ADVANCES IN RNAI THERAPEUTICS PLATFORM - VASANT JADHAV, PHD 3RD INTERNATIONAL CONFERENCE ON THE LONG AND THE SHORT OF NON-CODING RNAS - ALNYLAM ...
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Advances in RNAi Therapeutics Platform
3rd International Conference on the Long and the Short of Non-Coding RNAs
Vasant Jadhav, PhD
22nd June, 2019
© 2018 Alnylam Pharmaceuticals, Inc.
1
Outline
• Introduction to RNAi Platform
• New Frontiers for RNAi Therapeutics: CNS and Ocular Delivery
• Mechanistic Understanding: Durability of RNAi Therapeutics
• RNAi Therapeutics Towards Non-Parenteral Dosing
2
Alnylam Pharmaceuticals
Founded on the Bold Promise of Turning Nobel Prize Winning Science into a New Class of Medicine
2002: In vitro data by our scientific co-founders that started Alnylam
Discovery of RNAi in
mammalian cells
Elbashir et al., Nature,
2001;411:494-98
16-years later
2018: Approval of first ever RNAi-based therapeutic by FDA and EMA
3
Making Drugs Out of siRNAs
The Challenge
Characteristics
• M.W 12,000-14,000
• Size: 2 turns of helix
• 40 negative charges
• Hydrophilic
• Hydrated heavily
• ca. 5.5 nm X 2 nm
• Biostability
Sense strand 2 bp (double) 3’ overhangs
21-23 base pairs long
dsRNA
Seed
sequence
(residues 2-8)
Anti-sense or
‘Guide’ strand
4 Structure adapted from Klosterman, P. S.; Shah, S. A.; Steitz, T. A Biochemistry (1999), 38, 14784-14792.
Addressing the Delivery Challenge
Mechanisms for siRNA Delivery to Liver
siRNA
Current Clinical
Patisiran siRNAs
Lipid Nanoparticles (LNPs) GalNAc-siRNA Conjugates
▪ siRNA (limited modifications) ▪ Single chemical entity
in a multi-component lipid ▪ Tri-GalNAc ligand conjugated
to sense strand of extensively
formulation (LNP; ~85 nm)
modified siRNA
▪ Targeted delivery to liver
▪ Targeted delivery to liver
▪ Intravenous (IV) ▪ Subcutaneous (SC)
administration administration
5
GalNAc-siRNA Conjugates: SC-Administered Platform For Targeted Delivery To Hepatocytes
siRNA Asialoglycoprotein
Metabolic stability Receptor (ASGPR)
Intrinsic potency Highly expressed in hepatocytes
Duration of effect High turnover (recycling time ~15 min)
Safety Conserved across species
CMC
Ligand
Receptor affinity specificity
Metabolic stability
Safety
CMC
6
Alnylam Clinical Development Pipeline
Focused in 4 Strategic Therapeutic Areas (STArs):
Genetic Medicines Cardio-Metabolic Diseases HUMAN BREAKTHROUGH EARLY STAGE LATE STAGE REGISTRATION/ COMMERCIAL
Hepatic Infectious Diseases CNS/Ocular Diseases POC1 DESIGNATION (IND or CTA Filed-Phase 2) (Phase 2-Phase 4) COMMERCIAL3 RIGHTS
hATTR Amyloidosis2
● Global
Givosiran Acute Hepatic Porphyria
● Global
Patisiran
ATTR Amyloidosis
Label Expansion ● Global
Fitusiran
Hemophilia and Rare
Bleeding Disorders ● 15-30% royalties
Inclisiran Hypercholesterolemia
● Milestones & up to
20% royalties
Lumasiran Primary Hyperoxaluria Type 1
● Global
Vutrisiran ATTR Amyloidosis
● Global
Cemdisiran
Complement-Mediated
Diseases ● 50-50
Cemdisiran/Pozelimab
Combo4
Complement-Mediated
Diseases ● Milestone/Royalty
ALN-AAT02 Alpha-1 Liver Disease
● Global
ALN-HBV02
(VIR-2218)
Hepatitis B Virus Infection
● 50-50 option rights
post-Phase 2
ALN-AGT Hypertension
● Global
1 POC, proof of concept – defined as having demonstrated target gene knockdown and/or additional evidence of activity in clinical studies
2 Approved in the U.S. for the polyneuropathy of hATTR amyloidosis in adults, and in the EU for the treatment of hATTR amyloidosis in adults with stage 1 or stage 2 polyneuropathy
3 Includes marketing application submissions
4 Cemdisiran is currently in Phase 2 development and pozelimab is currently in Phase 1 development; Alnylam and Regeneron are evaluating potential combinations of these two investigational therapeutics
7 As of May 2019
Outline
• Introduction: RNAi Platform
• New Frontiers for RNAi Therapeutics: CNS and Ocular Delivery
• Mechanistic Understanding: Durability of RNAi Therapeutics
• RNAi Therapeutics Towards Non-Parenteral Dosing
8
RNAi Therapeutics for CNS and Ocular Diseases
• Many dominantly inherited • Many dominantly inherited eye
neurodegenerative diseases diseases
Enormous unmet medical need across the CNS and Ocular spaces
9
SOD1 siRNA Conjugates Demonstrate Superior Silencing in Rat CNS
Control
Superior silencing to parent at 10-fold lower dose SOD1 parent- 0.9 mg
SOD1 modified 0.9 mg
SOD1 modified 0.3 mg
Rest of brain Frontal SOD1 modified 0.07 mg
Cortex
Cerebellum
Lumbar Cerebellum
Frontal Cortex
Thoracic
Cervical Temporal Cortex
10Robust Silencing Throughout the CNS
Up to 6 months of silencing in some regions of the CNS following a single dose
Durable Silencing - 0.9 mg
1.50
Single IT dose- 0.9 mg
Lumbar spinal cord
1.25
Thoracic spinal cord
Cervical spinal cord
1.00
Message Remaining
Cerebellum
Hippocampus
0.75
Temporal Cortex
Frontal Cortex
0.50
0.25
0.00
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
Time (Days)
• Durable lowering across animals in most regions of the brain for up to 6 months
• Silencing in spine maintained close to NADIR through 6 months
• PD comparison in liver across species together with extended duration seen in rodents expected to support
infrequent dosing in human
11siRNA vs ASO in hSOD1 (SOD1G93A) Rats
Day 7 or 28 collection after single IT dose
Study Purpose
• Head-to-head comparison of siRNA and ASO
– siRNAs selected from mouse AAV-hsSOD1 screen
– ASO 1, based on McCampbell et al. (2018)
◦ Demonstrated ~75% maximum silencing at 2 weeks in the same rat model
Study Design
• Single IT injection of 0.9 mg assayed at day 7
– Same dose used for siRNA and ASO
• Single IT injection of 0.45 mg assayed at day 28
0.45 mg 28 day
0.9 mg 7 day
Study Day 0 7 28
Tissues collected Tissues collected
IT injection siRNA across spinal cord spinal cord
& ASO and brain
12
McCampbell et al, J Clin Invest. 2018;128(8):3558-3567siRNA vs ASO in hSOD1 (SOD1G93A) Rats
Improved hSOD1 mRNA reduction with siRNA compared to ASO in CNS at day 7
120
% hSOD1 message remaining
100
relative to aCSF
80
60
40
20
0
ASO
siRNA
ASO
siRNA
ASO
siRNA
ASO
siRNA
ASO
siRNA
ASO
siRNA
ASO
siRNA
ASO
siRNA
aCSF
Temporal Brain Frontal
Lumbar Thoracic Cervical Cerebellum Hippo
Cortex Stem Cortex
Greater silencing in all regions of the brain and spinal cord was observed using an
siRNA targeting hSOD1 in this model at day 7
13 Single IT dose of 0.9 mgsiRNA vs ASO in hSOD1 (SOD1G93A) Rats
Improved hSOD1 mRNA reduction with siRNA compared to ASO in the spine at day 28
120
% hSOD1 message remaining
100
relative to aCSF
80
60
40
20
0
aCSF ASO siRNA ASO siRNA ASO siRNA
Lumbar Thoracic Cervical
Greater silencing in all regions of the spinal cord were observed using an
siRNA targeting hSOD1 in this model at day 28
14 Single IT dose of 0.45 mgOcular TTR Silencing by Differentially Modified siRNA Conjugates in Rat
After Single Intravitreal Injection
Rat TTR mRNA
Day 14, 50 mg siRNA conjugate
256
% m e s s a g e r e m a in in g
128
64
32
16
8
4
2
1
0 .5
0 .2 5
C
S
d
d
e
ie
B
S
iz
E
P
if
m
d
o
ti
M
p
o
y
ll
H
ia
E
rt
a
P
15Dose Response and Duration of Activity of Ocular siRNA Conjugates in Mice
After Single Intravitreal Injection
O c u la r m T T R d o s e r e s p o n s e
O c u la r m T T R d u r a tio n 5 m o n th s
IV T D a y 1 3
s in g le 1 5 u g IV T in je c tio n
% m e s s a g e r e m a in in g
150
% m e s s a g e r e m a in in g
150
( r e la t iv e t o P B S )
( r e la t iv e t o P B S )
100
100
50
50
0
0
2
5
8
6
4
S
1
3
2
5
8
g
g
g
S
B
1
1
D
D
D
u
u
u
B
P
D
D
P
.6
.5
1
1
7
1
Excellent duration observed for siRNA conjugates in eye
16Current Ocular Design Shows Impressive Potency and Duration in NHP
% T T R R e m a in in g ( T T R A D - 2 9 1 8 4 5 )
% inT gT R
% T T R R e m a in ( TR
TRe ms aiRinNin
Agd(oTsTeRd AgDr o- 2u 9p1s8) 4 5 )
Day 28 Aqueous Hum or
D a y 2
Day 84 Aqueous Hum or 8 A q u e o u s H u m or
125
125
R e la t iv e t o P B S C o n t r o l
125
R e la t iv e t o P B S C o n t r o l
R e la t iv e t o P B S C o n t r o l
% T T R R e m a in in g
100
% T T R R e m a in in g
% T T R R e m a in in g
100 100
75 75
75
50 50
50
25 25 25
M in M in M in
0 % R e m a in in g 0 0 % R e m a in in%gR e m a in
PBS A D -2 9 1 8 4 5 A D -2 9 1 8 4 5 A D -2 9 1 8 4 5 A D -2 9 1 8 4 5 PBS AP
DB - 2S9 1 8 4 5 A DA- 2D9-12 89 41 58 4 5 A DA- 2
D9- 2
1981
485 4 5 A DA- 2
D9- 2
1981
485 4 5 A D -2 9 1 8 4 5
0 .0 0 3 m g 0 .0 3 m g 0 .1 m g 0 .3 m g 0 .0 0 3 m g 0 .0 00 .0
3 3m m g g 0 .0 0
3 .1
mm g g 0 .10 .3
m gm g 0 .3 m g
Excellent duration observed for siRNA conjugates in NHP eye
17
% T T R R e m a in in g ( T T R s iR N A d o s e d g r o u p s )Alnylam-Regeneron Alliance*
Landmark Alliance Focused on CNS & Ocular RNAi Therapeutics
• Partnership of two leading biopharmaceutical companies committed to innovation
‒ Alnylam R&D expertise and scientific excellence in RNAi therapeutics with emerging global commercial presence
‒ Regeneron scientific excellence, world-leading capabilities in human genetics, and industry-leading commercial presence in
ophthalmology and other large markets
• Broad, multi-product alliance across CNS, ocular, and select liver targets
‒ Both companies fully participate in value creation with 50-50 structure in CNS and select liver programs
‒ Milestone/royalty structure for ocular disease programs
• Accelerates Alnylam CNS and ocular programs, driving significant pipeline expansion
‒ Robust, highly durable, and widely distributed RNAi knockdown of key targets in CNS/ocular pre-clinical models
‒ Adds 1-2 new planned INDs/year toward CNS or ocular targets to previously planned 1-2 new INDs/year in liver beginning in 2020
• Significantly bolsters Alnylam balance sheet to >$2B pro forma for increased pipeline investment and future growth
18 * Alliance and equity agreements with Regeneron expected to close during Q2 2019Outline
• Introduction: RNAi Platform
• New Frontiers for RNAi Therapeutics: CNS and Ocular Delivery
• Mechanistic Understanding: Durability of RNAi Therapeutics
• RNAi Therapeutics Towards Non-Parenteral Dosing
19Extended Duration of Activity by ESC Conjugates
Human pharmacodynamic response* of two siRNAs with the same sequence, different chemistry
STC: Standard Template Chemistry Advanced ESC: Enhanced Stability Chemistry
2’-Fluoro 2’-OMe PS- Phosphorothioate
Relative levels of serum biomarker
STC: 500 mg (qDx5, qWx5)
Advanced ESC: 50 mg (single
dose)
STC qD x 5, qWx5 Days
*Phase 1 data in healthy volunteers from separate studies
Advanced ESC Single dose
20Depot Effect Hypothesis for Conjugate Extended Duration of Effect
Hepatocyte uptake, intracellular
trafficking and release
Circulation
GalNAc-siRNA
conjugate
Liver
SC
Injection site
• Sustained release of conjugate from SC injection site to liver?
• Increased half-life of siRNA-loaded RISC?
• Continuous supply of siRNA from an intracellular depot?
21The SC Injection Site Is Not A Depot For GalNAc-siRNA - IV Dosing Of Potent
Compounds Shows Similar Profile
vs
SC IV
Depot? No potential SC depot
Increase
stability
Nair et al., NAR, 2017
22In Vivo Duration Of Silencing In Mice Is Dependent On Delivery Modality
And Stability
• Unlike GalNAc-siRNA conjugates, LNP designed to promote efficient endosomal escape of siRNAs
• Doses selected to get similar level of KD and thus similar level of RISC loading expected
• Faster onset and recovery of activity with LNP
• Slower onset but substantially extended duration with GalNAc-conjugate
• Overall data suggests that RISC half-life alone can not explain the duration of activity for conjugates
23Functional siRNA Released From Acidic Compartments Up To Three Weeks
Post-Dose
GalNAc-siRNA
TTR Knockdown
Advanced ESC - 0.5 mg/kg
70
60
Percent Serum TTR
(Rel. to Pre-Dose)
8h, days 50
11 or 21
40 No Peptide
GalNAc Endolytic
Peptide 30 8h Peptide
D11 Peptide
20
D21 Peptide
10
siRNA 0
Lysopainter 0 10 20 30 40 50
Study Day
siRNA
release
24Weeks After Conjugate Dosing, Ectopically Expressed Tagged Ago2 Continues
To Load siRNA
GalNAc-siRNA
kD
160 7 days
FLAG-mAgo2 AAV
90
*
Antisense RISC Loading
Day 30
0.5
23 days
Antisense Strand
0.4
0.3
(ng/g)
0.2
0.1
0
No continuous Continuous
loading loading
25Outline
• Introduction: RNAi Platform
• New Frontiers for RNAi Therapeutics: CNS and Ocular Delivery
• Mechanistic Understanding: Durability of RNAi Therapeutics
• RNAi Therapeutics Towards Non-Parenteral Dosing
26Needle Free Delivery of GalNAc-siRNA via Lung Presented at OTS 2015
PoC Demonstrated in Mice Using Microsprayer®
Microsprayer® - A high pressure syringe for direct administration Microsprayer® Mediated Dosing Achieves Comparable Potency and Duration of
of aerosol at the junction of trachea for delivery in lung Activity to SC Delivered ESC Conjugates in Mouse Liver
1.4
Saline
Normalized serum TTR levels
SC Microsprayer®
1.2
1 1 mg/kg
0.8
0.6 3 mg/kg
0.4
0.2
0
Microsprayer® developed by PennCentury 0 7 14 21
Days Post Dose
Given the superior potency, metabolic stability and durable activity of GalNAc-siRNA conjugates, would they
also work via Oral Dosing- The least invasive method? 2
7
27
Systemic exposure of siRNA and LNA-antisense oligos by intra-tracheal dosing. Molecular Therapy 2011 19 (12), 2163–2168PoC for Oral Dosing of GalNAc-siRNA in Mice Delivered via Gavage Tube
The feeding tube is passed gently through the mouth and Study design
pharynx into the esophagus to deposit solution in stomach
▪ ESC siRNA +/- GalNAc
▪ Formulation containing permeation enhancer
▪ Single or 3 doses at Day 1, 2 and 5
Flexible Plastic Feeding Tubes
Instech’s plastic gavage tubes are
flexible to reduce trauma
Day 0 5 7 14 21
Serum collection for biomarker analysis
The Laboratory Mouse (2nd Edition)
2012, Pages 709-725
28Robust and Durable Activity Seen by Oral Dosing of GalNAc-siRNA in Mice
Serum biomarker levels post siRNA dosing in mice
1.6
1.4
1.2
1 PBS
Relative F12 levels
0.8
0.6
0.4
0.2 3 x 10 mg/kg Gavage
0 1 x 0.75 mg/kg SC
-0.2
0 5 10 15 20 25 30 35 40 45
Days
Oral gavage
29Dose Dependent Activity Seen by Oral Dosing of GalNAc-siRNA in Mice
1.2
Serum biomarker levels post siRNA dosing in mice
1
Relative Levels of Biomarker
0.8
3 mg/kg
0.6
0.4
10 mg/kg
0.2
3 X 3 mg/kg
3 X 10 mg/kg
0
0 5 10 15 20 25
Days
Oral gavage
30GalNAc Conjugation and Formulation are Important for siRNA Activity via
Oral Dosing
Serum biomarker levels post siRNA dosing in mice
1.6
1.4 Unconjugated siRNA
3 x 10 mg/kg
Relative biomarker levels
1.2
1
0.8
0.6
Without formulation
0.4 GalNAc-siRNA
3 X 10 mg/kg
0.2
With formulation
0
0 5 10 Days 15 20 25
Oral gavage
31Summary
RNAi therapeutics emerging as high impact, transformational medicines
◦ ONPATTRO® as 1st RNAi therapeutic is now in market serving patients
◦ Multiple RNAi therapeutics are in advanced stages of clinical development
New frontiers for future expansion of RNAi therapeutics opportunity
◦ Delivery of RNAi therapeutics to CNS and eye achieved
◦ Our learnings in the liver apply!!
Preclinical data suggests durability of GalNAc-siRNAs likely from continuous supply of siRNA from intracellular depot
Achieved PoC for oral dosing of GalNAc-siRNA conjugates- the least invasive method of drug administration
◦ Convenience of conventional dosing for modern medicine
32Acknowledgements
Our volunteers, patients and patient families
Participating volunteers,
patients and their families
Alnylam colleagues:
Research
Department
Early Development
RNAi Platform
MGH:
Dr. Brown lab
Thank you!
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