Moderna, Inc. · NASDAQ:MRNA

Moderna — Technical Presentation (May 2019)

135 slides from Moderna's technical presentation, May 7, 2019

1. Slide 1: Scientific Symposium May 7, 2019

   Scientific Symposium May 7, 2019 Confidential and Proprietary · © 2018 Moderna Therapeutics Exhibit 99.1

2. Slide 2: Forward-looking statements

   Confidential and Proprietary · © 2018 Moderna Therapeutics Forward-looking statements This presentation contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended. In some cases, forward-looking statements can be identified by terminolo

3. Slide 3: Welcome

   Welcome Stéphane Bancel Chief Executive Officer

4. Slide 4: mRNA as a potential new class of medicines

   Large product opportunity Higher probability of technical success Accelerated research and development timelines Greater capital efficiency over time vs. recombinant technology mRNA as a potential new class of medicines

5. Slide 5: Moderna's Mission

   Moderna’s Mission Deliver on the promise of mRNA science to create a new generation of transformative medicines for patients. Slide

6. Slide 6: mRNA sciences has dozen of vectors

   Confidential and Proprietary · © 2018 Moderna Therapeutics mRNA sciences has dozen of vectors

7. Slide 7: Our commitment to be the best at mRNA science

   Our commitment to be the best at mRNA science is core to who we are Product Performance 2011 Inception Time 2019 © 2018 Moderna Therapeutics Investing: For the long-term & At scale

8. Slide 8: Our commitment to be the best at mRNA science

   Our commitment to be the best at mRNA science is core to who we are Product Performance 2011 Inception Time 2019 © 2018 Moderna Therapeutics Capturing future product performance improvements requires investments 1) for the long-term 2) at scale 3) with the right team We have advanced mRNA science to

9. Slide 9: Expanding frontiers: Introduction to the immune nanoparticle program

   Expanding frontiers: Introduction to the immune nanoparticle program Confidential and Proprietary · © 2019 Moderna Therapeutics Stephen Hoge, MD President

10. Slide 10: Immune system

    Immune system Tissue resident Waste disposal, maintenance Initiation & resolution of inflammation Many types Confidential and Proprietary · © 2019 Moderna Therapeutics Mucosa associated (MALT) Non-encapsulated; epithelium Defense & homeostasis Regulatory CD25 CD4 Helper CD4 Cytotoxic CD8 >90% of mye

11. Slide 11: Immune system

    Immune system Tissue resident Waste disposal, maintenance Initiation & resolution of inflammation Many types Confidential and Proprietary · © 2019 Moderna Therapeutics Mucosa associated (MALT) Non-encapsulated; epithelium Defense & homeostasis Regulatory CD25 CD4 Helper CD4 Cytotoxic CD8 >90% of mye

12. Slide 12: Identifying a lead Immune Nanoparticle

    Identifying a lead Immune Nanoparticle Confidential and Proprietary · © 2019 Moderna Therapeutics

13. Slide 13: Policeman mRNA-based immune system therapeutics

    Policeman mRNA-based immune system therapeutics

14. Slide 14: Transient protein expression to reprogram cells

    Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types (system) Leverage programable trafficking to ensure desired interactions mRNA software to select cell type(s) and ensure safety

15. Slide 15: Setting the objective

    Setting the objective Dose-dependent in vivo pharmacology to all major cell types (system) How much expression do we need (how many cells)? Healthy CMV seropositive adults can have 5-10% of their peripheral CD8+ T cell pool responsive to the virus Human cell therapies (CART) dose ~300 million T-cell

16. Slide 16: Ex vivo performance of immune NP in human PBMC

    Human PBMC Ex vivo performance of immune NP Collect multiple donors Expose to Immune NP at same concentrations achieved in blood following IV administration (100ng per 2x105 cells) Use an mRNA encoding a transmembrane reporter (e.g., mouse OX40L)

17. Slide 17: Human PBMC reporter expression by cell type

    Human PBMC Percent of human PBMC cells expressing reporter (by type, 24h) FSC Transmembrane reporter (murine OX40L) 5%

18. Slide 18: T cells

    Confidential and Proprietary · © 2019 Moderna Therapeutics T cells NKT CD1r CD3 TCR CD4 Helper Cytotoxic CD8 Regulatory CD25 CD4 28% 18% 26% 19%

19. Slide 19: Does repeat dosing increase the effect?

    Human PBMC T cells Does repeat dosing increase the effect?

20. Slide 20: Repeat dose pharmacology

    Human PBMC Repeat dose pharmacology Dose 1 (24 hours) Dose 2 (48 hours) Dose 3 (72 hours) Immune NP Control T cells CD3+ Amount of protein per cell (MFI) also increases linearly Percent of human CD3+ cells expressing reporter, 24h after each dose

21. Slide 21: Does it translate in vivo?

    Does it translate in vivo? Administer a standard mRNA dose intravenously (0.3 mpk) using Immune NP Collect splenocytes & PBMCs at 24 hours Measure expression through FACS using transiently expressed transmembrane reporter (confirmatory imaging)

22. Slide 22: Mouse in vivo: T cells transfected upon single IV dose

    Mouse in vivo T cells transfected upon single IV dose Control Reporter (Immune NP) Reporter (mOX40L) positive cells among CD3+ splenocytes Reporter (Immune NP) Control C57Bl6 strain Balb/c strain Control Reporter (Immune NP) Reporter (mOX40L) positive cells among CD3+CD4+ splenocytes Reporter (Immun

23. Slide 23: Mouse in vivo: B cells transfected upon single IV dose

    Mouse in vivo B cells transfected upon single IV dose Control Reporter (Immune NP) Reporter (mOX40L) positive cells among CD19+ splenocytes Reporter (Immune NP) Control C57Bl6 strain Balb/c strain Approximately 30% of CD19+ splenocytes express reporter 24 hours after single dose Spleen includes matu

24. Slide 24: Mouse in vivo Summary

    Mouse in vivo Summary T cells B cells Monocytes Cell type 5% Average reporter (mOX40L) positive cells by cell type, splenocytes (n=20) Low/no staining at 24 hours in peripheral lymph nodes Lower staining in peripheral blood, particularly for lymphocytes, suggesting dilution from re-trafficking (data

25. Slide 25: Mouse in vivo: Investigating cell trafficking

    Mouse in vivo Investigating cell trafficking Kauffman, Kevin J., et al. "Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse." Molecular Therapy-Nucleic Acids 10 (2018): 55-63. Immune NP with mRNA encoding for Cre recombinase In

26. Slide 26: Blood cell types in ROSA (Cre-Lox) mice

    Mouse in vivo Blood cell types in ROSA (Cre-Lox) mice T cells CD4+ CD8+ B cells Monocytes Eosinophils Neutrophils Granulocytes 25% 25% 24% 26% 63% 83% 18% 32% CD4 Helper Cytotoxic CD8 Percent reporter positive cells by cell type 48-hours after a single IV dose of mRNA encoding Cre recombinase in Imm

27. Slide 27: Distribution of cells by lymphoid organ

    Mouse in vivo Distribution of cells by lymphoid organ T cells B cells Myeloid (various) Cell type Blood Spleen Lymph node (inguinal) Bone marrow 25% 15% 6% 9% 26% 8% 6% 8% 63% 44% 36% 7% Percent reporter positive cells by cell type 48-hours after a single IV dose of mRNA encoding Cre recombinase in

28. Slide 28: NHP in vivo T cells

    NHP in vivo T cells (all) 12% of T cells in vivo Buffer Control mRNA (Immune NP) Reporter mRNA (Immune NP) Reporter (mOX40L) positive cells among CD3+ splenocytes 24 hours post-dose, transmembrane reporter

29. Slide 29: NHP in vivo B cells

    NHP in vivo B cells 13% of B cells in vivo Buffer Control mRNA (Immune NP) Reporter (mOX40L) positive cells among CD3+ splenocytes Reporter mRNA (Immune NP) 24 hours post-dose, transmembrane reporter

30. Slide 30: NHP in vivo Monocytes

    NHP in vivo Monocytes 22% of monocytes Buffer Control mRNA (Immune NP) Reporter (mOX40L) positive cells among CD3+ splenocytes 24 hours post-dose, transmembrane reporter

31. Slide 31: NHP in vivo Summary

    NHP in vivo Summary T cells B cells Monocytes 12% 13% 22% ~9 billion (109) ~1.5 billion (109) ~2 billion (109) Cell type Percentage (single dose) Est. whole body number* *Estimate for 70kg human and 5L blood volume; based on extrapolating observed NHP splenic cell percentage to whole body pool (by c

32. Slide 32: Transient protein expression to reprogram cells

    Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types (system) Leverage programable trafficking to ensure desired interactions mRNA software to select cell type(s) and ensure safety

33. Slide 33: mRNA as software

    mRNA as software mRNA software to select cell type(s) and ensure safety Specific microRNA signatures exist in different most cell lineages Targeting sequences can be used as logic-gates to ensure translation is limited to target cells

34. Slide 34: mRNA as software

    mRNA as software Jain, et al. MicroRNAs Enable mRNA Therapeutics to Selectively Program Cancer Cells to Self-Destruct. Nucleic Acid Therapeutics, 2018 Systemic delivery- IV injection miR3x122ts Caspase-6 3x122ts CTRL (no 122ts) 3x122ts CTRL (no 122ts) ALT 0 1500 3000 15000 30000 U/L 1500 3000 15000

35. Slide 35: Software targeting miR TS

    Software targeting miR TS Targeting sequence 1 (not disclosed) Purpose Effect “Express in T cells, not monocytes” Percent of transfection preserved, normalized to a miR-less version of the same reporter construct in the same cell type Targeting sequence 2 (not disclosed) “Express in monocytes, not T

36. Slide 36: Software targeting miR TS

    Software targeting miR TS Targeting sequence 1 (not disclosed) Purpose Effect “Express in T cells, not monocytes” Percent of transfection preserved, normalized to a miR-less version of the same reporter construct in the same cell type Targeting sequence 2 (not disclosed) “Express in monocytes, not T

37. Slide 37: Software targeting miR TS

    Software targeting miR TS Targeting sequence 1 (not disclosed) Purpose “Express in T cells, not monocytes” Percent of transfection preserved, normalized to a miR-less version of the same reporter construct in the same cell type Targeting sequence 2 (not disclosed) “Express in monocytes, not T cells”

38. Slide 38: Software targeting miR TS

    Software targeting miR TS Targeting sequence 1 (not disclosed) Purpose Effect “Express in T cells, not monocytes” Targeting sequence 2 (not disclosed) “Express in monocytes, not T cells”

39. Slide 39: Software targeting miR TS

    Software targeting miR TS Targeting sequence 1 (not disclosed) Purpose “Express in T cells, not monocytes” Targeting sequence 2 (not disclosed) Illustrative protein “Express in monocytes, not T cells”

40. Slide 40: Software targeting

    Software targeting Create novel and specific signaling between cells by leveraging both sides of the immune synapse

41. Slide 41: Transient protein expression to reprogram cells

    Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types (system) Leverage programable trafficking to ensure desired interactions mRNA software to select cell type(s) and ensure safety

42. Slide 42: Putting it all together

    Putting it all together Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types (system) mRNA software to select cell type(s) and ensure safety Can we achieve in vivo dose-dependent immune cell-based pharmacology l

43. Slide 43: Putting it all together

    Putting it all together Can we achieve in vivo dose-dependent immune cell-based pharmacology leveraging multiple immune cells types*? C57Bl/6 wild-type mice IV dose of immune NP every 2 days x 4 doses, containing either: non-translating (control) mRNA or CD19-CAR mRNA Monitor CD19+ cells in PBMC for

44. Slide 44: Putting it all together

    Putting it all together This is not a CD19-CART, in fact expression will be on multiple cell types Expression with mRNA is transient and self-limiting CD19 is only used as a reporter to monitor activity against normal B cells (not diseased animals) T-cell Mac NK CD19 CD19 CD19

45. Slide 45: In vivo activity: targeted depletion of CD19+ cells

    In vivo activity Targeted depletion of CD19+ cells * ** *** **** p<0.05 p<0.01 p<0.001 p<0.0001 T-test unpaired, 2-tailed 48h (1 dose) **** 96h (2 doses) 8 days (4 doses) * ns ** 76% 73% Cells in peripheral blood Cells in spleen (day 8 only) T cells (control) B cells (CD19+)

46. Slide 46: Immune summary

    Immune summary Confidential and Proprietary · © 2019 Moderna Therapeutics

47. Slide 47: Putting it all together

    Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types Leverage programable trafficking to ensure desired interactions mRNA software to select cell type(s) and ensure safety Putting it all together

48. Slide 48: Policeman mRNA-based immune system therapeutics

    Transient protein expression to reprogram cells into new functions and phenotypes Dose-dependent in vivo pharmacology to all major cell types Leverage programable trafficking to ensure desired interactions mRNA software to select cell type(s) and ensure safety Policeman mRNA-based immune system ther

49. Slide 49: Why do we incorporate modified nucleotides into our mRNAs?

    Why do we incorporate modified nucleotides into our mRNAs and are they really necessary? Confidential and Proprietary · © 2019 Moderna Therapeutics

50. Slide 50: Innate vs adaptive immunity

    Innate vs adaptive immunity Innate immunity (rapid response) Adaptive immunity (slow response) T cell Natural killer T cell Natural killer cell Macrophage Neutrophil Eosinophil Basophil (Primary white blood cell) CD8+ T cell CD4+ T cell Pathogen B cell Antibodies Dendritic cell Confidential and Prop

51. Slide 51: Sentinels of the Innate Immune System

    Sentinels of the Innate Immune System Toll-like Receptor (TLR) RIG-I Confidential and Proprietary · © 2019 Moderna Therapeutics

52. Slide 52: Dual consequences of triggering the innate immune system

    Confidential and Proprietary · © 2019 Moderna Therapeutics Dual consequences of triggering the innate immune system Translation Immune Signaling Ribosome Innate immune cell Low High Adaptive immune cell B cell

53. Slide 53: Uridine is the critical nucleoside for TLR detection of ssRNA

    Confidential and Proprietary · © 2019 Moderna Therapeutics Uridine (U) is the critical nucleoside for TLR detection of ssRNA Toll-like Receptor (TLR)

54. Slide 54: Uridine binding is critical for TLR8 homodimerization

    Uridine (U) binding is critical for TLR8 homodimerization and activation Confidential and Proprietary · © 2019 Moderna Therapeutics Tanji … Shimizu (2015) Nat Struc Mol Biol

55. Slide 55: Uridine base modifications impair TLR8 activation

    Uridine (U) base modifications are predicted to impair TLR8 homodimerization and activation 1-Methylpseudouridine (1mY) H N O O N Unmodified Uridine (U) H N O O HN Confidential and Proprietary · © 2019 Moderna Therapeutics

56. Slide 56: Systemic administration of U19 activates splenic B-cells, 1mY19 does not

    Systemic administration of U19 activates splenic B-cells, whereas 1mY19 does not C57BL6 mice IV administration 0.5 mg/kg Analyzed at 6 hrs. post-dose Activated B-cell Frequency Confidential and Proprietary · © 2019 Moderna Therapeutics B cell Antibodies IgG

57. Slide 57: U19 changes splenic gene expression, 1mY19 does not

    Confidential and Proprietary · © 2019 Moderna Therapeutics C57BL6 mice IV administration 0.5 mg/kg Analyzed 6 hrs post-dose Genes regulated by innate immune stimulation PBS U19 1mY19 Systemic administration of U19 changes splenic gene expression profiles, whereas 1mY19 does not

58. Slide 58: What about mRNA? dsRNA byproducts

    Confidential and Proprietary · © 2019 Moderna Therapeutics dsRNA BOTTOM strand minor byproducts Transcription 1000's of copies TOP strand TOP strand BOTTOM strand DNA RNA polymerase What about mRNA? A, C, G, (U or 1mY)

59. Slide 59: Moderna's process results in very little dsRNA

    Confidential and Proprietary · © 2019 Moderna Therapeutics Moderna's process for making mRNA results in very little dsRNA Highly sensitive assay for detecting dsRNA

60. Slide 60: Minimizing dsRNA decreases innate immune activation

    Minimizing dsRNA substantially decreases innate immune activation, but… Confidential and Proprietary · © 2019 Moderna Therapeutics U 1mY19 Human Peripheral Blood Mononuclear Cells (PBMCs) Isolate Monocytes Differentiate to Macrophages Transfect with mRNA Measure IP-10 mRNA levels

61. Slide 61: Incorporating 1mY decreases activation even more

    … incorporating 1mY decreases it even more Confidential and Proprietary · © 2019 Moderna Therapeutics Human Peripheral Blood Mononuclear Cells (PBMCs) Isolate Monocytes Differentiate to Macrophages Transfect with mRNA Measure IP-10 mRNA levels U 1mY

62. Slide 62: Splenic B-cell activation: chemical & process optimization

    Splenic B-cell activation demonstrates the advantage of both chemical & process optimization C57BL6 mice IV administration 0.5 mg/kg Analyzed at 6 hrs. post-dose Confidential and Proprietary · © 2019 Moderna Therapeutics B cell Antibodies IgG Activated B-cell Frequency 1mY19

63. Slide 63: Splenic B-cell activation: chemical & process optimization

    Splenic B-cell activation demonstrates the advantage of both chemical & process optimization C57BL6 mice IV administration 0.5 mg/kg Analyzed at 6 hrs. post-dose Confidential and Proprietary · © 2019 Moderna Therapeutics B cell Antibodies IgG 1mY Activated B-cell Frequency Moderna process U 1mY19

64. Slide 64: Almost no splenic gene expression changes with 1mY Moderna mRNA

    Confidential and Proprietary · © 2019 Moderna Therapeutics C57BL6 mice IV administration 0.5 mg/kg Analyzed 6 hrs post-dose Genes regulated by innate immune stimulation PBS U Legacy U Moderna Almost no splenic gene expression changes upon systemic administration of 1mY Moderna mRNA 1mY19 Moderna

65. Slide 65: Summary: avoiding innate immune sensors

    Effective mRNA therapeutics must avoid detection by innate immune sensors Global substitution of U with 1mY results in immune silent mRNA Moderna's synthesis process results in highly pure mRNA Summary Confidential and Proprietary · © 2019 Moderna Therapeutics

66. Slide 66: Desired mRNA features

    Confidential and Proprietary · © 2019 Moderna Therapeutics 5′ Cap Poly (A) tail Coding sequence 3′ UTR 5′ UTR Translation initiation fidelity Protein per unit time Functional mRNA half-life Desired mRNA features Desired cell type Tailored to protein type Start at the right place Faithful decoding St

67. Slide 67: Coding sequence engineering

    Confidential and Proprietary · © 2019 Moderna Therapeutics Coding sequence engineering

68. Slide 68: Translation

    Confidential and Proprietary · © 2019 Moderna Therapeutics Translation

69. Slide 69: Coding sequence length is dictated by protein length

    Coding sequence length is dictated by the length of the encoded protein 0 500 1000 1500 2000 2500 3000 3500 4000 Length (nt) Moderna's experimental medicines 5′ Cap Poly (A) tail Coding sequence 3′ UTR 5′ UTR Confidential and Proprietary · © 2019 Moderna Therapeutics

70. Slide 70: Coding sequence engineering: a needle in a haystack

    Confidential and Proprietary · © 2019 Moderna Therapeutics Coding sequence engineering: discovering a needle in a haystack Amino acids: Lys Pro Thr Glu Asn Codons: AAG CCC ACC GAG AAC AAA CCU ACA GAA AAU CCA ACU CCG ACG Number of amino acids # of codon choices All valid! 2 4 4 2 2 * * * * = 128 choi

71. Slide 71: How does one choose a CDS?

    Confidential and Proprietary · © 2019 Moderna Therapeutics How does one choose a CDS? Randomly choose synonymous codons? Weight synonymous codon choice by use frequencies in human mRNAs? Use only the 20 most "optimal" human codons? What about secondary structure? Should we minimize, maximize, or som

72. Slide 72: Codon optimality

    Confidential and Proprietary · © 2019 Moderna Therapeutics Codon optimality tRNAs compete with each other for binding. The more abundant the tRNA – the more optimal the codon. Fast: Optimal codons Sub-optimal codons Slow:

73. Slide 73: Many ribosomes per mRNA = Polysome

    Many ribosomes per mRNA = Polysome Modified from Kiseleva (1989) FEBS Letters Confidential and Proprietary · © 2019 Moderna Therapeutics

74. Slide 74: Ribosome collisions lead to traffic jams

    Ribosome collisions lead to ribosome traffic jams Slow codons Polysome Ribosome traffic pileup mRNA cleavage Cleaved mRNA Fast codons Structure of two collided ribosomes Ikeuchi … Inada EMBO J 2019 Confidential and Proprietary · © 2019 Moderna Therapeutics

75. Slide 75: mRNA secondary structure

    Confidential and Proprietary · © 2019 Moderna Therapeutics 5′ Cap Poly (A) tail Coding sequence 3′ UTR 5′ UTR Another key feature of mRNA is its secondary structure Backbone Bases

76. Slide 76: What's the right balance to maximize protein output?

    What's the right balance to maximize protein output? Slower translation Faster translation mRNA secondary structure Strong secondary structure Weak secondary structure Codon optimality Low tRNA concentration High tRNA concentration after Gorochowski…Roubos (2014) NAR fast slow Confidential and Propr

77. Slide 77: Codon optimality and secondary structure can be calculated

    Codon optimality and secondary structure strength can be calculated Codon optimality Folding energy (DG) -297 kcal/mol -370 kcal/mol -463 kcal/mol Confidential and Proprietary · © 2019 Moderna Therapeutics low structure high structure med structure Slow Optimal

78. Slide 78: Computational design explores codon optimality vs structure

    Confidential and Proprietary · © 2019 Moderna Therapeutics Relative Synonymous Codon Usage (RSCU) 0.5 0.6 0.7 0.8 0.9 1.0 -250 -300 -350 -400 -450 -500 Folding Energy (DG) Random codon choice Human genome weighted choice Moderna algorithm Optimal Slow Low Structure High Structure eGFPd coding sequen

79. Slide 79: GFPd1 mRNA time-lapse in HeLa

    Confidential and Proprietary · © 2019 Moderna Therapeutics GFPd1 mRNA GFP Nuclei 72 h time-lapse, HeLa GFPd1 degron tag Short-lived GFP allows us to follow translation and mRNA decay kinetics in cells

80. Slide 80: Different coding sequences display different expression kinetics

    Confidential and Proprietary · © 2019 Moderna Therapeutics Different coding sequences display different expression kinetics GFPd1 mRNA GFPd2 mRNA GFP Nuclei 72 h time-lapse, HeLa eGFPd2 degron tag GFPd1 degron tag

81. Slide 81: Initial translation rate is not a good predictor of expression

    AUC Confidential and Proprietary · © 2019 Moderna Therapeutics The initial rate of translation is not a good predicter of overall eGFP expression, … R = 0.45 Relative AUC Rate of translation (kTrans)

82. Slide 82: Expression correlates with functional mRNA half-life

    AUC Confidential and Proprietary · © 2019 Moderna Therapeutics … but overall eGFP expression strongly correlates with functional mRNA half-life Relative AUC

83. Slide 83: Codon optimality and structure impact functional half-life

    AUC Confidential and Proprietary · © 2019 Moderna Therapeutics Both codon optimality and CDS structure positively impact functional mRNA half-life

84. Slide 84: Optimizing codons and structure increases protein production >10-fold

    AUC Optimizing codons and maximizing coding sequence structure can increase total protein production by >10-fold Relative AUC Structure Codons Slow Total protein Confidential and Proprietary · © 2019 Moderna Therapeutics

85. Slide 85: Summary: computational design of codon optimality and structure

    We used computational design to thoroughly explore the relationship between codon optimality and mRNA secondary structure. Mauger … MacFadyen (2019) BioRxiv 549022 We now have a new tool for dialing in desired protein output Confidential and Proprietary · © 2019 Moderna Therapeutics Summary

86. Slide 86: Start at the right place

    Confidential and Proprietary · © 2019 Moderna Therapeutics 5′ Cap Poly (A) tail 3′ UTR AUG 5′ UTR UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Coding sequence Translation initiation fidelity Protein per unit time Functional mRNA half-life Desired mRNA features Desir

87. Slide 87: Desired mRNA features

    Confidential and Proprietary · © 2019 Moderna Therapeutics 5′ Cap Poly (A) tail AUG UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Coding sequence 3′ UTR 5′ UTR Translation initiation fidelity Protein per unit time Functional mRNA half-life Desired mRNA features Desir

88. Slide 88: UTRs contain key regulatory information

    Confidential and Proprietary · © 2019 Moderna Therapeutics UTRs contain key regulatory information 5′ Cap Poly (A) tail Coding sequence 3′ UTR AUG 5′ UTR UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Controls speed at which ribosomes load in different cell types & un

89. Slide 89: UTR optimization

    Confidential and Proprietary · © 2019 Moderna Therapeutics UTR optimization

90. Slide 90: Power of sequence engineering

    Confidential and Proprietary · © 2019 Moderna Therapeutics Power of sequence engineering …Can we do better? mRNA expression 5′ Cap Poly (A) tail Coding sequence 3′ UTR AUG 5′ UTR UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Moderna mRNA sequence: Optimized coding se

91. Slide 91: Translation initiation

    Translation initiation mRNA 1 Small subunit recruitment Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail Confidential and Proprietary · © 2019 Moderna Therapeutics

92. Slide 92: Translation initiation

    Translation initiation mRNA 1 Small subunit recruitment 2 Start codon recognition and large subunit recruitment Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail Confidential and Proprietary · © 2019 Moderna Therapeutics

93. Slide 93: Translation initiation

    Translation initiation mRNA 1 Small subunit recruitment 3 Translation and elongation 2 Start codon recognition and large subunit recruitment Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail Confidential and Proprietary · © 2019 Moderna Therapeutics

94. Slide 94: Translation initiation

    Translation initiation mRNA 1 Small subunit recruitment 3 Translation and elongation 4 Termination Functional protein 2 Start codon recognition and large subunit recruitment Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail Confidential and Proprietary · © 2019 Moderna Therapeutics

95. Slide 95: Neural networks to develop 5′ UTRs with high ribosome loading

    Coding sequence 3′ UTR 5′ UTR Transfect mRNA library ~300,000 Neural Network Using neural networks to develop 5′ UTRs with high ribosome loading Polysome profiling ribosome load Ribosome load random human evolved Collaboration with Seelig lab, University of Washington Sample et al. bioRxiv doi: 10.1

96. Slide 96: Translation initiation: leaky scanning

    Translation initiation 1 Small subunit recruitment mRNA Leaky scanning Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail AUG Confidential and Proprietary · © 2019 Moderna Therapeutics

97. Slide 97: Translation initiation: leaky scanning

    Translation initiation Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail AUG 1 Small subunit recruitment mRNA 3 Start codon recognition and large subunit recruitment 2 Start codon missed Leaky scanning Confidential and Proprietary · © 2019 Moderna Therapeutics

98. Slide 98: Translation initiation: leaky scanning

    Translation initiation Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail AUG 1 Small subunit recruitment mRNA 3 Start codon recognition and large subunit recruitment 4 Translation & elongation 2 Start codon missed Leaky scanning Confidential and Proprietary · © 2019 Moderna Therapeutics

99. Slide 99: Truncated protein from leaky scanning

    Truncated protein Translation initiation Coding sequence 3′ UTR 5′ UTR AUG 5′ Cap Poly (A) tail AUG 1 Small subunit recruitment mRNA 3 Start codon recognition and large subunit recruitment 4 Translation & elongation 5 Termination 2 Start codon missed Leaky scanning Confidential and Proprietary · © 2

100. Slide 100: Designing 5′ UTRs with desired features

     Designing 5′ UTRs with desired features Random library 2 x 109 5′ UTRs 5′ UTR lead sequences In vitro analysis of sequences and identification of lead candidates In vivo validation of lead candidates in rodents Disease-relevant animal model Confidential and Proprietary · © 2019 Moderna Therapeutics

101. Slide 101: 5′ UTR screening: expression vs. initiation fidelity

     5′ UTR screening: protein expression vs. initiation fidelity Confidential and Proprietary · © 2019 Moderna Therapeutics 5′ Cap Poly (A) tail Coding sequence 3′ UTR 5′ UTR Initiation fidelity Expression HeLa (human cancer cells) AML12 (mouse liver cells) log2 transformed New 5′ UTRs with HIGH express

102. Slide 102: 3′ UTR optimization can increase mRNA potency

     3′ UTR optimization can increase mRNA potency 5′ Cap Poly (A) tail Coding sequence 3′ UTR AUG 5′ UTR UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Confidential and Proprietary · © 2019 Moderna Therapeutics

103. Slide 103: 3′ UTR leads show higher potency

     3′ UTR leads shows higher potency HeLa mouse hepatocytes …what does this mean for our future medicines? mRNA expression Confidential and Proprietary · © 2019 Moderna Therapeutics

104. Slide 104: 3′ UTR lead in disease mouse model

     3′ UTR lead in disease mouse model 1 Confidential and Proprietary · © 2019 Moderna Therapeutics 7 14 Day 0 Day 15 15 Protein -3 Biomarker -2 0 2 4 6 0 40 80 120 160 Days % Control biomarker 1 -3 Biomarker Editable Prism Graph Control Moderna standard sequence Moderna improved sequence

105. Slide 105: 5′/3′ UTR lead combination in disease model 2

     Confidential and Proprietary · © 2019 Moderna Therapeutics RNA Protein Activity Day 32 16 21 23 25 28 32 Day 7 14 0 5′/3′ UTR lead combination in disease model 2

106. Slide 106: 5′/3′ UTR lead combination in disease model 2

     0 7 14 21 28 80 90 100 110 120 Days post Injection % Change in body weight Confidential and Proprietary · © 2019 Moderna Therapeutics Body weight Biomarker Body weight Biomarker 16 21 23 25 28 32 Day 7 14 0 Editable Prism Graph 5′/3′ UTR lead combination in disease model 2 Control Moderna standard s

107. Slide 107: UTR optimization enables increased potency & duration of efficacy

     UTR optimization enables increased potency & duration of efficacy 5′ Cap Poly (A) tail Coding sequence 3′ UTR AUG 5′ UTR UAA UAG UGA Start at the right place Faithful decoding Stop at the right place Translation initiation fidelity Protein per unit time Functional mRNA half-life Increased mRNA poten

108. Slide 108: We target different tissues via multiple routes of administration

     We target different tissues via multiple routes of administration (ROAs) Confidential and Proprietary · © 2019 Moderna Therapeutics

109. Slide 109: Most ROAs require mRNA encapsulation: Lipid Nanoparticles

     Most ROAs require mRNA encapsulation: Lipid Nanoparticles (LNPs) PEG lipid Ionizable lipid Cholesterol Phospholipid mRNA Different LNPs for different delivery goals: Enhanced IV delivery to liver allows repeat dosing for treatment of rare diseases Enhanced IM delivery for vaccines Sabnis … Benenato

110. Slide 110: Enabling rational structure-based design for LNPs

     How do we enable rational structure-based design for LNPs? components function structure Δ molecules Δ composition Δ process Chemical stability Physical stability Biodistribution Cellular uptake Endosomal escape Protein expression Confidential and Proprietary · © 2019 Moderna Therapeutics

111. Slide 111: How are we tackling LNP structure?

     How are we tackling LNP structure? Cryogenic Electron Microscopy (CryoEM) Physics! Nuclear Magnetic Resonance (NMR) Atomic Force Microscopy (AFM) Small-Angle X-ray Scattering (SAXS) Small-Angle Neutron Scattering (SANS) Confidential and Proprietary · © 2019 Moderna Therapeutics

112. Slide 112: Enabling rational structure-based design for LNPs

     How do we enable rational structure-based design for LNPs? components function structure Δ molecules Δ composition Δ process Chemical stability Physical stability Biodistribution Cellular uptake Endosomal escape Protein expression Confidential and Proprietary · © 2019 Moderna Therapeutics

113. Slide 113: How can we elucidate the assembly process?

     structure function Chemical stability Physical stability Biodistribution Cellular uptake Endosomal escape Protein expression How can we elucidate the assembly process? components Δ molecules Δ composition Δ process Confidential and Proprietary · © 2019 Moderna Therapeutics black box

114. Slide 114: Watching LNP assembly in real time

     Confidential and Proprietary · © 2019 Moderna Therapeutics Watching LNP assembly in real time

115. Slide 115: LNP assembly

     Confidential and Proprietary · © 2019 Moderna Therapeutics LNP assembly 1x10-12 1x10-11 1x10-10 1x10-9 1x10-9 1x10-8 1x10-7 1x10-6 1x10-5 1x10-4 1x10-3 1x10-2 1x10-1 1x100 1x101 1x102 1x10-3 1 second 1 minute 1 hour 1x10-12 1x10-11 1x10-10 1x10-9 1x10-8 1x10-7 1x10-6 1x10-5 0.0001 0.001 0.01 0.1 1 1

116. Slide 116: LNP assembly via coarse-grained molecular dynamics

     Confidential and Proprietary · © 2019 Moderna Therapeutics LNP assembly via coarse-grained molecular dynamics

117. Slide 117: Molecular modeling

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular modeling Volpon … Borden (2006) EMBO

118. Slide 118: Central Dogma of Molecular Modeling

     Central Dogma of Molecular Modeling “The fundamental laws necessary for the mathematical treatment of the whole of chemistry are thus completely known, and the difficulty lies only in the fact that application of these laws leads to equations that are too complex to be solved.” Time-dependent Schröd

119. Slide 119: Molecular modeling

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular modeling 5 nm 80 nm to scale ~ 2-6 mRNA molecules of each component ~ 6k phospholipid ~ 30k ionizable lipid ~ 20k cholesterol ~ 1k PEG lipid

120. Slide 120: Molecular simulations

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular simulations Adapted from Perilla … Schulten (2013) Curr. Opin. Struct. Biol. 1Å 1nm 10nm 100nm 1µm Quantum mechanical simulations All-atom molecular dynamics Lipids mRNA Proteins Small molecules Atoms

121. Slide 121: Molecular simulations

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular simulations Adapted from Perilla … Schulten (2013) Curr. Opin. Struct. Biol. 1Å 1nm 10nm 100nm 1µm Quantum mechanical simulations All-atom molecular dynamics Lipids mRNA Proteins Small molecules Atoms Small organelles Bacterium Vir

122. Slide 122: Molecular simulations

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular simulations Adapted from Perilla … Schulten (2013) Curr. Opin. Struct. Biol. 1Å 1nm 10nm 100nm 1µm Quantum mechanical simulations All-atom molecular dynamics Virus capsids Lipids mRNA Proteins Small molecules Atoms Small organelles

123. Slide 123: Molecular simulations

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular simulations Adapted from Perilla … Schulten (2013) Curr. Opin. Struct. Biol. 1Å 1nm 10nm 100nm 1µm Quantum mechanical simulations All-atom molecular dynamics Lipid nanoparticles Lipids mRNA Proteins Small molecules Atoms Small orga

124. Slide 124: Molecular simulations

     Confidential and Proprietary · © 2019 Moderna Therapeutics Molecular simulations Adapted from Perilla … Schulten (2013) Curr. Opin. Struct. Biol. Lipid nanoparticles Lipids mRNA 1Å 1nm 10nm 100nm 1µm Quantum mechanical simulations All-atom molecular dynamics Coarse-grained molecular dynamics Protein

125. Slide 125: Coarse-grained modeling

     Coarse-grained modeling igem.org “all-atom” representation Confidential and Proprietary · © 2019 Moderna Therapeutics

126. Slide 126: Coarse-grained modeling

     Coarse-grained modeling igem.org “coarse-grain” representation “all-atom” representation Confidential and Proprietary · © 2019 Moderna Therapeutics

127. Slide 127: Coarse-grained modeling

     Confidential and Proprietary · © 2019 Moderna Therapeutics Coarse-grained modeling Risselada … Grubmüller (2014) PNAS Pinot … Barelli (2014) Science Barneda … Christian (2015) eLife

128. Slide 128: LNP components

     Confidential and Proprietary · © 2019 Moderna Therapeutics LNP components 6 µs 700 hours Proto LNP ~ 2-6 mRNA Components ~ 6k phospholipid ~ 30k ionizable lipid ~ 20k cholesterol ~ 1k PEG lipid 15 nm

129. Slide 129: Simulation of proto LNP assembly

     Confidential and Proprietary · © 2019 Moderna Therapeutics Simulation of “proto LNP” assembly cholesterol: head tail phospholipid: head tail ionizable lipid: head tail RNA oligomer: backbone nucleoside 25 nm 5.6 µs 5.4 µs 1.3 µs 1 µs 300 ns 5 nm 10 nm 15-35 nm

130. Slide 130: Simulation of RNA encapsulation

     Simulation of RNA encapsulation 1.3 µs 1 µs cholesterol: head tail phospholipid: head tail ionizable lipid: head tail RNA oligomer: backbone nucleoside 15 nm Confidential and Proprietary · © 2019 Moderna Therapeutics

131. Slide 131: Simulation of RNA encapsulation

     Simulation of RNA encapsulation 1.3 µs 1 µs cholesterol: head tail phospholipid: head tail ionizable lipid: head tail RNA oligomer: backbone nucleoside 15 nm Confidential and Proprietary · © 2019 Moderna Therapeutics

132. Slide 132: LNP assembly

     Confidential and Proprietary · © 2019 Moderna Therapeutics LNP assembly 1 second 1 minute 1 hour 1x10-12 1x10-11 1x10-10 1x10-9 1x10-9 1x10-8 1x10-7 1x10-6 1x10-5 1x10-4 1x10-3 1x10-2 1x10-1 1x100 1x101 1x102 1x10-3 1x10-12 1x10-11 1x10-10 1x10-9 1x10-8 1x10-7 1x10-6 1x10-5 0.0001 0.001 0.01 0.1 1 1

133. Slide 133: Enabling rational structure-based design for LNPs

     How do we enable rational structure-based design for LNPs? components function structure Δ chemistry Δ composition Δ process Chemical stability Physical stability Biodistribution Cellular uptake Endosomal escape Protein expression Confidential and Proprietary · © 2019 Moderna Therapeutics

134. Slide 134: Expressing RALDH in skin-draining lymph nodes

     Expressing RALDH in skin-draining lymph nodes Skin Skindraining LN Vaccination Reporter Protein (mCitrine) T cells (CD3) Wait 24h 1.18 0.86 1.26 Reporter Protein CD45 B cells T cells Control modRNA

135. Slide 135: Peripheral vaccination with RALDH mRNA immune NP induces mucosal memory cells

     Ethanol Am80 Small intestine 7 days post vaccination RALDH mRNA immune NP Peripheral vaccination with immune NP containing RALDH mRNA induces mucosal memory cells Spleen IEL LPL 102 103 104 105 106 107 Total OT-I recovered ETOH AM80 RALDH+retinal Vaccine only Vaccine + AM80 Vaccine + RALDH mRNA immu

Moderna — Technical Presentation (May 2019) on 6ix