drug metabolism and pharmacokinetic (dmpk) properties of ...2 presentation topics • rnai...
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1
Drug Metabolism and Pharmacokinetic
(DMPK) Properties of siRNA-GalNAc
Conjugates
Anshul Gupta Lead Scientist
DSM Alnylam Pharmaceuticals Cambridge MA
DIAFDA Oligonucleotide Based Therapeutic Conference September 09 -11 2015
2
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
3
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
4
RNA Interference (RNAi)
Mechanism of Action
mRNA
mRNA degradation
dsRNA dicer
Cleavage
Complementary pairing
Cleavage
Natural Process of RNAi
Synthetic siRNA
Targeted Gene
Silencing
RISC
Confidential
Strand separation
5
siRNA-GalNAc Conjugates
Asialoglycoprotein Receptor
(ASGPR)
Binds to GalNAc Ligand
bull Highly expressed in hepatocytes
(05-1 million copies per cell)
bull Low to no expression in other
tissues
bull High rate of uptake
bull Recycling time ~15 minutes
bull Conserved across species
Subcutaneous Delivery of RNAi Therapeutics
Nucleus
ASGPR
(pHgt5)
GalNAc-siRNA
conjugate
Clathrin-coated pit
Clathrin-coated
vesicle
Endosome
Recycling
ASGPR
mRNA
protein
RISC
GalNAc3
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
2
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
3
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
4
RNA Interference (RNAi)
Mechanism of Action
mRNA
mRNA degradation
dsRNA dicer
Cleavage
Complementary pairing
Cleavage
Natural Process of RNAi
Synthetic siRNA
Targeted Gene
Silencing
RISC
Confidential
Strand separation
5
siRNA-GalNAc Conjugates
Asialoglycoprotein Receptor
(ASGPR)
Binds to GalNAc Ligand
bull Highly expressed in hepatocytes
(05-1 million copies per cell)
bull Low to no expression in other
tissues
bull High rate of uptake
bull Recycling time ~15 minutes
bull Conserved across species
Subcutaneous Delivery of RNAi Therapeutics
Nucleus
ASGPR
(pHgt5)
GalNAc-siRNA
conjugate
Clathrin-coated pit
Clathrin-coated
vesicle
Endosome
Recycling
ASGPR
mRNA
protein
RISC
GalNAc3
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
3
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
4
RNA Interference (RNAi)
Mechanism of Action
mRNA
mRNA degradation
dsRNA dicer
Cleavage
Complementary pairing
Cleavage
Natural Process of RNAi
Synthetic siRNA
Targeted Gene
Silencing
RISC
Confidential
Strand separation
5
siRNA-GalNAc Conjugates
Asialoglycoprotein Receptor
(ASGPR)
Binds to GalNAc Ligand
bull Highly expressed in hepatocytes
(05-1 million copies per cell)
bull Low to no expression in other
tissues
bull High rate of uptake
bull Recycling time ~15 minutes
bull Conserved across species
Subcutaneous Delivery of RNAi Therapeutics
Nucleus
ASGPR
(pHgt5)
GalNAc-siRNA
conjugate
Clathrin-coated pit
Clathrin-coated
vesicle
Endosome
Recycling
ASGPR
mRNA
protein
RISC
GalNAc3
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
4
RNA Interference (RNAi)
Mechanism of Action
mRNA
mRNA degradation
dsRNA dicer
Cleavage
Complementary pairing
Cleavage
Natural Process of RNAi
Synthetic siRNA
Targeted Gene
Silencing
RISC
Confidential
Strand separation
5
siRNA-GalNAc Conjugates
Asialoglycoprotein Receptor
(ASGPR)
Binds to GalNAc Ligand
bull Highly expressed in hepatocytes
(05-1 million copies per cell)
bull Low to no expression in other
tissues
bull High rate of uptake
bull Recycling time ~15 minutes
bull Conserved across species
Subcutaneous Delivery of RNAi Therapeutics
Nucleus
ASGPR
(pHgt5)
GalNAc-siRNA
conjugate
Clathrin-coated pit
Clathrin-coated
vesicle
Endosome
Recycling
ASGPR
mRNA
protein
RISC
GalNAc3
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
5
siRNA-GalNAc Conjugates
Asialoglycoprotein Receptor
(ASGPR)
Binds to GalNAc Ligand
bull Highly expressed in hepatocytes
(05-1 million copies per cell)
bull Low to no expression in other
tissues
bull High rate of uptake
bull Recycling time ~15 minutes
bull Conserved across species
Subcutaneous Delivery of RNAi Therapeutics
Nucleus
ASGPR
(pHgt5)
GalNAc-siRNA
conjugate
Clathrin-coated pit
Clathrin-coated
vesicle
Endosome
Recycling
ASGPR
mRNA
protein
RISC
GalNAc3
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
6
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
7
Presentation Topics
bull RNAi Therapeutics
Mechanism of RNAi
Introduction to siRNA-GalNAc conjugates
bull Pharmacokinetic Behavior and ADME Properties of siRNA-GalNAc
Conjugates
Absorption and PK profile- plasma and liver kinetics
Distribution in tissues and Elimination
Metabolism Improved stability with ESC vs STC
Exposure vs efficacy relationship
Clinical translation
Enhanced potency of second generation conjugates in the clinic
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
8
SC Dosing Enhances Targeted Liver Exposure
Route-dependent PK Profile differences for typical ALNY molecule
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
L i v e r
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
0 1 2 3 4 5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
P l a s m a
T i m e ( h )
co
nc
en
tr
at
io
n (
ng
m
l)
I n t r a v e n o u s
S u b c u t a n e o u s
Plasma Parameter (IV) (SC)
Tmax (h) 0083 1
Cmax (microgml) 586 03
AUC0-t (hmicrogml) 201 067
t12β (h) 027 1
Liver Parameter (IV) (SC)
Tmax (h) 1 4
Cmax (microgg) 185 244
AUC0-t (hmicrogg) 622 1246
t12β (h) 548 597
Comparison between SC and IV dosing- Rat 3 mgKg
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
9
Liver Uptake Efficiency of siRNA-GalNAc
Conjugates Decreases with Increasing Dose
bull Lower dose provides higher uptake in liver ndash indicative of ASGPR saturation
prior to receptor turnover at higher doses
Theo van Berkel
Collaboration
1 2 5I - T y r - G a l N A c
3- s i R N A
0 1 0 2 0 3 0
0
2 5
5 0
7 5
1 0 0
l i v e r + 1 0 m g k g
l i v e r c o n t r o l
l i v e r + 1 0 0 m g k g
l i v e r u p t a k e
l i v e r + 1 m g k g ( 3 0 )
l i v e r + 5 m g k g
l i v e r + 1 m g k g ( 1 0 )
l i v e r c o n t r o l + N A c G a l
A D 2 6 1 1 5
t i m e ( m i n )
of
in
jec
te
d
do
se
A
dm
inis
tere
d D
ose
05 mgkg
15mgkg
100mgkg
10mgkg
5mgkg
Liver Uptake 125I labeled 3prime GalNAc3
Time
(hr)
Dose Group
25
mgkg
50
mgkg
75
mgkg
100
mgkg
8 198 206 124 128
Percent administered dose measured in
NHP liver after single SC dose of ALN-TTRsc
Mouse (IV dose)
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
10
Loss of Dose-Proportionality in Drug Liver
Concentration at ge 30mgkg siRNA-GalNAc
Representative Pharmacological Doses 01 ndash 30mgkg
bull Toxicity Doses 30 - 300 mgkg- QW x 5
bull Dose multiples 30x or greater Liver tissue exposure multiples up to 100X
2 5
5 0
30
0
10
0 0
30
0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
g
g)
0 1 0 0 2 0 0 3 0 0 4 0 0
1 0 0
1 0 0 0
D o s e ( m g k g )
Li
ve
r (
g
g)
Rat Liver concentrations after multiple SC doses
Group dosed at 5 mgKg is single dose
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
11
1 51
03
0
10
0
30
0
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
D o s e ( m g k g )
Co
nc
en
tr
at
io
n (
gg
)
at
t
=2
4h
L i v e r
K i d n e y
Rat Liver to Kidney Ratio of siRNA-GalNAc
Decreases with Increasing Doses
Kidney concentrations approach liver concentrations at higher doses
reflecting shift to increased renal clearance vs liver uptake
Doses 1 5 10 30 100 300
Liver 0051 014 009 036 06 16
Kidney 0002 001 002 007 04 11
Ratio 25 14 45 5 15 15
Doses 1 5 10 30 100 300
Liver 23 86 88 370 771 211
Kidney 028 11 28 111 590 248
Ratio 82 78 31 33 13 085
Cmax (mgg) AUC0-t (hmgg)
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
12
Tissue Distribution of siRNA-GalNAc
Conjugates Near Pharmacological Doses
Exposure is the highest in the target organ (liver) followed by kidney
Rest of the tissues examined typically have cumulative levels lt1-5 of
delivered dose deposited
Typically lt1-5
Delivered dose
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
13
Metabolism
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
14
Nuclease-Mediated Metabolism
3rsquo amp 5rsquo-Exonuclease
bull End products are mononucleotides
Endonuclease
bull Cleaves internally
Localization
bull Exo- and endonucleases are ubiquitously distributed in both plasma and tissues
Metabolic hot spots on siRNA-GalNAc
Manoharan TIDES May 2014
(GalNAc)3
S 5prime
Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5rsquo 3rsquo
5rsquo Exo- 3rsquo Exo-
Endo-
Metabolite
profiling in
liver 8h post
dose
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
15
8 hr - Peak Intensity 24 hr ndash Peak Intensity
Full Length Sense
Full Length AS
Sense Strand (5prime-3prime)
Antisense Strand (5prime-3prime)
Metabolite 1
Metabolite 35
Metabolite 1
Metabolite 29
Metabolic Profile Time Course In vivo NHP liver (10mgkg SC)
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
16
ESC Leads to Higher Liver Exposure
Manoharan TIDES May 2014
Liver Exposure and Metabolic Stability Single SC Dose 25 mgkg in Mice
Metabolic profiling in liver 8h post dose
(GalNAc)3
S 5prime
= Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed)
Standard Template Chemistry (STC)
AS 5prime
5prime AS
S 5prime
Enhanced Stabilization Chemistry (ESC)
Liver Exposure
Target Compound Tmax
(h)
Cmax
(microgg)
AUC0-t
(hmiddotmicrogg)
AUC0-48
(hmiddotmicrogg)
AT3 STC 2 595 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200
Liv
er
Co
ncen
trati
on
(n
gg
)
Time (h)
SC
ESC
Liver Exposure
STC
Liver t frac12
1-2 days for STC
1-2 weeks for ESC
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
17
Enhanced Stabilization Chemistry (ESC)
Improves Efficacy
bull ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
mTTRsc-STC mTTRsc-ESC
0
20
40
60
80
100
120
PBS 25 mgkg 5 mgkg 1 mgkg 5 mgkg 1 mgkg 02 mgkg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
of
PB
S C
on
tro
l
(mT
TR
mG
ap
dh
)
Subcutaneous Single Dose
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
18
Exposure versus Efficacy
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
19
Rat PKPD Liver Concentration vs PD
Single amp Multiple SC Dose
bull 10 mgkg SD and 5 mgkg MD schedule achieved similar maximum KD (~ 92)
bull Minimum threshold liver concentrations to maintain maximum KD- ~ 4 microgg
achieved at a dose of 5 mgkg no gain in PD at doses gt 5 mgkg
Single Dose (SD)- 10 mgkg Multi Dose (MD)- 5mgkg (2xW 6 doses)
0 2 0 4 0 6 0 8 0
0
1 0
2 0
3 0
4 0
0
5 0
1 0 0
T i m e ( D a y s )
Co
nc
en
tr
at
io
n (
gg
)
ta
rg
et
m
RN
A s
ile
nc
in
g
L i v e r c o n c e n t r a t i o n
t a r g e t m R N A s i l e n c i n g
Sparse sampling scheme (concentration measured at trough only following
1st through 5th dose full concentration-time profile post last dose)
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
20
Low Proportion of Effective Drug
Concentration in Liver Driving Efficacy
bull Initial data suggests Total tissue siRNA levels are generally ~100 - 1000 fold higher than RISC-loaded siRNA
bull Limited gain in efficacy with increased dose or dosing frequency
0 1 0 0 2 0 0 3 0 0 4 0 0
1
1 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
T i m e ( h )
Co
nc
en
tr
at
io
n (
ng
g
)
R I S C l o a d e d
A g o 2 a s s o c i a t e d
T o t a l l i v e r
Grinding Tissue
Liver Lysate
In Lysis IP buffer
IP Incubation with
Ago2 Ab + Beads
Detection
GalNAc-siRNA
Application Measured by quantifying Ago2 associated protein
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
21
Pharmacodynamic Effect in Non-Human Primate
100
80
60
40
20
0
-20
-10 0 10 20 30 40
Days
PBS 125 mgkg 25 mgkg 50 mgkg
Sil
en
cin
g A
LA
S-1
mR
NA
ALAS-1 GalNAc-siRNAqd x5 q2d x12
ALN-AS1 ALN-CC5
000
020
040
060
080
100
120
-5 45 95 145
Rela
tive
AA
T l
eve
ls
(pre
do
se
=1
)
Days
1mgkg q1w x12
1mgkg q4w X3
3mgkg q4w x3
3mgkg x1 1mgkg q4w x2
ALN-AAT ALN-AT3
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
22
Clinical Translation NHP to Man
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
23
Revusiran Phase 1 Study Results
bull Rapid (up to 94) dose-dependent consistent and durable knockdown of serum TTR
bull Generally well tolerated
bull Excellent correlation of human to non-human primate TTR knockdown
Duration of effect longer in human vs NHP
Zimmermann HFSA Sep 2013 Manoharan TIDES May 2014
Human POC for GalNAc-siRNA Conjugates
100
80
60
40
20
0
-20
Days Revusiran (mgkg) qd x5 qw x5
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
plusmn S
EM
)
25 (n=3) 50 (n=3)
100 (n=3)
Placebo (n=3)
Revusiran
dose groups
Human
Days
M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e
100
80
60
40
20
0
0 10 20 30 40 50 60
Human
NHP
Revusiran Single 100 mgkg
Injection
Human vs NHP
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
24
ALN-AT3 Phase 1 Study (MAD)
Potent dose-dependent and durable AT knockdown at low microgramkilogram (mcgkg) SC doses
bull Mean maximum AT knockdown of 59 plusmn 7 (plt005) durable lasting ~60 days
bull ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3) p lt 005 relative to baseline
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
25
ALN-AT3 Phase 1 Study (MAD)
bull Good correlation between NHP and human knockdown
bull Potency in humans significantly enhanced over NHP 5-10x improved
potency in humans with same slope of KD
Data as of 2 June 2015
Pharmacodynamics and Clinical Activity AT Knockdown
A
T K
no
ck
do
wn
100
80
60
40
20
0
-20
Time (Days)
0 10 20 30 40 50 60 70
15 mcgkg (N=3)
45 mcgkg (N=6)
75 mcgkg (N=3)
Human Human vs NHP
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
26
ALN-PCSsc Phase 1 Study Results (SAD)
bull Potent dose-dependent and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 plusmn 2 (plt005) durable lasting ~4 months (120 days)
bull ALN-PCSsc generally well tolerated
bull Enhanced potency observed in man compared to NHP rapid onset of effect and longer duration
Data in database as of 04 August 2015
Highly durable PCSK9 knockdown in both human and NHP
DayTreatment combinations where N=1 not displayed
Months
100
80
60
40
20
0
-20
-40
0 1 2 3 4 5
Me
an
(S
EM
)
PC
SK
9 K
no
ck
do
wn
Placebo
25 mg
100 mg
300 mg
500 mg 800 mg
Treatment
Days
0 20 40 60 80 100
10 30 60 100
P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
) 100
80
60
40
20
0
-20
ALN-PCSsc (mgkg)
NHP SAD Human SAD
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
27
Summary
bull siRNA-GalNAc molecules can successfully be delivered predominantly to the liver at
high exposure levels by virtue of GalNAc binding to ASGPR
bull Pharmacokinetic properties of siRNA-GalNAc molecules developed so far appear
similar across programs including
Short plasma half-life (plasma Tmax ~1 hour upon SC dosing) but long liver half-life high liver
partitioning and exposure corresponding to durable PD effects
At toxicological doses exposure in liver and kidney become disproportional potentially due to
reduced efficiency of receptor mediated uptake Maximum PD effects are achieved at doses lt
5mgkg
bull Several siRNA-GalNAc conjugates administered to humans ( eg ALN-TTRsc ALN-
AT3 ALN-CC5 ALN-PCS) have been shown to achieve a rapid dose-dependent and
durable knockdown of target protein
bull Data achieved so far confirm human translation of the siRNA-GalNAc conjugate
platform and demonstrates PoC for Alnylamrsquos approach of developing siRNA-GalNAc
as RNAi therapeutics
- Enhanced potency observed in the clinic for second generation molecules
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
28
Acknowledgements
Program Advisors
bull Akshay Vaishnaw
bull Rachel Meyers
bull Muthiah Manoharan
bull Sara Nochur
Program Teams
bull Tracy Zimmermann
bull Alfica Sehgal
bull Kevin Fitzgerald
bull Akin Akinc
bull Benny Sorensen
bull Vasant Jadhav
Chemistry
Klaus Charisse
Martin Maier
Rubina Parmar
Drug Safety amp Metabolism
bull Michael Placke
bull Renta Hutabarat
bull Yuanxin Xu
bull Prasoon Chaturvedi
bull Natalie Keirstead
bull Luke Utley
bull Mustafa Varoglu
bull Husain Attarwala
bull Ju Liu
bull Minggeng Gao
bull Valerie Clausen
bull Qianfan Wang
bull Xuemei Zhang
bull Krishna Aluri
bull Sean Dennin
29
Thank You
29
Thank You