doi:10.1038/nature04688
`
`LETTERS
`
`RNAi-mediated gene silencing in non-human
`primates
`Tracy S. Zimmermann1, Amy C. H. Lee2, Akin Akinc1, Birgit Bramlage3, David Bumcrot1, Matthew N. Fedoruk2,
`Jens Harborth1, James A. Heyes2, Lloyd B. Jeffs2, Matthias John3, Adam D. Judge2, Kieu Lam2,
`Kevin McClintock2, Lubomir V. Nechev1, Lorne R. Palmer2, Timothy Racie1, Ingo Ro¨hl3, Stephan Seiffert3,
`Sumi Shanmugam1, Vandana Sood2, Ju¨rgen Soutschek3, Ivanka Toudjarska1, Amanda J. Wheat2, Ed Yaworski2,
`William Zedalis1, Victor Koteliansky1, Muthiah Manoharan1, Hans-Peter Vornlocher3 & Ian MacLachlan2
`
`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 medicines1,2. Although
`there are numerous examples of in vivo silencing of target
`genes after local delivery of small interfering RNAs (siRNAs)3–5,
`there remain only a few reports of RNAi-mediated silencing in
`response to systemic delivery of siRNA6–8, and there are no
`reports of systemic efficacy in non-rodent species. Here we show
`that siRNAs, when delivered systemically in a liposomal formu-
`lation, 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 kg21. 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 demonstrat-
`ing 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 thera-
`peutics as a potential new class of drugs.
`ApoB is expressed predominantly in the liver and jejunum, and is
`an essential protein for the assembly and secretion of very-low-
`density lipoprotein (VLDL) and low-density lipoprotein (LDL),
`which are required for the transport and metabolism of cholesterol9.
`As a large, lipid-associated protein, ApoB is not accessible to target-
`ing with conventional therapies, but it is a highly relevant and
`validated disease target. Elevated ApoB and LDL levels are correlated
`with increased risk of coronary artery disease, and inadequate control
`of LDL–cholesterol after acute coronary syndromes results in
`increased risk of recurrent cardiac events or death10,11. Approaches
`targeting ApoB with second-generation antisense oligonucleotides
`have progressed to pre-clinical and clinical studies12. Despite progress
`in the management of hypercholesterolaemia using HMG-CoA
`reductase inhibitors and other drugs that affect dietary cholesterol,
`there remains a significant need for new therapeutic approaches.
`We have previously demonstrated silencing of Apob in rodents
`using cholesterol-conjugated siRNAs6. In the current study, we used
`a liposomal formulation of SNALP to evaluate systemic delivery
`of siRNA directed towards APOB. Preliminary evaluations were
`conducted in mice. Whereas administration of the Apob-specific
`siRNA siApoB-1, without formulation or chemical conjugation, at
`doses higher than 50 mg kg21 was previously shown to have no
`
`Figure 1 | SNALP–siRNA-mediated silencing of murine Apob is potent,
`specific, dose-dependent and long-lasting. a, Liver Apob mRNA levels
`normalized to Gapdh mRNA and serum ApoB-100 protein levels measured
`two days after single i.v. injections of saline, SNALP–siApoB-1 (1 mg kg21),
`mismatched SNALP–siApoB-MM (1 mg kg21) or empty SNALP vesicles
`(25 mg kg21) (n ¼ 5 per group). b, Liver Apob mRNA levels normalized to
`1Alnylam Pharmaceuticals Inc., 300 Third Street, Cambridge, Massachusetts 02142, USA. 2Protiva Biotherapeutics Inc., 100-3480 Gilmore Way, Burnaby, British Columbia
`V5G 4YI, Canada. 3Alnylam Europe AG, Fritz-Hornschuch-Str. 9, 95326 Kulmbach, Germany.
`© 2006 Nature Publishing Group
`
`Gapdh mRNA, assessed three days after i.v. administration of saline or 5, 2.5,
`1 or 0.5 mg kg21 SNALP–siApoB-2 (n ¼ 4 per group). c, Serum ApoB-100
`levels after i.v. administration of either saline or 2.5 mg kg21 SNALP–
`siApoB-2 (n ¼ 6 per group). Serum ApoB-100 levels for SNALP–siApoB-2-
`treated animals are relative to the saline-treated group for the same time
`point. Data show mean ^ s.d.
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`Figure 2 | Systemic silencing of APOB mRNA in non-human primates.
`a, b, Liver APOB mRNA levels for 12 biopsies (three isolated from each of
`four liver lobes) were quantified relative to GAPDH mRNA either 48 h
`(a, n ¼ 4 animals per group) or 11 days (b, n ¼ 2) after treatment with
`SNALP–siApoB-2. Data shown are mean APOB/GAPDH mRNA
`
`levels ^ s.d. for each animal. Mean values (^s.d.) of the per cent APOB
`mRNA reduction relative to the saline treatment group are shown above
`each group. Asterisks indicate statistical significance compared with the
`saline-treated group (P , 0.005; ANOVA).
`
`0
`
`5
`rapid amplification of cDNA ends (RACE) analysis and identifi-
`cation of the predicted cleavage site, exactly ten nucleotides from the
`0
`end of the antisense strand of siApoB-2 (Supplementary Fig. 5).
`5
`Notably, APOB mRNA silencing was maintained for 11 days after the
`single 2.5 mg kg21 treatment, with APOB mRNA levels still reduced
`by 91 ^ 1.5% (Fig. 2b). Monkeys treated with the 1 mg kg21 dose
`showed varying degrees of recovery from ApoB silencing at the day 11
`time point. Although APOB mRNA was efficiently silenced in the
`liver, SNALP–siApoB-2 showed no silencing of APOB expressed in
`the jejunum (Supplementary Fig. 6), consistent with the absence of
`significant biodistribution of SNALP-formulated siRNAs to intesti-
`nal tissues in mice (Supplementary Fig. 3b).
`The degree and persistence of RNAi-mediated silencing observed
`in cynomolgus monkeys far exceeds the results obtained with
`rodents. The lasting RNAi-mediated effects in vivo are consistent
`with observed long-lasting silencing by siRNAs in other studies13,14,
`and the longer duration observed in primates may relate to species
`differences in the efficiency and stability of the RNA-induced
`
`in vivo silencing activity6, ,80% silencing of liver Apob mRNA and
`ApoB-100 protein was achieved with a single 1 mg kg21 dose of
`SNALP-formulated siApoB-1 (Fig. 1a). In contrast, no detectable
`reduction was observed with a SNALP-formulated mismatched
`siRNA (siApoB-MM) or empty SNALP vesicles, indicating that
`silencing is specific to the siRNA and is not caused by the lipo-
`somal carrier. This silencing effect of SNALP-formulated siRNA
`represents more than a 100-fold improvement in potency compared
`with systemic administration of cholesterol-conjugated siApoB-1
`(chol–siApoB-1) (Supplementary Fig. 1). Moreover, liposomal for-
`mulation of siRNA seems to be a general strategy for silencing
`hepatocyte targets, as demonstrated in mice for coagulation factor
`VII, green fluorescent protein and cyclophilin B (A.A., R. Constien
`and M.N.F., unpublished results).
`As siApoB-1 was originally designed to be cross-reactive to both
`mouse and human ApoB genes, and we planned to conduct RNAi
`studies in non-human primates, a second ApoB-specific siRNA,
`siApoB-2, was designed to be cross-reactive with mouse, human
`and cynomolgus monkey ApoB genes. siApoB-2 was also selected on
`the basis of in vitro gene silencing activity and the absence of
`immunostimulatory activity (data not shown). Murine studies
`showed that encapsulated siApoB-2 showed a dose-dependent
`reduction in Apob mRNA, with .90% silencing achieved at the
`highest (5 mg kg21) dose (Fig. 1b). After a single 2.5 mg kg21 dose of
`SNALP–siApoB-2, 80% silencing of liver Apob mRNA was associated
`with a 72% reduction in serum ApoB-100 protein. The silencing
`effect was detected for up to nine days, and was followed by recovery
`to normal protein levels by day 13 after treatment (Fig. 1c).
`To address the therapeutic potential of this systemic RNAi
`approach, we evaluated the pharmacokinetics, efficacy and safety
`of SNALP-formulated siApoB-2 in cynomolgus monkeys. We first
`determined the circulating half-life of SNALP–siApoB-2 in plasma
`samples collected from cynomolgus monkeys (n ¼ 2) receiving a
`single 2.5 mg kg21 intravenous (i.v.) injection of the siRNA. An
`elimination half-life of 72 min was measured for the siRNA (Sup-
`plementary Fig. 2), compared with a 38-min half-life in mice
`(Supplementary Fig. 3a).
`To evaluate efficacy, cynomolgus monkeys were treated with saline
`or SNALP-formulated siApoB-2 at doses of 1 or 2.5 mg kg21 (n ¼ 6
`Figure 3 | Phenotypic effects of RNAi-mediated silencing of APOB mRNA in
`per group). siApoB-2 treatment was associated with a clear and
`non-human primates. a–d, Serial plasma samples were obtained from
`statistically significant dose-dependent gene-silencing effect on
`cynomolgus monkeys treated with saline or 1 or 2.5 mg kg21 SNALP–
`cynomolgus liver APOB mRNA. Forty-eight hours after treatment,
`APOB mRNA was reduced by 68 ^ 12% (mean ^ s.d., n ¼ 4,
`siApoB-2, and measured for ApoB-100 (a), total serum cholesterol (b), LDL
`P ¼ 0.004) and 90 ^ 1% (n ¼ 4, P ¼ 0.002) for the 1 mg kg21 and
`(c) and HDL (d) levels. Data show levels as a percentage of pre-dose values
`and are expressed as mean ^ s.d. Data sets collected at 0, 12, 24 and 48 h
`2.5 mg kg21 groups, respectively (Fig. 2a). Gene silencing was found
`have a group size of six, and data sets collected at later time points have a
`to be consistent across the liver and correlated with detectable tissue
`group size of two. Data points marked with asterisks are statistically
`levels of siApoB-2 (Supplementary Fig. 4). We also confirmed this
`significant compared with saline-treated animals (*P , 0.05, **P , 0.005;
`APOB mRNA silencing to be mediated by RNAi, as demonstrated by
`ANOVA).
`© 2006 Nature Publishing Group
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`after 11 days, which prevented full evaluation of the time course for
`RNAi-mediated effects. Although further optimization of treatment
`regimen and safety profile characterization may be required, our data
`suggest that systemic delivery of siRNAs for targeting hepatocyte-
`specific genes in a higher species is possible. Furthermore, the rapid
`and long-lasting silencing of APOB using RNAi may represent a new
`strategy for reducing LDL–cholesterol in several relevant clinical
`settings.
`
`METHODS
`Additional details of the methods used are provided in the Supplementary
`Information.
`siRNA formulation. The SNALP formulation contained the lipids 3-N-[(q -
`methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine
`(PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),
`1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a
`2:40:10:48 molar per cent ratio.
`In vivo experiments. Saline and siRNA preparations were administered by tail
`vein injection under normal pressure and low volume (0.01 ml g21) for all
`rodent experiments. Cynomolgus monkeys (n ¼ 6 per group) received either
`2 ml kg21 phosphate buffered saline or 1 or 2.5 mg kg21 SNALP–siApoB-2 at a
`dose volume of 1.25 ml kg21 as bolus i.v. injections via the saphenous vein. For
`mRNA measurements, three liver biopsies per lobe were collected 48 h (n ¼ 4)
`or 264 h (n ¼ 2) after siRNA administration.
`Bioanalytical methods. The QuantiGene assay (Genospectra) was used to
`quantify reduction in APOB mRNA levels relative to the housekeeping gene
`GAPDH in lysates prepared from mouse liver or cynomolgus monkey liver and
`jejunum as previously described6 but with minor variations. Mouse6 and
`cynomolgus monkey ApoB-100 protein levels were quantified by enzyme-linked
`immunosorbent assay (ELISA). LDL and HDL lipoprotein content were
`determined for plasma samples (250 m l) as described previously6.
`Statistical analysis. P-values were calculated for comparison of SNALP–siApoB-
`2-treated animals with saline-treated animals using analysis of variance
`(ANOVA, two-factor without replication) with an alpha value of 0.05. P-values
`less than 0.05 were considered significant.
`
`Received 12 January; accepted 6 March 2006.
`Published online 26 March 2006.
`
`2.
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`3.
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`4.
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`silencing complex (RISC), the mitotic state of hepatocytes and/or the
`tissue stability of the siRNA.
`The expected biological effects resulting from APOB mRNA
`silencing include reduction in the blood levels of ApoB-100 protein,
`total cholesterol and LDL. To evaluate the kinetics of these down-
`stream effects, we analysed plasma sampled serially from individual
`monkeys before and during the 11-day time course of the single-dose
`siApoB-2 study. Plasma ApoB-100 protein levels were reduced as
`early as 12 h after administration of 1 or 2.5 mg kg21 SNALP–
`siApoB-2, reaching nadirs of 35 ^ 2% and 22 ^ 9% of pre-treat-
`ment levels, respectively, 72 h after treatment (Fig. 3a). Animals that
`received the higher siRNA dose maintained a marked reduction in
`ApoB protein between 2 and 11 days after treatment, consistent with
`the lasting effect on mRNA silencing. Monkeys that received the
`lower siRNA dose showed an intermediate degree of ApoB protein
`reduction that returned to pre-dose levels by day 11, consistent with
`the observed recovery in APOB mRNA.
`Serum cholesterol
`levels were similarly reduced, in a dose-
`dependent manner and with comparable kinetics (Fig. 3b). The
`maximum cholesterol reduction of 62 ^ 5.5% (n ¼ 2, P ¼ 0.006)
`observed for the high dose siRNA group would be considered
`clinically significant for patients with hypercholesterolaemia,
`and exceeds levels of cholesterol reduction reported clinically for
`currently approved cholesterol-lowering drugs.
`Administration of SNALP–siApoB-2 also resulted in dramatic and
`rapid dose-dependent reduction in the ApoB-containing lipoprotein
`particle LDL. Reduction in LDL relative to pre-dose levels was
`observed as early as 24 h after treatment for both doses of SNALP–
`siApoB-2 (Fig. 3c). In contrast, there were no significant changes in
`circulating levels of the non-ApoB-containing high-density lipopro-
`tein particle (HDL, Fig. 3d). The reduction in LDL persisted over the
`11-day study for both siApoB-2 treatment groups, with a maximum
`82 ^ 7% decrease compared to pre-treatment levels observed for the
`high-dose group at day 11 (n ¼ 2, P ¼ 0.003). The time required for
`the biological effects to return to pre-dose levels was not determined
`for the high-dose group because the endpoint for this study was
`defined using rodent data, which indicated a faster rate of recovery.
`The rapid onset and lasting effect on lipoprotein metabolism suggest
`that siRNAs targeting APOB may be a valuable therapeutic strategy
`for achieving plaque stabilization in acute coronary syndromes10,11,
`as HMG-CoA reductase inhibitors can require up to 4–6 weeks to
`have the desired clinical effects15.
`An important consideration for the therapeutic application of
`siRNA relates to its general safety, as well as to the safety profile
`associated with specific delivery technologies. General tolerability
`as well as specific toxicities (such as activation of complement,
`coagulation and cytokines) were evaluated for all monkeys in this
`study. We observed no treatment-related effects on the appearance or
`behaviour of animals treated with SNALP–siApoB-2 compared with
`saline-treated animals. There was no evidence for complement
`activation, delayed coagulation, pro-inflammatory cytokine pro-
`duction (Supplementary Table 1) or changes in haematology pa-
`rameters (data not shown), toxicities that have been observed
`previously with treatments using related approaches16–19. Across a
`systematic evaluation, the only detected change in primates treated
`with SNALP–siApoB-2 was a transient increase in liver enzymes in
`monkeys that received the high dose of SNALP–siApoB-2. The
`observed transaminosis peaked 48 h after treatment and was highly
`variable across individual animals. These effects, which were
`observed only at the highest dose of SNALP–siApoB-2, were com-
`pletely reversible, with normalization by day 6 notwithstanding
`continued biological efficacy.
`Our study highlights the potential for therapeutic gene silencing
`using systemic RNAi in non-human primates. A single, low dose of
`APOB-specific siRNA resulted in rapid and lasting RNAi-mediated
`gene silencing, with associated and profound phenotypic changes.
`The study was limited by the premature termination of the protocol
`© 2006 Nature Publishing Group
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`5.
`
`6.
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`17. Chonn, A., Cullis, P. R. & Devine, D. V. The role of surface charge in the
`activation of the classical and alternative pathways of complement by
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`19.
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`Supplementary Information is linked to the online version of the paper at
`www.nature.com/nature.
`
`Acknowledgements We are grateful to P. Sharp, J. Maraganore and
`N. Mahanthappa for their assistance and support in this study. We would also
`like to thank W. J. Schneider, J. Frohlich, M. Hayden and J. E. Vance for
`
`discussions. We acknowledge the technical assistance of C. Woppmann and
`A. Wetzel, and thank V. Kesavan and G. Wang for preparation of the
`cholesterol-conjugated siRNA used in this study. Finally, we thank S. Young for
`providing anti-ApoB antibodies. This work was supported by grants from the
`National Science and Engineering Research Council of Canada (to A.J.W. and
`M.N.F.).
`
`Author Contributions This work represents the outcome of a collaboration
`between scientists at Alnylam Pharmaceuticals and Protiva Biotherapeutics Inc.
`
`Author Information Reprints and permissions information is available at
`npg.nature.com/reprintsandpermissions. The authors declare competing
`financial interests: details accompany the paper at www.nature.com/nature.
`Correspondence and requests for materials should be addressed to T.S.Z.
`(tzimmermann@alnylam.com) or I.M. (ian@protivabio.com).
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`Supplementary Information: Methods
`“RNAi-mediated Gene Silencing in Non-Human Primates”
`Zimmermann. T.S. et al.
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`Supplementary Methods
`
`Synthesis of siRNAs. The siRNAs used in this study each consisted of a 21 nucleotide
`
`(nt) sense strand and a 23 nt antisense strand resulting in a single 2 nt overhang at the 3'-
`
`end of the antisense strand of the annealed duplex. siApoB-1 (position 10167-10187,
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`NM_000384) sense: 5'-GUCAUCACACUGAAUACCAAU-3', antisense: 5'-
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`AUUGGUAUUCAGUGUGAUGACAC-3', siApoB-MM sense: 5’-
`
`GUGAUCAGACUCAAUACGAAU-3’, antisense: 5’-
`
`AUUCGUAUUGAGUCUGAUCACAC-3’. The sequence and synthesis of Chol-
`siApoB-1 are as previously described1. siApoB-2 (position 2098-2118, NM_000384)
`sense: 5’-GGAAUCuuAuAuuuGAUCcA*A-3’, antisense: 5’-
`
`uuGGAUcAAAuAuAAGAuUCc*c*U-3’. 2’O-Methyl modified nucleotides are in
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`lower case and phosphorothioate linkages are represented by asterisks. Sense and
`antisense strands for siApoB-2 were synthesized as described previously1 except that
`N4-Acetylcytidine phosphoramidites were used for cytidine residues. These
`oligonucleotides were characterized by ESMS and anion-exchange HPLC. siRNAs
`
`were generated by annealing equimolar amounts of complementary sense and antisense
`
`strands.
`
`Encapsulation of siRNA. siRNAs were encapsulated by an adaptation of the method
`of Jeffs et al2. The SNALP formulation contained the lipids PEG-C-
`DMA:DLinDMA:DSPC:Cholesterol (2:40:10:48 molar percent). 3-N-[(ω-Methoxy
`poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA)
`MW 2524 and 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) MW 616
`were prepared by Protiva Biotherapeutics3, 1,2-Distearoyl-sn-glycero-3-phosphocholine
`(DSPC) MW 790 was obtained from Avanti Polar Lipids (Alabaster, AL), and
`
`cholesterol MW 387 was obtained from Sigma (Oakville, ON). The particle sizes of the
`
`SNALP samples used in this study were 77-83 nm with a polydispersity range of 0.09-
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`0.15. Nucleic acid encapsulation efficiencies were 92-97%. Particle size was
`
`determined using a Malvern Instruments Zetasizer 3000HSA (Malvern, UK) and
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`nucleic acid encapsulation efficiency was determined using the Ribogreen assay as
`described elsewhere3.
`
`In vivo rodent experiments. All siRNAs were administered via tail vein injection
`
`under normal pressure and at a dosing volume of 0.01 ml/g. Female 8-10 week old
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`C57BL/6 mice (Charles River Laboratories, MA) were used for the Chol-siApoB-1 and
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`SNALP siApoB-1 dose response experiments. For the Chol-siApoB-1 experiment mice
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`received either saline or Chol-siApoB-1 at doses of 100, 50, 25 or 12.5 mg/kg and for
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`the SNALP siApoB-1 dose response mice received either saline or SNALP siApoB-1 at
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`siRNA doses of 1, 0.5, 0.25 or 0.1 mg/kg. Liver mRNA levels were assessed 72 h after
`
`injection. For determination of specificity of apoB knockdown, five week old female
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`BALB/c mice (Harlan Labs, IN) received either saline, SNALP siApoB-1 or SNALP
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`siApoB-MM at a siRNA dose of 1 mg/kg or empty SNALP vehicle at an equivalent
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`lipid dosage (25 mg/kg). Animals were sacrificed two days after treatment for liver
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`mRNA and serum apoB-100 protein measures. For the dose response and duration of
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`effect experiments, eight week old female C57BL/6 mice (Charles River Laboratories,
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`MA) received either saline or SNALP siApoB-2. Liver mRNA levels were determined
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`72 h after injection of 5, 2.5, 1 or 0.5 mg/kg SNALP siApoB-2 for the dose response
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`experiment. For the duration of effect experiment, animals received either saline or 2.5
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`mg/kg SNALP siApoB-2 and were anesthetized using isofluorine for collection of
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`serum samples by retro-orbital bleed pre-dose (d0) and 3, 6, 9 and 13 days post-dose for
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`measurement of apoB-100 protein. All procedures used in animal studies conducted at
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`Alnylam were approved by the Institutional Animal Care and Use Committee (IACUC)
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`and were consistent with local, state, and federal regulations as applicable. Animal
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`studies conducted at Protiva were performed under the oversight of Protiva’s
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`Institutional Animal Care and Use Committee (IACUC) in accordance with the
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`Canadian Council on Animal Care guidelines.
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`In vivo non-human primate experiment. For determination of the rate of plasma
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`clearance of SNALP siApoB-2 in cynomolgus monkeys, two animals received 2.5
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`mg/kg SNALP siApoB-2 via bolus i.v. injection in the saphenous vein and plasma
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`samples were collected 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post-dose. In addition, blood
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`samples were taken pre-dose, 6 and 24 h post-dose for Interferon-γ and IL-6 measures
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`and liver samples (three punch biopsies per lobe, twelve total) were collected 24 h post-
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`dose for determination of siRNA distribution and apoB mRNA levels. Eight jejunum
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`sections were isolated from animals treated with saline, 1 or 2.5 mg/kg SNALP siApoB-
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`2 48 h (n = 4) or 264 h (n = 2) post-dose for mRNA measurements. Plasma samples
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`from animals treated with saline, 1 or 2.5 mg/kg SNALP siApoB-2 were collected pre-
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`dose, 12, 24 and 48 h post-dose for all animals and 72, 96, 144, 192 and 264 h post-dose
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`for two animals per group for apoB-100 protein measurements. Animals were fasted for
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`16 h prior to blood sampling for total serum cholesterol and lipoprotein collections pre-
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`dose, 24 and 48 h post-dose for all animals and 144 and 264 h post-dose for two animals
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`per group. Blood samples were collected pre-dose, 0.25 and 48 h post-dose for
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`determination of complement Bb, CH50 and activated partial thromboplastin time
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`(APTT) (n = 6 per treatment group) and pre-dose, 24, 48 (n = 6 per treatment group),
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`144 and 264 h post-dose (n = 2 per treatment group) for determination of alanine
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`aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and urea nitrogen
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`levels. All procedures using cynomolgus monkeys were conducted by a certified
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`contract research organization using protocols consistent with local, state, and federal
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`regulations as applicable and approved by the Institutional Animal Care and Use
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`Committee (IACUC).
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`In vivo bioanalytical methods for non-human primate experiments. For
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`determination of the circulation half-life of SNALP siApoB-2 in primates, 30 µl plasma
`(one per animal and time point) was incubated at 42oC for 20 min in a buffer containing
`1.7 mg/ml proteinase K, 0.1 M Tris-Cl pH 7.5, 12.2 mM EDTA, 0.15 M NaCl, 1%
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`SDS, 4.2 µM 40mer RNA internal standard in a 96-well plate. Samples were spin-
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`filtered using a 0.2 µm filter plate and the filter was washed two times with 30 µl water
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`(Varian, CA). Washes were combined with the initial filtrate and analyzed by ion
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`exchange HPLC under denaturing conditions at pH 8 and 80°C with detection at 260
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`nm. Under these conditions, the siRNA eluted as two well-separated single strands.
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`The internal standard, used for normalization of small differences in recovered filtrate
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`volume, eluted at a later time than the two single strands of the siRNA. The amount of
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`full length sense and antisense strand was determined relative to an external calibration
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`curve derived from SNALP-formulated siApoB-2 and was calibrated over a linear range
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`of 5-200 pmoles RNA. An estimated constant of 40 ml plasma per kg animal weight
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`was used for calculation of the total amount of siRNA and the percent injected dose for
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`each time point was calculated as the ratio of the amount of the sense or antisense strand
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`to the amount of injected siRNA. Each data point represents the group mean ± s.d.
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`The QuantiGene® assay (Genospectra, CA) was performed to quantitate
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`reduction of apoB mRNA relative to the house keeping gene GAPDH in lysates
`prepared from mouse liver or cynomolgus liver and jejunum as previously described1
`with minor variations. For detection of cynomolgus mRNA, the apoB probe set was
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`specific to human apoB (positions 13870 to 14110, NM_000384) and cross-reactive to
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`the Macaque fascicularis apoB sequence (positions 380 to 615, CO775384) and the
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`GAPDH probe set was specific for human GAPDH (positions 224 to 444, NM_002046)
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`and cross-reactive to M. fasciularis GAPDH (positions 142 to 362, AB158631).
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`siRNA distribution in cynomolgus liver samples was detected using a
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`ribonuclease protection assay with a radiolabeled probe complementary to the antisense
`strand of siApoB-2 using methods previously described1. The assay was performed on
`total RNA isolated from each of the twelve liver biopsies.
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`5’ RACE analysis was performed using total RNA (5 µg) from pooled liver
`biopsies (4 biopsies, 1 per lobe) as described previously1. Ligated RNA was reverse
`transcribed using a gene specific primer (GSP: 5’-
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`TGGAAGAAGTTGGTGTTCATCTGGA-3’). Two rounds of PCR were performed.
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`First round PCR using primers complementary to the RNA adaptor (GeneRacer,
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`Invitrogen, CA) and apoB mRNA (Rev1: 5’-TCTTTGGTATAGCCAAAGTGGTCCA-
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`3’). Second round PCR using nested primers complementary to the RNA adaptor and
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`apoB mRNA (Rev2: 5’-AAAGCTTTGTTGACACTGTCTGGGAA-3’). The cleavage
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`site at 2108/2109 of the apoB mRNA (NM_000384) was confirmed by sequencing of
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`the PCR products.
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`Cynomolgus apoB-100 was detected from plasma samples using a sandwich
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`ELISA consisting of a polyclonal goat anti-human apoB capture antibody (Chemicon
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`International, CA) and a horseradish peroxidase-conjugated goat anti-human apoB-100
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`polyclonal detection antibody (Academy Bio-Medical Company, TX).
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`In vivo rodent PK experiment. Radiolabeled SNALP was prepared for plasma
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`clearance and tissue distribution studies by incorporation of 2.7 µCi/mg total lipid of the
`non-exchangeable lipid label 3H-CHE4. SNALP was administered at a siRNA dose of 2
`mg/kg via lateral tail vein injection in eight week old female BALB/c mice (Harlan
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`Labs, IN) and blood was collected via tail vein nick over a 24 h period. At 24 h after
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`injection, mice were euthanized and harvested tissues were homogenized in FastPrep
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`Lysing Matrix Tubes (MP Biomedicals, CA) containing distilled water. Tissue
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`homogenates were assayed for radioactivity by liquid scintillation counting with
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`Picofluor 40 and whole blood was assayed using Picofluor 15 (Perkin-Elmer, Boston,
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`MA).
`
`1. Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic
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`administration of modified siRNAs. Nature 432, 173-8 (2004).
`
`2. Jeffs, L. B. et al. A scalable, extrusion-free method for efficient liposomal
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`encapsulation of plasmid DNA. Pharm. Res. 22, 362-72 (2005).
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`3. Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I. Cationic lipid saturation
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`influences intracellular delivery of encapsulated nucleic acids. J. Control Release 107,
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`276-87 (2005).
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`4. Stein, Y., Halperin, G. & Stein, O. Biological stability of [3H]cholesteryl oleyl ether
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`in cultured fibroblasts and intact rat. FEBS Lett. 111, 104-6 (1980).
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`Supplementary Information: Figures 1-6 and Table 1
`“RNAi-Mediated Gene Silencing in Non-Human Primate”
`Zimmermann, T.