IONIS-ANGPTL3-L Rx , an antisense inhibitor to angiopoietin-like - PowerPoint PPT Presentation

IONIS-ANGPTL3-L Rx , an antisense inhibitor to angiopoietin-like protein 3 [ANGPTL3] reduces plasma ANGPTL3 and lipids in healthy volunteers with elevated triglycerides Teresa A. Brandt 1 , Li-Jung Tai 1 , Joseph L. Witztum 2 , Steven G. Hughes 1 , Eunju Hurh 3 , Brad McEvoy 1 , Rosie Yu 1 , Andres Digenio 3 , Richard Lee 1 , Mark Graham 1 , Rosanne Crooke 1 , Sotirios Tsimikas 1,4 1 Ionis Pharmaceuticals, Inc., Carlsbad, California, 2 University of California, San Diego, Department of Medicine, Division of Endocrinology & Metabolism, 3 Akcea Therapeutics, Cambridge, Massachusetts, 4 University of California, San Diego, Department of Medicine, Division of Cardiovascular Diseases

Disclosures: Teresa A. Brandt, Li-Jung Tai, Steven G. Hughes, Eunju Hurh, Brad McEvoy, Rosie Yu, Andres Digenio, Richard Lee, Mark Graham, Rosanne Crooke, and Sotirios Tsimikas are employees of Ionis Pharmaceuticals or Akcea Therapeutics Joseph L. Witztum is a consultant to Ionis Pharmaceuticals, Intercept, CymaBay and Prometheus. Joseph L. Witztum, Sotirios Tsimikas are co-inventors and receive royalties from patents owned by the University of California San Diego on oxidation- specific antibodies

Angiopoietin-Like 3 (ANGPTL3) is a Genetically Validated Lipid and Metabolic Target in Humans • Genome-wide association and exome sequencing studies have identified ANGPTL3 genetic variations that are associated with very low plasma LDL-C, HDL-C and TG 1-3 • ANGPTL3 complete loss-of-function mutations result in familial combined hypolipidemia (FHBL2), which is manifested by a reduction of all lipoproteins, except Lp(a) 4 • Loss of function of ANGPTL3 results in increased lipoprotein lipase and endothelial lipase activities, enhanced insulin sensitivity and decreased serum FFAs 5 1) Willer et al. Nat Gen , 2008; 2) Teslovich TM, et al. Nature , 2010; 3) Musunuru K, et al. NEJM , 2010; 4) Minicocci I, et al. JLR , 2013; 5) Robciuc et al. ATVB , 2013.

Effect of ANGPTL3 LOF Mutations on Lipid Phenotypes Pooled analysis in 115 subjects with FHBL2 with 13 different mutations % Difference Compared to Noncarrier Control Group (N=402) Heterozygotes Homozygotes or (N=93) Compound Hets. (N=22) % Difference p-value % Difference p-value Parameter -8.6% 0.007 -67.2% p<0.001 LDL-C -21.1% 0.005 -71.2% p<0.001 TG -16.8% p<0.001 -39.0% p<0.001 HDL-C -7.2% 0.008 -48.4% p<0.001 ApoB -13.1% 0.001 -95.1% p<0.001 ApoA1 • Other clinical characteristics of homozygotes • No increase in prevalence of fatty liver compared to controls • No increased atherosclerosis or other manifestations of CVD • Improved insulin sensitivity, lower plasma glucose, and lower incidence of T2DM Minicocci I, et al. JLR , 2013

Inhibition of ANGPTL3 May Have Multiple Beneficial Effects in Lipoprotein Metabolism Tikka et al Endocrine (2016) 52:187–193

RNA-Targeted Antisense Drugs Block the Translation of ANGPTL3 Protein 6 Disease-associated DNA mRNA Protein Traditional Drug Transcription Translation RNase H1 Degrades mRNA=No Transcription Translation Antisense Drug No Disease-associated ( Single stranded, DNA- Proteins Produced like) (Oligonucleotide) Adapted from: Crooke ST, ed. Antisense Drug Technology: Principles, Strategies and Applications. 2nd ed. Boca Raton, FL: CRC Press; 2007:601-639.

The Antisense Drug-Receptor Interaction Occurs by by Classical Watson-Crick Hybridization Antisense RNA Target Oligonucleotide H 2 N O 5' end N 3' end OH N NH G N O C N O N O NH 2 O O S P O NH 2 O O N O O P OH HN O N A N O U N O N O O O S H 3 C O P O O H 2 N O O N P NH OH O T N A N O N O N O O O S NH 2 P O O O O O N P N OH O N HN C O G N O N O H 2 N O O 3' end 5' end • Unmodified DNA and RNA do not make good drugs due to insufficient stability and distribution, and rapid degradation in plasma • Chemical modification of ribose backbone can convert oligonucleotides to therapeutic agents • Specificity and optimal binding usually requires a length of 15-20 bases

Significant Advances in Medicinal Chemistry Improve Potency and Tolerability O O O O O O Base Base Base O O O O O S S S P O P O P O O O O O LIC LIC LIC Gen Gen LICA 1 st A A 2/2+ A 2.5 Gen GalNac Design MOE Gapmer Design cEt Gapmer Design P-S ↑10X ↑10X ↑10X

IONIS-ANGPTL3 Rx and IONIS-ANGPTL3-L Rx Sequences and Gapmer Technology- GalNAc Gen 2+ IONIS-ANGPTL3-L Rx contains GalNAc and 6 PO ANGPTL3 Rx Gen 2+ Chimera / Gapmer RNase H1 Substrate MOE Deoxy MOE G G A C A T T G C C A G T A T C G C A A GsGsAsCsAsTsTsGsCsCsAsGsTsAsAsTsCsGsCsA affinity stability ANGPTL3-L Rx GalNAc Generation 2+ tolerability 5’-THA- 5' GalNac O B O O C H3 O O PS O MOE MOE P S O B O O O C H3 O PO O O O GsGoAoCoAoTsTsGsCsCsAsGsTsAsAsToCoGsCsA P S O O

Comparison of Dose-Response Curves of IONIS-ANGPTL3 Rx vs. IONIS-ANGPTL3-L Rx Following 6 Weeks of SC Administration in Humans IONIS- IONIS- Parameter ANGPTL3 Rx ANGPTL3-L Rx ~21X higher potency ED 50 (mg) 210 ± 1.1 10.4 ± 1.2 γ -2.57 ± 0.46 -1.11 ± 0.20 Note, the study for IONIS-ANGPTL3 Rx had two additional loading doses (on Days 3 and 5) compared to IONIS-ANGPTL3-L rx.

Pre-Clinical Data: ANGPTL3 Rx Significantly Reduces Plasma Cholesterol and Plasma and Liver TGs in Ldlr -/- Mice 11 Plasma TG Angptl3 Plasma Protein 800 600 600 400 -50% mg/dl ng/mL 400 * * * * -53% -76% 200 200 * -80% * * * * 0 0 0 5 10 15 20 Week Total Cholesterol Liver Triglyceride 1500 150 TG (mg/g liver WW) 1000 * 100 * mg/dl * -46% 500 * * 50 -66% * * -85% 0 0 0 5 10 15 20 Week * Indicates significantly different ( p< 0.05) when compared to control ASO treatment

Pre-Clinical Data: ANGPTL3 Rx Reduces Liver Triglyceride Accumulation in a Diet-Induced Obesity Mouse Model Control ASO MTP ASO ANGPTL3 ASO MTP/ANGPTL3 ASO Liver TG Lipogenic Gene Expression 500 150 -68% SREBP1c * ACC1 400 mg/ g liver WW FASN * Denotes significantly % control 100 300 different when * * 200 compared to control φ 50 ASO φ * 100 ᶲ Denotes significantly 0 0 Control ASO MTP ASO Angptl3 ASO MTP ASO / Angptl3 ASO O O O O O O O O O O O O different when S S S S S S S S S S S S A A A A A A A A A A A A l P 3 3 l P 3 3 l P 3 3 o o o compared to MTP ASO l l l l l l T T T r t t r t t r t t p p p p p p t M t M t M n n n g g g g g g o o o n n n n n n C C C A A A A A A / / / O O O S S S A A A P P P T T T M M M

PRE-Clinical Data: Murine-Specific ANGPTL3 Rx ASO Significantly Reduced the Progression of Atherosclerosis in Ldlr -/- Mouse Model 13 En Face Atherosclerosis 15 % Lesion Area -37% Control 10 -53% * * 5 0 Control ASO (50) ANGPTL3 ASO (12.5) ANGPTL3 ASO (50) ANGPTL3 ASO (50 mg/kg) 16 weeks post treatment * Indicates significantly different ( p< 0.05) when compared to control ASO treatment

Background: Phase I Trial IONIS-ANGPTL3 Rx Reduces ANGPTL3 Protein in Healthy Volunteers * p<0.05 ** p<0.01 ** *** p<0.001 N=8 for Placebo, *** N=6 for each IONIS-ANGPTL3 Rx Dose Group *** • Triglycerides reduced by up to 63% (group means up to 49%) • Total cholesterol reduced by up to 46% (group means up to 28%) • Larger reductions observed in subjects with higher baseline lipid levels

Study Design Phase 1/2a Clinical Study IONIS-ANGPTL3-L Rx - SAD/MAD Single Ascending Dose (N=12) Multiple Ascending Dose (N=32) Treatment 1 SC dose 6 once-weekly SC doses Subjects N = 4 per dose cohort N = 8 per dose cohort (active:placebo) (3:1) (6:2) Dose levels 20, 40 or 80 mg 10 mg, 20 mg, 40 mg or 60 mg (dose volume) (0.2, 0.4, 0.8 mL per dose) (0.1, 0.2, 0.4, 0.6 mL per dose) Post-treatment 30-90 days 13 weeks Inclusion Criteria: Healthy volunteers age 18-65, BMI <35 kg/m 2 • • MAD cohorts required TG >150 mg/dL and LDL-C >70 mg/dL Key Endpoints: • Safety and tolerability • Change in fasting LDL-C, HDL-C, VLDL-C, TC, TG, non-HDL-C, apoA-I, apoB, LDL:HDL ratio, TC:HDL ratio, Lp(a), apoC-III, and ANGPTL3 • PK of ANGPTL3-L Rx

RESULTS: IONIS ANGPTL3-L Rx 80 mg Single Dose Mean % Change in Plasma ANGPTL3 Levels (N=3) (N=3)

RESULTS: IONIS ANGPTL3-L Rx 80 mg Single Dose Mean % Change in Lipid Parameters 30 Mean % change from Baseline ±SEM 20 10 0 -10 -20 Day 3 -30 Day 8 Day 15 -40 -50 -60 -70 -80 Plasma TC nonHDL-C LDL-C HDL-C TG VLDL-C ANGPTL3