With delivery continuing to be a key hurdle for the RNAi therapeutics field, Copernicus Therapeutics remains confident that its DNA nanoparticle technology, originally developed for gene therapy applications, can be used with siRNAs.
Earlier this month, at the American Society of Gene Therapy’s annual meeting, the company presented in vitro data showing that the technology can help protect siRNAs from nuclease degradation.
Copernicus President and CEO Robert Moen told RNAi News this week that these data suggest that the work the company has done with DNA and can be successfully extended to RNAi, but he conceded that “we [still] have work to do,” including in vivo studies to build upon the cell-culture experiments.
However, as a small company that employs 14 people and has been “98 percent focused” on its lead cystic fibrosis gene-replacement therapy, Copernicus is unlikely to begin that follow-on work alone, he noted.
Studies evaluating the use of the DNA nanoparticle technology for RNAi are expected to be done with “corporate partners … who have [RNAi-related] interests” and intellectual property, Moen said.
He said that negotiations with potential collaborators are ongoing but declined to offer a timeline for when a deal might be finalized.
”We do have intellectual property for nucleic acids, including DNA and RNA,” such as IP covering composition of matter claims that cite the use of RNA as a payload for the firm’s technology, according to Mark Cooper, Copernicus co-founder and senior vice president of science and medical affairs. “But we don’t have additional IP in the area of siRNA.”
Should Copernicus begin looking at getting involved in any RNAi drug programs as opposed to simply out-licensing its technology, Moen said, “the most likely scenario” would be one in which the company does the work as part of a partnership.
’Connect the Dots’ to RNAi
Copernicus’ technology, which was initially developed at Case Western Reserve University, involves compressing and complexing “single molecules of nucleic acid, which could be DNA or siRNA, with polycations,” Cooper told RNAi News.
Having been optimized for the firm’s cystic fibrosis program, those polycations “consist of a 30-mer of lysine and N-terminal cysteine, which is covalently modified with polyethylene glycol,” he added.
In a bid to eke some value out of its delivery technology, Copernicus last year began exploring whether this technology could be used to deliver siRNAs in addition to DNA (see RNAi News, 7/13/2006).
Although Copernicus had expected to have in vivo data available to present at the ASGT meeting, time and financial constraints have limited the company’s efforts in RNAi to in vitro experiments.
However, the firm’s management is encouraged by the results of these studies.
According to a poster presented at ASGT, Copernicus researchers compared the effects of increasing RNase A levels on naked siRNAs and siRNAs protected with its nanoparticle technology.
“After incubation at 37 [degrees Celsius], polylysine was removed from [the] siRNA, [which] was analyzed by agarose gel electrophoresis,” the poster states. “Complete digestion of naked siRNA occurred at an RNase A concentration of 0.01 [microgram per microliter], although at 0.001 [microgram per microliter] and lower enzyme concentrations the siRNA was intact.”
The complexed siRNA, however, “was completely intact in the presence of up to 1 [microgram per microliter] of RNase A and required 10 [micrograms per microliter] … of the enzyme to be digested,” the poster adds, noting that this level of RNase A is 1000 times higher than the level at which the naked siRNA was degraded.
Further analysis indicated that complexed siRNAs, prepared in either water or saline, were protected against degradation for 20 hours after incubation with RNase A. The oligos, however, were degraded after 24 hours.
Additional studies will be conducted with “corporate partners … who have interests and [intellectual property in the RNAi] area.”
Oligos complexed with Copernicus’ nanoparticle technology bind to a cell membrane protein called nucleolin, which transports the resulting nanoparticles into the nucleus. Although nucleolin is present in most cells, it resides on the surface of only certain kinds such as ocular, neural, and pulmonary cells, which somewhat limits the scope of the delivery technology.
But in these types of tissues, “we’re getting gene transfer efficiencies that are … very similar to what has been observed with viral vectors,” Cooper said. “So it’s easy to connect the dots and envision interesting clinical indications with RNAi technology for disorders in the lung, eye, and brain.”
Additionally, Copernicus has recently launched a program to target the liver with its nanoparticle technology, although this work is currently limited to DNA.
Copernicus collaborators at Case Western Reserve University presented mouse data at ASGT showing that the firm’s DNA nanoparticles could be delivered to the liver after intravenous administration when conjugated to the peptide ligand C105Y.
To test the uptake of targeted particles in hepatocytes in culture, the Case Western researchers exposed HuH7 cells to a compacted CMV-luciferase expression plasmid for 30 minutes and assayed luciferase activity 24 and 48 hours later, according to the poster presented at the meeting. “Compared to controls … [the] targeted DNA nanoparticles effected a 0.5-[to]-1 log increase in gene expression.”
Whole-body imaging, meanwhile, showed 1-to-2-log higher levels of expression in the livers of mice that received targeted particles compared with those that received non-targeted particles or saline only, the poster added.
After the nanoparticles were modified with an imaging agent, the researchers performed imaging analyses and found that the nanoparticles were localized in the liver, correlating with the observed gene expression.
Should Copernicus’ efforts to further develop this liver-targeting method be successful, the company sees opportunities for its delivery technology in a host of other indications.
“I’m sure that people who have an interest in hepatitis with RNAi applications would be interested if we could target the liver” with our technology, Moen said.