Alnylam Pharmaceuticals, Inc. · NASDAQ:ALNY
Alnylam Pharmaceuticals — MIT Center for Biomedical Innovation's Biomanufacturing Summit (November 2010)
28 slides from Alnylam Pharmaceuticals's conference presentation, Nov 17, 2010
Slide 1: Application of RNAi to Block Adventitious Infections in Bioprocessing
MIT Center for Biomedical Innovation’s Biomanufacturing Summit Application of RNAi to Block Adventitious Infections in Bioprocessing Anthony J. Rossomando, Ph.D. November 17, 2010
Slide 2: Alnylam RNAi Technology
Application of RNAi to Block Adventitious Infections in Bioprocessing Alnylam RNAi Technology 2
Slide 3: RNA Interference
RNA Interference Synthetic siRNA dsRNA dicer Cleavage Natural Strand separation Process of RNAi RISC Complementary pairing Targeted Gene Silencing mRNA (A) n Cleavage mRNA degradation (A)n 3
Slide 4: Alnylam RNAi Product Platform
Alnylam RNAi Product Platform An Integrated Capability Lead Selection Lead Optimization Delivery conjugation formulation Introduce chemical modifications for “drug-like” properties siRNA design Stabilization PK/PD Selectivity screen Potency Biodistribution » Off target effects Selectiv
Slide 5: Efficient Identification of siRNAs
Efficient Identification of siRNAs High Potency, Selectivity and Stability •In Vitro and Informatic Screens and chemical modification 5
Slide 6: Combinatorial Chemistry Approach for Novel Materials
Combinatorial Chemistry Approach for Novel Materials Alnylam-MIT Collaboration Library Components In Vitro Screen O10 O12 O13 O14 O15 O18 N12 N14 N15 N16 N18 Q10 Q12 Q13 Q14 Q18 1 O NH2 33 HO NH2 O NH2 O10 O O NH2 82 N NH2 NO TEST 1 O 6 O 34 HO 86 HO N NH2 110 H2N N 0-20 6 O12 O NH2 NH2 O 7 O HO HO
Slide 7: Alnylam Biotherapeutics
Application of RNAi to Block Adventitious Infections in Bioprocessing Alnylam Biotherapeutics 7
Slide 8: Application of RNAi at Scale
Application of RNAi at Scale Leveraging Alnylam Capability and Asset Base Transfer and Application of Delivery, “Know How,” I.P. and Technology 8
Slide 9: Many Key Pathways are Well Characterized
Many Key Pathways are Well Characterized Enables a Candidate Gene Approach Viral Load & Engineered Cell Line Infection Glycosylation DNA Product Stress & Apoptosis mRNA mRNA Target Critical Metabolism Pathways via & RNAi Host Recombinant Growth Protein Protein Secretion & PTM • Quality • Quantity Id
Slide 10: Alnylam Biotherapeutics
Alnylam Biotherapeutics Achieving RNAi at bioprocessing scale » Identification of novel delivery lipids » RNAi Efficiency Scales Effectively Inhibiting Vesivirus 2117 Replication in CHO Cells Conclusions 10
Slide 11: Achieving RNAi at Scale
Achieving RNAi at Scale Novel Delivery Lipids (NDLs) Desired Attributes of NDLs Efficacy Tolerability Low cost Detection/ability to assay for clearance Stability 11
Slide 12: Novel Delivery Lipids Developed at Alnylam
Novel Delivery Lipids Developed at Alnylam Developed for CHO Cell Applications Suspension CHO cells (250 mL shake flasks) Day 5 viable cell density Seed density 200,000/mL 3.0 600 cont Rel. LDH activity Cell density (x106) 2.5 500 (U/106 cells) 2.0 400 1.5 300 -52% 1.0 200 -69% -83% 0.5 100 0 0 Cell
Slide 13: Alnylam NDLs are Better Tolerated than Commercial Control
Novel Delivery Lipids Developed at Alnylam Alnylam NDLs are Better Tolerated than Commercial Control Suspension CHO cells (50 mL shake flasks) Day 3 viable cell density Seed density 200,000/mL 2.5 1000 cont Cell density (x106) 2.0 LDH activity 750 (U/106 cells) 1.5 500 1.0 -65% -63% 0.5 250 0 0 20 m
Slide 14: Alnylam NDLs are Better Tolerated than Commercial Control
Novel Delivery Lipids Developed at Alnylam Alnylam NDLs are Better Tolerated than Commercial Control Cell Density Percent Viability 3.25 Cells only 105 3.00 100 1.5 l/ml RNAiMax 2.75 95 2.50 0.8 g/ml P8 90 2.25 85 106 cells/ml % Viability 2.00 80 1.75 Cells only 75 1.50 70 1.5 l/ml RNAiMax 1.25 65 0
Slide 15: Durable Target Gene Knockdown over 6 Days
Novel Delivery Lipids Developed at Alnylam Durable Target Gene Knockdown over 6 Days Using the P8 NDL an siRNA directed against Lactate Dehydrogenase (LDH) achieves 80-90% knockdown of LDH activity for 6 days with a single 1nM dose in a 1 L bioreactor LDH Activity (Units/106 cells) 0.8 0.7 Control s
Slide 16: Novel Delivery Lipids (NDLs)
Novel Delivery Lipids (NDLs) Highly effective NDLs have been identified for CHO cells Optimal NDLs for a given cell can vary exhibiting cell line specificity » Some lipids are active across multiple cell types NDLs with efficient uptake and tolerabilities are rare Optimal NDLs for CHO cells have bee
Slide 17: RNAi Efficiency Scales Effectively
RNAi Efficiency Scales Effectively Comparison of 40L vs. 3L CHO DG44 Batch Cultured Cells 0.8 (Units/106 cells) 0.7 LDH Activity 0.6 0.5 Control siRNA 0.4 3.50 0.3 100.0 0.2 Viable Cell Density (x106 cells/mL) LDH siRNA 0.1 0.0 90.0 3.00 0.0 2.0 4.0 6.0 8.0 Elapsed Time (Days) 80.0 2.50 70.0 % LDH A
Slide 18: RNAi Efficiency Scales Effectively: Summary
RNAi Efficiency Scales Effectively Summary In scale up we see linearity that suggests efficiency is very similar at small and larger scales » Due to more consistent conditions (oxygen, pH etc) and cell/siRNA exposure in the reactors efficiency may be better at larger scales Multi-dose protocols can
Slide 19: Alnylam Biotherapeutics
Alnylam Biotherapeutics Achieving RNAi at bioprocessing scale » Identification of novel delivery lipids » RNAi Efficiency Scales Effectively Inhibiting Vesivirus 2117 Replication in CHO Cells Conclusions 19
Slide 20: Vesivirus (Calicivirus) Strain 2117
Vesivirus (Calicivirus) Strain 2117 http://www.expasy.ch/viralzone/all_by_species/197.html 20
Slide 21: Vesivirus (Calicivirus) Strain 2117 siRNA Duplexes
Vesivirus (Calicivirus) Strain 2117 siRNA Duplexes 24 siRNA duplexes (each) designed and synthesized to target two of the key viral enzymes used for replication. Duplexes chosen to maximize predicted efficacy, minimize off-targeting in CHO genome RNA helicase RNA polymerase Genome coordinates: 1 151
Slide 22: Treatment of CHO Cells Grown Suspension
Treatment of CHO Cells Grown Suspension With siRNA Directed Against Vesivirus 2117 to Block Infection CHO cells not treated with Vesivirus-directed siRNA Vesivirus replication occurs Vesivirus No RNAi infected Vesivirus CHO cell CHO cell CHO cells are infected with Vesivirus followed by transfection
Slide 23: Vesivirus qPCR Standard Curve
Vesivirus qPCR Standard Curve Slope -3.2024982 Y-Inter: 37.77677 R2: 0.9975183 Collaboration with Goodfellow Lab, Imperial College London 23
Slide 24: Vesivirus siRNA Duplex Screen
Vesivirus siRNA Duplex Screen qPCR Assay H P H P H P H P H P H P H P H P H - Helicase P - Polymerase Final Concentration = 10 nM Collaboration with Goodfellow Lab, Imperial College London 24
Slide 25: RNAi Blocks Vesivirus 2117 Infection & Replication
RNAi Blocks Vesivirus 2117 Infection & Replication CHO DG44 Cells NS – non Specific siRNA Polymerase Helicase Polymerase 1. Infect CHO DG44 cells with Vesivirus for 3 hours – Spin down cells and remove unbound virus 2. Transfect siRNA at indicated concentration using Lipofectamine 2000 3. Extract RN
Slide 26: Alnylam Biotherapeutics
Alnylam Biotherapeutics Achieving RNAi at bioprocessing scale » Identification of novel delivery lipids » RNAi Efficiency Scales Effectively Inhibiting Vesivirus 2117 Replication in CHO Cells Conclusions 26
Slide 27: Conclusions
Conclusions Novel delivery lipids have been identified that efficiently deliver siRNA to manufacturing cell lines with no effect on cell density or viability compared to commercially available lipids RNAi effect is potent, durable following a single dose, and scalable RNAi can be used at scale in bi
Slide 28: Thank You
Thank You