advancing the safety and reach of rnai therapeutics
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CONFIDENTIAL1 © 2020 Alnylam Pharmaceuticals, Inc.
Maja Janas De Angelis, PhD, DABT
Associate Director, Early Development
OTS 2020
Advancing the Safety and Reach
of RNAi Therapeutics
CONFIDENTIAL2
Thank You Alnylam Team, Past and Present
Medicinal Chemistry
RNA Synthesis and Annealing
RNAi Lead Discovery
RNAi Biology and Bioinformatics
In Vivo Sciences
Pathology
Bioanalytical Sciences and DMPK
Martin Maier Vasant Jadhav Scott Barros Natalie Keirstead Jing-Tao Wu Pete Smith Kevin Fitzgerald
Mark Schlegel Ivan Zlatev
Yongfeng Jiang Saket Agarwal Lei Johnson Onur Yilmaz Sam Chigas Annabelle SangreeShreya Jadhav
CONFIDENTIAL3
Agenda
Optimizing the safety of liver-targeted GalNAc-siRNA conjugates
Translating liver learnings to CNS-targeted siRNA conjugates
CONFIDENTIAL4
Safety Profile of GalNAc-siRNAs to DateORION-10+11 Pooled Data: Safety and Tolerability of InclisiranNo Evidence of Liver, Kidney, Muscle or Platelet Toxicity
1 Safety population includes all patients who received at least 1 dose of study medication and individual patients may be counted in more than one category2 No cases met Hy’s Law
CONFIDENTIAL5
Characteristics of the Human LFT Signal with Subset of GalNAc Conjugates:
Only Observed with Few Compounds Suggesting Sequence SpecificityALN-AAT01 Phase 1/2 Interim Results
Me
an
[+
/-S
EM
] A
AT
Re
lative
to
Ba
se
line
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
Days since dose
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
Placebo (N=5) 0.1 mg/kg (N=3) 0.3 mg/kg (N=3)
1.0 mg/kg (N=3) 3.0 mg/kg (N=3) 6.0 mg/kg (N=3)
PD: Serum AAT Protein Levels Placebo
1.0 mg/kg
3.0 mg/kg
6.0 mg/kg
Safety: Serum ALT Levels
-10 0 10 20 30 40 50 60 70
Days Since Dose
1
4
8
1
4
8
1
4
8
1
4
8
• Anecdotal evidence for RISC-mediated mechanism:
◦ Onset of LFT elevations coincides with onset of maximum RNAi activity
◦ High target knockdown appears necessary (but not sufficient) for LFT elevations
• Similar profile seen in few other programs with sporadic LFT elevations
CONFIDENTIAL6
Safety Considerations for RNAi Therapeutics
(1) Non-natural chemical modifications,
e.g. 2’-fluoro, phosphorothioate
(3) Hybridization-based off-target effects,
e.g. microRNA-like seed-based activity
(2) Dysregulation of natural RNAi pathways,
e.g. microRNAs
CONFIDENTIAL7
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
0
5
1 0
1 5
2 0
L iv e r - P u r in e s
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -A R e v u s ira n
2 '-F -A T T R S C 0 2
2 '-F -G R e v u s ira n
2 '-F -G T T R S C 0 2
2 '-F -I R e v u s ira n
2 '-F -I T T R S C 0 2
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
0
5
1 0
1 5
2 0
L iv e r - P y r im id in e s
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -U R e v u s ira n
2 '-F -U T T R S C 0 2
2 '-F -C R e v u s ira n
2 '-F -C T T R S C 0 2
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
0
1
2
3
4
P la s m a - P u r in e s
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -A R e v u s ira n
2 '-F -A T T R S C 0 2
2 '-F -G R e v u s ira n
2 '-F -G T T R S C 0 2
2 '-F -I R e v u s ira n
2 '-F -I T T R S C 0 2
0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2
0
1
2
3
4
P la s m a - P y r im id in e s
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -U R e v u s ira n
2 '-F -U T T R S C 0 2
2 '-F -C R e v u s ira n
2 '-F -C T T R S C 0 2
Monomer Generation Is Minimized with the ESC Design In Vivo
Rat, 30 mg/kg Single Subcutaneous Dose of Revusiran or ALN-TTRSC02
• 2'-F-monomer half-life of 1-2 days indicates
that no accumulation is expected with weekly
or less frequent dosing
• Only 2'-F-pyrimidines appear to re-distribute
systemically in plasma
CONFIDENTIAL8
2'-F-Monomers Are Not Detectable in Human Plasma or Urine at
Therapeutically-Relevant Doses of ESC-Conjugate
Revusiran: 7.5 mg/kg qd x 5 to healthy volunteers
ALN-TTRSC02: 50 mg (~0.83 mg/kg) single dose to healthy volunteers
• 2'-F-monomers reached steady state in plasma and
urine by Day 4, indicating a half-life of ~ 1 day and
therefore no accumulation with weekly or less frequent
dosing
0 4 8 1 2 1 6 2 0 2 4
0
2
4
6
P la s m a
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -U R e v u s ira n
2 '-F -I R e v u s ira n
2 '-F -U T T R S C 0 2
2 '-F -I T T R S C 0 2
0 -6 6 -1 2 1 2 -2 4
0
2
4
6
8
U r in e
T im e p o s t-d o s e (h rs )
mg
2 '-F -U R e v u s ira n
2 '-F -I R e v u s ira n
2 '-F -G R e v u s ira n
T T R S C 02
• Up to ~15 % of total monomer dose is excreted
in urine, and therefore even revusiran is likely
mainly excreted in oligomeric forms
0 4 8 1 2 1 6 2 0 2 4
0
2
4
6
P la s m a
T im e p o s t-d o s e (h rs )
Co
nc
en
tra
tio
n (
M)
2 '-F -U R e v u s ira n
2 '-F -I R e v u s ira n
2 '-F -U T T R S C 0 2
2 '-F -I T T R S C 0 2
0 -6 6 -1 2 1 2 -2 4
0
2
4
6
8
U r in e
T im e p o s t-d o s e (h rs )
mg
2 '-F -U R e v u s ira n
2 '-F -I R e v u s ira n
2 '-F -G R e v u s ira n
T T R S C 02
CONFIDENTIAL9 Janas MM, Zlatev, I et al. Nucleic Acids Res. 2019 Apr 23;47(7):3306-3320
2'-F-Monomer De-Risking
Monomer generation from STC siRNA in rat and human Low
Monomer generation from ESC siRNA in rat and human Mostly undetectable
Polymerase inhibition (Pol-γ, Pol-α, Pol-β, POLRMT) No
Polymerase substrate (Pol-γ, POLRMT) Poor
Obligate chain termination No
Non-obligate chain termination No
Cytotoxicity and mtDNA effects in vitroIn a subset of cell lines at concentrations > 16-fold higher
than generated in vivo from STC siRNA
2-year rat carcinogenicity study with STC siRNAsNo effects on survival or tumor incidence;
no apparent effects on mitochondrial function
CONFIDENTIAL10 Janas MM et al. Nucleic Acid Ther. 2017 Feb;27(1):11-22
Phosphorothioate Modification Is Well-Tolerated in
Double-Stranded Oligonucleotides In Vitro
DNA double stranded break assessment after transfection into HeLa cells:
Mock
CONFIDENTIAL11
*Nat Struct Mol Biol. 2013 Jul;20(7):789-95; Mol Biol Cell. 2010 May 1;21(9):1462-9; RNA. 2013 May;19(5):605-12
Potential Reasons:
• Relatively low % of cellular RISC pool occupied by siRNA
• Stabilization of additional RISC complexes in the presence of siRNA*
No Evidence of a Class Effect of siRNAs on microRNA Activity
- 5 10 15 30 45 60 90 120 - 5 10 15 30 45 60 90 120
100 nM PPIB 1 siRNA Transfection Mock Transfection
let-7 activity (HeLa cells)
Let-7 cleavage
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 5 10 15 30 45 60 90 120
Time (minutes)
% C
leavag
e
100 nM siRNA transfection
Mock Transfection
B A
D-6
49
58
G A
D-5
86
43
B A
D-5
77
27
B A
D-5
86
42
G A
D-6
81
11
B A
D-5
86
41
G A
D-7
23
92
G A
D-6
39
68
E+
AD
-72
78
8
G A
D-6
72
48
B A
D-6
42
72
G A
D-5
86
81
B A
D-6
68
08
G A
D-6
72
46
B A
D-6
54
21
G A
D-6
08
35
Ctr
l
G A
D-6
56
95
G A
D-6
68
10
G A
D-6
69
20
B A
D-6
68
16
G A
D-6
72
44
E+
AD
-75
99
5
G A
D-6
54
03
G A
D-6
87
26
B A
D-6
09
40
Ctr
l R
vr
G A
D-6
76
37
G A
D-6
73
27
B A
D-6
69
21
B A
D-6
66
78
B A
D-6
45
46
B A
D-6
69
22
G A
D-6
68
83
G A
D-6
14
44
Rv
r
G A
D-6
73
35
B A
D-6
66
86
B A
D-6
68
14
G A
D-6
40
08
G A
D-6
87
30
B A
D-6
45
43
B A
D-6
56
44
-8 0
-6 0
-4 0
-2 0
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
2 2 0
m iR -2 2 -3 p - S e e d A c tiv ity
% t
arg
et
re
ma
inin
g
Increased
No impact
Decreased
Endogenous microRNA activity assessment after siRNA transfection:
miR-22 activity (COS cells)
siRNA ID
CONFIDENTIAL12 Janas MM, Schlegel, MK et al. Nat Commun. 2018 Feb 19;9(1):723
Seed-Based Off-Target Effects Are Important Drivers of
Rodent Hepatotoxicity for Subset of GalNAc-siRNAs
3’-UTR
miRNA-like seed mediated
binding to off-targets
Translation repression,
mRNA destabilization
Min/Marked hepatocellular degen, Mod
SCN, Min coagulative necrosis, Mod
↑mitoses, Min BDH, Mod vacuolation
Rat NOAEL: 10 mg/kgSeed enriched off-
target effects observed
CONFIDENTIAL13
ESC+ Seed Pairing Destabilization Strategy to Improve
Specificity and Therapeutic Index
Bramsen et. al. Nucleic Acids Res. 2010
Vaish et. al. Nucleic Acids Res. 2011
Lee et. al. Nat. Comm. 2015
Janas, Schlegel et al. Nat. Comm. 2018
Utilize seed-pairing
destabilization via novel
chemical modifications
to selectively destabilize
off-target binding
Off-target bindingPartial sequence match
Off-target mRNA 3’-UTR
Antisense loaded RISCpos. 2-8
Undesired off-
target activity Important Considerations
1. On-target potency must be maintained in vivo
2. Off-target activity should be minimized
(S)-GNA
CONFIDENTIAL14
ESC+ Demonstrates More Quiescent Off-Target Signature
Across Dose Levels in Rat Liver with Comparable On-Target KD
ES
CE
SC
+
3 mg/kg 10 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg
DEGs (Differentially Expressed Genes), significant, 3’UTR match, significant, 3’UTR match, not significant
Dose: qw x 3
Necropsy: Day 16
Histopath
for 30 mg/kg dose
Parent ESC
AS7-GNA
= target mRNA
CONFIDENTIAL15
ALN- AAT01 ALN-AAT02
Structure*
Efficacy Up to 89% KD Up to 89% KD
Liver Safety
(ALT >3x ULN)1/15 (up to 6 mg/kg dose) 0/18 (up to 6 mg/kg dose)
Positive ESC+ Human POCALN-AAT02 Clinical Activity and Safety
* Images are representative
GalNAcGalNAc
GalNAc
GalNAcGalNAc
GalNAc
0 2 4 6 8 1 0 1 2
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
S t u d y W e e k
Se
ru
m A
AT
(g
/L)
0 . 3 m g / k g
1 . 0 m g / k g
3 . 0 m g / k g
P L A C E B O
L L O Q
S i n g l e D o s e A L N - A A T 0 2
0 2 4 6 8 1 0 1 2
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
S t u d y W e e k
Se
ru
m A
AT
(g
/L)
0 . 3 m g / k g
1 . 0 m g / k g
3 . 0 m g / k g
P L A C E B O
L L O Q
S i n g l e D o s e A L N - A A T 0 2
0 2 4 6 8 1 0 1 2
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
S t u d y W e e k
Se
ru
m A
AT
(g
/L)
0 . 3 m g / k g
1 . 0 m g / k g
3 . 0 m g / k g
L L O Q
P L A C E B O
S i n g l e D o s e A L N - A A T 0 1
CONFIDENTIAL16
GalNAc-siRNA Safety Summary
• Antisense strand-driven RNAi-mediated off-target effects are important drivers of hepatotoxicity
◦ No evidence for impact of chemical modifications on observed toxicity
◦ No evidence for microRNA competition
• ESC+ strategy mitigates seed-mediated off-target effects, improves specificity and further expands therapeutic window of siRNA conjugates
◦ Utilizes thermally destabilizing nucleotide, such as glycol nucleic acid (GNA), in seed region of antisense strand
◦ Multiple ESC+ conjugates have advanced into clinical development
◦ Positive human POC achieved
◦ Some sequences show an inherent ESC+ profile and may not require further chemical modification
Hybridization-based off-target effects, e.g.
microRNA-like seed-based activity
CONFIDENTIAL17
Agenda
Optimizing the safety of liver-targeted GalNAc-siRNA conjugates
Translating liver learnings to CNS-targeted siRNA conjugates
CONFIDENTIAL18
Knockdown Observed in Deep Brain Regions after a Single IT Injection
siRNA Conjugate Is Active Across CNS Regions
NHPTemporal
Cortex
Prefrontal
Cortex
Striatum
Lu
mb
ar
Sp
ine
Cerv
ical S
pin
e
Pre
fro
nta
l C
or t
ex
Tem
po
ral C
or t
ex
Str
iatu
m
0
2 0
4 0
6 0
8 0
1 0 0
T o o lk it A P P s iR N A
7 2 m g , D a y 2 9
AP
P m
RN
A R
em
ain
ing
(%
)
Lu
mb
ar
Sp
ine
Cerv
ical S
pin
e
Fro
nta
l C
or t
ex
Tem
po
ral C
or t
ex
Str
iatu
m
0
2 0
4 0
6 0
8 0
1 0 0
T o o lk it S O D 1 s iR N A
0 .9 m g , D a y 7 -8
SO
D1
mR
NA
Re
ma
inin
g (
%)
Rat
CONFIDENTIAL19
Lowest Activity in the Endothelium
siRNA Conjugate Is Active in Most CNS Cell Types in Rat
Neuron Astrocyte MicrogliaEndothelium
Green: MAP2
Purple: Duplex
Yellow: GFAP
Purple: Duplex
Yellow: IBA1
Purple: DuplexYellow: CD31
Purple: Duplex
OligodendrocyteYellow: MBP
Purple: Duplex
siR
NA
ac
cu
mu
lati
on
siR
NA
ac
tivit
y
GFAP siRNA IBA1 siRNAPECAM1 siRNA MBP siRNA
Lu
mb
ar
Sp
ine
Cerv
ical S
pin
e
Fro
nta
l C
or t
ex
Tem
po
ral C
or t
ex
Str
iatu
m
0
3 0
6 0
9 0
1 2 0
1 5 0
D a y 3 2
GF
AP
mR
NA
Re
ma
inin
g (
%)
Lu
mb
ar
Sp
ine
Th
ora
cic
Sp
ine
Cerv
ical S
pin
e
Cere
bellu
m
Rem
ain
ing
Hem
isp
here
0
3 0
6 0
9 0
1 2 0
1 5 0
D a y 1 4
PE
CA
M1
mR
NA
Re
ma
inin
g (
%)
Lu
mb
ar
Sp
ine
Cerv
ical S
pin
e
Hip
po
cam
pu
s
Tem
po
ral C
or t
ex
Bra
inste
m
0
3 0
6 0
9 0
1 2 0
1 5 0
D a y 3 2
IBA
1 m
RN
A R
em
ain
ing
(%
)
Lu
mb
ar
Sp
ine
Cerv
ical S
pin
e
Bra
inste
m
Cere
bellu
m
Rig
ht
Hem
isp
here
0
3 0
6 0
9 0
1 2 0
1 5 0
D a y 1 4
MB
P m
RN
A R
em
ain
ing
(%
)
MAP2 siRNA
Lu
m
ba
r S
pi n
e
Ce
rv
i ca
l S
pi n
e
Fro
nta
l C
orte
x
Te
m
po
ra
l C
orte
x
Stri a
tu
m
0
3 0
6 0
9 0
1 2 0
1 5 0
D a y 2 8
MA
P2
m
RN
A
Re
ma
in
in
g
(%
)
CONFIDENTIAL20
Single IT Injection, 60 mg
siRNA Conjugate Is Durable for > 6 Months in NHP
0
2 0
4 0
6 0
8 0
1 0 0
A D -4 5 4 8 4 4
T im e p o in t , (d a y s )
AP
P R
em
ain
ing
fro
m B
as
eli
ne
(%
)
1 0 0 1 -s A P P
1 0 0 2 -s A P P
1 0 0 1 -s A P P
1 0 0 2 -s A P P
1 7 13 29 55 93 120 153 184
Toolkit APP siRNA
CONFIDENTIAL21
• siRNA conjugates are active across CNS regions, including deep brain structures
• siRNA conjugates are active in most CNS cell types, including neurons, microglia, astrocytes, and
oligodendrocytes
• siRNA conjugates have long half-life in the CNS, resulting in durable activity (> 6 months in
monkey)
• No test article-related safety findings to date in rodent or monkey nonclinical studies
CNS Summary
CONFIDENTIAL22
Our Innovative Work Continues…with Physical Distancing
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