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Researcher Develops RNA-DNA Hybrid That May Offer Advantages Over siRNA

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Call it a new form of hybrid vigor. Allen Christian, a Lawrence Livermore National Laboratory researcher, has come up with a new type of RNAi molecule that he says is not only able to silence target genes with greater specificity than siRNAs, but is capable of being introduced into cells without transfection and may be effective against bacterial genes.

The molecules — termed siHybrids — consist of two annealed strands, one DNA and one RNA, with a 2-base overhang at each 3’ end. According to Christian, they are the result of academic curiosity rather than a focused research effort.

“One young [undergraduate] that was working for me [named Janelle Lamberton] was doing a senior honors thesis on cancer … and we had to knock out some tumor suppressor genes,” Christian told RNAi News. “We thought we would do it temporally using antisense.”

At that time, however, RNAi was becoming well-known as an effective gene-silencing mechanism, Christian said, so he and Lamberton decided to use siRNAs. “We did it and it worked,” he said.

Later on, “we were sitting around the lab late one night wondering how all this stuff worked — nobody had published a mechanism, nobody had any idea,” Christian said. “We thought: ‘Does it have to be siRNA? What if it’s siDNA?’”

While the siDNAs were able to silence target genes, they weren’t able to do it as well as siRNAs, he said. However, “we were pretty fascinated at the thought that the siDNA would work so we decided that, since we had the DNA and we had the RNA, we would try the DNA-RNA hybrid. It turned out that it worked a lot better for us” than siRNAs, Christian said.

“We were just screwing around” when making the hybrid molecules, he said. “We really had no idea” that they’d be so effective.

The experiments, which were published in the June 2003 issue of Molecular Biotechnology, showed that siHybrids were able to silence the glucose-6-phosphate dehydrogenase gene in normal and cancerous cells from both humans and hamsters, and were more potent and had a longer duration of action than siRNAs and siDNAs.

Additional research, not found in the paper, has shown that siHybrids work in mammalian cells without being transfected. “We just drop them on the cells without lipofectamine,” Christian said. “We don’t know how they get in, but they get in pretty nicely.”

He noted that these findings, which are supported by data from cancer researchers at the University of California, Davis, working with the siHybrids, have recently been submitted for publication.

Christian also said that early research indicates that siHybrids can be used to silence bacterial genes, something that siRNAs are unable to do effectively.

Rockefeller University researcher Thomas Tuschl explained to RNAi News in an e-mail that “bacteria use [an] antisense RNA mechanism to regulate their genes, but they don’t have the conserved protein machinery like Dicer, argonaute, and so on for this purpose.”

Christian said it remains unclear why siHybrids can silence bacterial genes, but declined to comment further, noting that the findings haven’t been published and are too preliminary to support any claims of efficacy. He added that his lab is currently conducting additional experiments in this area.

Despite Christian’s reticence to discuss the use of siHybrids against bacterial genes, a recently published patent application covering the molecules (see IP Update, p. 3) offers a few details.

According to that application, which lists Christian as the lead inventor, “the remarkable efficacy of gene silencing using siHybrids on mammalian cells suggested its use on bacterial cells, under the logical premise that its remarkable longevity in mammalian cells would allow it to act similarly in bacteria.”

The application states that Christian’s first experimental targets were “antibiotic resistance genes located on plasmids transformed into the bacteria.” Escherichia coli was used for all the tests, the application notes.

“In a series of experiments, genes providing resistance to ampecillin and chloramphenicol, two common antibiotics, were silenced,” the application states. “In all cases, bacteria that were able to grow readily in antibiotic-containing media died when siHybrids against the antibiotic resistance gene were added to the media. No transforming steps, nor any transfection media or agents, were necessary; simply adding the siHybrids to the media was sufficient.”

The application notes that siHybrids not targeted to an expressed gene had no effect on the bacteria, indicating that a direct effect had been observed, “not a wholesale, nonspecific killing of the cells.”

Additional experiments targeting the folA gene were conducted and confirmed that the siHybrid silencing was specific and non-lethal, and not possible with conventional siRNAs, according to the application. “By selecting a unique gene, necessary for bacterial vitality, for specific bacteria, siHybrids can act as an antibiotic for those specific bacteria by silencing the gene,” it adds.

The siHybrids are owned by the University of California, which operates the Lawrence Livermore National Laboratory. Christian said that there has been some interest in the technology from commercial entities, but he declined to comment further due to “non-disclosure agreements.”

Rupurt Xu, business development executive with Industrial Partnership and Commercialization, the technology transfer arm of the Lawrence Livermore National Laboratory, told RNAi News that licensing discussions are ongoing with companies considering siHybrids for development as reagents and as potential therapeutics.

He said that he expects a deal, most likely with a reagents firm, to be forged within a couple of months, adding that any licensing arrangement would likely include an upfront fee to IPAC, as well as milestones and royalties.

He declined to comment more specifically on the licensing talks or the financial terms of any resultant deal.

—DM

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