4788–4797 Nucleic Acids Research, 2010, Vol. 38, No. 14
`doi:10.1093/nar/gkq206
`
`Published online 31 March 2010
`
`The siRNA sequence and guide strand overhangs
`are determinants of in vivo duration of silencing
`
`Walter R. Strapps*, Victoria Pickering, Gladys T. Muiru, Julie Rice, Stacey Orsborn,
`Barry A. Polisky, Alan Sachs and Steven R. Bartz
`
`Sirna Therapeutics Inc., A Wholly Owned Subsidiary of Merck & Co., Inc., 1700 Owens Street, San Francisco,
`CA 94158, USA
`
`Received September 3, 2009; Revised March 10, 2010; Accepted March 11, 2010
`
`ABSTRACT
`
`The use of short interfering RNAs (siRNA) in animals
`for target validation or as potential therapeutics is
`hindered by the short physical half-life when de-
`livered as unencapsulated material and in turn the
`short active half-life of siRNAs in vivo. Here we dem-
`onstrate that the character of the two 30-overhang
`nucleotides of the guide strand of siRNAs is a de-
`terminant of the duration of silencing by siRNAs
`both in vivo and in tissue culture cells. We demon-
`strate that deoxyribonucleotides in the guide strand
`overhang of siRNAs have a negative impact on
`maintenance of both the in vitro and in vivo activity
`of siRNAs over
`time. Overhangs that contain
`ribonucleotides or 20-O-methyl modified nucleotides
`do not demonstrate this same impairment. We also
`demonstrate that the sequence of an siRNA is a
`determinant of the duration of silencing of siRNAs
`directed against the same target even when those
`siRNAs have equivalent activities in vitro. Our
`experiments have determined that a measurable
`duration parameter exists, distinct
`from both
`maximum silencing ability and the potency of
`siRNAs. Our
`findings provide information on
`incorporating chemically modified nucleotides into
`siRNAs for potent, durable therapeutics and also
`inform on methods used to select siRNAs for thera-
`peutic and research purposes.
`
`INTRODUCTION
`
`Over the past quarter century, several generations of
`potential therapeutic nucleic acid technologies have been
`brought forward for clinical development. These include
`antisense oligonucleotides, ribozymes, aptamers and now
`siRNAs. The ubiquitous presence of nucleases in biologic-
`al settings and consequent physical instability of RNA and
`DNA outside cells has led most nucleic acid drug
`
`developers to pursue chemical modification of the active
`nucleic acid entity. These modifications—either to the
`phosphodiester backbone, the base, or the sugar—can
`result in dramatic improvement in physical stability in
`animal or human serum (1,2). However, naked siRNAs,
`even when extensively chemically modified and highly
`active in vitro, have been shown to have unacceptable
`pharmacokinetic properties in vivo, being rapidly cleared
`through the kidney in rodents (3). These properties
`severely limit the use of siRNA for systemic applications
`without a more sophisticated mode of delivery. A variety of
`delivery technologies that minimize renal clearance have
`been explored,
`including conjugation of ligands to the
`siRNA to facilitate cellular uptake (4), polymeric cages to
`encapsulate siRNA and lipid-based nanoparticles, which
`are designed to be taken up into endosomes and subse-
`quently escape, releasing their cargo into the cytoplasm
`where the siRNA responsive machinery is located.
`Recent approaches with siRNA given systemically have
`utilized sugar-modified siRNAs encapsulated in lipid-
`based nanoparticles to efficiently deliver siRNA to thera-
`peutic targets of interest in the liver (5,6). An important
`question raised by these studies was the interplay between
`chemical modification of the siRNA and the type of
`delivery vehicle
`in determining efficiency of
`target
`knockdown in vivo. For example, if a siRNA is efficiently
`encapsulated and protected from serum nucleases, does
`chemical modification have an impact on duration of
`silencing in vivo? Also, siRNAs have been shown to
`trigger unwanted inflammatory responses in vitro and
`in vivo via interaction with toll-like receptors (TLRs) or
`other pattern recognition receptors (7). These receptors
`are located on plasma or endosomal membranes as well
`as in the cytoplasm where they monitor the presence of
`pathogen-derived macromolecules. Do chemical modifica-
`tions that abrogate activation of these receptors (5,8) also
`have an impact on duration of silencing in vivo?
`Previous reports have indicated both positive and
`negative effects of patterns of chemical modification on
`specific properties of siRNAs such as resistance to serum
`nucleases, reduction of target mRNA target levels and
`
`*To whom correspondence should be addressed. Tel: +1 415 814 8423; Fax: +1 415 552 5499; Email: walter_strapps@merck.com
`
`ß The Author(s) 2010. Published by Oxford University Press.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
`by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Alnylam Exh. 1084
`
`

`

`induction of cytokines (2–4,7). The experiments reported
`below focused specifically on the two 30-overhang nucleo-
`tides of the guide strand of the siRNA to examine the
`effect of chemically modified nucleotides on the silencing
`and duration of silencing both in vitro and in vivo.
`Within the field,
`the original motivations for using
`deoxythymidine nucleotides as the overhang nucleotides
`in siRNAs were related to cost, availability and ease of
`synthesis of the siRNAs when utilizing these nucleotides,
`as well as increased stability of the oligos to serum nucle-
`ases (9). In addition, the authors reported no detectable
`loss of activity of the siRNAs that was dependent upon
`the character of the nucleotides as long as the length of the
`overhang was two nucleotides. However, other reports
`have demonstrated that the character of the 30-overhang
`of the guide strand is a determinant of strand selection
`(10), and that the 30-overhang on the guide strand inter-
`acts with the PAZ domain of RISC in the RNA-binding
`pocket (11), suggesting that the overhang does in fact have
`a possible contribution to siRNA activity. In contradic-
`tion to the original observation that deoxythymidine nu-
`cleotides in the guide strand overhangs have little negative
`consequence on silencing, other reports have made the
`observation that these overhangs can reduce maximal
`silencing activity of siRNAs (12,13).
`We demonstrate that the nucleotide content of the guide
`strand overhang appears to have little or no effect on
`maximum silencing in vitro. However, we also show that
`minor differences in the character of the overhang nucleo-
`tides of a siRNA guide strand have a profound effect on
`the duration of silencing. Finally, we demonstrate that
`siRNAs that have very similar capacities for reduction
`of mRNA levels in tissue culture cells and in vivo have
`distinct durations of silencing that are appear dependent
`upon the composition of the siRNA.
`
`MATERIALS AND METHODS
`
`Design of siRNAs
`
`The siRNA sequences used in this article were designed
`using a previously described algorithm (14) developed to
`predict silencing efficacy in unmodified form with two
`deoxythymidine nucleotides as the overhang.
`The four siRNA sequences used for the in vivo studies
`have the following sequences (all in the 50–30 direction)—
`where XX indicates the various guide strand overhangs
`described in this work; Seq1 Passenger CUCUCACAUA
`CAAUUGAAATT, Seq1 Guide UUUCAAUUGUAUG
`UGAGAGUUXX; Seq25 Passenger CUCCUAUAAUG
`AAGCAAAATT, Seq25 UUUUGCUUCAUUAUAGG
`AGUUXX; Seq37 Passenger CUUUAACAAUUCCUG
`AAAUTT, Seq37 Guide AUUUCAGGAAUUGUUAA
`AGUUXX; Seq40 Passenger UCAUCACACUGAAUA
`CCAATT; Seq40 Guide UUGGUAUUCAGUGUGAU
`GAUUXX. All
`other
`siRNA sequences
`are
`in
`Supplementary Table S1.
`
`Cells and reagents
`
`The mouse hepatoma Hepa 1-6 cell line was obtained from
`the American Type Tissue Collection (Cat # CRL-1830).
`
`Nucleic Acids Research, 2010, Vol. 38, No. 14 4789
`
`Cells were grown in Dulbecco’s modified Eagle’s medium
`(Mediatech, Cat #10-013-CV) with 4 mM L-glutamine
`adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l
`glucose and supplemented with 10% of
`fetal bovine
`serum, 100 mg/ml of streptomycin and 100 U/ml penicillin.
`Cells were cultured at 37C in the presence of 5% CO2.
`
`Preparation of synthetic siRNAs (in vitro experiments)
`
`SiRNAs for the in vitro experiments were ordered from
`Sigma-Aldrich. A total of 40 sequences were ordered, each
`being synthesized with four different guide strand over-
`hangs; ribonucleotides complementary to positions 1
`and 2 in the target mRNA (rN-rN), 20-O-methylated
`complementary nucleotides
`(oN-oN), deoxythymidine
`overhangs (dT-dT) and 20-O-methyl-uridine overhangs
`(oU-oU).
`
`Preparation of synthetic siRNAs (in vivo experiments)
`
`The siRNAs used in the in vivo studies were synthesized by
`methods previously described (15). For each oligonucleo-
`tide, the two individual, complementary strands of the
`siRNA were synthesized separately using solid phase syn-
`thesis, then purified separately by ion exchange chroma-
`tography. The complementary strands were annealed to
`form the double strand siRNA (duplex). The duplex was
`then ultrafiltered and lyophilized to form the solid drug
`substance. The duplex material was tested for the presence
`of endotoxin by standard methods.
`
`Preparation of siRNA–lipid nanoparticle complex
`
`Lipid nanoparticles (LNPs) were made using the cationic
`lipid CLinDMA (2-{4-[(3b)-cholest-5-en-3-yloxy]-butoxy}-
`N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-
`propan-1-amine),
`cholesterol,
`and
`PEG-DMG
`(monomethoxy(polyethyleneglycol)-1,2-dimyristoylglycerol)
`in 50.3 : 44.3 : 5.4 molar ratio. siRNAs were incorporated
`in the LNPs with high encapsulation efficiency by mixing
`siRNA in buffer into an ethanolic solution of the lipid
`mixture, followed by a stepwise diafiltration process.
`Cholesterol was purchased from Northern Lipids,
`PEG-DMG was purchased from NOF Corporation and
`CLinDMA was synthesized by Merck and Co. The encap-
`sulation efficiency was determined using a SYBR Gold
`fluorescence assay and the particle size measurements
`were performed using a Wyatt DynaPro plate reader.
`The siRNA and lipid concentrations in the LNP were
`quantified by a HPLC method, developed in house,
`using a PDA detector.
`
`Transfection of siRNAs for screening
`
`Cells were plated in 96-well plates at 5000 cells/well and
`transfected at a final siRNA concentration of 10 nM using
`RNAiMax (Invitrogen, Cat #13778150) according to the
`manufacturers specifications. Cells were lysed 24 h after
`transfection.
`RNA was isolated by preparing lystates from the cells
`on a Biomek FX liquid handler using Cells-to-Ct Bulk
`Lysis and Stop Reagents (ABI, Cat #4391851C) according
`to the manufacturer’s instructions.
`
`

`

`4790 Nucleic Acids Research, 2010, Vol. 38, No. 14
`
`In vitro duration assay (RNAiMax transfection)
`
`Taqman data analysis
`
`A plate for transfection of the cells was prepared such that
`each siRNA was represented in six different random loca-
`tions on the plate. This was done to address concerns
`about well-to-well variability in transfection and/or
`growth of cells over
`the long time course of
`the
`experiment.
`Cells were plated in 96-well plates at 1200 cells/well and
`transfected at a final siRNA concentration of 10 nM using
`RNAiMax (Invitrogen, Cat #13778150) according to the
`manufacturers specifications. Cells were lysed 24, 48, 72,
`96 and 120 h after transfection.
`
`Transfection of LNP formulated siRNAs into tissue
`culture cells
`
`Transfections of LNPs were performed in six replicates in
`two replicate experiments (three replicates/experiment).
`Cells were plated in 96-well plates at 3500 cells/well. The
`final concentration of siRNA in each well was 120 nM.
`The time of incubation with the LNP-siRNA complexes
`was 24 and 120 h. Media change was performed every 48 h
`from the time cells were plated by replacing 75 ml with
`fresh complete growth culture media until the 120 h time
`point.
`RNA was isolated by preparing lystates from the cells
`on a Biomek FX liquid handler using Cells-to-Ct Bulk
`Lysis and Stop Reagents (ABI, Cat #4391851C) according
`to the manufacturer’s instructions.
`
`RNA isolation from in vivo study samples
`
`C57BL/6 mice were dosed with 3 mg/kg siRNA (in LNP)
`and sacrificed at various time points. Cohorts for controls
`and each siRNA tested consisted of five animals.
`Blood and liver samples were collected immediately fol-
`lowing euthanasia.
`Total RNA was isolated from liver tissue using the
`RNeasy 96 Tissue Kit for high-throughput 96-well RNA
`minipreps (Qiagen, Cat #74881) and a QIAvac 96 vacuum
`manifold according to the manufacturer’s instructions. All
`RNA samples were treated with DNase I (Qiagen, Cat
`#79254) on column for 15 min at room temperature.
`Final RNA eluted was quantified and normalized to a
`concentration of 50 ng/ml.
`
`Reverse transcription and PCR
`cDNA was generated from lysates and RNA in a 20 ml
`reaction using reverse transcription reagents from the
`Ambion Cells-to-Ct Kit
`(Applied Biosystems, Cat
`#4368813) according to the manufacturer’s instructions.
`On an ABI 7900 HT real-time PCR System, quantitative
`real-time PCR was carried out
`in a 384-well plate.
`Reactions were set up in duplicate and one well was
`probed with the Apob Taqman reagents, the other with
`the GAPDH Taqman reagent in a final volume of 10 ml
`using TaqMan Gene Expression Master Mix (Applied
`Biosystems, Cat #4370074). All Taqman probes and
`primers were supplied as prevalidated sets by Applied
`Biosystems: mouse GAPDH, Cat #4352339E; mouse
`Apob, Assay ID Mm01545154_g1.
`
`The Taqman data were analyzed by standard methods on
`an ABI 7900 instrument. Within each experiment, the
`baseline was set in the exponential phase of the amplifica-
`tion curve, and based on the intersection point of the base-
`lines with the amplification curve; a Ct value is assigned by
`the instrument. The expression level of the gene of interest
`and percentage knockdown was calculated using com-
`parative Ct method:
` Ct ¼ CtTarget CtGAPDH
` Ct ¼ CtðTarget siRNAÞ CtðNTCÞ
`Relative expression level ¼ 2
` Ct
` CtÞ
`% KD ¼ 100  ð1 2
`
`The mRNA knockdown was calculated relative to a
`non-targeting control siRNA in each experiment.
`
`Cytokine quantitation
`
`Cytokine levels were measured in mouse serum using the
`SearchLight Mouse IR Cytokine Array, 12-plex assay
`(Thermo Fisher Scientific, Waltham, MA) according
`to the manufacturer’s instructions. Data were acquired
`with the Odyssey Infrared Imaging System (LI-COR
`Biosciences,
`Lincoln, NE)
`and
`analyzed with
`SearchLight Array Analyst Software.
`
`Statistical analysis
`
`Unless otherwise noted, datasets were compared using a
`two-tailed Student’s t-test to generate P-values. Also,
`unless otherwise noted, statistical analyses were performed
`using the data expressed as log2-fold change rather than in
`percent expression or percent knockdown of the mRNA
`transcript, as the logarithm of Taqman error is uniformly
`distributed, and the logarithm of
`target-normalized
`siRNA silencing is normally distributed.
`
`Calculation of retention rate of silencing
`
`To calculate the retention rate of silencing both in vitro
`and in vivo, we converted the percent knock-down values
`at the relevant timepoints to log2-fold change (ddCt
`values from Taqman), subtracted the early timepoint
`value from the later timepoint value and divided by the
`number of days between the two timepoints. This gave us
`a value for the fraction of silencing seen at the first
`timepoint
`that was lost per day up to the second
`timepoint.
`
`RESULTS
`
`In vitro efficacy of chemically modified siRNAs
`
`In this study, we examined the effects of varying the over-
`hanging nucleotides of the guide strand of a standard
`21 nucleotide duplex siRNA in several assays. We chose
`to examine four different guide strand overhangs. We
`selected deoxythymidime overhangs because they are the
`de facto industry standard (9), ribonucleotides which
`are complementary to positions 1 and 2 relative to
`
`

`

`the target site in the mRNA as those would form the
`overhang if
`the siRNA being examined were to be
`produced naturally within a cell, and 20-O-methyl
`modified nucleotides, either uridines to correspond to
`the thymidines, or complementary nucleotides.
`First, we examined the effect of different guide strand
`overhang modifications on the extent of knockdown of the
`target mRNA in tissue culture cells 24 h post-transfection.
`We compared overhangs of ribonucleotides complemen-
`tary to positions 1 and 2 in the target mRNA (rN-rN),
`20-O-methyl modified versions of those complementary
`nucleotides
`(oN-oN),
`dexoxythymidine
`overhangs
`(dT-dT) and to 20-O-methyl-uridine (oU-oU) overhangs.
`In all cases, regardless of the guide strand overhang, the
`passenger strand overhang was maintained as dT-dT to
`experimentally isolate the effect of
`the guide strand
`overhang. Forty siRNA sequences directed against the
`murine Apob gene were synthesized with these four
`guide strand overhangs and tested in Hepa1-6 cells, a
`mouse hepatoma-derived cell
`line, for their ability to
`reduce the level of Apob mRNA.
`The average degree of knockdown seen with the dT-dT
`overhangs was 97.7 ± 1.8%; with oU-oU, 97.0 ± 4.4%;
`with oN-oN, 98.3 ± .4; and with rN-rN, 97.2 ± 2.3%.
`These data can be found in Supplementary Table S1.
`This similarity in knockdown between dT-dT overhangs
`and rN-rN overhangs is consistent with previously pub-
`lished reports on the activity of siRNA duplexes with
`deoxyribonucleotides
`in guide strand overhangs
`(9)
`though in disagreement with other published articles
`which showed a detrimental effect of dT-dT overhangs
`compared specifically to rN-rN guide strand overhangs
`(12) or to rU-rU overhangs (13). To compare the data
`statistically, we converted the knockdown results to log2-
`fold change to obtain a normal distribution of variance
`across silencing levels allowing us to perform standard
`parametric statistics. The data showed no systematic dif-
`ference in the ability of siRNAs with these four different
`guide strand overhangs to reduce the target mRNA level
`in tissue culture cells at 24 h post-transfection (Student’s t-
`test: oU-oU versus dT-dT, P = 0.20; rN-rN versus dT-dT,
`P = 0.18; oN-oN versus dT-dT, P = 0.12).
`
`In vitro duration assays
`
`In order to assess any differential effect the sequences and
`the guide strand overhangs had on the rate of recovery of
`the expression of the Apob mRNA levels in vitro, we per-
`formed assessments of the levels of mRNA reduction after
`treatment with each of the 40 siRNA sequences in four
`chemistries, for a total of 160 siRNAs each day from 1 to
`5 days post-transfection. These data are summarized in
`Supplementary Table S1.
`We observed that even though all 40 sequences showed
`extremely similar maximum mRNA reductions, the rate at
`which the silencing activity was lost in vitro appeared to
`have an siRNA-specific component as well as an
`overhang-specific component. However, we were unable
`to identify sequence motifs associated with increased or
`decreased duration of silencing in this data set, perhaps
`due to the limited number of sequences.
`
`Nucleic Acids Research, 2010, Vol. 38, No. 14 4791
`
`The data from these duration assays are summarized in
`Figure 1. siRNAs with dT-dT overhangs consistently per-
`formed more poorly over time than siRNAs with any of
`the other
`tested overhangs. We quantified this by
`comparing the knockdown results in log2-fold change
`space at each of the individual timepoints (Figure 1).
`Even by 2 days post-transfection the mean silencing of
`siRNAs with dT-dT overhangs is significantly reduced
`relative to silencing of the same sequences to the oN-oN
`overhang (P = 0.013). The difference is increased by day 3
`and now shows significance to all
`three overahangs
`(to rN-rN P < 0.001; to oU-oU P = 0.003; to oN-oN
`P < 0.001) this significant difference is retained at day 4
`(to rN-rN P < 0.001; to oU-oU P < 0.001; to oN-oN
`P < 0.001) and is slightly reduced by day 5 (to rN-rN
`P = 0.001; to oU-oU P = 0.017; to oN-oN P = 0.01),
`probably attributable to an increasing number of se-
`quences having no activity regardless of the guide strand
`overhang at the latest timepoint.
`
`In vitro dose response curves
`
`To further evaluate the efficacies of siRNAs with different
`guide strand overhangs, we selected four siRNA sequences
`(Seq1, Seq25, Seq37 and Seq40) each with dT-dT, rN-rN,
`oN-oN and oU-oU overhangs. Based on our in vitro ex-
`periments, we expected rN-rN, oN-oN and oU-oU over-
`hangs to behave equivalently. These four sequences were
`selected on the basis of having comparable degrees of
`mRNA reduction in our initial screens regardless of the
`guide strand overhang tested (see Table 1). We also
`determined in our
`subsequent experiments
`that
`the
`sequences demonstrated a range of in vitro durations.
`Seq40 appeared to have the most stable duration in our
`in vitro assays, both Seq25 and Seq37 appeared to be
`among the least durable siRNAs, and Seq1 fell between
`those two extremes. We were interested in differences in
`
`Figure 1. siRNA with different guide strand overhangs were tested
`over a time course in mouse Hepa1–6 cells. Each data point represents
`results from: dT-dT n = 39, oU-oU n = 39, rN-rN n = 40 and oN-oN
`n = 40 unique sequences. Asterisk indicates that the dT-dT data point
`at that particular timepoint has a P-value of <0.05 compared with the
`data points for each of the other three overhangs tested. Data are
`expressed as mean ± SEM.
`
`

`

`4792 Nucleic Acids Research, 2010, Vol. 38, No. 14
`
`Table 1. Comparison of siRNA activities in vitro and in vivo
`
`Sequence and
`overhang
`
`Percentage mRNA
`reduction in vitro
`
`Maximum percentage
`mRNA reduction in vivo
`
`Retention rate
`(in vivo) Days 1 to 7 (%)
`
`Retention rate
`(in vitro) Days 1 to 5 (%)
`
`Time to 50%
`knock-down (days)
`
`Seq1 dT-dT
`Seq1 oU-oU
`Seq1 rN-rN
`Seq25 dT-dT
`Seq25 oU-oU
`Seq25 rN-rN
`Seq37 dT-dT
`Seq37 oU-oU
`Seq37 rN-rN
`Seq40 dT-dT
`Seq40 oU-oU
`Seq40 rN-rN
`
`96.4 ± 2.3
`97.6 ± 1.8
`99.1 ± 0.3
`99.4 ± 0.4
`99.5 ± 0.1
`99.3 ± 0.2
`99.2 ± 0.1
`97.3 ± 1.0
`96.4 ± 0.6
`97.2 ± 1.0
`96.9 ± 1.3
`94.5 ± 1.5
`
`81.4 ± 17.4
`92.0 ± 7.1
`89.2 ± 14.4
`78.0 ± 3.8
`89.3 ± 2.8
`85.7 ± 7.5
`51.0 ± 8.8
`78.5 ± 6.5
`85.9 ± 3.3
`87.9 ± 18.9
`91.0 ± 8.1
`91.9 ± 14.1
`
`85.2
`88.8
`91.9
`91.8
`91.4
`93.6
`86.3
`91.2
`95.9
`92.3
`98.9
`94.8
`
`79.3
`85.9
`88.7
`78.0
`83.8
`83.9
`76.0
`83.1
`86.7
`89.2
`97.6
`95.3
`
`5
`10
`10.5
`9
`11.5
`10
`<3
`6.5
`13
`11.5
`>14
`>14
`
`the concentration required to produce half the maximum
`degree of reduction of the target and the relationship
`if any of concentration dependence of
`silencing to
`siRNA sequence or guide strand overhang. As shown in
`Figure 2, siRNAs with the same target sequence but dif-
`ferent guide strand overhangs shared similar IC50 values.
`We did observe differences in the IC50 values between se-
`quences. Notably Seq25 had an IC50 in all chemistries that
`was 20-fold higher than that seen with the Seq40, and
`4 times higher than that seen with Seq37 (Figure 2).
`
`In vitro duration assay with LNP formulated siRNAs
`
`The four sequences in three chemistries described above
`were
`formulated in a LNP-delivery
`vehicle. The
`LNP-formulated siRNAs were transfected into cells, and
`the cells were harvested 24 and 120 h post-transfection
`(Figure 3). The 12 tested siRNAs showed an average of
`96.2 ± 1.3% reduction of the target mRNA at 24 h, but by
`120 h a large range in the degree of knockdown was
`observed.
`Comparing the amount of activity lost between the 24-h
`and 120-h timepoints in siRNAs with dT-dT overhangs,
`one of the sequences lost 8.9% (from 94.5% mRNA re-
`duction at 24 h to 85.6% mRNA reduction at 120 h) of
`activity (Seq40), while another (Seq37) showed a loss of
`76.2% of activity (from 95.6% mRNA reduction at 24 h to
`19.4% mRNA reduction at 120 h) over the same time
`period. The remaining two sequences (Seq1 and Seq25)
`showed a loss of activity of 46 and 54%, respectively.
`Comparing the sequences with oU-oU overhangs, we
`observed losses of 16 (Seq1), 18.9 (Seq25), 25.1%
`(Seq37) and no detectable loss in Seq40. When the over-
`hangs were rN-rN, we observed losses of 8 (Seq1), 19.1
`(Seq25), 11.2% (Seq37) and again no measurable loss in
`Seq40. These data indicate that siRNA sequence and
`overhang chemistry both likely play a role in determining
`silencing duration.
`An overall comparison of the loss of silencing activity
`dependent on chemistry showed that the oU-oU and
`rN-rN overhangs
`showed
`comparable
`losses
`of
`14.9 ± 10.9 and 9.9 ± 7.6%, respectively between 24 and
`120 h, while the siRNAs containing dT-dT overhangs
`showed a loss of 46.5 ± 28.0%. The dT-dT loss of
`activity had a P-value of 0.03 compared to oU-oU and
`
`0.05 to rN-rN. The loss of activity with oU-oU overhangs
`compared to the loss of activity with rN-rN overhangs had
`a P-value of 0.23. In all cases dT-dT overhangs were dem-
`onstrably worse in their ability to maintain silencing over
`the time range tested in this assay.
`
`In vivo efficacy and duration
`
`We had verified in vitro that
`the LNP-encapsulated
`siRNAs retained the ability to comparably reduce Apob
`mRNA levels
`24 h
`post-transfection
`(Figure
`3).
`Furthermore, the IC50s were virtually identical across
`three guide strand overhangs, although showing some
`sequence dependence (Figure 2).
`To test duration of silencing in vivo and investigate the
`contribution of
`siRNA sequence and guide strand
`overhang modification to in vivo duration, a single dose
`of formulated siRNAs were delivered via the tail vein and
`the degree of knockdown of Apob mRNA in liver was
`examined at 1, 3, 7 and 14 days (Figure 4). In most
`cases, siRNAs with all three of the guide strand overhangs
`showed comparable degrees of knockdown at the shortest
`timepoint measured (1-day post-treatment). The exception
`was Seq37, which showed reduced day 1 knockdown
`in vivo with the dT-dT overhang. This sequence had also
`shown the largest degree of loss in the vitro duration assays
`when the dT-dT overhang (Figure 3) was compared to
`oU-oU or to rN-rN. As expected, in all cases there was
`a loss of activity of the siRNAs over time. Differences in
`the rate of loss of activity were in part due to siRNA
`sequence. An example of this sequence dependent range
`is shown in Figure 5, where the dT-dT versions of all four
`sequences are compared, and the difference between the
`durations of the sequences is clear. This difference is also
`seen when comparing the four sequences in the other two
`tested chemistries, though as we appear to approach the
`maximum possible duration of action of the siRNAs
`in vivo, the differences become less obvious.
`We looked at several parameters of the siRNA activity
`in vivo as a means to discern the differences between the
`various guide strand overhangs (Table 1). We examined
`the maximum degree of mRNA reduction and retention
`rate of silencing activity per day. The values for these par-
`ameters appear
`to be worse for dT-dT overhangs
`compared to the other two overhangs for each of the
`
`

`

`Nucleic Acids Research, 2010, Vol. 38, No. 14 4793
`
`Figure 2. Testing of siRNAs for maximum reduction of mRNA levels and the potency. siRNAs were tested in a range of concentrations on mouse
`Hepa1–6 cells to compare both the maximum reduction of mRNA levels with each of the siRNAs and the potency. In each panel, the filled circles
`are for siRNAs with dT-dT overhangs, filled squares are siRNAs with oU-oU overhangs, filled diamonds are siRNAs with rN-rN overhangs and
`filled triangles are siRNAs with oN-oN overhangs. Panel (A) is Seq1, (B) Seq25, (C) Seq37 and (D) Seq40.
`
`individual sequences. These parameters are expected to be
`independent, but are both critically important for thera-
`peutic dosing. We attempted to estimate the duration of
`therapeutic effect by calculating the length of time for
`which 50% knockdown was achieved, which is a param-
`eter which includes both the maximum knockdown
`achieved and the amount of silencing lost each day. This
`last parameter demonstrates a clear difference between the
`siRNAs with dT-dT overhangs and siRNAs with rN-rN
`and oU-oU overhangs. For all the sequences rN-rN and
`oU-oU overhangs yielded similar results,
`in all cases
`having a longer duration than the respective siRNAs
`with dT-dT overhangs. The improvement in duration
`relative to the same sequence with dT-dT overhangs
`varied from 11 to >100% in the length of time for
`which 50% knockdown was achieved.
`
`Comparison of in vitro and in vivo data
`
`We compared the level of mRNA expression at the four
`in vivo timepoints to the level of mRNA expression at both
`timepoints tested in tissue culture cells to determine how
`well the in vitro assay was capable of predicting the
`
`duration of siRNA activity in vivo. Comparing any of
`the in vivo timepoints to the mRNA expression at the
`24-h timepoint of the in vitro assay produced no obvious
`correlation between the two datasets (data not shown). A
`comparison of the in vitro 120-h data with each of the
`available sets from the in vivo experiment showed an
`R2 value of 0.65 at 1 day, 0.89 at 3 days, 0.84 at 7 days
`and 0.54 at 14 days. This indicates that the in vitro assay as
`performed here provides a very strong correlation with
`observed data from the in vivo studies. Additionally, the
`percent of silencing retained per day, calculated over Days
`1–7 in vivo and from 24 to 120 h in vitro, was well
`correlated (R2 = 0.60; P < 0.003).
`
`Effect of guide strand overhangs on cytokine release
`
`An additional advantage of using chemically modified
`siRNAs is the ability of those siRNAs to prevent or
`reduce the release of cytokines by the treated cells (8).
`We wanted to be certain that
`the differences we
`observed in the loss of activity that were dependent on
`chemistry or sequence were not the result of variable
`cytokine responses.
`
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`

`4794 Nucleic Acids Research, 2010, Vol. 38, No. 14
`
`Figure 3. Comparison of activity of siRNAs at 24 and 120 h post-transfection. LNP-encapsulated siRNAs were tested in mouse Hepa1–6 cells to
`compare the reduction of mRNA levels for each of the siRNAs over time. In all panels the filled circles indicate dT-dT overhangs, open circles
`oU-oU overhangs and filled triangles indicate rN-rN overhangs. (A) Seq1, (B) Seq25, (C) Seq37 and (D) Seq40.
`
`We compared the circulating levels of five different cyto-
`(IL-1a, KC,
`IFNg and TNFa) at 4 h
`kines
`IL-6,
`post-dosing with the 12 duplexes tested in vivo in LNP
`forumation, to determine if the variation in the guide
`strand overhangs had any effect on immunostimulation
`(Supplementary Figure S1). For the siRNAs examined
`in this study, there was no apparent dependence of the
`degree of cytokine release on the guide strand overhangs
`or on the underlying sequence of the siRNAs.
`
`DISCUSSION
`
`A critical aspect of chemical modifications of siRNAs is to
`improve their drug-like properties. To date, almost all
`siRNAs used in animals have been chemically modified
`in some form (3–6), but the exact impact of many of
`these modifications on performance in vivo relative to un-
`modified siRNAs has not been established. While most
`studies have focused on maximal silencing achieved by
`in vitro (16,17),
`siRNAs
`for
`therapeutic utility an
`extended duration of action is in practice a required cor-
`ollary. Currently available delivery mechanisms necessi-
`tate intravenous delivery of
`the therapeutic material,
`thereby increasing the need for maximizing the dosing
`interval.
`
`We first investigated a set of 40 sequences directed
`against the mouse Apob gene in assays in tissue culture
`cells. From these experiments, we were able to demon-
`strate that the guide strand overhang has no measurable
`effect on the maximal capacity of a given siRNA to reduce
`the
`expression of
`the
`target. However, we
`also
`demonstrated that over time, differences in the capablities
`of the siRNAs to mediate silencing begin to manifest
`themselves. Specifically,
`the dT-dT overhangs, which
`are a common industry standard, have a consistently
`negative effect on the duration of action of siRNAs in
`tissue culture cells. Interestingly, there are reports in the
`literature that see no effect of the guide strand overhang at
`the tested timepoint (9) and those where a difference is
`clearly evident (12,13). We suggest that both of these
`results can be explained as the result of the timepoints
`under which
`the
`experiments were
`carried
`out.
`Essentially, the dependence of silencing on guide strand
`overhangs is a direct result of the timepoints used in the
`experiments.
`We noted that duration of silencing has some sequence
`dependence, and sequences with naturally long durations
`of silencing may not show much overhang dependence of
`silencing in vitro. We observed that an siRNA with natur-
`ally long duration of silencing lost virtually no activity
`
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`Nucleic Acids Research, 2010, Vol. 38, No. 14 4795
`
`Figure 4. Liver Apob target mRNA reduction by siRNAs with different guide strand overhangs. C57Bl/6 mice were intravenously injected with LNP
`formulated siRNAs at a dose of 3 mg/kg. Livers were har