Researchers at Alnylam Pharmaceuticals have used an intravenously delivered siRNAs to knock down in rats a gene encoding apolipoprotein B, a protein essential for the formation of low-density lipoproteins and associated with cholesterol metabolism, according to an article in this week’s Nature.
Alnylam researcher Jurgen Soutschek and colleagues targeted aopB using synthetic siRNAs with partial phosphorothioate backbone and 2’-O-methyl sugar modifications on the sense and antisense strands. These modifications appear to confer resistance to degradation by exo- and endonucleases in serum and tissue homogenates, the scientists wrote in the paper.
The siRNAs were further modified through the conjugation of cholesterol to the 3’ ends of their sense strands using a pyrrolidine linker. The resultant chol-siRNAs showed improved in vivo pharmacokinetic properties after being administered intravenuously into rats, but did not impair gene silencing activity in cell culture, according to the researchers.
Administering the siRNAs intravenously enabled the researchers to silence apoB messenger RNA in the liver and jejunum, decrease plasma levels of apoB protein, and reduce total cholesterol, the team wrote. The scientists further demonstrated that the siRNAs could be used to silence human apoB in a transgenic mouse model.
The effects were shown to be a result of RNAi-mediated mRNA degradation, with mRNA cleavage occurring specifically at a predicted site, Soutschek and his colleagues noted in the paper.
In an accompanying Nature article, Beckman Research Institute’s John Rossi noted that the method of siRNA delivery employed by the Alnylam researchers “did not require expensive lipid complexes or other macromolecular carriers, but merely a single cholesterol conjugate per RNA duplex.” He added that the cholesterol group was key to the researchers’ success, as it helped the cells take up the siRNAs.
Rossi stressed, though, that Alnylam’s method is still far from applicable in humans.
“The treatment of high-cholesterol levels in humans might require lifetime use of cholesterol-lowering compounds,” he wrote. “But the consequences of long-term siRNA therapy are not known.”
Additionally, if the dosage required to achieve the knockdown effect in mice is translated to humans, “gram quantities of siRNA-cholesterol conjugates would be required in regular infusions, which would be enormously expensive,” Rossi added.
During a conference call this week announcing the findings, Alnylam President and CEO John Maraganore noted that while the Nature paper supports the company’s ability to advance its program of systemically delivered siRNA-based medicines, “we haven’t made a formal commitment yet to apoB as a clinical program.”