Continuing a streak of publishing its early RNAi work in key peer-reviewed journals, Alnylam Pharmaceuticals this week reported data showing that siRNAs can be used to knock down production of angiopoietin 2, an angiogenic growth factor, in the lungs of mice.
The data, which were published by researchers from Alnylam and Yale University this week in the online version Nature Medicine, shows that siRNAs were able to silence the Ang2 protein, allowing the animals to live longer with decreased lung injury compared to controls.
“These results showed that we were able to design and synthesize siRNAs that, following pulmonary delivery in vivo, effectively silence Ang2, a very important target associated with acute lung injury,” Antonin de Fougerolles, senior director of research at Alnylam and a co-author of the paper, said in a statement.
“The findings demonstrate a critical role for Ang2 in the pathogenesis of hyperoxic acute lung injury and that silencing the Ang2 gene with RNAi may be therapeutically relevant,” he added.
According to the paper, exposure to high concentrations of oxygen for prolonged periods can result in acute lung injury characterized by endothelial and epithelial injury and enhanced alveolar capillary protein leak.
While the mechanisms of this kind of injury and the resultant cell death “have not been adequately defined … we considered that Ang2 might have a critical role in the pathogenesis of HALI … [given its ability to] destabilize blood vessels, enhance vascular leak, antagonize [the related growth factor] Ang1 and, in the absence of other angiogenic stimuli, induce vascular regression and endothelial cell apoptosis,” the researchers wrote in Nature Medicine.
To test their hypothesis, the researchers “characterized both the expression of angiopoietins during HALI and the effects of hyperoxia in mice that produce Ang2 normally, in mice with null Ang2 mutations, and in mice treated with siRNA targeted to Ang2,” they wrote. The team added that they also “defined the effects of recombinant Ang2 on mice in hyperoxia, and epithelial cells in normal and hyperoxic in vitro culture.”
According to Alnylam, the siRNAs were administered to the mice intranasally.
“Data showed that … administration in mice of an siRNA targeting Ang2 resulted in specific inhibition of messenger RNA by more than 60 percent,” the company said. “A proportionate drop in Ang2 protein levels was also observed … [while] acute lung injury was reduced to near non-injury levels as measured by … reductions in lung inflammation and cell death.”
“The findings demonstrate a critical role for Ang2 in the pathogenesis of hyperoxic acute lung injury and that silencing the Ang2 gene with RNAi may be therapeutically relevant.”
Alnylam noted that mRNA levels of other genes such as VEGF and Bcl-2 were not affected by the siRNAs.
According to the Nature Medicine paper, the data indicated that Ang2 is stimulated during HALI and contributes to the pathogenesis of oxidant injury, DNA injury, apoptosis, inflammation, edema, and mortality induced by hyperoxia in vivo.
“They also show that Ang2 induces epithelial cell necrosis in hyperoxia and that increased amounts of plasma, alveolar edema fluid, and tracheal aspirate Ang2 are present in human adults and neonates with [acute lung injury] and pulmonary edema,” the researchers wrote.
“Our observations suggest that Ang2 is an important mediator of the injury, edema, and/or cell death responses in [HALI and ALI] and that Ang2 regulators may thus be therapeutically useful,” they concluded in the paper. “Additional investigation of the effector profile of Ang2 in health, healing, and disease, [as well as] the consequences of Ang2 manipulation in these settings, is warranted.”
“Through this study, we have learned that Ang2 seems to be a mediator of cell death in the settings of high oxygen concentrations in the lung causing acute lung injury and pulmonary edema,” Jack Elias, a researcher at the Yale University School of Medicine and study co-author, said in a statement. “In applying an RNAi approach, we were able to observe in mice that silencing of the Ang2 protein resulted in animals that lived longer and had evidence of decreased lung injury compared to animals with the protein intact.”