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Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 5% methanol (mobile phase A) and 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50x2.1mm i.d., 2.5microm particle size), and separation was performed at 60 degrees C. Quantitation was achieved with ultraviolet (UV) detection at 260nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110microg/ml. Accuracy of the method was determined by replicate analysis (n=5) at four concentrations (R(2)>0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

Delivery of small interfering RNAs (siRNAs) in vivo , using clinically relevant modes of administration, is critical for the advancement of RNA interference (RNAi) therapeutics. In this work, we demonstrate systemic delivery of siRNAs and potent in vivo down-modulation of two important disease targets, apolipoprotein B (apoB) and proprotein convertase subtilisin kexin 9 (PCSK9). A single injection of liposomal siRNA resulted in >90% silencing of apoB mRNA expression in the liver 48 h after administration. The effect was demonstrated to occur through cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism. Reductions in apoB protein, cholesterol, and low-density lipoprotein (LDLc) levels were observed in 48 hours that lasted for at least 23 days, thus demonstrating an immediate, potent and durable biological effect. In addition to apoB we have also demonstrated the ability to down-modulate other important liver targets such as PCSK9. PCSK9 has been closely implicated in LDLc regulation. We have demonstrated PCSK9 down-modulation in several animal models including, mouse, humanized mouse, rat, and non-human primate. Down-modulation of PCSK9 levels resulted in significant lowering of cholesterol (20 – 60%) in all animal models tested. These findings strongly support the potential of RNAi therapeutics as a new class of drug for metabolic and cardiovascular diseases. Our next steps include selecting the most potent lead molecule and moving it into GLP safety studies.