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Danish Research Team Publishes Data Showing Fewer Off-Target Effects from Nicked siRNAs

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A team of Danish researchers last month published in vitro data in Nucleic Acids Research demonstrating that a novel triple-stranded RNA can silence a target gene through the RNAi pathways as well as conventional siRNA while avoiding off-target effects triggered by the helix’s sense strand.
 
These so-called small internally segmented interfering RNA, or sisiRNA, also allow for functional antisense strand modifications, which could potentially improve the RNAi molecule’s pharmacokinetic properties in vivo.
 
Although animal studies of the sisiRNA are still ongoing, the early data is positive, suggesting that these molecules might have benefits over other RNAi agents in a therapeutic setting, Jesper Wengel, director of the Nucleic Acid Center at the University of Southern Denmark and co-author of the Nucleic Acids Research paper, told RNAi News this week.
 
Being able to modify both the sense and antisense strands of an RNAi molecule “is a very promising way forward to promote the gene-silencing technology for therapeutic applications,” he said. “To modify the molecules, to do additional chemistry to siRNA oligos … this is the way forward.”
 
Additionally, the unique design of the sisiRNA might put them outside the bulk of the RNAi field’s intellectual property, which focuses on double-stranded molecules, creating an opportunity to work in the field without having to worry about patents held by other companies.
 
How the development of therapeutic sisiRNA will occur remains unclear. Wengel said that RiboTask, a company he co-founded last year, has a license to manufacture and sell sisiRNA for research purposes. The therapeutic rights, however, lie with Santaris Pharma through a licensing deal with the University of Southern Denmark and the University of Aarhus, which co-developed the technology.
 
Although RiboTask could possibly be interested in pursuing development of therapeutic sisiRNA, the company is small and still looking to raise the funding necessary to ramp up its operations. As such, it is keeping its focus on the reagent market.
 
Santaris’ primary focus, meanwhile, is on single-stranded locked nucleic acid molecules, indicating that the company might be more interested in out-licensing the technology than developing it in-house.
 
Officials from Santaris were not available for comment.
 
Specific Knockdown
 
The sisiRNA molecule “is a co-invention of Jorgen Kjems … and myself,” Wengel said. “The idea was to design molecules that would reduce off-target effects … [by] loading only the antisense … strand of an siRNA-like complex into RISC.”
 
The sisiRNA molecules are characterized by an intact antisense strand and two shorter sense strands — or a nicked sense strand — approximately 9 to 13 nucleotides long.
 
In initial experiments, the construct was further modified with locked nucleic acids at the two and four positions in the sense 5’ and 3’ half-strands, respectively, and near the 3’ end of the antisense strand, according to the paper.
 
Later work showed that the position of the LNA modifications does not need to be fixed in the sisiRNA, and that the position of the nick in the sense strand does not need to mimic the molecule’s natural cleavage site. Increasing the gap size of the sense strands does negatively impact silencing activity, however.
 
In experiments detailed in Nucleic Acids Research, the Danish team tested sisiRNA targeting enhanced green fluorescent protein in lung carcinoma cells that express destabilized EGFP. The researchers also evaluated unmodified siRNAs against EGFP and control siRNAs.
 
Cells treated with the sisiRNA showed a 10-fold target knockdown after 48 hours, a result similar to that observed in cells transfected with the siRNAs. Additional experimentation showed that sisiRNA were capable of knocking down the endogenous gene GAPDh in the cells at levels similar to unmodified or LNA-modified siRNAs.
 
When comparing the serum stability of LNA-modified siRNAs and sisiRNA with unmodified siRNAs, the investigators found that unmodified sisiRNA resulted in no significant target inhibition, “suggesting that LNA modifications are not only beneficial but also essential for the integrity of the sisiRNA design in biological fluids,” they wrote.
 
Additionally, the sisiRNA did not appear to trigger cellular interferon responses compared with traditional siRNAs, indicating that “at least in cell culture, sisiRNA appear to be immunologically similar to standard siRNAs,” they added.
 
In order to assess whether the use of a nicked sense strand eliminated its guide activity, the investigators inserted the EGFP target sequence for either the siRNA antisense or sense strand within the 3’ UTR of a firefly luciferase reporter construct.
 
“This strategy allowed us to differentially assess the knockdown effect derived from the antisense and the sense strand incorporation into activated RISC,” they wrote. “As predicted, the LNA-modified sisiRNA construct was significantly more specific than the equivalent siRNA and LNA-modified siRNA duplexes since [around] 50 percent knockdown was constantly seen from the sense strand with standard siRNA design.”
 

“The sisiRNA design can accommodate a wide variety of bulky chemical modifications that otherwise are incompatible with the activity of standard duplex siRNAs.”

By comparison, the sisiRNA design “completely abrogated” the silencing of the sense strand target compared with controls without affecting knockdown mediated by the antisense strand, they added.
 
A key aspect of the sisiRNA is their ability to support functional antisense modifications, the researchers noted in Nucleic Acids Research.
 
“We and others have observed that extensive chemical modifications in the antisense strand of siRNAs generally are incompatible with their function in gene silencing,” they wrote. However, the sisiRNA design is able to enhance the efficiency of heavy antisense modifications.
Specifically, the researchers found that sisiRNA incorporating adamentyl and pyrenyl modifications resulted in 40 percent to 60 percent knockdown of EGFP expression. By comparison, similarly modified siRNAs were almost entirely non-functional.
 
“Hence, the sisiRNA design can accommodate a wide variety of bulky chemical modifications that otherwise are incompatible with the activity of standard duplex siRNAs,” they wrote.
 
While it is not entirely clear why the sisiRNA design allows for functional antisense modifications, Wengel speculated that nicked sense strands ease the loading of the antisense strand into RISC “because of increased flexibility and the fact that cleavage of the passenger strand likely is not necessary.”
 
With the development of sisiRNA, “we have developed a radical new siRNA design … [that] is fully functional and … has several advantages over the standard 21 [nucleotide] duplex siRNA designs,” including enhanced stability in serum with high potency, the paper’s authors concluded.
 
Additionally, “the segmented nature of the passenger strand completely alleviates its contribution to unwarranted gene knockdown thereby greatly increasing targeting specificity and expectably reducing off-target effects.”
 
Lastly, the sisiRNA design has “the ability to rescue the function of chemically modified antisense strands that are non-functional in the context of a standard siRNA duplex … [and] has six terminal ends compared to four in normal siRNA, which can conveniently be used for tethering functional chemical groups to enhance … cellular delivery,” they wrote.
 
“The possibility to incorporate more extensive chemical modifications into the sisiRNA design as compared to standard siRNAs may have beneficial properties for steps both upstream and downstream of RISC activation in the RNAi pathway,” the authors added.

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