Recent Advances in the Lipid Nanoparticle-Mediated Delivery of Messenger RNA Ian MacLachlan, Ph.D. Executive Vice President and Chief Technology Officer AsiaTIDES Conference February 25, 2014 Tokyo, Japan 1 Tekmira’s Lipid Nanoparticle Technology Components Amino Lipid Structural Lipid PEG - Lipid Nucleic Acid Jeffs et al. Pharm. Res. 2005. 40-140 nm diameter 2 1 Tekmira’s Lipid Nanoparticle Technology Components Amino Lipid Structural Lipid PEG - Lipid Nucleic Acid = 100 nm ~90 nm diameter Jeffs et al. Pharm. Res. 2005. 3 Tekmira’s Lipid Nanoparticle Technology Components Amino Lipid Structural Lipid PEG - Lipid Nucleic Acid = 100 nm ~80 nm diameter Jeffs et al. Pharm. Res. 2005. 4 2 Methods of Lipid Nanoparticle Manufacture Detergent Dialysis Spontaneous Vesicle Formation By Ethanol Injection Lipids Nucleic Acid Buffer with Detergent Lipids in EtOH Nucleic Acid Mixed Micelles in Detergent LNP in EtOH Dialysis TFU Detergent Ethanol LNP LNP Wheeler et al, Gene Therapy, 6: 271-281, 1999. Fenske et al, Methods in Enzymology, 346: 36-71, Academic Press, San Diego, 2002. 5 Ethanol Dilution LNP Manufacturing Process Flow • Simple controlled mixing process • No physical particle sizing step • Applicable broadly for nucleic acid encapsulation • Highly scalable and reproducible • Efficient (high encapsulation) • CGMP; protected by IP Jeffs et al. Pharm. Res. 2005. 6 3 Comparison of LNP Particle Morphologies Electron Dense Lipid Nanoparticles Preferred for Therapeutic Applications A & B Manufactured By Ethanol Injection A B = 50 nm C D C & D Manufactured By Ethanol Dilution 7 cGMP LNP Manufacturing Has Been Scaled 0.4 g 50 g 100 g 1000 g Scale/g Test Method 0.4 2 2 10 10 100* 1000* Appearance Visual Pass Pass Pass Pass Pass Pass Pass pH pH Meter 7.3 7.4 7.4 7.4 7.4 7.4 7.2 siRNA Assay AX HPLC 2.2 2.0 1.9 2.0 1.9 2.0 2.2 siRNA Purity AX HPLC ND ND 86.7 89.8 88.4 88.8 95.6 siRNA Encapsulation Fluorometric assay 96 95 94 93 97 95 92 Dynamic Light Scattering Particle Size Analysis 79 0.04 78 0.04 81 0.04 78 0.04 81 0.02 78 0.04 80 0.03 Particle Size: Z-average Polydispersity *Demonstration runs 8 4 cGMP LNP Manufacturing at Tekmira Particle Formation Skid in Tekmira cGMP Facility (suitable for 100g+ batches) Tangential Flow Ultrafiltration Skid 9 Lyophilized LNP Products Tekmira has developed lyophilized LNP to: • Eliminate cold-chain requirements • Facilitate use in tropical climates • Allow use of “meta-stable” formulation components Current product characteristics: • • • • • Reconstitution time <15 sec. Particle size <80 nm >90% encapsulation Stable at 40°C Bioequivalent to wet formulation (efficacy, tolerability) 10 5 Function of Amino Lipids Formulating: Low pH, Charged Storage: pH ~7, Neutral Circulation: pH ~7.4, Neutral Endosome: pH 5 - 6, Charged • Ionizable amino head group allows lipid to take advantage of surrounding pH • Linoleyl chains prevent tight packing and promote fusion 11 12 6 Lipid Nanoparticles (LNP) – Experience in Man Seven LNP Products have entered Clinical Development Product Phase Indication Treated* Comments ALN-TTR01 1 Amyloidosis 24 First demonstration of RNAi silencing of disease causing protein in man ALN-TTR02 2 Amyloidosis 13 Potent pharmacodynamic effect demonstrated, well tolerated TKM-Ebola 1 Ebola Infection >1 (ongoing) TKM-ApoB 1 Hyperlipidemia 17 TKM-PLK1 1 Oncology >23 (ongoing) Tumor responses reported (data collection ongoing) ALN-PCS 1 Hyperlipidemia 24 Potent pharmacodynamic effect demonstrated, well tolerated ALN-VSP 1 Oncology 41 Two patients treated longer than a year Results not yet reported Early LNP formulation used • Numbers include only publicly reported data, and do not include placebo controls • All LNP products have been manufactured by Tekmira 13 Clinical Validation of LNP Technology and RNAi Alnylam’s TTR Program Using Tekmira’s LNP Improvements in LNP Formulation Technology Translate from The Lab to The Clinic TTR01 Phase 1 Data 120 100 100 80 >10x 60 40 Placebo 20 TTR01 1.0 mg/kg 0 0 5 10 15 Day 20 Mean Serum TTR Relative to Baseline Mean Serum TTR Relative to Baseline TTR02 Phase 1 Data 120 80 Placebo 60 TTR02 0.3 mg/kg 40 20 25 0 30 XIIIth International Symposium on Amyloidosis, May 2012 Single LNP Dose 0 5 10 15 Day 20 25 30 B.U. Med Center July, 2012 “Second Generation LNP” “First Generation LNP” Both Products Contain the Same TTR siRNA LNP Formulation Improvements Result in More than 10 Fold Increase in Potency 14 7 Evolution in LNP Formulation Technology Ongoing Formulation Improvements Translate from the Lab to the Clinic LNP 10000 8000 6000 4000 Therapeutic Window Potency Lipid Conjugate Fold Increase in Potency 14000 12000 Second Generation 2012 Tolerability TI ~1000 Third Generation MC3 First Generation LNP Third Generation LNP 2000 0.01 Year TI ~5 First Gen. 2006 0 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 TI ~150 Second Generation 2009 0.1 1 siRNA Dose (mg/kg) 10 Mean Serum TTR Remaining Relative to Baseline 100 80 Clinical Data Confirms that LNP NHP 60 Formulation Improvements Translate Human 40 from Non-Clinical Species to Man 20 0 Single LNP Dose 0 5 10 15 Day 20 25 30 B.U. Med Center July, 2012 15 Observations from Clinical Development Efficacy Observations Safety Observations • Multiple POC for RNAi in human subjects • Absence of significant hepatotoxicity • Multiple POC for LNP in human subjects • Multiple doses are well tolerated • Formulation improvements translate from the lab to the clinic • Immune mediated effects avoided with generational improvements • PK is maintained in multiple dosing • Profound knockdown at dose levels below the inflammatory threshold LNP enabled RNAi drugs are a viable therapeutic modality, capable of delivering products with favorable PK and PD characteristics and a clinically and commercially viable therapeutic index 16 8 Alternative LNP Payloads LNP Enable Essentially Any Nucleic Acid Payload Small ‘RNA’ Payloads • Canonical siRNA • UsiRNA • MV-RNA • miRNA Small ‘DNA’ Payloads • Antisense Oligonucleotides • LNA Larger Nucleic Acids • Plasmid DNA • Messenger RNA 17 Alternative LNP Payloads LNP Enable Essentially Any Nucleic Acid Payload Small ‘RNA’ Payloads • Canonical siRNA • UsiRNA • MV-RNA • miRNA Small ‘DNA’ Payloads • Antisense Oligonucleotides • LNA Larger Nucleic Acids • Plasmid DNA • Messenger RNA 18 9 mRNA-LNP Formulations: • Tekmira has developed a broad library that features thousands of lipid compounds and LNP formulations. • The formulations described here, while drawing from our experience with other nucleic acids, are tailored specifically for mRNA. • Liver LNP – Liver directed formulation for mRNA delivery • Tumor LNP – Distal tumor directed formulation for mRNA delivery • LNP described here contain commercially available mRNA, which is: • • • • Capped Polyadenylated Fully substituted with pseudouridine and 5-methylcytidine Purified by silica spin column. 19 mRNA-LNP Formulations: • mRNA are readily encapsulated using LNP methodology, though early work resulted in reduced encapsulation efficiency. • Physicochemical characteristics are similar to LNP currently in the clinic. • mRNA-LNP stable at 4°C for >1 year, measured by: • Size • Polydispersity • mRNA Encapsulation • No toxicities at the doses described in this work. 20 10 Advancements in mRNA Formulation Increasing Encapsulation Efficiency to Levels that Support Product Development Earlier Process = Less Efficient Encapsulation Composition Amino Lipid Ratios Method Zavg PDI % Encaps 2nd Gen 1 A 93 0.09 48% 1 A 86 0.08 62% 1 B 82 0.10 93% 2 B 92 0.05 95% 3 B 82 0.11 92% 3rd Gen Refined Process = Improved Encapsulation 21 Why Does mRNA Encapsulation Efficiency Matter? Luciferase Gene Expression in Liver 6 hr after IV Administration of mLuc LNP (0.05mg/kg) in Balb/C Mice • Free or surface associated mRNA is available to nucleases and subject to degradation, COGs. • Surface associated mRNA is more effectively presented to TLRs and can be immune stimulatory. Luciferase Activity (ng/g tissue) undermining potency, therapeutic index and 150 125 100 75 50 25 0 • >90% encapsulation is typically required for 62% 93% mRNA Encapsulation GMP and product development purposes. 22 11 Advancements in mRNA Formulation Optimizing LNP Composition to Support Product Development 1st Generation LNP Amino Lipid A Ratios Method Zavg PDI % Encaps DLinDMA 124 0.07 58 B C2K 128 0.10 66 C MC3 158 0.11 50 D XXX 79 0.06 97 E YYY 86 0.10 95 2nd Generation 2 B 3rd Generation 23 Why Does Size and LNP Composition Matter? mLuc mRNA Expression in Liver 6 hrs (0.5 mg/kg) in Mice composition result in optimal efficacy of mRNA LNP 3rd Generation 3000 2500 2000 1500 1000 1st Generation Optimal size and LNP Luciferase Activity (ng/g liver) 3500 2nd Generation 500 0 DLinDMA A C2K B MC3 C XXX D YYY E LNP Composition 24 12 mRNA Lipid Nanoparticle Morphology Electron Dense Lipid Nanoparticles Containing mRNA • Electron dense lipid nanoparticles have been shown to be highly efficacious in delivering an array of nucleic acid payloads and are the preferred particle morphology for commercialization. • Using adapted formulation conditions and lipid compositions integrating third generation LNP technology we have successfully prepared electron 100 nm dense LNP fully encapsulating mRNA. 25 mRNA LNP Activity In Vivo Enabling Highly Effective mRNA Delivery and Expression Luciferase Gene Expression in Balb/C Mice 6 & 24 hrs after IV Administration of mLuc mRNA Liver LNP (0.5 mg/kg, ~10ug) 10000000 mLuc-LNP (6hr) Luciferase Activity (pg/g organ) 1000000 4 Orders of Magnitude Greater Than Previously Described* Positive Control mLuc-LNP (24hr) TransIT-mLuc (6hr) 100000 Luciferase expression normalized to mass quantities of recombinant Luc assayed in buffer. 10000 1000 100 10 Liver Spleen Lung Kidney Heart * Kariko et al. Mol Ther. 2012 May;20(5):948-53. 26 13 mRNA LNP Activity In Vivo Linear mRNA Dose Response Over Three Orders of Magnitude Luciferase Gene Expression in Balb/C Mice Liver 6 hrs after IV Administration of mLuc mRNA Liver LNP Luciferase Activity (ng/g Liver) 10000 1000 100 10 1 0.1 0.001 ~200ng 0.01 0.1 mLuc Dose (mg/kg) 1 ~20 ug 27 mRNA LNP Activity In Vivo mCherry mRNA Delivery and Expression in the Liver – Negative Control 28 14 mRNA LNP Activity In Vivo mCherry mRNA Delivery and Expression in the Liver – 24h 29 Liver Delivery of mCherry mRNA Impact of LNP Formulation Composition PBS Control mLuc LNP Control Second Generation LNP Third Generation LNP 30 15 mRNA-LNP Delivery to Orthotopic Liver Tumors Time Course and Biodistribution of Gene Expression in Scid Mice Bearing Orthotopic Hep3B Tumors Following 0.5 mg/kg (~10ug) of mLuc-Liver LNP (n=5) Luciferase Activity (pg/g organ) 1000000 100000 10000 1000 100 10 Liver Tumor Spleen 6h 24 h 48 h 31 mRNA-LNP Delivery to Orthotopic Liver Tumors Orthotopic Hep3B Liver Tumors H&E Liver Tumour 32 16 mRNA-LNP Delivery to Orthotopic Liver Tumors mCherry Expression @ 24h Liver Tumour 33 Biodistribution of LNP-mRNA Expression in Tumor-bearing Mice Distal Tumor Model –Tumor Directed LNP Luciferase mRNA Expression Following IV Administration of mLuc-Tumor LNP in SubQ Hep3B Tumor-bearing Scid Mice (n=4, 1.0mg/kg (~20ug) luc mRNA ) Luciferase Activity (pg/g Tissue) 1000000 Liver Spleen Lung Kidney Heart Tumor 100000 10000 1000 100 2h 4h 6h 8h 16 h 24 h 48 h 34 17 LNP-mRNA Expression in Tumor-bearing Mice Tumor-mRNA Expression Is Long Lived Relative to Other Tissues Luciferase mRNA Expression Following IV Administration of mLuc-Tumor LNP in SubQ Hep3B Tumor-bearing Scid Mice (n=4, 1.0mg/kg (~20ug) luc mRNA ) Luciferase Activity (pg/g Tissue) 1000000 100000 Tumour Liver Spleen 10000 1000 100 0 5 10 15 20 25 30 Time Point (h) 35 40 45 50 35 Summary • LNP delivered RNA are a clinically and commercially viable technology enabling the development of RNA therapeutics. • Using Tekmira’s proprietary scalable manufacturing methodology, mRNA are readily encapsulated in electron dense LNP, integrating third generation LNP technology. • mRNA encapsulation efficiencies support product development. • Tekmira has designed LNP for hepatic delivery, oncology applications, inhalation and delivery to immune cells. • The results suggest a role for LNP in the development of mRNA therapeutics. 36 18 Acknowledgements James Heyes Lorne Palmer Mark Wood Alan Martin Aaron Lyons Amy Lee Adam Judge Lloyd Jeffs Peter Lutwyche Marjorie Robbins Sean Semple Ed Yaworski Kevin McClintock Ellen Ambegia Janet Shaw Nancy Fuselli AWARD/CONTRACT No. W9113M-10-C-0057 37 Recent Advances in the Lipid Nanoparticle-Mediated Delivery of Messenger RNA Ian MacLachlan, Ph.D. Executive Vice President and Chief Technology Officer AsiaTIDES Conference February 25, 2014 Tokyo, Japan 38 19
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