cell lines that are typically difficult to introduce siRNAs into — T cells, human umbilical vein endothelial cells, and human embryonic stem cells — and got similar results for all the different cell types.
"This is an entirely different approach than current siRNA delivery approaches, which are predominantly nanoparticles and liposomal particles," Dowdy says. "This looks very promising. It's very early [and] we have a lot of work ahead to see how it will compare," but he notes several important differences between this method and using liposomes: the pharmokinetics are better, it's non-cytotoxic, the siRNA complex is degraded through known pathways, and, most importantly, the fusion protein targets knockdown in the entire population of cells. "And when you're talking about cancer, it's all or nothing," Dowdy says. "Getting 90 percent of the cells in cancer doesn't count. You've got to get the entire population."
Dowdy is also the scientific founder of Traversa Therapeutics, which is developing his technology for therapeutic applications against cancer. To his mind, Dowdy says, "RNA interference is the only cancer treatment on the table for personalized medicine" as these drugs could be made to evolve along with the genetic mutations in tumors.