GalNAc-DsiRNA-EX Conjugate Development. Bob D. Brown, Ph.D. CSO, SVP Research Dicerna Pharmaceuticals TIDES 2016, USA

Transcription

1 GalNAc-DsiRNA-EX Conjugate Development Bob D. Brown, Ph.D. CS, SVP Research Dicerna Pharmaceuticals TIDES 216, 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 3 Dicerna GalNAc-DsiRNA-EX Conjugate Formats: A Flexible Platform

4 DsiRNA-EX Conjugate Development Timeline License of DsiRNA and invention of DsiRNA-EX structures, EnCore, nascent conjugates 28 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 Q3 213 expand internal chemistry effort Q2 214 initial SC conjugate knockdown results of >1 mg/kg Q4 214 announcement of SC knockdown of ~2mg/kg 214 All future liver disease programs to be DsiRNA-EX Conjugates instead of lipid nanoparticles, oncology remains LNP-based Q2 215 translation of activity from rodents to non-human primates 215 Systematic generation of GalNAc conjugates, SC or IV PC in monkeys for four therapeutic targets Q3 215 announcement of SC knockdown of ~.6 mg/kg EC 5 >1 mg/kg EC 5 ~2 mg/kg EC 5 <1. mg/kg 216 Q1 216 Last chemical optimizations, key monkey data for 3 targets Q2-Q4 216 Clinical Lead declarations for three DRNA programs EC 5.3 mg/kg 4 EC 5 = 5% knockdown of mrna levels in mice

5 5 Solid Phase Synthesis of GalNAc-DsiRNA-EX Tetraloop Conjugates 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 = Nucleobases: Uracil, N4-Acetylcytosine, N6-Benzoyladenine, N2-Isobutyroylguanine R = Me, F, or any 2'-modification B 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 Phosphoramidite 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 Characterization: 2 -H to GalNAc Linker Length SAR 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 (PEGequivalents) PBS DP2554P: DP255G DP3692P: DP4387G DP551P: DP4387G DP5513P: DP4387G Hao1 GalNAc-DsiRNA-EX Conjugate DP2554P: DP4387G 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 Discovery and ptimization In vitro screen: intrinsic potency of sequences Standard DsiRNA, light modification Dicer cuts Tetraloop Pattern Stamping = Statistics Matter 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 1 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 Each interaction suggests features that streamlines the process

8 8 HT Screening to Reveal Significant and Modification Elements In Vitro Screening Results: vs Chemical Modification Pattern 65 sequences x 31 modification patterns 125 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/M86 M583/M887 M66/M71 M649/M79 M72/M79 M72/M886 M624/M71 M689/M72 M689/M481 M624/M78 M576/M859 M624/M77 M65/M78 M624/M861 M649/M861 M649/M157 M916/M133 M583/M729 M72/M71 M575/M721 M576/M538 M582/M538 M583/M494 M66/M79 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 and passenger mod combination Data mining is necessary to reveal subtle but important features

9 Primary DsiRNA-EX Activity versus Chemical Modifications HT screening of thousands of sequences and thousands of chemical modification patterns and combinations In Vitro Screening of Chemically Modified DsiRNA-EX Tetraloops Against ne Gene 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 ( ) Permissive sequence and tolerated pattern features are emerging

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

11 11 Hepatic Accumulation Correlates with Increased Target mrna Knockdown ng/g GalNAc-DsiRNA-EX / Liver GalNAc-DsiRNA-EX PK-PD Correlation in Mouse Liver High uptake Low activity Stable but inactive modifications Active in vitro, but too unstable in vivo Low uptake Low activity mrna Knockdown High uptake High activity Highly potent in vitro, but too unstable in vivo Low uptake High activity Ideal balance of nuclease resistance and retained intrinsic potency In vivo activity appears to have a critical mass threshold SC bioavailability is nonlinear, unlike IV

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 Conjugate Comparison, Multiple-Dose Relative Serum A1AT Protein Concentration 5 Multidose mpk Study Day Relative Serum A1AT Protein Concentration Dose Response, Single-Dose PBS 5 mpk 1 mpk 25 mpk Single- Dose Study Day. SERPINA1-DP349P:DP2518G SERPINA1-DP3552P:DP2532G = = = 5 mg/kg 1 mg/kg 25 mg/kg Hit: SERPINA1-DsiRNA-EX DP349P:DP2567G Reference Prototype 13 SERPINA1-DP349P:DP2567G Clear dose response and long duration of action with DP349P:DP2567G

14 Very Long Duration of Action in Non-Human Primates (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 nly partial recovery 18.5 weeks after last dose 14 weeks after last dose Study Day 14 Note this symbol (this conjugate) on previous slides SERPINA1-DsiRNA-EX DP349P:DP2567G = = This is the prototype hit identified on Slide 13 not a med-chem optimized form >75% reduction of serum A1AT and effects lasting >7 weeks post-last dose

15 15 Screening of SERPINA1 GalNAc-DsiRNAs-EX Tetraloops In Vivo GalNAc-DsiRNA-EX A1AT Protein KD in PiZ Mice = Previous Hit: SERPINA1-DsiRNA-EX DP349P:DP2567G 12 = DP4618P:DP4617G --- Primary #2: Killed by a new mod pattern A1AT Serum Protein PBS DP349P: DP4612P: DP4614P: DP4616P: DP4618P: DP462P: DP2567G DP4611G DP4613G DP4615G DP4617G 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 DP462P:DP4619G, #6 Four new sequences, higher potency hits created by stamping a specific modification patterns on in vitro hits These hits are being optimized for both SC and IV administration

16 16 >1X Potency Improvement for SERPINA1-DsiRNA-EX Conjugates Potency optimization in PiZ mice yields candidates for testing in NHP DCR-AAT Leads: In Vivo IC 5 << 1. mg/kg in PiZ Mice Initial hit, 75% KD at Day in NHP Hit from 15-RES-18 is 5X more potent SERPINA1 hits were screened in mice % SERPINA1 mrna (PiZ mrna) Duplex shown in prior slide PBS DP349P DP2567G DP462P DP4619G DP462P DP58G Newest hits are >2X more potent again All short-linker GalNAc-phosphoramidites DP462P DP581G DP462P DP582G DP583P DP58G DP583P DP581G DP583P DP582G The most potent prototype hit sequence yielded an IC5 of 5 mg/kg Chemical optimization on one new sequence yielded hits >1X more potent in vivo in PiZ GEMM mice DP462P: DP4619G DP462P: DP581G DP583P: DP581G Three tetraloop conjugates were tested in dose-response curves, next slide 5 mg/kg SERPINA1 prototype 1 mg/kg SERPINA1-optimized

17 17 SERPINA1-DsiRNA-EX Dose-Response: EC 5 <.3 mg/kg Dose Response Curves, DCR-AATsc Hits in PiZ GEMM Mice single dose, sc, mg/kg % Serum hpiz Protein EC 5 1. mg/kg from previous slide reproduced EC 5 <.3 mg/kg EC 5.3 mg/kg Single-dose, dose response results confirm an EC 5 of <.3 mg/kg for the best lead targeting SERPINA1 These duplexes were scaled up for testing in monkeys PBS DP462P: DP4619G DP462P: DP581G DP583P: DP581G SERPINA1-DsiRNA-EX Retested for IC 5 (selected from hits shown on the previous slide)

18 18 SERPINA1 mrna Knockdown in Monkeys - ngoing Day 32, intermediate time point, one effective dose for Q4W group Day 32 Snapshot: NHP SERPINA1 mrna Knockdown 16-RES-43 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 P re- 1 m pk 1 m pk 2 m pk 3 m pk dose Q1W x12 Q1W x5 2 m pk Q4W x2 Q2W x7 Q4W x4 Interim results: Treatment Regimen A single dose of 3 mg/kg yields mrna knockdown of approximately 8% 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 HA1 mrna Knockdown in Monkeys: 1 mg/kg IC 5 for HA1-DsiRNA-EX Single-dose, sc, 3 mg/kg comparison, biopsies on Day 14 post-dose Hao1 mrna Knockdown, Single-dose Comparison Novel GalNAc-DsiRNA-EX modifications Liver [HA1 mrna] Day 14 % Hao1 Remaining, NHP (rel to time-matched PBS) Dose mg/kg PBS 5 DP3692P: DP4387G DP4621P: DP439G DP3692P: DP5452G DP3692P: DP5455G DP5844P: DP5843G ne dose of 3 mg/kg achieves 88% Hao1 mrna knockdown in nonhuman primates 3 mg/kg single-injection dosing is feasible in humans Equal or better potency is expected in humans DCR-PH1sc Candidate

20 2 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

21 21 State of the Art RNAi Platform Building upon the Long History of GalNAc The Tetraloop GalNAc-DsiRNA-EX configuration offers - High potency DsiRNA sequences can be chemically modified with no loss of activity - Molecules can be efficiently designed by stamping some patterns on permissive sequences - The platform is remarkably tolerant of conjugate linker lengths and chemistries - Direct combination of potency, stability, ease of chemical modification Potent in vivo activity in rodents and monkeys - 1 mg/kg in vivo IC 5 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 We are on track to declare our DCR-PH1 program lead

22 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

23 Thank you