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IDT Aims to Develop U1 Adaptors as Gene-Silencing Alterative, siRNA Adjunct

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By Doug Macron

Integrated DNA Technologies is developing a new gene-silencing technology that it expects could be used to boost the inhibitory effects of siRNAs and provide an alternative to the RNAi molecules in cases where they are unable to produce the desired level of target knockdown, a company official told RNAi News this week.

And while IDT hopes to eventually commercialize the technology, it is still not quite ready for prime time, according to IDT CSO Mark Behlke. Meanwhile, the company is currently waiting for word from the National Institutes of Health on whether it has received a phase II Small Business Innovation Research grant that would provide the funding necessary to further refine the approach.

Developed by Rutgers University’s Sam Gunderson, the single-stranded oligonucleotides are comprised of a target-gene binding domain and a U1 domain that attracts and inhibits the cellular splicing apparatus. Together, these block the pre-mRNA maturation step of polyA tail addition, according to Rutgers.

Although the initial work on the oligos was conducted by Gunderson, his lab lacked the necessary medicinal and nucleic acid chemistry expertise to develop the technology fully, IDT CSO Mark Behlke said. “So we started a collaboration about two years ago to test out different oligonucleotide designs, different lengths, [and] different chemical modification patterns to try to figure out how to make the gene-knockdown process work efficiently.”

The result was a next-generation version of the oligos dubbed U1 adaptors.

In March, Behlke and Gunderson, along with Rutgers researcher and collaborator Rafal Goraczniak, published the details of the U1 adaptors in Nature Biotechnology. According to that paper, the development process began with a gene-silencing approach called U1 small nuclear RNA interference, or U1i.

“In this method, a plasmid vector is used to express a U1 snRNA in which the natural U1 targeting domain is replaced by a ten-nucleotide sequence complementary to the target’s terminal exon,” according to the paper. “The U1i snRNA assembles into a U1 snRNP complex that hybridizes to the target’s pre-mRNA and inhibits polyA tail addition, an obligatory RNA processing step for nearly all eukaryotic mRNA. Without poly-adenylation, the pre-mRNA fails to mature and is degraded in the nucleus, thereby reducing cytoplasmic mRNA levels of the target gene.”

Still, the approach failed to catch on because of the “inconvenience of preparing custom U1i targeting plasmids and concerns over specificity,” the paper notes. “Furthermore, the U1i snRNA must be expressed from a plasmid or viral vector and attempts to make it amenable to chemical synthesis by shortening it have failed.”

With its target domain hybridizing to the target gene’s pre-mRNA in the 3’ terminal exon and the U1 domain that binds the U1 snRNP to the target pre-mRNA, proper 3’-end formation is inhibited, leading to RNA degradation, the paper states.

With the U1 adaptors, “you can easily get 10- to 20-fold knockdown of a target RNA, much like if you are using antisense or RNAi,” Behlke said. Potency, meanwhile, appears on par with antisense approaches, with certain oligos achieving an IC50 in the range of 0.5 to 1 nanomolar, he added.

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Still, the U1 adaptors aren’t as potent as IDT’s top-performing Dicer-substrates, which Behlke said have an IC50 as low as 1 picomolar for certain targets. Additionally, they are likely to be more expensive than an siRNA since they are fully modified compounds. As such, he doesn’t envision the new technology replacing RNAi.

Rather, Behlke sees U1 adaptors as providing a solution for researchers not finding success with an siRNA or Dicer-substrate.

“Maybe you haven’t been able to find an siRNA that works as well,” which sometimes is a target-specific, rather than sequence-specific, issue, he said. “This gives you an opportunity to target that structure when it is a pre-mRNA in the nucleus … so it may open a place where you can target a message that may be hard to knock down when it’s a mature mRNA.”

At the same time, the U1 adaptors may be used to enhance an RNAi effect, Behlke said.

“Because it [relies on] an entirely separate mechanism of action, [the new technology] will work additively with siRNA,” he said. “Some genes may be really dosage sensitive, and to get a real phenotype, you may have to go to better-than-95 percent knockdown. If you’re only getting 85-to-90 percent knockdown with siRNA, to get that extra kick, you may need to add something else.

“The U1 adaptors could be added to the mix” to achieve the desired result.

But it is still early days for the technology, and Behlke indicated that it could be some time before IDT is in a position to begin marketing the oligos.

“To truly commercialize [this technology], we’re going to need to be able to come up with good design rules for site selection, which means testing a lot of U1 adaptors and a lot of different targets,” he explained. “That’s a somewhat lengthy, expensive process, so … we’re going to need help from a phase II SBIR” grant.

About one year ago, IDT received a one-year, $170,000 grant from the NIH to develop the U1 adaptors. And this August, the company received a $99,750 extension that will buy an additional six months, according to Behlke.

“The extension will keep us working on our current experiments, which are looking at further optimizations of the chemistry,” he said. But the phase II funding will be required to develop the design algorithms that will make the technology “reliable and robust enough for people to use without undo experimentation on their part.”

The phase II grant application has already been reviewed by the NIH and received “a good score,” Behlke said. “We’re just waiting now for NIH to decide what score levels get funded. We should know that within a month.”

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