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Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA. Correspondence should be addressed to V.J. (vjadhav@alnylam.com).

Received 2 June 2017; accepted 12 March 2018; published online 14 May 2018; doi:10.1038/nbt.4136

Safety and efficacy data from a number of clinical studies have generated growing evidence for the therapeutic potential of RNAi1–4. The development of the GalNAc (trivalent N-acetylgalactosamine)–siRNA conjugate platform has led to substantial improvements in efficacy with potent mRNA knockdown in the liver sustained for several months5,6. With this extended duration, RNAi therapeutics may benefit from a technology that enables reversal of target mRNA knockdown and provides finer control over pharmacology, a desired attribute for therapeutic entities.

One way to modulate the RNA-induced silencing complex (RISC)-mediated activity is to direct a complementary oligonucleotide as a synthetic target with high affinity against the RISC-loaded siRNA guide (antisense) strand7,8. Similar approaches targeting micro-RNAs (miRs) have advanced in clinical studies9–12. Antidotes for exogenous oligonucleotide therapeutic modalities, such as antisense13 and aptam-ers14, have also been described, but so far, the high doses required in the absence of targeted delivery systems have limited the scope of such applications13. Here, we describe an approach, termed REVERSIR, to reverse siRNA silencing using probes that hybridize to RISC-loaded guide strands with high affinity, rendering the functional RISC inac-tive (Supplementary Fig. 1).

We synthesized oligonucleotides with a variety of chemical modifications and, to achieve selective delivery to hepatocytes, the same trivalent GalNAc ligand as in the GalNAc–siRNA15 (Fig. 1a). For high affinity and metabolic stability, 2′-O-methyl and locked

reversal of sirna-mediated gene silencing in vivo

Ivan Zlatev , Adam Castoreno, Christopher R Brown, June Qin, Scott Waldron, Mark K Schlegel , Rohan Degaonkar, Svetlana Shulga-Morskaya, Huilei Xu, Swati Gupta , Shigeo Matsuda, Akin Akinc, Kallanthottathil G Rajeev, Muthiah Manoharan, Martin A Maier & Vasant Jadhav

We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc–siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.

nucleic acid (LNA) modifications along with a uniform phospho-rothioate (PS) backbone were used for sequences complementary to the guide strand of GalNAc–siRNAs targeting the mRNA of transthyretin (Ttr) or coagulation factor VII (F7) (Supplementary Table 1). We measured the thermal melting temperatures (Tm) of the REVERSIR molecules annealed with the siRNA guide strand as a measure of hybridization affinity. As expected, increasing the LNA content resulted in a steady increase in Tm and was associated with improved reversal of siRNA-mediated target RNA knockdown in vitro (Supplementary Tables 2 and 3 and Supplementary Fig. 2). For in vivo evaluation, mice were treated with a single subcutaneous (SC) dose of TTR–siRNA (3 mg/kg) to achieve robust knock-down, followed 7 days later by a single SC dose of TTR-REVERSIR (0.1 mg/kg). In agreement with the in vitro findings, in vivo activ-ity was dependent on LNA content, with the five LNA-containing REVERSIRs conferring rapid and full reversal of siRNA activity by day four after the REVERSIR dose (Fig. 1b).

We next evaluated the LNA positional effect, where every nucle-otide was changed from a 2′-O-methyl to a single LNA in a 15-mer REVERSIR. The most striking benefit in vitro was seen with an LNA complementary to position 6 of the target guide strand (g6) (Supplementary Fig. 2d). We evaluated this position in vivo by select-ing molecules containing five LNAs at identical positions, except for shifting a single LNA away from the position opposite g6 to g7. This change resulted in reduced reversal of siRNA activity, which further confirmed the position-specific importance of LNA at g6 (Fig. 1c). Based on the crystal structure data of miR-Ago2-loaded guide strands, it has been proposed that mRNA target binding is initiated by the recognition of nucleotides g2–g5 (step 1), and upon binding, Helix α-7 shifts to relax the kink between g6 and g7, allowing pairing to the remainder of the target (step 2)16. Our findings suggest that a high-affinity-modification LNA opposite g6 may be important in facili-tating step 2 and maintaining the ‘bound’ guide–REVERSIR state, preventing the reintroduction of the kink and REVERSIR removal.

The length-dependence of REVERSIR activity was evaluated in sequences containing five LNAs at identical positions while preserv-ing binding to the full seed region (g2–g8) of the guide (Fig. 2a). We found that in mice shorter-length REVERSIR molecules (8- and 9-mers) showed maximal reversal of siRNA activity in contrast to moderate reversal for the longer molecules (15-and 22-mers) at same molar equivalent dose (Fig. 2b). The 7-mer REVERSIR was inactive, and these findings were generalizable to other targets (Supplementary Fig. 3). Shorter REVERSIRs, with lower Tm to target guide and comparable asialoglycoprotein receptor (ASGPR) binding to longer versions, had consistently higher potency under conditions of GalNAc-ASGPR-mediated uptake in vitro, but had lower potency when delivered by transfection (Supplementary Figs. 4 and 5). The benefit seen by GalNAc-ASGPR-mediated endocytosis of the shorter length REVERSIR was not due to improved bulk uptake, as longer

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REVERSIR molecules showed better uptake and still showed poor reversal of siRNA activity (Supplementary Fig. 5). We hypothesize that the higher potency of shorter REVERSIR molecules is likely due to enhanced release into the cytosol.

In accordance with trends published for anti-miRs17 and consistent with the pre-organization of nucleotides g2–g5 (ref. 16), our results indicate that full coverage of the entire guide seed region is required for optimal REVERSIR activity (Supplementary Fig. 6). In addition, we confirmed the necessity of targeted delivery and the benefit of an intracellular nuclease-cleavable linker between ligand and oligonu-cleotide18 (Supplementary Fig. 7). As expected, REVERSIR activity was sequence-specific, as even a single mismatch was detrimental to the reversal of siRNA activity (Supplementary Table 2). Also, no statistically significant changes were found in global RNA expres-sion deriving from any potential REVERSIR off-target binding events (Supplementary Fig. 8).

We probed the mechanism by which REVERSIR engages and abrogates the activity of GalNAc–siRNA in mice by quantifying siRNA levels in whole liver and Argonaute 2 (Ago2) (Fig. 2c,d and Supplementary Fig. 9). The administration of either sequence-spe-cific or scrambled REVERSIR of varying length did not affect the amount of guide strand in total liver or associated with immuno-precipitated Ago2 (Supplementary Fig. 9). In addition, REVERSIR was recovered in Ago2 immunoprecipitations only when the guide strand was present, thus providing direct evidence for REVERSIR engagement with the sequence-complementary guide strand in RISC (Fig. 2e and Supplementary Fig. 10).

In summary, we report a potent, generalizable REVERSIR template design (9-mer with five LNAs), enabling reversal of siRNA activity with a single low-dose SC administration (Supplementary Fig. 11).

Our studies provide comprehensive insights applicable for other oligonucleotide therapeutics, such as anti-miRs. Recently, we reported that a subset of GalNAc–siRNAs showed rat hepatotoxic-ity at supraphysiological doses19. Many of these hepatotoxic find-ings by GalNAc–siRNAs were attenuated with REVERSIR treatment, suggesting that guide-strand-driven, RNAi-mediated, hybridization -based, off-target effects, not the chemical modifications or competition for RISC loading with endogenous small RNAs, appear to be an important driver of rat hepatotoxicity. REVERSIR technology could prove therapeutically beneficial in providing finer control over the duration of action of GalNAc–siRNAs, particularly in the context of acute indications.

MEthOdSMethods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACKnoWleDGMentSWe thank J. Maraganore, R. Meyers, K. Charisse, and K. Fitzgerald for helpful discussions. We thank J. Barry for help with RNA-seq.

AUtHoR ContRIBUtIonSI.Z., A.C., M.M., A.A., K.G.R., M.A.M., and V.J. designed the research. I.Z., S.W., and M.K.S. synthesized oligonucleotides and generated Tm data. A.C. generated in vitro data in PMHs. J.Q. generated in vivo data. R.D. and S.G. generated ASGPR binding and uptake data. C.R.B. quantified siRNA levels and REVERSIR association with Ago2. S.S.-M. generated and H.X. analyzed RNA-seq data. S.M. contributed reagents. I.Z. and V.J. wrote the manuscript with input from all authors.

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Figure 1 REVERSIR design and activity. (a) Schematic of F7–siRNA guide strand (top) hybridized with REVERSIR molecule. REVERSIR molecules start at a position complementarity to g2 of the guide strand. The GalNAc ligand is connected at the 3′ end through a 2′-deoxyadenosine nucleotide via a phosphodiester linkage (indicated as a nuclease-cleavable linker and not included in the length of REVERSIR). Modifications: OMe: 2′-O-Methyl, PS: phosphorothioate, LNA: locked nucleic acid, and F: 2′-F. (b) In vivo reversal of SC-dosed TTR–siRNA (3 mg/kg, day 0) activity in mice (n = 3) by SC-dosed REVERSIR (0.1 mg/kg; day 7) containing either two (R-3) or five (R-6) LNAs (Supplementary tables 2 and 3). Measure of center is mean for each data point. Error bars are s.d. (n = 3). (c) Experimental conditions same as in b but REVERSIRs containing five LNAs with key change at g6; either LNA (R-31) or 2′-OMe (R-32). Measure of center is mean for each data point. Error bars are s.d. (n = 3).

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CoMPetInG InteReStSAll authors are or were employees of Alnylam Pharmaceuticals when they contributed to this work. REVERSIR is a trademark of Alnylam Pharmaceuticals.

reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Publisher’s note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1. Coelho, T. et al. N. Engl. J. Med. 369, 819–829 (2013).2. Fitzgerald, K. et al. Lancet 383, 60–68 (2014).3. Fitzgerald, K. et al. N. Engl. J. Med. 376, 41–51 (2017).4. Zimmermann, T.S. et al. Mol. Ther. 25, 71–78 (2017).5. Nair, J.K. et al. Nucleic Acids Res. 45, 10969–10977 (2017).6. Foster, D.J. et al. Mol. Ther. 26, 708–717 (2018).

7. Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. RNA 10, 544–550 (2004).8. Hutvágner, G., Simard, M.J., Mello, C.C. & Zamore, P.D. PLoS Biol. 2, e98 (2004).9. van der Ree, M.H. et al. Lancet 389, 709–717 (2017).10. Rottiers, V. et al. Sci. Transl. Med. 5, 212ra162 (2013).11. Janssen, H.L.A. et al. N. Engl. J. Med. 368, 1685–1694 (2013).12. Krützfeldt, J. et al. Nature 438, 685–689 (2005).13. Crosby, J.R. et al. Nucleic Acid Ther. 25, 297–305 (2015).14. Rusconi, C.P. et al. Nat. Biotechnol. 22, 1423–1428 (2004).15. Nair, J.K. et al. J. Am. Chem. Soc. 136, 16958–16961 (2014).16. Schirle, N.T., Sheu-Gruttadauria, J. & MacRae, I.J. Science 346, 608–613

(2014).17. Obad, S. et al. Nat. Genet. 43, 371–378 (2011).18. Neben, S. et al. The 51st International Liver Congress, EASL, Barcelona, Spain

http://www.natap.org/2016/EASL/EASL_158.htm (2016).19. Janas, M.M. et al. Nat. Commun. 9, 723 (2018).

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Figure 2 Evaluation of REVERSIR design features. (a) Schematic representation of a TTR–siRNA guide strand with REVERSIR molecules of varying length. The 3′-DNA-A linker and GalNAc are not shown in the schematic and not included in the total length assignment. (b) In vivo reversal of TTR–siRNA activity in mice (n = 3) using a 0.025 molar equivalent dose of TTR-REVERSIR molecules with different lengths (normalized to the molar equivalent of the 0.03 mg/kg dose of 15-mer REVERSIR). Measure of center is mean for each data point. Error bars are s.d. (n = 3). (c–e) 15-mer REVERSIR R-6 (Supplementary table 2). (c) Reversal of TTR–siRNA knockdown activity (10 mg/kg, day 0) by a TTR–REVERSIR administered at 1 mg/kg (day 3, n = 3). Ttr expression analysis was performed on day 7. Individual points and mean values plotted. Error bars are s.d. (n = 3). (d) Levels (ng/g) of TTR–siRNA guide (black) and passenger (gray) strands loaded into Ago2 from whole mouse liver collected on day 7. Ago2 protein was immunoprecipitated (IP) and loaded siRNA was quantified by RT-qPCR. Individual points and mean values plotted. Error bars are s.d. (n = 3). (e) Northern blot analysis of Ago2 and control IPs (Ctl) from liver samples used in c and d. IP elutions were split and run on two gels, probing either for the guide strand (top) or 15-mer REVERSIR (bottom). Standards include guide strand (with or without 5′ phosphate, both without GalNAc) and 15-mer REVERSIR (without GalNAc). The Ago2 levels in samples used for e were quantified by western blot (full scans of all gels in Supplementary Fig. 10). The northern and western blot experiments were performed independently twice with similar results.

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ONLINE MEthOdSOligonucleotide synthesis. All oligonucleotides were prepared on a MerMade 192 synthesizer on a 1-µmole scale using custom GalNAc supports15. LNA phosphoramidites were purchased from Exiqon. Cy5 phosphoramidite was pur-chased from Glen Research. All phosphoramidites were used at a concentration of 100 mM in 100% acetonitrile, 9:1 acetonitrile:DMF (2′-OMe-C, 2′-OMe-U), or 1:1 DCM:acetonitrile (LNA-5-Me-C) with a standard protocol for 2-cyanoethyl phosphoramidites and 5-(Ethylthio)-1H-tetrazole (ETT) activator, except that the coupling time was extended to 400 s. Phosphite oxidation to phosphate or sulfurization to phosphorothioate was achieved using a solution of 50 mM iodine in 9:1 acetonitrile:water or 100 mM 1,2,4-dithiazole-5-thione (DDTT) in 9:1 pyridine:acetonitrile, respectively. After the trityl-off synthesis, columns were incubated with 150 µL of 40% aqueous methylamine for 30 min at room temper-ature and the solution was drained via vacuum into a 96-well plate. After repeat-ing the incubation and draining with a fresh portion of aqueous methylamine, the plate containing crude oligonucleotides solution was sealed and shaken at 60 °C for an additional 30 min to completely remove all protecting groups. Precipitation of the crude oligonucleotides was accomplished via the addition of 1.2 mL of 9:1 acetonitrile:EtOH to each well, followed by incubation at −20 °C overnight. The plate was then centrifuged at 3,000 r.p.m. for 45 min at 4 °C, the supernatant removed from each well, and the pellets resuspended in 950 µL of 20 mM aqueous NaOAc. Those REVERSIR molecules that did not precipitate (shorter than ~10 nucleotides) were concentrated in vacuo and redissolved in 1.0 mL of 20 mM aqueous NaOAc. Each crude solution was finally desalted over a GE Hi-Trap desalting column (Sephadex G25 Superfine) using water to elute the final oligonucleotide products. Cy5-labeled oligonucleotides were processed according to the Glen Research protocols. The identities and purities of all oligonucleotides were confirmed by electrospray ionization mass spec-troscopy (ESI-MS) and ion exchange high-performance liquid chromatography (IEX-HPLC), respectively.

Thermal melting experiments. These studies were performed in 1-cm-path-length quartz cells on a Beckman DU800 spectrophotometer equipped with a thermoprogrammer. Each cuvette contained 200 µL of sample solution (duplex at a concentration of 1 µM) covered by 125 µL of light mineral oil. Samples were initially annealed in the instrument by heating at a rate of 5 °C/min from 15–95 °C followed by cooling at the same rate from 95–15 °C. After a waiting period of 5 min, melting curves were monitored at 260 nm with a heating rate of 1 °C/min from 15–95 °C. Melting temperatures (Tm) were calculated from the first derivatives of the heating curves and the reported values are the result of at least two independent measurements.

In vivo mouse studies. All procedures were conducted by certified labora-tory personnel using protocols consistent with local, state, and federal regula-tions, as applicable, and approved by the (i) Institutional Animal Care and Use Committee; (ii) AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) – accreditation number: 001345. C57BL/6 female mice, aged 6–8 weeks, acquired from Charles River Laboratories (n = 3 per group) were dosed subcutaneously at a volume of 10 µL GalNAc conjugate (siRNA or REVERSIR) per gram of body weight. The control group was dosed with phosphate buffered saline (PBS). Serum samples were collected and analyzed for siRNA activity for specific target proteins as described below. Serum TTR protein was quantified by enzyme-linked immunosorbent assay (ELISA) from serum isolated from whole blood. ELISA was performed according to manufacturer protocol (ALPCO, 41-PALMS-E01) after a 3,025-fold dilution of the serum samples. Data were normalized to pre-bleed TTR levels. Factor 9 activity levels were measured using the Aniara Biophen Factor 9 activity kit. Serum levels of Factor 7 protein were determined using an activity-based chromogenic assay (Biophen FVII; Aniara, Mason, OH). Group averages are depicted with +/− s.d. All samples were assayed in duplicate and each data point is the average of all the mice within each cohort (n = 3).

In vitro screening in primary mouse hepatocytes (PMH). PMHs were transfected first with siRNA by adding 4.9 µL of Opti-MEM plus 0.1 µL of Lipofectamine RNAiMAX (Invitrogen) to 5 µL of 10 nM siRNA per well in a 384-well Biocoat Collagen I-coated plate (Corning). Following a 15-min room

temperature incubation, 40 µL of William’s E Medium (Life Tech) containing 5 × 103 cells was added to the siRNA–lipofectamine mixture (1 nM siRNA final concentration). Cells were subsequently incubated for 4 h, followed by a single PBS wash, media change, and addition of 5 µL REVERSIR molecules directly to 45 µL William’s media (at final doses ranging from 100 nM–50 pM). After a 48-h incubation, cells were lysed and processed for RNA isolation, cDNA synthesis, and quantitative PCR analysis.

Co-transfection of siRNA and REVERSIR. PMHs were co-transfected with siRNA and REVERSIR by combining 4.8 µl of Opti-MEM, 0.2 µL of Lipofectamine 2000 (Invitrogen), 5 µL of siRNA, and 5 µL REVERSIR per well in a 384-well Biocoat Collagen I-coated plate. After a 15-min incubation at room temperature, 35 µL of William’s E Medium (Life Tech) containing 5 × 103 cells was added to the siRNA-REVERSIR-Lipofectamine mixture (1 nM siRNA and 100 nm–0.4 pM REVERSIR final concentrations). After a 24-h incubation, cells were lysed and processed for RNA isolation, cDNA synthesis, and quantitative PCR analysis.

RNA isolation, cDNA synthesis, and quantitative PCR. Automated mRNA isolation was performed on a BioTek-EL406 using the Dynabeads mRNA DIRECT Kit (ThermoFisher) followed by cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was then used for qPCR in a 10-µL reaction containing 2 µL cDNA, 0.5 µL of mouse Gapdh TaqMan VIC Probe (4352339E, ThermoFisher), and 0.5 µL mouse target FAM probe (F9 Mm01302526_m1, F7 Mm00487329_m1, and Ttr Mm00443267_m1, ThermoFisher) in a white 384-well plate (Roche). qPCR was performed in a LightCycler 480 Real Time PCR system (Roche). Samples were performed in quadruplicate, and data were normalized to cells transfected with a non-targeting control siRNA. To calculate relative fold-change, real time data were analyzed using the ∆∆Ct method and nor-malized to assays performed with cells transfected with a non-targeting control siRNA.

Bioinformatics. No perfect sequence match against REVERSIR molecules was found among experimentally validated human and mouse microRNA guide strands ‘seed’ regions (positions g2–g8). The experimentally validated miR list was consulted through miRBase: the microRNA database (http://www.mirbase.org/).

RNA-seq analysis. PMH were treated with 1 nM TTR–siRNA and 10 nM scrambled or TTR-REVERSIR by in vitro free uptake for 24 h. RNA extracted with the Purelink RNA kit (ThermoFisher) was used for cDNA library prepa-ration with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the NextSeq500 desktop sequencer (Illumina), all according to manufacturers’ instructions. Raw RNA-seq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf20. Filtered reads were aligned to the Mus musculus genome (NCBIM37) using STAR with default parameters21. Uniquely aligned reads were counted by featureCounts22. Differential gene expression (DEG) analysis was performed by the R package DESeq2 (ref. 23).

Mechanistic evaluation of REVERSIR mode of action. C57BL/6 female mice, aged 6–8 weeks, were acquired from Charles River Laboratories and were dosed subcutaneously (day 0) with a volume of 10 µL per gram of body weight (n = 3 per time point) at 10 mg/kg (TTR GalNAc–siRNA conjugate). Control groups were dosed with phosphate buffered saline (PBS). REVERSIR molecules (9-mer, 15-mer, 22-mer) were dosed subcutaneously on day 3 at 1.0, 1.0, and 2.5 mg/kg, respectively. Mice were euthanized on day 7 post-siRNA dose (4 days after REVERSIR dose), and livers were snap-frozen in liquid nitrogen and ground into powder for further analysis. Ttr mRNA levels were quantified using methods described earlier15. Total siRNA liver levels and Ago2-bound siRNA was quantified by SL-RT QPCR as described previ-ously24–26. For northern blot analysis, lysates for Ago2 immunoprecipitation (IP) were prepared as described previously24 with the following exceptions: to aid in the removal of non-specifically associated REVERSIR molecules, samples were incubated with an equal volume of 20 mg/mL of anion exchange resin (QAE Sephadex A-50, GE Healthcare) for 15 min at 4 °C. Resin was

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removed before overnight IP by passing samples through cellulose acetate filters (Pierce Spin Cups, Thermo Scientific). Mouse Ago2 antibody (Wako Chemicals, Clone 2D4) and control mouse IgG (Santa Cruz Biotechnology, sc-2025) were used for IPs in conjunction with Protein G Dynabeads (Life Technologies). Post-IP washes were performed under high salt (1 M NaCl) conditions to further reduce REVERSIR background. Ago2-associated siRNAs were eluted by heating (100 µL PBS, 0.25% Triton; 95 °C, 5 min). Elutions were treated with 0.6 mg/mL Proteinase K (Invitrogen) and further cleaned up by running through spin cup filters (Nanosep 30K Omega, Pall). Samples were ethanol-precipitated and resuspended directly in RNA denaturing gel load-ing buffer (New England BioLabs). Samples were split in two and loaded on two 15% TBE-Urea gels (Criterion Precast Gels, BioRad). Standards loaded onto each gel include 20 fmol of 15-mer REVERSIR and 20 fmol of guide strand (with or without a 5′ phosphate, all without GalNAc). Following the gel run, samples were transferred to Biodyne B Nylon Membranes (Pall) using a Trans-Blot SD. Semi-Dry Transfer Cell (BioRad). Membranes were UV-cross-linked using the autocrosslink function (CX-2000 UV Crosslinker, UVP). Membranes were hybridized with P32-end-labeled probes for either the guide strand (5′-aaaacaguguucuug(mC)(T)(mC)(T)a(T)aa-3′) or the REVERSIR (5′-ua(T)agag(mC)aa(G)(A)ac(A)-3′) in ULTRAhyb-Oligo Hybridization Buffer (ThermoFisher) overnight at 37 °C. Uppercase nucleotides in brack-ets indicate LNA, while lowercase letters represent 2′-OMe. Membranes were washed with 2× SSC, 0.1% SDS followed by phosphorscreen exposure to obtain images. For western blot analysis, Ago2 IPs were performed as described for the northern blot analysis except that after the wash steps, samples were eluted from the Protein G Dynabeads by adding 30 mL NuPAGE LDS Sample Buffer (ThermoFisher Scientific) and heated to 95 °C for 5 min. Half of the sample was run on a 4–12% Bis-Tris Protein Gel (ThermoFisher Scientific), using 1× NuPAGE MOPS SDS running buffer (ThermoFisher Scientific) with a Chameleon Duo Pre-Stained Protein Ladder (Li-Cor). For detection of Ago2 a rabbit monoclonal anti-Ago2 antibody was used at 1:1,000 (Cell Signaling, C34C6) followed by a goat anti-rabbit secondary antibody (IRDye 800CW goat anti-rabbit, 1:5,000, Li-Cor).

ASGPR binding assay. Freshly isolated primary mouse hepatocytes were resuspended at 1 million cells per mL in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) with 2% Bovine Serum Albumin (BSA, Sigma-Aldrich) and subjected to a flow-cytometry-based competitive binding assay27. A GalNAc-conjugated, Alexa647-labeled siRNA substrate was diluted to a final concentration of 20 nM and was premixed with the corresponding competing unlabeled REVERSIR compound at an appropriate range of final concentra-tions from 3 µM to 0.5 nM in 2% BSA/DMEM. 100,000 hepatocytes per well were added to the siRNA-REVERSIR mixture and incubated at 4 °C for 15 min. Cells were washed two times with 2% BSA/1 × Dulbecco’s Phosphate-Buffered Saline with Mg/Ca (DPBS, Life Technologies) using centrifugation at 50 G for 3 min to pellet cells between washes. Cells were resuspended in a solution of 2% BSA/1 × DPBS with 2 µg/mL propidium iodide and analyzed on an LSRII flow cytometer instrument (BD Biosciences). Compensation was performed using Diva software (BDBiosciences). Hepatocytes were gated by size using

forward scatter and side scatter and dead cells were excluded from analysis by propidium iodide staining. Median fluorescent intensity of the Alexa647 siRNA was quantified. Data were analyzed using FlowJo v10 software (FlowJo, LLC) and GraphPad Prism 7 software (GraphPad Software, Inc.).

Free uptake of Cyanine5-labeled REVERSIR compounds in primary mouse hepatocytes. Cryopreserved primary mouse hepatocytes (#FGF BioreclamationIVT) were resuspended at 2 million cells per mL in InVitroGRO CP Rodent medium (#C24087A BioreclamationIVT) and used for flow-cytometry-based free uptake assay. The hepatocyte cell suspension was plated onto 96-well collagen-coated plates at 20 × 103 cells/well to final volume of 100 µL/well. The GalNAc-conjugated, Cyanine5-labeled REVERSIR com-pounds were treated at range of final concentrations from 1,000 nM, 100 nM, and 10 nM. The cells were incubated for 48 h at 37 °C with 5% CO2. Cells were washed two times with 2% BSA/1 × Dulbecco’s Phosphate-Buffered Saline with Mg/Ca (DPBS, Life Technologies) using centrifugation at 50 G for 3 min to pellet the cells between washes. Cells were resuspended in a solution of 2% BSA/1 × DPBS and analyzed on an LSRII flow cytometer instrument (BD Biosciences). Hepatocytes were gated by size using forward scatter and side scatter. Median fluorescent intensity of the Cyanine5-REVERSIR was quantified. Each compound was run in triplicate. Data were analyzed using the FlowJo software (FlowJo, LLC).

Statistical analysis. Statistical analyses were performed using GraphPad Prism 7. Data are presented as mean ± s.d. with sample numbers n noted in the figure legends along with the number of times each experiment was replicated. P values in figures are reported as exact values. The statistical analysis was performed using multiple t-test one per row (one-sided). For detailed informa-tion, please also refer to the Life Sciences Reporting Summary.

Life Science Reporting Summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data availability. The data discussed in this publication have been deposited in NCBI′s Gene Expression Omnibus28 and are accessible through GEO Series accession number GSE110620.

Software. The following software and versions were used for data analysis: Excel 2016, FlowJo v10, GraphPad Prism 7, R package DESeq2, featureCounts v1.5.0. No custom software was used.

20. Aronesty, E. https://expressionanalysis.github.io/ea-utils/ (2011).21. Dobin, A. et al. Bioinformatics 29, 15–21 (2013).22. Liao, Y., Smyth, G.K. & Shi, W. Bioinformatics 30, 923–930 (2014).23. Love, M.I., Huber, W. & Anders, S. Genome Biol. 15, 550 (2014).24. Elkayam, E. et al. Nucleic Acids Res. 45, 3528–3536 (2017).25. Pei, Y. et al. RNA 16, 2553–2563 (2010).26. Chen, C. et al. Nucleic Acids Res. 33, e179 (2005).27. Rajeev, K.G. et al. ChemBioChem 16, 903–908 (2015).28. Edgar, R., Domrachev, M. & Lash, A.E. Nucleic Acids Res. 30, 207–210

(2002).

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Corresponding author(s): Vasant Jadhav, Ph. D.

Initial submission Revised version Final submission

Life Sciences Reporting SummaryNature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity.

For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

Experimental design1. Sample size

Describe how sample size was determined. Use of n=3/group for in vivo mouse work and flow cytometry and RNA seq (in vitro free uptake) as well as an n=4/treatment for all other in vitro RNAi knockdown experiments is standard operating procedure that gives reproducible results with the targets and test articles used herein. No specific statistical methods were employed to select the n numbers.

2. Data exclusions

Describe any data exclusions. No results were excluded for any studies described in this work.

3. Replication

Describe whether the experimental findings were reliably reproduced.

Every experiment had a control parent group to observe knockdown in the absence of Reversir, these results were consistent across three experiments. All attempts at replication of these experiments were successful.

4. Randomization

Describe how samples/organisms/participants were allocated into experimental groups.

Mice were randomized upon arrival at the facility and split into the experimental groups.

5. Blinding

Describe whether the investigators were blinded to group allocation during data collection and/or analysis.

Animals were not blinded as one person dosed, sacrificed, processed samples and performed the analysis.

Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

6. Statistical parameters For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed).

n/a Confirmed

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)

A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

A statement indicating how many times each experiment was replicated

The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section)

A description of any assumptions or corrections, such as an adjustment for multiple comparisons

The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted

A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)

Clearly defined error bars

See the web collection on statistics for biologists for further resources and guidance.

Nature Biotechnology: doi:10.1038/nbt.4136

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SoftwarePolicy information about availability of computer code

7. Software

Describe the software used to analyze the data in this study.

Excel 2016, FlowJo v10, GraphPad Prism 7, R package DESeq2, featureCounts v1.5.0 used for data analysis. No custom softwares were utilized.

For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic.

Materials and reagentsPolicy information about availability of materials

8. Materials availability

Indicate whether there are restrictions on availability of unique materials or if these materials are only available for distribution by a for-profit company.

The unique materials are proprietary and not available for distribution.

9. Antibodies

Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species).

Anti-mouse Ago2 antibody (Wako Chemicals, 018-22021, Clone 2D4, Lot LKE1001, 3ug per IP). Normal mouse IgG (Santa Cruz Biotechnology, sc-2025, Lot E3117, 3ug per IP). Anti-Ago2 rabbit mAb (Cell Signaling Technology, 2897S, Clone C34C6, Lot 5, 1:1000 dilution for Western). Goat anti-rabbit secondary antibody (Li-COR, 926-32211, Lot C70426-05, 1:5000 dilution for Western). These antibodies have been validated for their use in the work by several previous studies that are appropriately referenced in the paper, in addition to validation statements on the manufacturer's websites

10. Eukaryotic cell linesa. State the source of each eukaryotic cell line used. Plateable cryopreserved CD-1 male mouse hepatocytes (ThermoFisher Cat#

MSCP10).

b. Describe the method of cell line authentication used. Vendor tests each hepatocyte lot for: - Phase I and II metabolizing enzyme activities - morphology - viability >75% - attachment efficiency and monolayer formation

c. Report whether the cell lines were tested for mycoplasma contamination.

N/A - Fresh vial of hepatocytes (~4-6 million) thawed for each experiment.

d. If any of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use.

None of the cell lines used are listed in the ICLAC database.

Animals and human research participantsPolicy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines

11. Description of research animalsProvide details on animals and/or animal-derived materials used in the study.

C57BL/6 female mice, aged 6-8 weeks with weight range of 18-21 grams were acquired from Charles River Laboratories

Policy information about studies involving human research participants

12. Description of human research participantsDescribe the covariate-relevant population characteristics of the human research participants.

The studies described here did not involve human research participants.

Nature Biotechnology: doi:10.1038/nbt.4136

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Corresponding author(s): Vasant Jadhav, Ph. D.

Initial submission Revised version Final submission

Flow Cytometry Reporting Summary Form fields will expand as needed. Please do not leave fields blank.

Data presentationFor all flow cytometry data, confirm that:

1. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

2. The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

3. All plots are contour plots with outliers or pseudocolor plots.

4. A numerical value for number of cells or percentage (with statistics) is provided.

Methodological details5. Describe the sample preparation. -The primary mouse hepatocyte were purchased from commercial vendor

BioreclamationIVT (Lot # FGF). -The cryopreserved cells were thawed at 37C and plated at 20000 cells/ well in a 96 well plate. -The REVERSIR compounds were treated with respective doses for 48h before performing the flow cytometry. - After 48h incubation time, cells were washed 1X with PBS and detached using trypsin-EDTA solution (0.25%). -The trypsin was neutralized with invitroGRO CP rodent medium (BioreclamationIVT) and cells were centrifuged for 3 minutes at 50*g . -The cells were washed 2X with 2% BSA /1X-DPBS containing Mg/Ca and resuspended with propidium iodide (2ug/ml) in2% BSA /1X DPBS containing Mg/Ca for flow cytometry analysis.

6. Identify the instrument used for data collection. The instrument used to collect the data was BD LSR II with HTS from BD Biosciences.

7. Describe the software used to collect and analyze the flow cytometry data.

Data was collected on BD FACSDiva software Version 6.1.1 Data was analyzed on FLOWJO V10.1r7

8. Describe the abundance of the relevant cell populations within post-sort fractions.

- The PMH we used for free uptake are cryopreserved mouse hepatocytes from commercial vendor. -Based on the forward (FSC-A) and side scatter (SSC-A) plots, we selected the hepatocyte population, which was around 80% of the total population. -The selected population of cells were consistent in all the treatment groups which confirms the purity of the cells.

9. Describe the gating strategy used. The analysis of the data was performed using FLOWJO software -First, we identified the hepatocyte population by selecting a gate on forward (FSC-A) and side scatter (SSC-A) plot. This gate was applied to all the specimens being tested for a given experiment. -On the selected gate, we created a contour plot for each condition being treated and quantified the MFI (median fluorescence intensity) of the plot. -The negative plot was observed for the cells with no reversir treated group where the MFI was around 28. (refer to blue contour plot on supplementary figure 5c) -The positive plot was observed for the cells treated with various dose ranges of reversir compounds where the MFI was higher than 500. (refer

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to the orange, red and maroon contour plots in the supplementary figure 5c).

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

Nature Biotechnology: doi:10.1038/nbt.4136

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