Researchers from Mirus Bio this week published data on a polymer-based systemic siRNA delivery system the company hopes will prove more effective in a therapeutic setting than competing non-viral technologies, namely Protiva Biotherapeutics’ SNALPs and RXi Pharmaceuticals’ interfering nanoparticles.
According to Jim Hagstrom, Mirus’ co-founder and vice president of scientific operations, the company’s so-called dynamic polyconjugate technology uses ligands to actively target specific cells. Additionally, the polyconjugates themselves are relatively small at around 10 nanomoles.
By comparison, the lipid-based SNALP and iNOP (which RXi also calls nanotransporters) technologies are passively targeted to the liver and form particles significantly larger than the polyconjugates.
With the polyconjugates, “we are talking about a defined polymer size range that has hydrophobic characteristics and is directly conjugated to the siRNAs — it’s a very simple system in that regard,” Hagstrom told RNAi News this week.
Given the promise of the early data, which were published online in the early edition of The Proceedings of the National Academy of Sciences, Mirus is now exploring the therapeutic potential of the polyconjugates in combination with siRNAs against cancer targets, Hagstrom said.
Although this work is ongoing, he noted that the company hopes to collaborate with a large industry partner to drive development of the delivery system.
“We are actively talking to potential partners … pharmaceutical companies, predominantly … for a strategic relationship for this technology,” Hagstrom said. “One of our goals would be to make a strong relationship” with a single company.
“We’ve been in contact with a number of [potential partners] right now, and we’re looking for someone that’s looking for broad scope in multiple tissues — someone that would be looking to use a system like this to achieve RNA interference in a variety of tissue in vivo,” he said.
He declined to elaborate on the discussions or comment on whether Pfizer, which recently formed a collaboration with Mirus to optimize gene-silencing methods (see RNAi News, 1/11/2007), was interested in the polyconjugate technology.
According to the PNAS paper, Mirus’ polyconjugate technology involves linking an siRNA to a pH-sensitive endosomolytic polymer through a disulfide linkage, which prevents premature displacement of the RNAi molecule from the delivery vehicle. The siRNA/polymer conjugate is then modified with the protectant polyethylene glycol and a targeting ligand.
Since the Mirus team aimed to transfect hepatocytes with siRNAs while avoiding uptake by Kupffer cells in order to sidestep toxicity issues, they modified the polyconjugates with the hepatocyte-targeting ligand N-acetylgalactosamine.
“The resulting siRNA polyconjugate is negatively charged, soluble, and non-aggregating under physiological conditions,” the researchers wrote in PNAS.
“After simple IV injection, the siRNA polyconjugate is designed to engage [NAG receptors] on hepatocytes and be taken into the cell via endocytosis,” they stated. As the endosome matures and its pH decreases, the bond attaching the PEG and NAG groups breaks, exposing the positively charged amine groups on the polymer.
“This release results in the activation of the endosomolytic capability of [the polymer] and the release of the siRNA into the reducing environment of the cytoplasm,” the authors noted. “Once there, the siRNA cargo can be cleaved from the polymer, allowing the siRNA to engage RISC and induce RNAi.”
To test the delivery technology, the Mirus investigators began with tissue culture experiments. According to the PNAS paper, they were able to completely knock down expression of their target gene, apolipoprotein B, which is involved in cholesterol transport.
“In vitro transfection reagents … deliver things to cells growing in a dish, but none of those work effectively in vivo,” Hagstrom explained. “The reverse shouldn’t be true: once an in vivo transfection reagent is made, it should work in vitro. So one of the first things we showed was that this polyconjugate works well in an in vitro setting with cells that have a galactose receptor.”
The next step was to test the ability of the polyconjugates to deliver their RNAi payload in vivo.
In mice, the researchers used siRNA polyconjugates targeting apoB and peroxisome proliferator-activated receptor alpha, a gene that plays a role in controlling fatty acid metabolism in the liver and is only expressed in hepatocytes.
The team was able to achieve an 80 percent to 90 percent knockdown of the targets using a 2.5 mg/kg dose of the polyconjugates with no observed liver toxicity.
“Delivery of apoB siRNA caused a phenotypic effect that was manifested by lowered serum cholesterol and ApoB protein levels, and by a fatty liver,” they wrote. “A single dose of siRNA polyconjugate resulted in decreased cholesterol levels for one week to 10 days.”
“We are talking about a defined polymer size range that has hydrophobic characteristics and is directly conjugated to the siRNAs — it’s a very simple system in that regard.”
Additionally, mice treated with ppara-targeting siRNA polyconjugates displayed a “gene-appropriate phenotype, characterized by a significant increase in serum triglycerides,” the authors noted.
The phenotype, they added, was less reproducible than the apoB knockdown phenotype, possibly due to the potency of the siRNAs used in the experiments.
The Mirus team also conducted experiments in which the NAG-targeting ligand was replaced with mannose, which redirected the polyconjugate away from hepatocytes to nonparenchymal cells in the liver including sinusoidal endothelial and Kupffer cells, which possess mannose receptors.
“When you change that ligand … you completely change the tropism of the polyconjugate,” Hagstrom said, which opens the door to the possibility of using the delivery technology with cell types other than those in the liver.
Doing so, however, “is going to depend on having the appropriate ligands,” he stressed, adding that “the chemistry will need to be worked out on how to attach each individual ligand.”
Working out that chemistry is something Mirus has already begun to do as part of an effort to develop the polyconjugates as carriers of therapeutic siRNAs for cancer.
“Tumor cells … are known to have a leaky vasculature, which is ideal for getting small particles out of the bloodstream,” Hagstrom said. “So ligands for tumor cells are something of interest to us, and some active research is going on at Mirus.”
Additionally, Mirus sees promise for use of the delivery technology in vitro with difficult-to-transfect cell types such as lymphoid cells, but this is something the company plans to tackle “down the road,” he noted.
“We’re still at an early stage [with in vitro] … and we’re focusing first on the in vivo side,” he said.