Apparent absence of toxicity and persistence of silencing may bode well for humans
By Charles Choi | March 27, 2006
A disease-causing gene was silenced in monkeys through RNA interference (RNAi) therapy delivered into the bloodstream, scientists reported online yesterday (March 26) in Nature. This is the first study to show that systemic administration of RNAi works in non-human primates, and the findings affirm the promise of this new type of therapy, scientists say.
In the study, gene silencing using the highest dose persisted for 11 days, without any observed toxicity, a finding that "bodes well for future human systemic trials of RNAi therapies," John Rossi of the Beckman Research Institute of the City of Hope in Duarte, Calif., who did not participate in this study, told The Scientist.
Scientists at Alnylam Pharmaceuticals and Protiva Biotherapeutics gave cynomolgus monkeys a single injection of small interfering RNAs (siRNAs) against the gene for apolipoprotein B (ApoB). This gene is involved in the assembly and secretion of low-density lipoprotein (LDL), and has not proven accessible to targeting with conventional small molecule or protein therapies. The researchers encapsulated the siRNAs in a liposomal formulation designed to target the liver, and modified their backbones to prevent siRNA degradation.
Gene silencing occurred in a dose-dependent manner. Within 48 hours, the maximum level of APOB silencing exceeded 90%, with a more than 75% reduction in plasma ApoB and a more than 80% drop in LDL. The researchers detected no reductions with empty liposomes or those containing mismatched siRNA.
Using 5' rapid amplification of cDNA ends (RACE) analysis, the scientists confirmed that silencing occurred via APOB mRNA cleavage at precisely the predicted site. APOB mRNA levels were substantially reduced for at least 11 days -- a silencing effect far more persistent than results previously obtained with rodents, which suggested APOB mRNA levels would recover within four to seven days. Alnylam president and chief executive officer John Maraganore said that metabolic differences between rodents and primates may explain why silencing persisted longer in this study.
The researchers saw no evidence of toxicity, such as pro-inflammatory cytokine production, a finding Maraganore attributed to in vitro toxicity screening beforehand and to the siRNA backbone modifications. The only detected change was a fleeting increase in liver enzymes in monkeys that received the high dose of the liposome-encased anti-APOB siRNAs, which peaked 48 hours after treatment and varied highly across individual animals. These effects normalized by the sixth day, and did not interfere with biological efficacy.
The researchers ended their study after 11 days because "the durability of the silencing far exceeded what we expected," Maraganore told The Scientist. They plan to continue longer-term experiments in primates, and improve the liposome formulations. Still, Maraganore said he believes they are ready for human clinical trials within the next 18 to 24 months.
However, scientists cautioned many details of the therapy should be investigated further. "The data is all from a single dosing, and it is going to be important to determine what happens after multiple dosing, as will be required for chronic liver problems," Rossi cautioned. While the liposome vectors the researchers used are most effective in delivery to the liver, they could also investigate whether the liposomes can deliver siRNAs to different targets, such as the kidneys or lungs, Rossi added.
Investigations should also tease apart how necessary the backbone modifications are to the therapy's effectiveness, Martin Woodle at RNAi therapeutic company Intradigm in Rockville, Md., who did not participate in this study, told The Scientist. "Nanoparticle and liposomal systems are now quite well accepted and very successful, without invoking the need for chemical modifications of the siRNAs themselves," he said. Future research should also investigate whether systemic delivery localizes siRNAs selectively to the liver, or if the silencer reaches other organs, as well, Woodle added.
Links within this article
T.S. Zimmermann et al. "RNAi-mediated gene silencing in non-human primates." Nature, published online March 26, 2006.
C. Choi, "Mice tolerate siRNAs," The Scientist, November 24, 2004.
A. Adams. "RNA therapeutics enter clinical trials." The Scientist, January 17, 2005.
J.F. Wilson. "The biological basis for atherosclerosis." The Scientist, October 30, 2000.
J. Soutschek et al. "Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs." Nature 432, 173-178, November 11, 2004.