12 November 2004. An important RNA interference (RNAi) milestone—the silencing of a therapeutically relevant endogenous gene in an animal model by systemic administration of short interfering (si) RNAs—was reported today in Nature. Researchers at the biotech company Alnylam in Kulmbach, Germany, and in Cambridge, Massachusetts, silenced the gene for apolipoprotein B (ApoB) by conjugating siRNAs with cholesterol to help them gain entry to cells.
Delivery to the right place is the biggest issue in designing therapeutic siRNAs, particularly gaining entry to cells. The two predominant solutions are to use lipid complexes or viral vectors (see ARF related news story). In a study published earlier this year, Alnylam scientists reported in vitro success with a simpler approach: RNAi conjugated to cholesterol was able to gain entry to liver cells and silence a reporter gene (Lorenz, 2004). How this occurs is not clear, though the scientists noted in that earlier paper that "siRNA modified with lipophilic moieties may enhance siRNA uptake via a receptor-mediated mechanism or by an increased membrane permeability of the otherwise negatively charged RNA."
The other critical issue in making RNAi therapy viable is protecting the RNA from nucleases, either inside or outside cells. Nucleases in the bloodstream are a particular problem, since the ability to deliver siRNAs systemically would be a great advantage. Fortunately, in the current Alnylam study Hans-Peter Vornlocher, first author Jürgen Soutschek, and colleagues were able to take advantage of well-tested methods to stabilize oligonucleotides against nucleases—the use of phosphorothioate backbone and methylated sugars. Plus, the cholesterol conjugation appeared to provide added protection for the siRNAs against nucleases in blood.
The researchers chose to target ApoB, a major structural component of the low-density lipoprotein (LDL) cholesterol complex, the "bad" sort that contributes to coronary artery disease. ApoB is the ligand for the LDL receptor, and is expressed primarily in liver and jejunum. In their first in vivo experiments, Soutschek and colleagues administered cholesterol-conjugated ApoB-siRNA (chol-ApoB-siRNA) systemically to normal C57BL/6 mice. The researchers detected the siRNAs in liver, jejunum, and other tissues (though apparently not in brain), and one of their constructs lowered ApoB mRNA by 57 +/- 6 percent in liver and 73 +/- 10 percent in jejunum (P < 0.0001). This, in turn, led to plasma reductions of ApoB protein of up to 68 +/- 14 percent (P < 0.0001).
Perhaps most impressive was the fact that these changes were reflected in blood cholesterol and lipoprotein profiles, including a lowering of total cholesterol by 37 +/- 11 percent (P < 0.0001). "In aggregate, the effects on cholesterol reduction and lipoprotein profiles would be considered highly clinically significant in patients with hypercholesterolemia, and actually exceed the level of cholesterol reduction observed in heterozygous ApoB knockout mice," the authors write.
Supporting evidence came from a transgenic mouse model expressing a human ApoB variant. In this case, the chol-ApoB-siRNA significantly reduced both endogenous ApoB mRNA and the human transgenic ApoB mRNA in liver. The authors did not report on whether the siRNA was able to prevent the atherosclerosis seen in these animals when fed a high-fat diet.
In a News and Views commentary, John Rossi of the Beckman Research Institute of the City of Hope in Duarte, California, extols the simplicity of the siRNA construct. "The system did not require expensive lipid complexes or other macromolecular carriers, but
merely a single cholesterol conjugate per RNA duplex," he writes. But Rossi does provide some of the usual warnings against undue optimism. Presumably, this therapy would have to be used by human patients for many years, and the long-term effects of siRNA would have to be investigated closely. Rossi also points out that the dosage used in the mice would require regular injections of gram quantity chol-ApoB-siRNAs in humans, perhaps a prohibitive expense.—Hakon Heimer.
Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie R, Rajeev KG, Röhl I, Toudjarska I, Wang G, Wuschko1 S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher H-P. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004 Nov 11;432(7014):173-8. Abstract