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1© 2018 Alnylam Pharmaceuticals, Inc.
22nd June, 2019
Advances in RNAi Therapeutics Platform
3rd International Conference on the Long and the Short of Non-Coding RNAs
Vasant Jadhav, PhD
2
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
3
Founded on the Bold Promise of Turning Nobel Prize Winning Science into a New Class of Medicine
Alnylam Pharmaceuticals
2018: Approval of first ever RNAi-based therapeutic by FDA and EMA
Discovery of RNAi in
mammalian cells
Elbashir et al., Nature,
2001;411:494-98
16-years later
2002: In vitro data by our scientific co-founders that started Alnylam
4 Structure adapted from Klosterman, P. S.; Shah, S. A.; Steitz, T. A Biochemistry (1999), 38, 14784-14792.
The Challenge
Making Drugs Out of siRNAs
21-23 base pairs long
dsRNA
2 bp (double) 3’ overhangs
Seed sequence
(residues 2-8)
Anti-sense or‘Guide’ strand
Sense strand
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
5
Lipid Nanoparticles (LNPs)
▪ siRNA (limited modifications)
in a multi-component lipid
formulation (LNP; ~85 nm)
▪ Targeted delivery to liver
▪ Intravenous (IV)
administration
Addressing the Delivery Challenge
GalNAc-siRNA Conjugates
▪ Single chemical entity
▪ Tri-GalNAc ligand conjugated
to sense strand of extensively
modified siRNA
▪ Targeted delivery to liver
▪ Subcutaneous (SC)
administration
Current Clinical
siRNAs
siRNA
Patisiran
Mechanisms for siRNA Delivery to Liver
6
GalNAc-siRNA Conjugates: SC-Administered Platform For Targeted Delivery To Hepatocytes
siRNAMetabolic stability
Intrinsic potency
Duration of effect
Safety
CMC
LigandReceptor affinity specificity
Metabolic stability
Safety
CMC
AsialoglycoproteinReceptor (ASGPR)
Highly expressed in hepatocytes
High turnover (recycling time ~15 min)
Conserved across species
7
Alnylam Clinical Development Pipeline
1 POC, proof of concept – defined as having demonstrated target gene knockdown and/or additional evidence of activity in clinical studies2 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 polyneuropathy3 Includes marketing application submissions4 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
As of May 2019
HUMAN
POC1
BREAKTHROUGH
DESIGNATIONEARLY STAGE
(IND or CTA Filed-Phase 2)
LATE STAGE (Phase 2-Phase 4)
REGISTRATION/
COMMERCIAL3
COMMERCIAL
RIGHTS
hATTR Amyloidosis2 ● Global
Givosiran Acute Hepatic Porphyria ● Global
PatisiranATTR Amyloidosis
Label Expansion ● Global
FitusiranHemophilia and Rare
Bleeding Disorders ● 15-30% royalties
Inclisiran Hypercholesterolemia ● Milestones & up to
20% royalties
Lumasiran Primary Hyperoxaluria Type 1 ● Global
Vutrisiran ATTR Amyloidosis ● Global
CemdisiranComplement-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
Focused in 4 Strategic Therapeutic Areas (STArs):Genetic Medicines Cardio-Metabolic Diseases
Hepatic Infectious Diseases CNS/Ocular Diseases
8
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
9
RNAi Therapeutics for CNS and Ocular Diseases
Enormous unmet medical need across the CNS and Ocular spaces
• Many dominantly inherited eye
diseases
• Many dominantly inherited
neurodegenerative diseases
10
Superior silencing to parent at 10-fold lower dose
SOD1 siRNA Conjugates Demonstrate Superior Silencing in Rat CNS
Lumbar
Thoracic
Cervical
Cerebellum
Frontal Cortex
Temporal Cortex
Control
SOD1 parent- 0.9 mg
SOD1 modified 0.9 mg
SOD1 modified 0.3 mg
SOD1 modified 0.07 mg
Cerebellum
Frontal
Cortex
Rest of brain
11
Single IT dose- 0.9 mg
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 1700.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
Temporal Cortex
Durable Silencing - 0.9 mg
Messag
e R
em
ain
ing
Lumbar spinal cord
Cerebellum
Hippocampus
Frontal Cortex
Thoracic spinal cord
Cervical spinal cord
Up to 6 months of silencing in some regions of the CNS following a single dose
Robust Silencing Throughout the CNS
• 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
12
siRNA vs ASO in hSOD1 (SOD1G93A) Rats
McCampbell et al, J Clin Invest. 2018;128(8):3558-3567
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
Day 7 or 28 collection after single IT dose
Study Day
Tissues collected
across spinal cord
and brain
0 7
IT injection siRNA
& ASO
Tissues collected
spinal cord
28
0.45 mg 28 day
0.9 mg 7 day
13
Improved hSOD1 mRNA reduction with siRNA compared to ASO in CNS at day 7
siRNA vs ASO in hSOD1 (SOD1G93A) Rats
aC
SF
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
0
20
40
60
80
100
120
% h
SO
D1 m
essage r
em
ain
ing
rela
tive to a
CS
F
Lumbar Thoracic CervicalBrain
StemCerebellum Hippo
Frontal
CortexTemporal
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
Single IT dose of 0.9 mg
14
Improved hSOD1 mRNA reduction with siRNA compared to ASO in the spine at day 28
siRNA vs ASO in hSOD1 (SOD1G93A) Rats
Greater silencing in all regions of the spinal cord were observed using an
siRNA targeting hSOD1 in this model at day 28
Single IT dose of 0.45 mg
aCSF ASO siRNA ASO siRNA ASO siRNA
0
20
40
60
80
100
120
% h
SO
D1 m
essage r
em
ain
ing
rela
tive to a
CS
F
Lumbar Thoracic Cervical
15
Ocular TTR Silencing by Differentially Modified siRNA Conjugates in Rat
After Single Intravitreal Injection
PB
S
Par t
ially M
od
ifie
d
ES
C
EH
op
tim
ized
0 .2 5
0 .5
1
2
4
8
1 6
3 2
6 4
1 2 8
2 5 6
% m
es
sa
ge
re
ma
inin
g
Rat TTR mRNA
Day 14, 50 mg siRNA conjugate
16
Dose Response and Duration of Activity of Ocular siRNA Conjugates in Mice After Single Intravitreal Injection
PB
S
1 u
g
7.5
ug
11.6
ug
0
5 0
1 0 0
1 5 0
O c u la r m T T R d o s e re s p o n s e
IV T D ay 13
% m
es
sa
ge
re
ma
inin
g
(re
lati
ve
to
PB
S)
PB
SD
28
D56
D84
D112
D135
0
5 0
1 0 0
1 5 0
O c u la r m T T R d u ra tio n 5 m o n th s
s in g le 1 5u g IV T in je c tion
% m
es
sa
ge
re
ma
inin
g
(re
lati
ve
to
PB
S)
Excellent duration observed for siRNA conjugates in eye
17
Current Ocular Design Shows Impressive Potency and Duration in NHP
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R A D -2 9 1 8 4 5 )
D a y 2 8 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R s iR N A d o s e d g ro u p s )
D a y 8 4 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R A D -2 9 1 8 4 5 )
D a y 2 8 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R s iR N A d o s e d g ro u p s )
D a y 8 4 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in gP B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R A D -2 9 1 8 4 5 )
D a y 2 8 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R s iR N A d o s e d g ro u p s )
D a y 8 4 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R A D -2 9 1 8 4 5 )
D a y 2 8 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
P B S A D -2 9 1 8 4 5
0 .0 0 3 m g
A D -2 9 1 8 4 5
0 .0 3 m g
A D -2 9 1 8 4 5
0 .1 m g
A D -2 9 1 8 4 5
0 .3 m g
0
2 5
5 0
7 5
1 0 0
1 2 5
% T T R R e m a in in g (T T R s iR N A d o s e d g ro u p s )
D a y 8 4 A q u e o u s H u m o r
% T
TR
Re
ma
inin
g
Re
lati
ve
to
PB
S C
on
tro
l
M in
% R e m a in in g
Excellent duration observed for siRNA conjugates in NHP eye
18
Alnylam-Regeneron Alliance*
* Alliance and equity agreements with Regeneron expected to close during Q2 2019
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
19
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
20
Extended Duration of Activity by ESC Conjugates
Rela
tive
le
ve
ls o
f s
eru
m b
iom
ark
er
Human pharmacodynamic response* of two siRNAs with the same sequence, different chemistry
DaysSTC qD x 5, qWx5
Advanced ESC Single dose
STC: 500 mg (qDx5, qWx5)
Advanced ESC: 50 mg (single
dose)
*Phase 1 data in healthy volunteers from separate studies
2’-Fluoro 2’-OMe PS- Phosphorothioate
Advanced ESC: Enhanced Stability ChemistrySTC: Standard Template Chemistry
21
• 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?
Depot Effect Hypothesis for Conjugate Extended Duration of Effect
SC
Injection site
GalNAc-siRNA
conjugate
Circulation
Liver
Hepatocyte uptake, intracellular
trafficking and release
22
The SC Injection Site Is Not A Depot For GalNAc-siRNA - IV Dosing Of Potent
Compounds Shows Similar Profile
SC
Depot?
IV
No potential SC depot
vs
Nair et al., NAR, 2017
Increase
stability
23
In 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
24
Functional siRNA Released From Acidic Compartments Up To Three Weeks
Post-Dose
GalNAc-siRNA
8h, days
11 or 21
siRNA
release
GalNAc Endolytic
Peptide
0
10
20
30
40
50
60
70
0 10 20 30 40 50
Pe
rce
nt
Seru
m T
TR
(Re
l. t
o P
re-D
ose
)
Study Day
TTR KnockdownAdvanced ESC - 0.5 mg/kg
No Peptide
8h Peptide
D11 Peptide
D21 Peptide
siRNA
Lysopainter
25
Weeks After Conjugate Dosing, Ectopically Expressed Tagged Ago2 Continues
To Load siRNA
GalNAc-siRNA
7 days
FLAG-mAgo2 AAV
23 days
No continuous
loading
Continuous
loading
160
90
kD
*
0
0.1
0.2
0.3
0.4
0.5
An
tisen
se S
tran
d
(ng
/g)
Antisense RISC LoadingDay 30
26
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
27
Needle Free Delivery of GalNAc-siRNA via LungPoC Demonstrated in Mice Using Microsprayer®
Microsprayer® - A high pressure syringe for direct administration
of aerosol at the junction of trachea for delivery in lung
2
7
Microsprayer® developed by PennCentury
Systemic exposure of siRNA and LNA-antisense oligos by intra-tracheal dosing. Molecular Therapy 2011 19 (12), 2163–2168
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 7 14 21
No
rma
lize
d s
eru
m T
TR
le
ve
ls
Days Post Dose
Microsprayer® Mediated Dosing Achieves Comparable Potency and Duration of
Activity to SC Delivered ESC Conjugates in Mouse Liver
Saline
1 mg/kg
3 mg/kg
Presented at OTS 2015
SC Microsprayer®
Given the superior potency, metabolic stability and durable activity of GalNAc-siRNA conjugates, would they
also work via Oral Dosing- The least invasive method?
28
PoC for Oral Dosing of GalNAc-siRNA in Mice Delivered via Gavage Tube
The Laboratory Mouse (2nd Edition)
2012, Pages 709-725
The feeding tube is passed gently through the mouth and
pharynx into the esophagus to deposit solution in stomach
Day 0 145 7
Serum collection for biomarker analysis
21
Study design
▪ ESC siRNA +/- GalNAc
▪ Formulation containing permeation enhancer
▪ Single or 3 doses at Day 1, 2 and 5
Flexible Plastic Feeding TubesInstech’s plastic gavage tubes are
flexible to reduce trauma
29
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 5 10 15 20 25 30 35 40 45
Days
Serum biomarker levels post siRNA dosing in mice
Robust and Durable Activity Seen by Oral Dosing of GalNAc-siRNA in Mice
PBS
3 x 10 mg/kg GavageRela
tive
F1
2 leve
ls
1 x 0.75 mg/kg SC
Oral gavage
30
Dose Dependent Activity Seen by Oral Dosing of GalNAc-siRNA in Mice
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
Re
lati
ve
Le
ve
ls o
f B
iom
ark
er
Days
3 X 10 mg/kg
3 X 3 mg/kg
10 mg/kg
3 mg/kg
Serum biomarker levels post siRNA dosing in mice
Oral gavage
31
GalNAc Conjugation and Formulation are Important for siRNA Activity via
Oral Dosing
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 5 10 15 20 25
Re
lati
ve
bio
ma
rke
r le
ve
ls
Days
Serum biomarker levels post siRNA dosing in mice
Without formulation
Unconjugated siRNA
3 x 10 mg/kg
With formulation
GalNAc-siRNA
3 X 10 mg/kg
Oral gavage
32
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
Summary
33
Acknowledgements
Our volunteers, patients and patient families
Thank you!
Participating volunteers, patients and their families
Alnylam colleagues: Research Department Early Development RNAi Platform
MGH: Dr. Brown lab