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Stanford Researcher Questions Alnylam Paper Touting Safety of siRNA Therapies

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A team led by investigators from Alnylam Pharmaceuticals this week published a study in Nature demonstrating that siRNAs could be used to knock down target genes in mice without interfering with the animal’s endogenous microRNA pathway.
 
Release of the findings comes about a year and a half after researchers from Stanford University published a paper in Nature showing that sustained, high-level shRNA expression in mouse livers can result in severe toxicity and death — most likely as a result of disruption of the miRNA processing machinery (see RNAi News, 5/25/2006).
 
Referencing the Stanford paper in a statement this week, Alnylam said that its new data dissociates “siRNA-mediated gene silencing effects from cellular processes related to microRNA biosynthesis and function in vivo,” and “represents an important de-risking event towards the translation of [RNAi] technology into innovative medicines."
 
The Stanford study was also cited in this week’s Nature paper.
 
But to Mark Kay, a professor at Stanford and senior author of last year’s Nature paper, a comparison between his paper and Alnylam’s isn’t so straightforward because of differences in thelevels of the RNAi oligos evaluated in the two studies.
In the Alnylam paper, “they were looking at 70 and 80 percent reductions” in the expression of two hepatocyte-specific genes using a single dose of siRNA, he said. Other experiments covered in the paper looked at three doses of siRNA given once weekly.
 
But in the experiments detailed in last year’s paper, Kay and his colleagues examined the effects of sustained expression of shRNA. Additionally, they were evaluating shRNA designed to inhibit the hepatitis B virus, and to “inhibit a virus enough to make a clinical difference [would require] two or three orders of magnitude or more knockdown,” Kay said.
 
“If you really wanted to … do a direct comparison [to the earlier paper] and say, ‘siRNA are safer than shRNA’ … you need to compare the two directly and measure the [cellular] concentration of the delivered oligonucleotides,” he added.
 
In an e-mail, an Alnylam spokesperson said that “while levels of siRNA in the cell were not quantified in the Alnylam study, we know that the siRNA concentrations achieved were sufficient to silence target genes by at least 80 percent for a period of a month with no effects on miRNA biogenesis or function."
 
The spokesperson was not available for further comment.
 
Ultimately, Kay views Alnylam’s paper as “reasonable … for pushing siRNA-based therapies forward,” but “not a conceptually new study” since it doesn’t provide much more evidence regarding the safety of siRNA drugs than had earlier Alnylam studies such as one published in Nature last March (see RNAi News, 3/30/2006).
 
Apples and Oranges?
 
In this week’s Nature paper, Alnylam investigators administered a single bolus injection of siRNA targeting apolipoprotein B and factor VII and formulated in novel liposomal nanoparticles to mice at one of three different dose levels.
 
The highest dose, 5 mg/kg, resulted in “marked silencing” of around 80 percent of the hepatocyte-expressed genes at day 2 compared with placebo. As expected, the gene-silencing effect was dose dependent, with animals receiving the lowest dose of 2 mg/kg showing “intermediate silencing effects.”
 
By day 30, gene-expression levels returned to normal, according to the paper.
 
To determine whether the gene silencing affected the animals’ miRNA pathway, the research team measured levels of miR-122, the most highly expressed miRNA in the liver, and the ubiquitously expressed miRNAs miR-16 and let-7a in liver tissue samples.
 

“If you really wanted to … do a direct comparison [to the earlier paper] and say, ‘siRNA are safer than shRNA’ … you need to compare the two directly and measure the concentration of the delivered oligonucleotides.”

“No significant differences were measured in miRNA levels in siRNA-treated animals,” the paper’s authors wrote. “Certainly, no reduction of miRNA levels to match the … level previously reported [in Kay’s paper] was observed with administration of synthetic siRNA.”
 
To determine whether multiple doses of siRNA would affect miRNA levels, the Alnylam-led team administered three weekly injections of siRNA targeting the hepatocyte-expressed gene SREBP cleaving-activating protein, or Scap, to hamsters.
 
This treatment resulted in a roughly 75 percent inhibition of Scap protein but did not affect miR-122 levels.
”Our findings demonstrate that robust gene silencing, both acute and longer-term, can be achieved in the liver without any detectable alteration of cellular miRNA biogenesis or function,” the authors concluded.
The work by Alnylam was done in collaboration with Roche, the Massachusetts Institute of Technology, the University of Texas Southwestern Medical Center, and the Swiss Federal Institute of Technology.
 
In the Nature paper published last year, Kay and his colleagues treated hepatitis B transgenic mice with shRNA-expression cassettes delivered with an optimized delivery vector based on duplex-DNA-containing adeno-associated virus type 8 and a U6 promoter.
 
“An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death,” according to that paper. “Morbidity was associated with the down-regulation of liver-derived microRNAs, indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs.”
 
But as Kay pointed out to RNAi News last year, his team was trying to use the highest possible shRNA dose in its studies.
 
“What gets lost a lot when people talk about [the paper from last year] is the fact that when we used weaker promoters, we had really high-level sustained knockdown without toxicity,” he said this week. “You can get reasonable levels of knockdown [with shRNAs] without interfering with the endogenous microRNA pathway.”
 
One shouldn’t be surprised to see toxicities associated with high levels of any kind of drug, whether an shRNA- or siRNA-based one, because “you can overdose on anything, even water,” Kay added.

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