Preclinical Comparison of GalNAc-Conjugated siRNA and ASO Platforms Amy Chan, Anshul Gupta, Tim Racie, Christopher Brown, Jason Gilbert, Kirk Brown, Yongli Gu, Carrie Mallozzi, Sean Dennin, Mark Schlegel, Don Foster, Adam Castoreno, Klaus Charisse, Brenda Carito, Carole Harbison, Jay Nair, Stuart Milstein, Tracy Zimmermann, Muthiah Manoharan, Rachel Meyers, Vasant Jadhav and Martin Maier Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA

AbstractThe ability to selectively suppress disease causing genes, either by engagement of the naturally occurring RNA interference pathway in the case of small interfering RNAs (siRNA), or by exploitation of RNAse H cleavage for antisense oligonucleotides (ASO), has transformed the field of oligonucleotide-based therapeutics. Although their mechanisms of actions are distinct, both technologies aim to reduce disease-causing RNA transcripts, thereby ablating expression of the downstream pathogenic proteins. For systemic, liver-directed delivery of RNAi therapeutics, we have developed a strategy which utilizes multivalent N-acetylgalactosamine (GalNAc) ligands, covalently attached to the siRNA. This GalNAc-conjugate platform enables specific, targeted uptake into hepatocytes, resulting in highly potent and durable target silencing after subcutaneous administration with infrequent dosing at low volume.1 Fully phosphorothioate-modified antisense oligonucleotides (ASOs) have been reported to exhibit a rather broad tissue distribution2 driven by a non-specific uptake mechanism, resulting in less potent compounds with reduced durability, thereby increasing frequency of dosing, and heightening the potential for toxicity. More recently, it was demonstrated that application of the identical multivalent GalNAc ligands to ASOs greatly improves hepatocyte specific-targeting, resulting in an improved efficacy profile across a number of liver-expressed targets.3 The data presented herein include select preclinical findings from comparative studies examining the performance of sequence-matched GalNAc-siRNA and GalNAc-ASO compounds. The collective data highlight the similarities and differences between these two gene silencing approaches.

SummaryFactor IX GalNAc-siRNA Factor IX GalNAc-ASO

In Vitro Potency

• High inherent potency by transfection

• ≥40%KD observed in 80% of screened compounds

• Low potency observed by transfection

• ≥40%KD observed in 15% of screened compounds

Preclinical Efficacy

• 40-80% max suppression following a single subcutaneous dose

• Potent and durable knockdown observed across multiple sequences

• 20-65% maximum suppression following a single subcutaneous dose

• Less durable PD effect

Plasma Protein Binding • Low affinity plasma binding (25-45%) • Highly bound in plasma (75-98%)

Tissue Concentration • ≥15 fold higher levels in liver than kidney

• Approximately 1:1 distribution to liver and kidneys

Test Article & mRNA Distribution

• Punctate cytoplasmic labeling in hepatocytes for GalNAc-siRNA

• Robust mRNA reduction by ISH & qPCR

• Strong cytoplasmic labeling of Kupffer cells with faint hepatocyte labelling

• Marginal mRNA reduction by ISH & qPCR

References1. Nair, JK, et al. (2014) J Am Chem Soc., 136:16958–16961.2. Bennett, C.F. (2007) Antisense Drug Technology: Principles, Strategies and AppGalNAc-ASOtions. pp. 273–304.3. Prakash TP, et al. (2014) Nucleic Acids Res. 42:8796–8807.

Figure 1. Antisense and RNAi Pathways – Overview

Activated RISC

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productsTarget mRNA

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A BAntisense (RNAse H1) RNAi

Figure 2. Chemical Modifications to Impart Drug-Like Properties in ASOs and siRNAs

Gapmer Design 5-10-5

• 10-nt DNA gapmer region – Essential for RNaseH1 activity

• 5-nt 2’-Methoxyethyl (MOE) region in 5’ & 3’ wings – Nuclease & thermal stability for improved target RNA recognition

• All 5-methyl-C bases• Full Phosphorothioate (PS) backbone

– Nuclease protection & cellular uptake through lipophilic nature of PS backbone

• Specific pattern of 2’-OMe and 2’-F in both strands

– Improves nuclease stability while retaining RNAi activity

– Minimizes immune stimulation• Dispersed site specific PS linkages

– Full PS protection not needed• Targeting agent required for efficient cellular

uptake

Enhanced Stabilization Chemistry (ESC)

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GalNAc Conjugates for Targeted Delivery to HepatocytesA. ASGPR Pathway• Highly expressed in hepatocytes

– 0.5-1x106 copies/cell• Clears serum glycoproteins via

clatherin-mediated endocytosis• High rate of uptake• Recycling time ~15 minutes• Conserved across speciesB. Carbohydrate conjugate Schematic representation of triantennary GalNAc ligand. C. Conjugation of GalNAc ligand for ASGPR mediated delivery to hepatocytes The triantennary ligand is attached to the 3’ end of the sense strand of the siRNA, or to the 3’ end of the ASO through the use of a cleavable linker.

Figure 3. Receptor-Targeted Conjugate Approach for Delivery to Hepatocytes

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GalNAc-ASO ConjugatesdA-GalNAc3 on 3’ end of ASO

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GalNAc-siRNA ConjugatesGalNAc3 on 3’-sense of siRNA

GalNAc-ASOGalNAc-ASO

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ASGPR Pathway Multivalent N-Acetylgalactosamine Ligand

Conjugation of Multivalent N-Acetylgalactosamine Ligand to ASOs and siRNAs

Figure 4. Strategy for Head-to-Head Comparison of GalNAc-ASO and GalNAc-siRNA Conjugates

Direct comparison of multiple, sequence matched siRNAs and ASOs targeting Factor IX• Design of ASO and siRNA against >30 different sites• In vitro screen by transfection to identify active sites• Direct comparison of most active GalNAc-ASO with sequence-matched GalNAc-siRNA conjugates

in vivo – Potency and duration of effect – Distribution in liver tissue

• Pharmacokinetics comparison

Figure 5. Factor IX-Targeting siRNAs Demonstrate Better In Vitro Potency Than Sequence-Matched Factor IX-Targeting ASOs

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In Vitro Potency of Sequence-Matched Factor IX-targeting ASOs and siRNAs by Transfection in Hep3b CellsActivity of sequence-matched unconjugated ASOs and siRNAs in Hep3b cells. mRNA KD measured relative to control ASO and siRNA at 24 hours post-transfection with 10nM and 100nM ASO or 10nM siRNA using Lipofectamine 2000. • ≥40% target reduction is observed in

– ~70% of ASOs at 100nM, ~15% at 10nM – ~80% of sequence-matched siRNAs at 10nM – Sequences 1, 3, and 11 selected for in vivo evaluation

Figure 6. More Robust and Durable Pharmacodynamic Effect Observed With GalNAc-siRNA Conjugates Than Sequence-Matched GalNAc-ASO Conjugates in Mice

Sequence 3 Sequence 11

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In Vivo Rodent Activity of Sequence-Matched Factor IX GalNAc-siRNA and Factor IX GalNAc-ASO ConjugatesC57BL/6 mice received a single subcutaneous dose of Factor IX GalNAc-siRNA conjugate at 2 mg/kg or Factor IX GalNAc-ASO conjugate at 2 and 10 mg/kg. Factor IX activity relative to pre-dose levels was measured at select time points out to 49 days post-dose by ELISA. A maximum of 40-80% activity suppression was achieved across the 3 GalNAc-siRNA conjugates by Day 7 post-dose, whereas approximately 20-65% maximum suppression was demonstrated with sequence-matched GalNAc-ASO conjugates. Differences in duration of effect were also observed, with more sustained suppression demonstrated by all GalNAc-siRNA conjugates.

Figure 7. Lower Plasma Protein Binding Observed With GalNAc-siRNA Conjugate Relative to GalNAc-ASO

GalNAc-ASO GalNAc-siRNAMW PBS 10 50 90 PBS 10 50 90

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Plasma Protein Binding Profiles of Sequence-Matched Factor IX GalNAc-siRNA and Factor IX GalNAc-ASO Conjugates by Electrophoretic Mobility Shift AssayFactor IX compounds based on sequence 1 were separated by (A) native gel electrophoresis following incubation with increasing plasma concentrations. Compounds were detected by SYBR gold staining, and (B) densiometric analysis completed using Image J Software. % unbound compound was calculated relative to PBS.• 25-45% Factor IX GalNAc-siRNA bound to plasma (55-75% unbound) • 75-98% plasma binding demonstrated by sequence-matched Factor IX GalNAc-ASO (2-25% unbound)

Figure 8. Higher Distribution to Liver Demonstrated With GalNAc-siRNA Than GalNAc-ASO in Mice

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Tissue Concentration Profiles of Sequence Matched Factor IX GalNAc-siRNA and Factor IX GalNAc-ASO ConjugatesC57BL/6 mice received a single subcutaneous dose of Sequence 1 Factor IX GalNAc-siRNA conjugate at 2 mg/kg or Factor IX GalNAc-ASO conjugate at 10 mg/kg. Quantitation of siRNAs and ASOs in liver and kidney were performed by stem-loop qPCR and hybridization enzyme-linked immunosorbant assay (ELISA), respectively. Higher liver:kidney ratios and more potent mRNA reductions are observed across multiple timepoints post-single dose for the Factor IX GalNAc-siRNA than the sequence-matched GalNAc-ASO compound.

Figure 9. Greater Hepatocyte-Specific Conjugate Distribution & Transcript Reduction in Histological Liver Sections Following GalNAc-siRNA Administration

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•No detection of test article

•Cytoplasmic, diffuse hepatocellular •RNA transcript staining

•Punctate cytoplasmic hepatocyte labeling•Scattered Kupffer cell labelling

•Near complete target RNA signal loss•78% mRNA reduction by qPCR

•Strong cytoplasmic Kupffer cell labelling•Faint punctate labeling of hepatocytes

•Residual target RNA across zones•35% mRNA reduction by qPCR

Hepatocyte

Kupffer cells

Detection of Factor IX Test Articles by In Situ Hybridization (ISH) and Transcript Silencing by RNAscope in Liver Tissue SectionsC57BL/6 mice received a single subcutaneous dose of Factor IX GalNAc-siRNA conjugate or Factor IX GalNAc-ASO conjugate at 2 mg/kg, and livers harvested at 7 days-post dose. Livers were fixed in 10% formalin for 72 hours, and processed into paraffin blocks. Test articles were detected in 4 micron tissue sections with a Factor IX sequence-specific LNA-enhanced ISH probe, using the Ventana Discovery XT/Ultra platform. Factor IX transcript was detected using ACD Bio RNAscope technology on the Ventana Discovery XT/Ultra Platform.

A BEMSA Gel: Sequence 1 % Unbound Compound in Presence of Plasma