Slide 1: Preclinical Development of AMX0114, an Antisense Oligonucleotide Targeting Calpain-2
Preclinical Development of AMX0114, an Antisense Oligonucleotide Targeting Calpain-2 Evan Mizerak Amylyx Pharmaceuticals, Inc. July 10, 2024 In memory of Mick, a husband and father, who was a gifted tattoo artist and musician.
Slide 2: Disclosures/Disclaimers
Disclosures/Disclaimers • Evan Mizerak is a full-time employee of and has stock ownership options in Amylyx Pharmaceuticals • This presentation is intended to provide scientific information about antisense oligonucleotides targeting calpain-2 • AMX0114 is an investigational agent not approved for us
Slide 3: Our mission is to one day end the suffering caused by neurodegenerative diseases
Our mission is to one day end the suffering caused by neurodegenerative diseases. Every day, we strive for better therapies.
Slide 4: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • Science supporting • Development of • Preclinical efficacy • Toxic
Slide 5: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • Science supporting • Development of • Preclinical efficacy • Toxic
Slide 6: ALS is a Relentlessly Progressive, Debilitating, and Universally Fatal Disease
ALS is a Relentlessly Progressive, Debilitating, and Universally Fatal Disease Caused by Motor Neuron Degeneration1 • ALS leads to deteriorating muscle function, inability to move and speak, respiratory paralysis, and death1,2 • Age of onset ranges from 40-65 years old3 • >90% of people with ALS hav
Slide 7: Complex ALS Pathophysiology Provides Multiple Pathways to Target
Complex ALS Pathophysiology Provides Multiple Pathways to Target Glial dysfunction Oxidative stress Excitotoxicity Neuroinflammation Dysregulated vesicle transport Mitochondrial dysfunction Impaired protein homeostasis and ER stress Impaired DNA damage repair Axonal Degeneration Dysregulated nucleoc
Slide 8: Axonal Degeneration Recognized as a Key Early Contributor to ALS
Axonal Degeneration Has Been Increasingly Recognized as a Key Early Contributor to the Clinical Presentation and Pathogenesis of ALS1,2 Glial dysfunction Oxidative stress Excitotoxicity Neuroinflammation Dysregulated vesicle transport Mitochondrial dysfunction Impaired protein homeostasis and ER str
Slide 9: Activation of Calpain-2 is Proposed as a Critical Effector of Axonal Degeneration
Activation of Calpain-2 is Proposed as One of the Critical Effectors of Axonal Degeneration1-3 Mechanisms of Axonal Degeneration 4 • Calpains are a family of calcium (Ca2+)-dependent proteases that target substrates within the axonal cytoskeleton2 • There are over a dozen calpain isoforms, but activ
Slide 10: Calpain-2 Levels Are Upregulated in ALS
Calpain-2 Levels Are Upregulated in ALS CAPN2 mRNA is upregulated in biopsied Ratio of active to inactive calpain-2 in muscle samples of people with ALS1 post-mortem brain tissue of people with ALS (P) is ~3x higher than that in healthy controls (C)2 CAPN2, calpain-2 gene encoding. 1. Ueyama H et al
Slide 11: Calpain-2 Activation Leads to Neurofilament Proteolysis
Calpain-2 Activation Leads to Neurofilament Proteolysis • Neurofilaments are broadly researched biomarkers in ALS related to axonal degeneration and neurofilament was reported as a substrate for calpain-2 proteolysis as early as 19821 Full length • Calpain-2 cleaves neurofilament to produce 22 kDa,
Slide 12: Calpain-2 Modulation Has Demonstrated Therapeutic Benefit in Preclinical Models
Calpain-2 Modulation Has Demonstrated Therapeutic Benefit in Preclinical Models (d) In the transgenic SOD1G93A mouse model of ALS, neuron-specific overexpression of calpastatin, an endogenous inhibitor of calpain-2 (hSOD1/CAST) increases overall survival (a-b), delays disease onset (c), and rescues
Slide 13: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • Science supporting • Development of • Preclinical efficacy • Toxic
Slide 14: AMX0114: An Antisense Oligonucleotide (ASO) Targeting Calpain-2
AMX0114: An Antisense Oligonucleotide (ASO) Targeting Calpain-2 Selectivity of the ASO modality offers distinct advantages over earlier, small molecule- based approaches to targeting calpain-2 • Specifically inhibits calpain-2 without disrupting the function of other calpains or calpastatin • Design
Slide 15: Advantages of RNase H Competent (Gapmer) Antisense Oligonucleotides
Advantages of RNase H Competent (Gapmer) Antisense Oligonucleotides Distinct practical advantages over small molecule approach • Highly targeted1 • Standardized, tunable approach to target evaluation and lead ID lends itself to platform building1 ASOs with comparable chemical modifications exhibit c
Slide 16: Key Design Features of AMX0114
AMX0114: An Antisense Oligonucleotide Targeting Calpain-2 Key Design Features of AMX0114 1 2 2′-O-methoxyethyl (2'MOE) 20-mer (5-10-5 gapmer) modifications on wing Convenient size for synthesis, long residues enough to ensure uniqueness in the human genome1 Improve nuclease stability and binding abi
Slide 17: AMX0114 Achieves Potent, Dose-Dependent, and Durable Knockdown of CAPN2 mRNA and Protein
AMX0114 Achieves Potent, Dose-Dependent, and Durable Knockdown of CAPN2 mRNA and Calpain-2 Protein CAPN2 mRNA Calpain-2 Protein Days After Knockdown Knockdown AMX0114 (Reduction vs. (Reduction vs. Removal Control) Control) 0 94.47% 3.25% 3 87.12% 23.32% 7 84.36% 45.47% 10 82.16% 31.39% 14 83.80% 51.
Slide 18: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • Science supporting • Development of • Preclinical efficacy • Toxic
Slide 19: AMX0114 Reduces Extracellular NfL Levels in Models of Neuronal Injury
AMX0114 Reduces Extracellular NfL Levels in Multiple Models of Trigger-Induced Neuronal Injury Induced pluripotent stem cell (iPSC)-derived motor neurons were exposed to varying concentrations of the neurotoxic compounds vincristine, rotenone, and colchicine after pretreatment with AMX0114 Cohen J,
Slide 20: AMX0114 Improves Survival in a Model of TDP-43 ALS
AMX0114 Improves Survival in a Model of TDP-43 ALS ALS model neurons (no AMX0114) Healthy neurons (no AMX0114) ALS model neurons (with AMX0114) • TDP-43(M337V) is an ALS-linked mutation resulting in increased risk of cell death • Treatment with as little as 0.01 µM AMX0114: o Reduced cell death by n
Slide 21: AMX0114 is Neuroprotective in a Model of Oxidant-Induced Cell Death
AMX0114 is Neuroprotective in a Model of Oxidant-Induced Cell Death Calpain-2 knockdown translates to improved survival following exposure to hydrogen peroxide (H2O2), a stressor meant to induce oxidative stress and cell death LESS neuronal death MORE neuronal death Survival analyses performed in th
Slide 22: AMX0114 is Neuroprotective in a Model of Oxidant-Induced Cell Death
AMX0114 is Neuroprotective in a Model of Oxidant-Induced Cell Death Representative images of control (untreated) and AMX0114-treated iPSC- derived motor neurons before (pre) and after (post) exposure to H2O2 Survival analyses performed in the lab of Dr. Giovanni Manfredi at Weill Cornell Medicine by
Slide 23: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • IND-enabling studies are concluding and reflect a favorable safety
Slide 24: AMX0114 Development Program in ALS
AMX0114 Development Program in ALS We Are Here Scientific Screening Preclinical IND-Enabling (Preparation First-in-Human Rationale, and Lead ID Proof-of- Studies and for) IND Filing Bioinformatics Concept CMC Scale-Up and Discovery • Targeting first participant dosed in first-in-human multiple ascen
Slide 25: Calpain-2 Inhibition is a Promising Therapeutic Strategy in Many Disease Areas
Calpain-2 Inhibition is a Promising Therapeutic Strategy in Many Disease Areas Current Status AMX0114 Has Potential Applications Beyond ALS Traumatic The scientific literature has Neurodegenerative Scientific Brain and Spinal Diseases1,2,3,4 identified a role for calpain-2 Rationale in Cord Injury5
Slide 26: AMX0114: An Antisense Oligonucleotide Targeting Calpain-2
AMX0114: An Antisense Oligonucleotide Targeting Calpain-2 • Activation of calpain-2 is a critical step in axonal degeneration, an early contributor to ALS • Scientific literature supports a role for calpain-2 in ALS pathophysiology, including in the proteolysis of NfL • Amylyx has developed AMX0114,
Slide 27: Thank you!
Thank you! YY AMYLYX