Update on GalNAc-DsiRNA-EX Conjugates. Bob D. Brown, Ph.D. CSO, SVP Research Dicerna Pharmaceuticals TIDES 2016, USA

Transcription

1 Update on GalNAc-DsiRNA-EX Conjugates Bob D. Brown, Ph.D. CS, SVP Research Dicerna Pharmaceuticals TIDES 2016, USA

2 2 Forward-looking statements This information may contain projections and other forward looking statements regarding future events, including regarding Dicerna s technology platform, product candidates, preclinical and clinical pipeline and milestones, regulatory objectives, market opportunities, and intellectual property. Such statements are predictions only and are subject to risks and uncertainties that could cause actual events or results to differ materially. These risks and uncertainties include, among others, the cost, timing and results of preclinical studies and clinical trials and other development activities; the unpredictability of the duration and results of regulatory review of New Drug Applications and Investigational NDAs; market acceptance for approved products and innovative therapeutic treatments; competition; the possible impairment of, inability to obtain and costs of obtaining intellectual property rights; and possible safety or efficacy concerns, general business, financial and accounting risks and litigation. More information concerning Dicerna and such risks and uncertainties is available on its website and in its press releases, and in its public filings with the U.S. Securities and Exchange Commission. Dicerna is providing this information as of its date and does not undertake any obligation to update or revise it, whether as a result of new information, future events or circumstances or otherwise. Additional information concerning Dicerna and its business may be available in press releases or other public announcements and public filings made after the date of this information.

3 DsiRNA-EX Conjugate Development Timeline License of DsiRNA and invention of DsiRNA-EX structures, EnCore, nascent conjugates 2008 Startup focus is oncology DCR-MYC candidate using DsiRNA and EnCore LNP is declared, developed, and the IND filed in Q Reduction to practice of conjugate structures Q expand internal chemistry effort Q initial SC conjugate knockdown results of >10 mg/kg Q announcement of SC knockdown of ~2mg/kg 2014 All future liver disease programs to be DsiRNA-EX Conjugates instead of lipid nanoparticles, oncology remains LNP-based Q translation of activity from rodents to non-human primates 2015 Systematic generation of GalNAc conjugates, SC or IV PC in monkeys for four therapeutic targets Q announcement of SC knockdown of ~0.6 mg/kg EC 50 >10 mg/kg EC 50 ~2 mg/kg EC 50 <1.0 mg/kg 2016 Q Last chemical optimizations, key monkey data for 3 targets Q2-Q Clinical Lead declarations for three DRNA programs EC mg/kg 3 EC 50 = 50% knockdown of mrna levels in mice

4 4 Dicerna GalNAc-DsiRNA-EX Conjugate Formats: A Flexible Platform

5 5 Solid Phase Synthesis of GalNAc-DsiRNA-EX Tetraloop Conjugates This is independent of the RNAi configuration Synthesis Solid phase synthesis using standard phosphoramidite chemistry GalNAc-phosphoramidites are compatible with standard synthesis and purification processes GalNAc-phosphoramidites are simple to synthesize and scale up GalNAc can be incorporated at any position and at any valency Functional across all DsiRNA-EX platforms DMTr C = CPG 1 R B B = Nucleobases: Uracil, N4-Acetylcytosine, N6-Benzoyladenine, N2-Isobutyroylguanine R = Me, F, or any 2'-modification DMTr = Dimethoxytrityl CE = Cyanoehtyl DCA = Dichloroacetic acid Detritylation DCA H H C P CE C 2 R 7 B 4) Detritylation DCA R R B B DMTr CE DMTr P CE B R P N(iPr)2 3 1) Coupling Tetrazole 2) Capping Ac2 C R B R or B DMTr CE DMTr CE P P N(iPr)2 C R 4 B GalNAc Phosphoroamidite B R AcHN B + 3) xidation I2/H2/Pyridine H 3 C Ac C Ac Ac B C R 5 6

6 6 GalNAc-DsiRNA-EX Structural Characterization: Linker Length SAR (In vivo knockdown of Hao1, 15-RES-189) Hao1 Knockdown vs. GalNAc-DsiRNA-EX Linker Length % Hao1 mrna Remaining (Rel to PBS) Prototype Hit 1x 3x 7x 11x 2 - to GalNAc Linker Length (PEG-equivalents) PBS DP2554P: DP2505G DP3692P: DP4387G DP5510P: DP4387G DP5513P: DP4387G DP2554P: DP4387G HA1 GalNAc-DsiRNA-EX Conjugate DP4968P: DP4387G Novel chemistry mg/kg The 2 -hydroxyl groups of the ribose rings in the GNRA tetraloop position the GalNAc to bind the ASGPR cooperatively, across a broad range of non-proprietary and proprietary linker lengths and chemistries

7 7 GalNAc-DsiRNA-EX Conjugate Lead Generation Each in vivo-active series of conjugates suggests features that streamlines the process, increases power In vitro screen: intrinsic potency of sequences Assay Development human and mouse, in silico DsiRNA design, synthesis and QC Standard DsiRNA, light modification Dicer cuts Tetraloop Pattern Stamping = Statistics Matter In vitro screening for potency: e.g. 12 patterns x 16 most potent sequences = 192 duplexes Tetraloop stamps: very heavy modification Nick 1 mg scale synthesis (high throughput) GalNAc-DsiRNA-EX conjugate screening in mice n-column, GalNAc-amidite-based synthesis Nick 10 mg to 2 g scale synthesis for lead optimization Conjugate optimization in mice, confirmation in NHP, further medchem optimization if needed Site-specific med-chem modifications Nick ur timeline to active GalNAc-DsiRNA-EX Conjugates in monkeys for new targets is 24 weeks or less

8 8 Primary DsiRNA-EX Activity versus Chemical Modifications HT screening of thousands of sequences and thousands of chemical modification patterns and combinations Patterns Eight DsiRNA-EX Tetraloop Modification Patterns vs. Twelve Primary DsiRNA s All sequences are selected for primary RNAi potency in vitro low to sub-picomolar hits are required Some sequences tolerate almost no chemical modification (7), others are extremely permissive to modification (9) Some patterns are poorly tolerated with strong exceptions ( ), others are generally well tolerated ( ) Design guidelines for permissive sequences and tolerated patterns are emerging 12

9 9 HT Screening to Tease out GalNAc-DsiRNA-EX and Modification Design Elements In vitro potency versus 65 sequences and 31 chemical modification patterns 125 In Vitro Screening Results: vs Chemical Modification Pattern Example 65 sequences x 31 modification patterns Each dot is a different DsiRNA-EX sequence being tested for tolerance of chemical modifications % mrna Remaining Serial Number M575/M538 M576/M565 M675/M859 M687/M860 M583/M887 M660/M710 M649/M709 M702/M709 M702/M886 M624/M710 M689/M720 M689/M481 M624/M708 M576/M859 M624/M707 M650/M708 M624/M861 M649/M861 M649/M1057 M916/M1033 M583/M729 M702/M710 M575/M721 M576/M538 M582/M538 M583/M494 M660/M709 M689/M447 M675/M874 M687/M874 M883/M932 Comparison of high- vs lowactivity sequences reveals useful sequence landmarks Clusters in this region reveal tolerated chemical modification features Each number is a different guide/passenger chemical modification combination Data mining is necessary to recognize many important design elements

10 10 Application of GalNAc-DsiRNA-EX Design Rules: Generalization Across s Single-dose, sc, 1mg/kg, 72 hours (16-RES-109) Three s versus Three Mod Patterns XYZ Gene Target Knockdown % hxyz Expression Remaining (Relative to PBS) = Pattern A = Pattern B = Pattern C DsiRNA-EX sequences permissive to chemical modifications are starting to yield predictable in vivo performance

11 11 Hepatic Accumulation Correlates with Increased Target mrna KD SAR table across different mod patterns Hepatic uptake and potency inform Pattern-Stamping GalNAc-DsiRNA-EX PK-PD Correlation in Mouse Liver High uptake Low activity High uptake High activity ng/g GalNAc-DsiRNA-EX / Liver Stable but inactive modifications Active in vitro, but too unstable in vivo Low uptake Low activity mrna Knockdown Low uptake High activity Ideal balance of nuclease resistance and retained intrinsic potency Highly potent in vitro, but too unstable in vivo In vivo activity appears to have a critical mass threshold Bioavailability is non-linear

12 GalNAc-DsiRNA-EX Discovery Case Study Application of GalNAc-DsiRNA-EX to a well-characterized hepatic disease gene, Alpha-1-antitrypsin (SERPINA1)

13 Reduction of Serum A1AT with GalNAc-DsiRNA-EX in PiZ Transgenic Mice Characterization of initial SC conjugate hits, focus in the lead prototype Conjugate Comparison, Multiple-Dose Relative Serum A1AT Protein Concentration Multidose mpk Study Day Relative Serum A1AT Protein Concentration Dose Response, Single-Dose PBS 5 mpk 10 mpk 25 mpk Single- Dose Study Day. SERPINA1-DP3490P:DP2518G SERPINA1-DP3552P:DP2532G = = = 5 mg/kg 10 mg/kg 25 mg/kg Hit: SERPINA1-DsiRNA-EX DP3490P:DP2567G Reference Prototype 13 SERPINA1-DP3490P:DP2567G Clear dose response and long duration of action with DP3490P:DP2567G

14 Very Long Duration of Action in Non-Human Primates (prototype conjugate) >75% reduction of serum A1AT and effects lasting >7 weeks post-last dose with a prototype conjugate Multiple-Dose PD in NHP, Prototype DsiRNA-EX GalNAc Conjugate % A1AT protein remaining (normalized to pre-dose) % Serum A1AT Protein (Relative to pre-dose) mg/kg qwx5 SC 14 weeks after last dose nly partial recovery 18.5 weeks after last dose Study Day Note this symbol (this conjugate) on previous slides SERPINA1-DsiRNA-EX DP3490P:DP2567G = = This is the prototype hit identified on Slide 13 not a med-chem optimized form 14 ur highest-potency DsiRNA-EX Conjugates are in on-going multi-dose duration studies

15 15 Screening of SERPINA1 GalNAc-DsiRNAs-EX Tetraloops These A1AT PiZ GEMM conjugate hits are below 1 mg/kg IC 50 in vivo, all different sequences GalNAc-DsiRNA-EX A1AT Protein KD in PiZ Mice = Previous Hit: SERPINA1-DsiRNA-EX DP3490P:DP2567G = DP4618P:DP4617G --- Primary #2: Killed by a new mod pattern A1AT Serum Protein PBS Each conjugate is a different sequence DP3490P: DP2567G DP4612P: DP4611G DP4614P: DP4613G DP4616P: DP4615G DP4618P: DP4617G DP4620P: DP4619G 5 mg/kg single dose, day 7 harvest. Protein normalized to individual animal pre-dose serum A1AT protein concentrations. = = = = DP4612P:DP4611G #3 DP4614P:DP4613G, #4 DP4616P:DP4615G: #5 DP4620P:DP4619G, #6 Four new sequences, higher potency hits created by stamping modification patterns on in vitro hits These conjugates are being optimized for both SC and IV administration

16 16 >10X Potency Improvement for SERPINA1-DsiRNA-EX Conjugates 15-RES-211: Reproduce DP4620P:DP4619G from Exp 15-RES-80 and optimize it in PiZ mice for NHP nomination DCR-AAT Leads: In Vivo IC 50 << 1.0 mg/kg in PiZ Mice Initial hit, 75% KD at Day 100 in NHP % SERPINA1 mrna (PiZ mrna) Duplex shown in prior slide PBS DP3490P DP2567G 5 mg/kg SERPINA1 prototype DP4620P DP4619G Hit from 15-RES-180 is 5X more potent DP4620P DP5080G Newest hits are >2X more potent again All short-linker GalNAc-phosphoramidites DP4620P DP5081G DP4620P DP5082G DP5083P DP5080G 1 mg/kg SERPINA1-optimized DP5083P DP5081G DP5083P DP5082G SERPINA1 hits were identified in mice The most potent prototype hit sequence yielded an IC 50 of 5 mg/kg in mice This prototype was scaled up and tested in monkeys: A 5 mg/kg qwx5 sc regimen of the prototype hit conjugate yielded 75% protein knockdown at Day 100 Med-chem optimization on one new sequence yielded hits >10X more potent in vivo in PiZ GEMM mice DP4620P: DP4619G DP4620P: DP5081G DP5083P: DP5081G Three tetraloop conjugates were tested in doseresponse curves, next slide

17 17 DCR-AATsc Dose-Response for Serum PiZ Protein: EC 50 <0.3 mg/kg SERPINA1-DsiRNA-EX dose response curves: EC mg/kg, single dose, sc (15-RES-248) Dose Response Curves, DCR-AATsc Hits in PiZ GEMM Mice single dose, sc, mg/kg % Serum hpiz Protein EC mg/kg from previous slide reproduced EC 50 <0.3 mg/kg EC mg/kg ur single-dose, dose response results confirm an EC 50 of <0.3 mg/kg for our best lead targeting SERPINA1 These duplexes are being scaled up for testing in monkeys 0 PBS DP4620P: DP4619G DP4620P: DP5081G DP5083P: DP5081G SERPINA1-DsiRNA-EX Retested for IC 50 (selected from hits shown two slides prior)

18 18 SERPINA1-DsiRNA-EX mrna Knockdown in Monkeys ngoing Day 32 biopsies, intermediate dosing regimen time point, one effective dose for Q4W group; 16-RES-043 Day 32 Snapshot: NHP SERPINA1 mrna Knockdown 16-RES-043 Study Design % SERPINA1 mrna R em aining Normalized to individual predose biopsies Groups A B C D Activity of these doses is minimal at this time point 0 Interim study results: P redose 1 m pk Q1W x12 1 m pk Q1W x5 2 m pk Q4W x2 2 m pk Q2W x7 3 m pk Q4W x4 A single dose of 3 mg/kg yields mrna knockdown of approximately 80% Treatment Regimen We estimate the NHP mrna EC 50 of our DCR-AATsc lead is 2.0 mg/kg Circulating alpha-1-antitrypsin protein is the primary molecular marker for this study, Analysis of the first serum samples for A1AT protein is underway

19 19 The Liver Target pportunity Space Example targets/indications across four therapeutic opportunity classes Rare Disease First in Class Chronic Liver Disease Liver Infectious Disease Rare FIC 1 Potent program leads identified Efficacy data in multiple animal models PD PC in NHP Lead selection in NHP underway Rare FIC 2 Hits in HDI mice PC hits for NHP selected Lead optimization underway Disease models being established Rare FIC 3 Hits in WT and HDI mice PC hits for NHP selected Efficacy markers observed in vivo Lead opt. underway Rare FIC 4 Hits in WT and HDI mice PD studies in mouse underway PC hits for NHP selected Lead opt. underway Rare FIC 5 Hits in WT and HDI mice PD studies in mouse underway ptimization underway Multiple Fibrosis/NASH Conjugate hits for multiple target genes with high in vivo potency Efficacy markers observed in multiple fibrosis models pt. underway HBV Hit conjugates identified for PC studies HBV surface antigen reductions achieved in vivo ptimization underway Rare Disease Fast Follower Cardiovascular Disease HA1 PH1 SERPINA1 A1AT liver disease SERPINC1 Hemophilia A & B C5 PNH, ahus TTR Amyloidosis PCSK9 Hypercholesterolemia APC3 Dyslipidemia Potent program leads identified Animal model efficacy data in PH1 mice PD PC in NHP Lead selection in NHP underway Potent program leads identified Animal model efficacy data in PiZ mice PD PC in NHP Lead selection in NHP underway Hits in HDI mice PD studies in mouse underway PC hits for NHP selected ptimization underway Hits in HDI mice Potent murine hits Human sequence optimization underway Efficacy in arthritis model Potent leads and conjugates identified n hold Screening and assay development initiated Test case for new conjugate design rules Screening and assay development initiated Test case for new conjugate design rules

20 20 State of the Art RNAi Platform Building upon the Long History of GalNAc The Tetraloop configuration of GalNAc-DsiRNA-EX offers specific advantages - High potency RNAi sequences can be chemically modified with no loss of activity - Direct combination of potency, stability, ease of chemical modification - Molecules can be efficiently designed by stamping some patterns on sequences - The platform is remarkably tolerant of conjugate linker lengths and chemistries Potent in vivo activity in rodents and monkeys - 1 mg/kg in vivo IC 50 GalNAc-DsiRNA-EX conjugates have been identified against multiple targets - Almost six months of A1AT protein knockdown in monkeys with an early prototype conjugate - Long-term testing in monkeys is underway against multiple therapeutic targets with high-potency leads GalNAc-DsiRNA-EX hits against several therapeutic targets have been developed - Non-human primate data for short and long-term knockdown will be released throughout 2016

21 Acknowledgments Cheng Lai, Ph.D. Translation Mike Dills Rohan Diwanji Jessica Gierut Natalie Pursell Wei Zhou Weimin Wang, Ph.D. Chemistry John Feinberg Boyoung Kim Venkata Krishnamurthy Rebecca Lescarbeau Qingyi Li Naim Nazef Juili Shelke Serena Shui Rachel Storr Jennifer Lockridge, Ph.D. Prog. Manag. Hank Dudek, Ph.D Discovery Luciano Apponi Utsav Saxena Anee Shah Nandini Venkat Jr-Shiuan Yang Marc Abrams, Ph.D. Pre-Pharm Edmond Chipumuro Girish Chopda Kevin Craig Chaitali Dutta Shanthi Ganesh Marty Koser Zakir Siddique David Miller, Ph.D. perations Geremew Desta Jeffrey H. Teckmann, M.D. Saint Louis University PiZ mice

22 Thank you