Platform Advances in RNAi Therapeutics
RNAi Roundtable
August 23, 2017
2
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
3
Reminders
Event will run for approximately 60-75 minutes
Q&A Session at end of presentation
• Submit questions at top of webcast screen
• Questions may be submitted at any time
Replay, slides and transcript available at www.alnylam.com/capella
4
Alnylam Forward Looking Statements
This presentation contains forward-looking statements, within the meaning of Section 27A of the Securities
Act of 1933 and Section 21E of the Securities Exchange Act of 1934. There are a number of important
factors that could cause actual results to differ materially from the results anticipated by these forward
looking statements. These important factors include our ability to discover and develop novel drug
candidates and delivery approaches and successfully demonstrate the efficacy and safety of our product
candidates; pre-clinical and clinical results for our product candidates; actions or advice of regulatory
agencies; delays, interruptions or failures in the manufacture and supply of our product candidates; our
ability to obtain, maintain and protect intellectual property, enforce our intellectual property rights and defend
our patent portfolio; our ability to obtain and maintain regulatory approval, pricing and reimbursement for
products; our progress in establishing a commercial and ex-United States infrastructure; competition from
others using similar technology and developing products for similar uses; our ability to manage our growth
and operating expenses, obtain additional funding to support our business activities and establish and
maintain business alliances; the outcome of litigation; and the risk of government investigations; as well as
those risks more fully discussed in our most recent quarterly report on Form 10-Q under the caption “Risk
Factors.” If one or more of these factors materialize, or if any underlying assumptions prove incorrect, our
actual results, performance or achievements may vary materially from any future results, performance or
achievements expressed or implied by these forward-looking statements. All forward-looking statements
speak only as of the date of this presentation and, except as required by law, we undertake no obligation to
update such statements.
5
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
6
HUMAN POC* EARLY STAGE(IND or CTA Filed-Phase 2)
LATE STAGE (Phase 2-Phase 3)
REGISTRATION/
COMMERCIAL
COMMERCIAL
RIGHTS
PatisiranHereditary ATTR
Amyloidosis ● US, Canada,
Western Europe
FitusiranHemophilia and Rare
Bleeding Disorders ●50%
US, Canada,
Western Europe
Inclisiran Hypercholesterolemia ● Milestones &
Royalties
GivosiranAcute Hepatic
Porphyrias ● Global
ALN-CC5Complement-
Mediated Diseases ● Global
ALN-GO1Primary
Hyperoxaluria Type 1 ●Subject to
partner option
rights
ALN-TTRsc02Hereditary ATTR
Amyloidosis ●Subject to
partner option
rights
ALN-HBVHepatitis B Virus
Infection ● Global
Focused in 3 Strategic Therapeutic Areas (STArs):
Genetic Medicines
Cardio-Metabolic Diseases
Hepatic Infectious Diseases
Alnylam Clinical Development Pipeline
*Proof of concept defined as having demonstrated target gene knockdown and/or additional evidence of activity in clinical studies
7
Alnylam Investigational RNAi Therapeutics PlatformExtensive Human Safety Experience
Number of
Programs
Number of
Clinical Studies
Total Patients or
Volunteers Dosed
Greatest Duration
of Exposure
>10 >20 >1000 ~36 months
Platform related findings*
• Low incidence (15.2%) of generally mild, transient injection site reactions
• Low incidence (2.2%) of generally mild, asymptomatic, reversible LFT increases >3x ULN
• No evidence of safety signals similar to revusiran program
Favorable emerging profile for ESC-GalNAc platform compared with
competing oligo platforms†
• No evidence of thrombocytopenia, renal toxicity, or systemic inflammatory effects
*All reported data as of December 2016† Based on reported study data - not based on direct comparative studies
8
GalNAc-Conjugate Platforms for Delivery to Liver
• Standard Template Chemistry
GalNAc conjugate
• SC administration
• Fitusiran
• Inclisiran
• Givosiran
STC-Conjugate ESC-Conjugate
• ALN-TTRSC02• ALN-GO1• ALN-CC5• ALN-HBV
Revusiran
• Enhanced Stability Chemistry
GalNAc conjugate
• SC administration
• 2018 INDs and CTAs
ESC+ Conjugate
• Enhanced Stability Chemistry
GalNAc conjugate, ↑ specificity
• SC administration
First generation
GalNAc conjugate,
Initial human POC
Second generation
GalNAc conjugate,
Human POC,
greater potency
and durability with
lower exposures
Next generation
GalNAc conjugate
with further
improvements to
specificity and
therapeutic index
9
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
10
Enhanced Stabilization Chemistry (ESC) GalNAc-siRNAsSC-Administered Conjugate Platform for Targeted Delivery to Hepatocytes
AsialoglycoproteinReceptor (ASGPR)• Highly expressed in
hepatocytes
• High capacity receptor
• Conserved across species
siRNA• Metabolic stability
• Intrinsic potency
Ligand• Trivalent GalNAc
• High affinity and specificity
11
Recap of STC to ESC Transition
Metabolic profiling of conjugates in mouse liver
Standard Template Chemistry (STC)
Enhanced Stabilization Chemistry (ESC)
5′-sense
5′-AS
5′-sense
5′-AS
= Phosphorothioate linkage (PS) = 2’-F = 2’-OMe
Region of nuclease
hotspots
Region of nuclease
hotspots
End stabilization with PS linkages
12
ESC Significantly Enhances Efficacy and DurationReduction of AT Protein After Single SC Dose in NHP
Potent and durable silencing achieved after single SC dose• >10-fold improvement in efficacy over standard template chemistry
• Substantially extended duration of effect
% K
no
ck
do
wn
se
rum
AT
(R
ela
tive
to
pre
-do
se
)
Day
100
80
60
40
20
1.0
1.2
-10 0 10 20 30 40
STC-AT3 (10 mg/kg)
ESC-AT3 (10 mg/kg)
ESC-AT3 (1 mg/kg)
Single SC dose
13
Objective• Identify new generalizable conjugate designs with improved in vivo potency through optimal
placement of 2’-F and 2’-OMe modifications
Approach• Begin with motifs identified by statistical analysis of large dataset of conjugate in vitro activity
• Apply screening platform using a set of multiple standard sequences (3 targets) and conditions for rapid and systematic evaluation of new designs
siRNA design
In vitro activityIn vivo efficacy /
exposure
Generalizability
(expanded screen)
Towards Advanced ESC DesignsIterative Screening Platform to Drive Continuous Design Improvements
14
Performance of Advanced ESC Conjugate in Mice
Advanced ESC Design
0.0001
0.001
0.01
0.1
1
10
4h 3 7 14 21 35
Liv
er
Exp
osu
re (
ug
/g)
Days Post-Dose
ESC Design
2’-F 2’-OMe PS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
4h 3 7 14 21 35
mR
NA
(n
orm
to
PB
S)
Advanced ESC (0.75 mg/kg)
ESC (2.5 mg/kg)
Target mRNA levels in Liver Total siRNA levels in Liver
Days Post-Dose
Advanced ESC (0.75 mg/kg)
ESC (2.5 mg/kg)
15
Performance of Advanced ESC Conjugate in NHP
ALN-TTRsc02 (Advanced ESC design) compared to Revusiran
Revusiran
ALN-TTRsc02
Days
Total
Dose
AUEC for %
TTR lowering
Revusiran 45 mg/kg 4154
ALN-TTRsc02 1 mg/kg 8139
Adjusting for dose difference (45-fold) and AUEC
(1.95-fold), ALN-TTRsc02 shows ~88-fold in vivo
potency improvement over Revusiran
AUEC= Area under effect curve
16
Me
an
[+
/- S
EM
] T
TR
Re
lative
to
Ba
se
line
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Days since first dose
0 40 80 120 160 200 240 280 320
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Cohort Placebo (N=20) TTRSC02 (5mg) (N=6)TTRSC02 (25mg) (N=6) TTRSC02 (Optional; 25mg) (N=6)TTRSC02 (Subjects of Japanese descent; 25mg) (N=6) TTRSC02 (50mg) (N=6)TTRSC02 (Optional; 50mg) (N=6) TTRSC02 (Subjects of Japanese descent; 50mg) (N=6)TTRSC02 (100mg) (N=6) TTRSC02 (200mg) (N=6)TTRSC02 (300mg) (N=6)
ALN-TTRsc02 Phase 1 Preliminary Study Results
Single Ascending Dose Study in Healthy Volunteers
• No SAEs and no discontinuations due to AEs
• All AEs mild or moderate in severity
◦ 14 AEs in 8 subjects considered possibly related to treatment; majority mild
◦ Events included injection site erythema, injection site pain, injection site bruising, rhinorrhea, pruritus, cough, nausea, fatigue, genital rash and abdominal pain
◦ No clinically significant changes in lab parameters, EKG or physical exam
As of data cutoff on 31May2017
17
Pushing the EnvelopeSteadfast Focus on Platform Research Driving Potency Improvements
Sharp’s Law
Conjugate potency improvements in mice (single dose ED50)
0.01
0.1
1
10
100
2005 2007 2009 2011 2013 2015 2017
ED
50 (
mg
/kg
)
Year
Endo-
light
STC
ESC
Advanced ESC
Projected
ESC
Advanced
ESC
Endo-light
STC
18
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
19
GalNAc-siRNAStudy
Study Population
Subjects Treated
Max Duration Treatment
ALT>3x ULN
Revusiran Phase 1 NHV 66 6 wks 1Revusiran Phase 2 ATTR cardiomyopathy 26 6 wks 1Revusiran Phase 2 OLE ATTR cardiomyopathy 25^ Up to 18 mos 0Fitusiran Phase 1, Part A NHV 3 Single dose 0
Fitusiran Phase 1, Parts B & CSevere and moderate
HA and HB w/o inhibitor 25 Up to 3 mos 1Fitusiran Phase 1 D HA and HB w/ inhibitor 16 Up to 3 mos 3Fitusiran Phase 2 (OLE) HA and HB w/ and w/o inhibitor 23^ Up to 14 mos 3ALN-CC5 Phase 1, Parts A & B NHV 33 Up to 3 wks 0ALN-CC5 Phase 1, Part C PNH 6 Up to 6 mos 1Inclisiran Phase 1 NHV w/ elevated LDL-C 51 Up to 2 mos 1Inclisiran Phase 2 High Risk CVD; elevated LDL 370 Single dose 1Givosiran Phase 1, Parts A & B ASHE 23 Up to 2 mos 1Givosiran Phase 1, Part C AIP 11 Up to 6 mos 1ALN-AAT Phase 1/2, Parts A & B NHV 19 Up to 4 mos 1ALN-GO1 Part A NHV 24 Single dose 0
ALT=alanine aminotransferase; ULN=upper limit of normal ^Subjects treated with drug in previous study
ALT Elevations in Clinical ProgramsData Transfer as of November 2016
LNP-siRNAStudy
Study Population
Subjects Treated
Max Duration Treatment
ALT>3x ULN
Patisiran Phase 1 NHV 22 Single dose 0Patisiran Phase 2 hATTR polyneuropathy 29 Up to 4 wks 0Patisiran Phase 2 OLE hATTR polyneuropathy 27^ Up to 25mos 1
STC
ESC
20
GalNAc-siRNAStudy
Study Population
Subjects Treated
Max Duration Treatment
ALT>3x ULN
Revusiran Phase 1 NHV 66 6 wks 1Revusiran Phase 2 ATTR cardiomyopathy 26 6 wks 1Revusiran Phase 2 OLE ATTR cardiomyopathy 25^ Up to 18 mos 0Fitusiran Phase 1, Part A NHV 3 Single dose 0
Fitusiran Phase 1, Parts B & CSevere and moderate
HA and HB w/o inhibitor 25 Up to 3 mos 1Fitusiran Phase 1 D HA and HB w/ inhibitor 16 Up to 3 mos 3Fitusiran Phase 2 (OLE) HA and HB w/ and w/o inhibitor 23^ Up to 14 mos 3ALN-CC5 Phase 1, Parts A & B NHV 33 Up to 3 wks 0ALN-CC5 Phase 1, Part C PNH 6 Up to 6 mos 1Inclisiran Phase 1 NHV w/ elevated LDL-C 51 Up to 2 mos 1Inclisiran Phase 2 High Risk CVD; elevated LDL 370 Single dose 1Givosiran Phase 1, Parts A & B ASHE 23 Up to 2 mos 1Givosiran Phase 1, Part C AIP 11 Up to 6 mos 1ALN-AAT Phase 1/2, Parts A & B NHV 19 Up to 4 mos 1ALN-GO1 Part A NHV 24 Single dose 0
ALT Elevations in Clinical ProgramsData Transfer as of November 2016
LNP-siRNAStudy
Study Population
Subjects Treated
Max Duration Treatment
ALT>3x ULN
Patisiran Phase 1 NHV 22 Single dose 0Patisiran Phase 2 hATTR polyneuropathy 29 Up to 4 wks 0Patisiran Phase 2 OLE hATTR polyneuropathy 27^ Up to 25mos 1
STC
ESC
Frequency of ALT >3x ULN = 2.2%
(16/724)
ALT=alanine aminotransferase; ULN=upper limit of normal ^Subjects treated with drug in previous study
21
GalNAc-siRNA ConjugatesWide Therapeutic Index from Non-Clinical GLP Studies
NOAEL1 4 or 7 weeks
(mg/kg)
NOAEL 13 weeks
(mg/kg)
NOAEL 6 mos Rat
(mg/kg)
NOAEL 9 mos
NHP (mg/kg)
Expected Human
Therapeutic Index (TI)2
Rat NHP Rat NHP
Revusiran3 30 ≥300 N/A N/A 30 ≥200 >80
ALN-TTRsc02 N/A N/A ≥120 ≥300 Planned Planned >500
Fitusiran3,4 15 0.35 N/A N/A 0.55
0.55
>105
Inclisiran ≥250 ≥250 ≥250 ≥250 ≥250 ≥300 >500
ALN-CC5 ≥50 ≥100 50 100 ≥50 ≥100 >200
Givosiran N/A N/A ≥30 ≥150 Ongoing Ongoing >500
ALN-AAT N/A N/A ≥50 ≥150 Ongoing Ongoing >500
ALN-HBV N/A N/A ≥60 ≥200 Planned Planned>500
1No Adverse Event Level (NOAEL)2TI calculated as NOAEL in NHP/Expected dose in man37 week studies4NOAEL in hemophilia mice >100 mg/kg, qW x 75 Secondary to exaggerated pharmacology
NHP
◦ Liver
– Basophilic granules in Kupffer cells
and hepatocytes; represents drug
accumulation
◦ Lymph nodes
– Vacuolated macrophages (with
basophilic stippling); represents
phagocytosis of drug
Rat
◦ Liver– Hepatocellular vacuolation: increased
number and size of normal rat hepatocellular vacuoles, most contain lipid
– Increased single cell necrosis
– Increased mitosis and regeneration
◦ Kidney– Basophilic granules in proximal tubular
epithelium; represents drug accumulation
22
In silico prediction &In vitro efficacy
In vitro screen for predicted off-targets
Rodent Knockdown
Rat Tox @ >100x PD dose
NHP Knockdown DC
Bad Actors (40%):
Show hepatotoxicity
Good Actors (60%):
No hepatotoxicity
Subset of Conjugates Shows Rat Hepatotoxicity at Toxicological
Doses and Drop Out of DC Selection Process
Single cell necrosis and/or
hepatocellular degeneration with
↑LFT 2x upper limit of normal
23
Potential Causes of Hepatotoxicity in Rodent Toxicity Studies
1. Non-RNAi drug effects e.g. protein binding
2. Competition for RISC
loading with miRNAs
RISC loading
mRNA
RISC
On-target bindingFull sequence match
Desired
on-target activity
3’-UTR
Off-target bindingPartial sequence match
3. Undesired off-target
activity
24
5’ Modifications on Antisense Strand Can Block
RISC Loading With No Impact on Liver Exposure
RISC loading
mRNA
Sense strand removal
Target RNA cleavage
Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
N o Y e s N o Y e s
1
1 0
1 0 0
R a t L iv e r E x p o s u re
ug
AS
/ g
liv
er
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ckN o Y e s N o Y e s
0 .0
0 .2
0 .4
0 .6
R a t R IS C L o a d in g
ng
AS
/ g
liv
er
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ck
25
Blocking Antisense RISC Loading Without Altering
2’F/2’OMe/PS Content Mitigates Hepatotoxicity
*
*^
#
No Yes
RISC loading block (siRNA-1)
Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
N o Y e s N o Y e s
0
1 0 0
2 0 0
3 0 0
R a t A L T
U/L
R e fe re n c e ra n g e
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ck
26
Accumulation of chemically-modified siRNAs in the liver is not
sufficient for rat hepatotoxicity.
27
ESC
Advanced ESC
Changing siRNA Chemical Modification Pattern
Does Not Reduce Liver Exposure or RISC Loading Dose: 100 mg/kg
Regimen: qw x 9
Necropsy: Day 58
E S C Ad v a n c e d E S C
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
R a t R IS C L o a d in g
ng
AS
/ g
liv
er
E S C Ad v a n c e d E S C
1
1 0
1 0 0
1 0 0 0
R a t L iv e r E x p o s u re
ug
AS
/ g
liv
er
28
Changing siRNA Chemical Modification Pattern
Does Not Mitigate Hepatotoxicity Dose: 100 mg/kg
Regimen: qw x 9
Necropsy: Day 58
S a lin e E S C Ad v a n c e d E S C
0
1 0 0
2 0 0
3 0 0
R a t A L T
U/L
R e fe re n c e ra n g e
ESC Advanced ESC
#
* #*
29
siRNA sequence, not the chemical modifications, is important for rat
hepatotoxicity.
30
Potential Causes of Hepatotoxicity in Rodent Toxicity Studies
1. Non-RNAi drug effects e.g. protein binding
2. Competition for RISC
loading with miRNAs
RISC loading
mRNA
RISC
On-target bindingFull sequence match
Desired
on-target activity
3’-UTR
Off-target bindingPartial sequence match
3. Undesired off-target
activity
31
Reversir to Abrogate RNAi Activity Without Changing siRNA
Chemistry or RISC Loading
0%
20%
40%
60%
80%
100%
120%
0 3 6 9 12 15 18 21 24
Rel
ativ
e Ta
rget
Pro
tein
Lev
el
No Reversir
Time (Days)
3 mg/kg
GalNAc-siRNA
0.1 mg/kg
GalNAc-Reversir
Reversir
Reversir
Reversir binds as a “synthetic target” to antisense strand in functional RISC:
32
Prevention
Treatment
Reversir
siRNA
Reversir
siRNA
( )
Blocking RISC-Loaded Antisense Strand with Reversir
Does Not Reduce Liver Exposure or RISC Loading
3-10 mg/kg
30 mg/kg
3-10 mg/kg
30 mg/kg
1
1 0
1 0 0
1 0 0 0
R a t L iv e r E x p o s u re
ug
A
S /
g l
ive
r
- Targeting Ctr - Targeting Ctr Targeting Ctr RVR
siRNA1 2 3
1
1 0
1 0 0
1 0 0 0
R a t R IS C L o a d in g
ng
A
S /
g l
ive
r
- Targeting Ctr - Targeting Ctr Targeting Ctr RVR
siRNA1 2 3
33
Reversing Activity of RISC-Loaded Antisense Strand
Mitigates Hepatotoxicity
Pre
ve
nti
on
+ Ctr RVR + Targeting RVR
#*
#
+
*
*#
**
*
^
Tre
atm
en
t
0
5 0
1 0 0
1 5 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 1
0
1 0
2 0
3 0
4 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 2
0
1 0
2 0
3 0
4 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 3
siRNA: 30 mg/kg qw x 3
Reversir: 3 or 10 mg/kg SD
Necropsy: Day 15 (Prevention)
Day 30 (Treatment)
34
Competition for RISC loading with endogenous miRNAs is not a major
contributor to rat hepatotoxicity; seed-mediated hybridization of the
RISC-loaded antisense strand appears to be important.
35
Potential Causes of Hepatotoxicity in Rodent Toxicity Studies
2. Competition for RISC
loading with miRNAs
RISC loading
mRNA
RISC
On-target bindingFull sequence match
Desired
on-target activity
3’-UTR
Off-target bindingPartial sequence match
3. Undesired off-target
activity
36
Seed-Mediated Off-Target Effects Observed at High Doses
RNAseq in Rat Hepatocytes (24 hrs, 10 nM)
siRNA-2siRNA-1
Downregulated genes Upregulated genes
Antisense seed
enrichment
Sense seed
enrichment
Antisense seed
enrichment
Sense seed
enrichment
siRNA-1 p < 2.2e-16 p = 0.05286 p = 0.667 0.2174
siRNA-2 p < 2.2e-16 p = 0.9679 p = 0.6345 p = 0.3364
65
199
160
190
p < 0.05
On-target mRNA
On-target mRNA
37
ESC GalNAc-siRNA Hepatotoxicity Summary
Antisense strand-driven RNAi-mediated hybridization-based off-target
effects, not chemical modifications or competition for RISC loading with
miRNAs, appear to be important drivers of rat hepatotoxicity.
3’-UTR
Off-target bindingPartial sequence match
Translation repression,
mRNA destabilization
3’-UTR
Undesired off-target activity
38
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
39
ESC+ Strategy for Improved Specificity and Therapeutic Index
Utilize seed-pairing destabilization via novel chemical
modifications to selectively destabilize off-target binding
Off-target mRNA 3’-UTR
Off-target bindingPartial sequence match
Antisense loaded RISCpos. 2-8
Undesired off-
target activity
40
ESC+ Conjugates
Design Considerations
Important Considerations
1. On-target potency must be maintained in vivo
2. Minimal impact on metabolic stability
3. Off-target activity should be minimized
Position and nature of modification important for all three
Incorporate modification(s) to
destabilize seed-pairing
41
ESC+ vs. ESCComparable Potency and Reduced Off-Target Activity In Vitro
AD-64958 IC50: 0.0115 nM
Concentration in [nM]1x10
-50.001 0.1 10
%T
arg
et
rem
ain
ing
0
20
40
60
80
100
120
AD-72788 IC50: 0.0041 nM
Concentration in [nM]1x10
-50.001 0.1 10
%T
arg
et
rem
ain
ing
0
20
40
60
80
100
120
ESC
ESC+
Potency Off-Target Activity
On-target
On-target
42
ESC+ vs. ESC
Comparable Potency in Rats
ED50 ~ 0.050 mg/kg ED50 ~ 0.075 mg/kg
ESC ESC+
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
Seru
m T
TR r
elat
ive
to D
ay 0
Day post-dose
0.075 mg/kg
0.15 mg/kg
0.3 mg/kg
1 mg/kg
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
Seru
m T
TR r
elat
ive
to D
ay 0
Day post-dose
0.075 mg/kg
0.15 mg/kg
0.3 mg/kg
1 mg/kg
43
ESC+ vs. ESC
Hepatotoxicity Mitigated in Rats
Degeneration 4/4 (2.5)
SCN 4/4, (1.25)
Coag Nec 1/4 (1)
Increased mitoses 3/4 (2.25)
Bile duct hyperplasia 1/4 (1)
Vacuolation 4/4 (2)
Degeneration 0/4
SCN 1/4, (1)
Coag Nec 0/4
Increased mitoses 0/4
Bile duct hyperplasia 0/4
Vacuolation 3/4 (1)
ESC ESC+
Dose: 30 mg/kg
Regimen: qw x 3
Necropsy: Day 15
44
ESC+ vs. ESC
≥6-Fold Improvement in Therapeutic Index in Rats
ESC
0.0
75
0.1
5
0.3
0.6 1 3
10
30
60
12
0
0
2 5
5 0
7 5
1 0 0
0
1
2
3
4
5
A D -6 4 9 5 8
D o s e (m g /k g )
% K
no
ck
do
wn
To
x g
ra
de
N O A E L
N O E L
E D8 0 T I =
66
ESC+
0.0
75
0.1
5
0.3
0.6 1 3
10
30
60
12
0
0
2 5
5 0
7 5
1 0 0
0
1
2
3
4
5
A D -7 2 7 8 8
D o s e (m g /k g )
% K
no
ck
do
wn
To
x g
ra
de
N O A E L
N O E L
E D8 0 T I 4 0 0
45
Placebo
0.1 mg/kg
0.3 mg/kg
1.0 mg/kg
3.0 mg/kg
6.0 mg/kg
ALT AST
-10 0 10 20 30 40 50 60 70
Days Since Dose
-10 0 10 20 30 40 50 60 70
Days Since Dose
1X ULN
ALN-AAT Phase I Single Dose in Healthy VolunteersSerum Transaminases Elevations at 3 and 6 mg/kg SD
N
5
3
3
3
3
3
1
4
8
1
4
8
1
4
8
1
4
8
1
4
8
1
4
8
Data transfer: 30Jun2016
Haslett et al., OTS, September 2016
46
ALN-AAT02: ESC+ Version of ALN-AAT with Reduced
Off-Target Activity In Vitro
ALN-AAT ALN-AAT02
-94%
0
52AAT mRNA
0
3AAT mRNA
RNAseq from Hep3B cells transfected with 10 nM siRNA, 16 hrs treatment
47
ALN-AAT02 Retains Potent Activity in NHP (1 mg/kg)
Plan to advance ESC+ ALN-AAT02 into clinical development in 2018
0 2 0 4 0 6 0 8 0 1 0 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
1 .2
1 .4
D a y s P o s t-D o s e
AA
T (
Re
lati
ve
to
Pre
-do
se
)A L N -A A T
A L N -A A T 0 2
48
Summary
• Platform advances continue to drive potency of GalNAc-siRNA conjugates,
potentially allowing quarterly or even bi-annual dosing at low doses in
humans
• Demonstrated RNAi-mediated off-target effects as important driver of
hepatotoxicity for subset of ESC conjugates in rodent toxicity studies
◦ No evidence for impact of chemical modifications on observed toxicity
• Developed ESC+ design for mitigating seed-mediated off-target effects
◦ Improves specificity and further expands therapeutic index of conjugates
• Plan to advance first ESC+ conjugate, ALN-AAT02, into clinical
development in 2018
• All future candidates planned to employ ESC+ design
49
Agenda
Welcome
• Josh Brodsky, Associate Director, Investor Relations & Corporate Communications
Introduction
• Akshay Vaishnaw, M.D., Ph.D., Executive Vice President of R&D
Platform advances in RNAi therapeutics
• Advanced ESC Design for Potency Improvements
◦ Vasant Jadhav, Ph.D., Senior Director, Research
• Selection of Well Tolerated GalNAc-siRNAs by Screening in Rodent Toxicity Studies
◦ Maja Janas, Ph.D., Senior Scientist, Early Development
• ESC+ Design with Improved Specificity and Therapeutic Index
◦ Mark Schlegel, Ph.D., Senior Scientist, Research
Q&A Session
50
Upcoming RNAi Roundtables
Givosiran, in development for the treatment of acute hepatic porphyrias
• Thursday, September 7, 10:30 am ET
Fitusiran, in development for the treatment of hemophilia and rare
bleeding disorders
• Tuesday, September 12, 10:30 am ET
Additional details for upcoming RNAi Roundtables, including speakers, dates and times, will be
provided on the Capella section of the Company's website, www.alnylam.com/capella.
Thank you