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Dharmacon Preparing to Launch Reverse-Transfection Platform, Transfection Agent

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LONDON — Although its development of RNAi-based reverse-transfection technology hit a roadblock when partner Akceli went out of business earlier this year (see RNAi News, 3/19/2004), Dharmacon didn’t give up on the effort.

Its persistence paying off, the company is now preparing to launch by year-end an as-yet unnamed reverse-transfection platform that will incorporate its modified siRNAs and a newly developed transfection reagent.

“Normal transfection is [where] you’ve got a … layer of cells growing on a plate surface, and then you … put on top [liposomes formulated with an oligonucleotide] — that’s called forward transfection because the formulated cationic liposome essentially falls down on the cells, then transfection occurs,” William Marshall, executive vice president of research and operations and site manager at Dharmacon, explained to RNAi News this week at the RNAi Europe 2004 meeting here.

“With reverse transfection, we essentially set it up in the opposite way where we have formulated oligonucleotides on the plate’s surface [and place] trypsinized cells on top,” he explained. The cells “then fall down on the surface and start growing, and you get uptake of the siRNA.” Researchers can then use the plate to run the assay of their choice.

According to Marshall, the process allows a researcher to side-step certain upstream activities, shaving up to a couple of days off an siRNA experiment. “Moreover, it’s very amenable to high-throughput automated platforms,” he noted.

Dharmacon has envisioned developing this kind of technology for years, and to help it do so, signed a deal in late 2003 to work with Akceli.

The technology being developed by Akceli was created by MIT’s David Sabatini and involved culturing cells on glass slides printed with nucleic acids. The cells growing on the printed areas take up the nucleic acids creating areas of localized transfection amid non-transfected cells.

“What Akceli had done, prior to our interaction with them, was to do [reverse transfection in a microarray format] with … plasmid-expressed siRNAs in the bottom of microtiter plates,” Marshall said. “We were looking at it with them with siRNAs.”

When Akceli shut down, effectively ending the companies’ collaboration, Dharmacon turned its attention to developing the reverse-transfection platform it is now planning to introduce, wherein “you basically have one siRNA per well” compared with Akceli’s microarray design, Marshall said.

Ahead of the Game

Dharmacon isn’t alone in its efforts to bring to market reverse-transfection technology, but the company appears to be the only one to have been able to overcome key barriers.

“People have wanted to do this for some time, but the main issue to date has been dose-limited toxicity,” Marshall said. Key to Dharmacon’s overcoming this problem, he said, is a new lipid-based delivery agent called Dharmafect, which has been designed specifically for siRNAs and is expected to be commercially available mid-November.

“Previous lipids [such as lipofectamine] are really optimized for delivering plasmids,” Marshall said. However, “the biophysical properties of a lipid [designed] to deliver siRNAs are different. When you adjust the … properties of the lipid to match the siRNA, you can get good uptake and minimize the toxicity.”

Other issues that have been stymieing the development reverse-transfection platforms, Marshall said, include finding the right cellular density and siRNA-to-lipid ratios. “Our goal was to find agents that could work across multiple cell densities and then have expanded siRNA-to-lipid rations, so that independent of the experiment you wanted to do, you’d be able to delivery [the siRNAs].” Here, again, is where Dharmafect comes into play.

Also expected to help along the reverse-transfection technology is a new line of siRNAs that feature dual-strand modifications. The siRNAs, which are being readied for a December launch but have not been given a brand name, are designed to “adjust the signal-to-noise ratio so that we can get [more efficient] RISC activation and target down-regulation,” Marshall noted.

Currently, Dharmacon has tested the reverse-transfection technology in more than 25 different cell types, Marshall said, including “cells that have been considered recalcitrant to uptake” such as primary cells.

However, the platform isn’t ready for primetime just yet. The company is currently conducting additional work and has placed the technology with a number of high-throughput screening groups in both the pharmaceutical industry and academia that are acting as beta-testers in different cell types including neurons and T-cells.

“We want to have … up to five … groups in [pharma], and another three to five additional users [in other areas], get back to us with good results” before the platform launches, Marshall said.

“Our focus is really on showing that you get minimal toxicity with maximum target down-regulation using quantitative RNA-dependent polymerase, and we’re working with [the beta-testers] … to put [the platform] through its paces in [highly] functional assays,” he said. And, there’s always the hope that the beta-testers will turn into customers once the evaluation period is over, he added.

“A lot of beta-testers are people that have bought products from us in the past and were happy with them,” Marshall said. “We have a good relationship with them and say, ‘Look, we have a new product that’s coming out. Would you like early access to it to give it a try?’ Most of them want to do that.”

Marshall declined to name any of the beta-testers, citing contractual confidentiality.

As for what the platform will look like exactly, Marshall said that the specifications have not been finalized, but noted that “we’re going to include several controls, as well as several open wells, and provide it in a format that makes it easy to do replicates.”

Dharmacon expects the commercial introduction of the reverse-transfection platform to help it expand its customer base into areas that have heretofore not had much experience with the gene-silencing technology, Marshall said.

“There’re a lot of entities that want to have collections to do multiple assays” and will embrace the technology, he said. “But there are some people that may want to do a single screen, and this provides them with the access to a large collection of siRNAs in an easy-to-use format at a [reasonable] price point.”

He noted that an exact pricing structure has not yet been worked out.

— DM