TIDES 2014 GalNAc-siRNA with Enhanced Stabilization ... · PDF fileMay 14, 2014 Muthiah...
Transcript of TIDES 2014 GalNAc-siRNA with Enhanced Stabilization ... · PDF fileMay 14, 2014 Muthiah...
May 14, 2014
Muthiah Manoharan
TIDES 2014
GalNAc-siRNA
with Enhanced Stabilization Chemistry:
ESC-GalNAc-siRNA
Agenda
siRNA-GalNAc Conjugates: Background
Standard Template Chemistry (STC)
» Human POC of ALN-TTRsc
Enhanced Stabilization Chemistry (ESC)
» mTTRsc
» ALN-AT3
» ALN-PCSsc
Summary
2
GalNAc-siRNA Conjugates for Systemic RNAi Receptor and the Ligand
ASGPR
(pH>5)
GalNAc-
siRNA
conjugate
Clathrin-coated pit
Clathrin-
coated vesicle
Endosome
Recycling
ASGPR
mRNA
Nucleus
protein
RISC
Asialoglycoprotein Receptor (ASGPR) Highly expressed in hepatocytes » 0.5-1 million copies/cell
Clears serum glycoproteins via
clathrin-mediated endocytosis
High rate of uptake
Recycling time ~15 minutes
Conserved across species
Valency Gal GalNAc
Mono- ~4 mM ~50 μM
Bi- ~3 μM ~24 nM
Tri- ~100 nM ~3 nM
Valency and Affinity
3
Making Drugs Out of siRNA Conjugates Role of Chemical Modifications
4
Ligand
GalNAc mediates binding and internalization of conjugate by hepatocytes
siRNA Chemistry
Stabilize conjugate in subcutaneous space, lymphatics, plasma, and
hepatocytes
Increase specificity
Reduce immune stimulation
Enhance cellular delivery
Improve RISC activity
siRNA
= 2′OH = 2′-O-methyl = PS
Ligand
5′-sense
= 2′-F (GalNAc)3
Schröder et. al., The Proteome of the Lysosome, Proteomics.10:4053-4076 (2010)
(adapted from Mark Cancilla, Merck)
siRNA Conjugates Encounter Various Metabolic
Enzymes En Route to Site of Action
5
SC
Injection site Capillary Uptake
Lymphatics
Central
Compartment
(systemic
circulation)
D/M
Kidney
D/M
Liver
Effect Site
Biliary excretion
Endosome
RISC
Lysosome1
Hepatocyte
Nucleases (5′ exo, 3′ exo, endo) Peptidases
Excretion
Phosphatases
Nucleases
Peptidases
N-acetylgalactosidase
many others
P-bodies and associated
exonucleases
Kinases - Clp1
Phosphatases
Cytosol
GalNAc-siRNA
conjugate
GalNAc-siRNA Conjugates for Systemic RNAi Molecular Targets Discussed Today
ALN-TTRsc, ALN-AT3, and ALN-PCSsc
6
Potent, rapid, dose-dependent, and durable target
knockdown with SC administration
Hepatocyte target genes
» ALN-TTRsc targeting TTR for treatment of transthyretin-
mediated amyloidosis
» ALN-AT3 targeting AT to restore thrombin generation in
hemophilia and rare bleeding disorders
» ALN-PCSsc targeting PCSK9 to lower cholesterol for
treatment of hypercholesterolemia
ED80 levels for target knockdown from 0.5 – 5 mg/kg
» Generally achieved with qw dosing regimen
More potent conjugates reflect advances in Enhanced
Stabilization Chemistry (ESC)
Used with ALN-AT3 and ALN-PCSsc
Significantly improved potency and durability
Maintains wide therapeutic index and safety
Agenda
siRNA-GalNAc Conjugates: Background
Standard Template Chemistry (STC)
» Human POC of ALN-TTRsc
Enhanced Stabilization Chemistry (ESC)
» mTTRsc
» ALN-AT3
» ALN-PCSsc
Summary
7
ALN-TTRsc Phase 1 Study Results Human POC for GalNAc-siRNA Conjugates
Randomized, double-blind, placebo-controlled SAD and MAD study in healthy volunteers Rapid, dose-dependent, consistent, and durable knockdown of serum TTR
» Significant knockdown of serum TTR (p<0.01) up to 94% TTR knockdown; mean knockdown up to 92.4%
Generally well tolerated » Only AEs associated with drug were generally mild ISRs resolved within ~2 hours of onset
Duration of effect longer in human vs. NHP
Zimmermann, Heart Failure Society of America, Sept. 2013
100
80
60
40
20
0
-20
Days ALN-TTRsc (mg/kg), qd x5; qw x5
% M
ean
TT
R K
no
ckd
ow
n
Rela
tive t
o B
aselin
e (
± S
EM
)
2.5 (n=3)
5.0 (n=3)
10.0 (n=3)
Placebo (n=3)
ALN-TTRsc dose groups
% 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
Days
Human
NHP
ALN-TTRsc Single 10.0 mg/kg
Injection
8
Conjugates More stable in Human than NHP In vitro Metabolic Profile at 24 hr
NHP – 24 hour (5µM)
Peak Intensity
Human – 24 hour (5µM)
Peak intensity
9
Metabolite -1
Metabolite -2
Metabolite -3
Metabolite -4
Metabolite -5
Metabolite -6
Metabolite -7
Metabolite -8
Metabolite -9
Metabolite -10
Metabolite -11
Metabolite -12
Metabolite -1
Metabolite -2
Metabolite -3
Metabolite -4
Metabolite -5
Metabolite -6
Metabolite -7
Metabolite -8
Metabolite -9
Metabolite -10
Metabolite -11
Metabolite -12
Full Length AS
Full Length Sense
Human vs. NHP metabolic profile explains extended
duration of action in human for TTRsc
5′-sense
= 2′-F = 2′-O-methyl (GalNAc)3
Standard Template Chemistry (ALN-TTRsc)
Metabolic Profile
Sense Strand (5′-3′)
Antisense Strand (5′-3′)
Agenda
siRNA-GalNAc Conjugates: Background
Standard Template Chemistry (STC)
» Human POC of ALN-TTRsc
Enhanced Stabilization Chemistry (ESC)
» mTTRsc
» ALN-AT3
» ALN-PCSsc
Summary
10
Conjugate Platform Transitioning Standard Template Chemistry (STC) to Enhanced Stabilization Chemistry (ESC)
5′-sense
5′-antisense
= 2′-F = 2′-O-methyl (GalNAc)3
Standard Template Chemistry, STC
(ALN-TTRsc)
5′-sense
5′-antisense
= 2′-F = 2′-O-methyl (GalNAc)3
Enhanced Stabilization Chemistry, ESC
(mTTR, ALN-AT3, ALN-PCS)
Lead
Optimization
by
Further
Stabilization
Chemistries
11
ESC Improves Efficacy of mTTRsc
12
ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc
STC ESC
0
20
40
60
80
100
120
PBS 25 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 0.2 mg/kg
TT
R m
RN
A l
eve
ls D
ep
icte
d
as
% o
f P
BS
Co
ntr
ol
(mT
TR
/mG
apd
h)
Subcutaneous Single Dose
GalNAc Conjugates Are a Repeat-Dose Platform Chronic Dosing in Mice with ESC-mTTRsc
Steady knockdown maintained with chronic dosing Sustained knockdown at both ED50 (1 mg/kg) and ED80 (2.5 mg/kg) dose levels
Absence of tachyphylaxis or sensitization
No changes in serum TTR levels in PBS control group
GalNAc Conjugates are a multi-dose platform, showing dose additive effect
13
% K
no
ck
do
wn
Se
rum
TT
R
(Fra
ction
P
re-d
ose
)
0 30 60 90 120 150 180 210 240 270
Long-Term Dosing: QW Dosing with ESC-mTTRsc
100
80
60
40
20
0
-20
PBS 1.0 mg/kg 2.5mg/kg
Day
siRNA-GalNAc Lead Optimization Tuning STC-siRNA-GalNAc to ESC-siRNA-GalNAc
siRNA
synthesis
In vitro
screening
in vivo exposure/
PD in rodent or hTG
Design chemically
modified siRNAs
Xn
ESC-siRNA-GalNAc
STC-siRNA-GalNAc
14
Select 3-6 conjugates
for high dose
rat toxicity screen
NHP PK/PD
ESC Leads to Higher Liver Exposure Liver Exposure and Metabolic Stability, Single SC Dose, 25 mg/kg in Mice
Metabolic profiling in liver 8h post dose
= 2′-F = 2′-O-methyl
(GalNAc)3
S 5′
= Enzymatic cleavage site (thickness reflects frequency
of corresponding cleavage products observed)
S 5′
Standard Template Chemistry (STC)
Enhanced Stabilization Chemistry (ESC)
AS 5′
AS 5′
Liver Exposure
Target Compound Tmax
(h)
Cmax
(µg/g)
AUC0-t
(h·µg/g)
AUC0-48
(h·µg/g)
AT3 STC 2 59.5 735 735
AT3 ESC 8 285 21546 9697
10
100
1000
10000
100000
1000000
0 50 100 150 200Liv
er
Co
ncen
trati
on
(n
g/g
)
Time (h)
SC
ESC
Liver Exposure
STC
15
ESC Significantly Enhances Efficacy and Duration Reduction of AT Protein After Single SC Dose in NHP
16
Potent and durable silencing achieved after single SC dose >10-fold improvement in efficacy over standard template chemistry
Substantially extended duration of effect
% K
no
ck
do
wn
Se
rum
AT
(R
ela
tive
to
Pre
-do
se
)
Day
100
80
60
40
20
1.0
1.2
-10 0 10 20 30 40
STC-AT3 (10 mg/kg)
ESC-AT3 (10 mg/kg)
ESC-AT3 (1 mg/kg)
Weekly SC administration of ALN-AT3 in WT mice results in potent and consistent AT suppression
Repeat dose ED50 for AT knockdown <0.75 mg/kg
Nadir reached at ~day 14
Maintenance of AT Knockdown with Repeat Dosing ALN-AT3 Pre-Clinical Studies
Akinc, WFH, July 2012 17
100
80
60
40
20
0
-20
4 8 12 16 20 24 28 32
Day
% A
nti
thro
mb
in K
no
ck
do
wn
Norm
aliz
ed
to
Pre
-Dose
an
d P
BS
0
3.00
1.50
0.75
ALN-AT3
(mg/kg)
ALN-AT3, qw x5
Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver)
ALN-AT3, dosing
(qw x 14)
Post-dose
(4 h)
Tissue
collection Pre- and post-
dose (4 h)
Pre- and post-
dose (4 h, 3 days)
Pre- and post-
dose (4 h)
Pre- and post-
dose (4 h)
18
10
100
1000
10000
0 10 20 30 40 50 60 70 80 90
Liv
er
siR
NA
(n
g/g
)
Day
Actual Data with Interpolated Data Added Actual Data
1.0 mg/kg qw
0.5 mg/kg qw
0.2 mg/kg qw
Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver)
Additional peak and trough concentration data added by interpolation; additional 3-day
post-dose data added by assuming fixed ratio relative to peak concentration
Liver levels are approximately dose proportional
Mean steady state liver drug load of ~3 g/g per mg/kg cumulative weekly dosing
ALN-AT3, dosing
(qw x 14)
Post-dose
(4 h)
Tissue
collection Pre- and post-
dose (4 h)
Pre- and post-
dose (4 h, 3 days)
Pre- and post-
dose (4 h)
Pre- and post-
dose (4 h)
19
10
100
1000
10000
0 10 20 30 40 50 60 70 80 90
Liv
er
siR
NA
(n
g/g
)
Day
1100 ng/g
400 ng/g
3400 ng/g
Actual Data with Interpolated Data Added Actual Data
1.0 mg/kg qw
0.5 mg/kg qw
0.2 mg/kg qw
ALN-AT3 Pre-Clinical Efficacy Potent AT Knockdown and Normalization of Thrombin Generation
20
ALN-AT3 achieves potent AT knockdown and fully corrects thrombin generation in non-human primates (NHP)
Weekly SC doses for >5 months result in potent, dose-dependent, and durable AT knockdown
In NHP hemophilia “inhibitor” model, ALN-AT3 fully restores thrombin generation to normal levels
Akinc, ISTH, July 2013
Recovery
Recovery 0.25 mg/kg qw x 12
Recovery 1.5 mg/kg qw x 5
100
80
60
40
20
0
-20 0 20 40 60 80 100 120 140 160
% M
ea
n A
T K
no
ck
do
wn
(P
re-d
ose
= 1
)
Day
1 mg/kg q2w x 4
0.125 mg/kg qw x 12 0.5 mg/kg qw x 8
Normal Hemophilia A (Induced)
Pre-dose
ALN-AT3 (mg/kg) qw
Rela
tive
Th
rom
bin
Ge
nera
tio
n
(Pe
ak T
hro
mb
in)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Saline
1.6
0.25
~60%
AT
KD
0.5
p<0.01
~80%
AT
KD
What About Species-Related Differences? ALN-AT3 Single Dose Efficacy and Recovery
21
Single-dose efficacy: mouse ≈ rat > NHP
Time to nadir and recovery kinetics: NHP > mouse > rat
Day
100
80
60
40
20
0
-20
-40
-10 0 10 20 30 40 50 60
Re
lati
ve
AT
Le
ve
l
Mouse 2.5 mg/kg
Rat 3.0 mg/kg
NHP 3.0 mg/kg
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fra
cti
on
PC
S
messag
e r
em
ain
ing
siRNA rank order +/- SD
0.1 nM, average activity across 3 human cell lines
Screen of ca. 200 PCSK9 siRNAs identified initial lead
Tested Lead as STC and ESC versions in vivo SD
ALN-PCSsc Lead Selection Use What You Know, Then Iterate
PCSsc
Lead
22
STC-PCS ESC-PCS 0.0
0.2
0.4
0.6
0.8
1.0
1.2
PBS
hP
CS
K9 p
rote
in
(Rela
tive t
o p
re-d
ose)
STC-PCS vs. ESC-PCS hPCSK9 tg, mice 72h, 3 mg/kg
ED90 of ESC-PCS ~1 mg/kg BIW (12 doses)
Reduction in PCSK9 results in >60% lowering of LDL-C (data not shown)
Duration of nadir PCSK9 post-last dose (BIW) is ~20 days
The compound potency was sufficient but stability/dose frequency not sufficient
to meet TPP Goal of once-monthly dosing
Another round of ESC lead optimization was used!
Multi-Dose ESC-PCS Results in >90% Lowering of PCSK9 But Duration can be Improved (NHP)
23
Day
0 20 40 60 80 100 120
100
80
0
-20
q2w
q1w
60
40
20
-40
-60
% K
no
ckd
ow
n P
CS
K9
(r
ela
tive t
o p
re-b
leed)
ESC-PCS
1 mg/kg
Dosing
Schedule BIW
siRNA-GalNAc Lead Optimization Tuning ESC-siRNA-GalNAc to Development Candidate for PCSK9
siRNA
synthesis
In vitro
screening
in vivo exposure/
PD in rodent or hTG
Design chemically
modified siRNAs
X2
Select 3-6 conjugates
for high dose
rat toxicity screen
ALN-PCSsc Development Candidate
NHP PK/PD
ESC-siRNA-GalNAc
24
ALN-PCSsc Achieves Potent, Highly Durable PD Pre-Clinical Efficacy in NHP with Single Dose
LDL-C PCSK9
Days
0 20 40 60 80 100
1.0 3.0 6.0 10.0
% P
CS
K9
Kn
oc
kd
ow
n
(re
lative
to
pre
-ble
ed
)
Days
0 20 40 60 80 100 120
% L
DL
-C L
ow
eri
ng
(re
lative
to
pre
-ble
ed
)
100
80
60
40
20
0
-20
80
60
40
20
0
-20
100
ALN-PCSsc (mg/kg)
ALN-PCSsc (mg/kg)
1.0 3.0 6.0 10.0
ALN-PCSsc achieves highly durable PCSK9 knockdown and LDL-C reduction with single dose Single SC dose 1-10 mg/kg
Up to 96% PCSK9 knockdown
Up to 77% LDL-C lowering in absence of statins
Highly durable effects, supports once-monthly and possibly once-quarterly dosing » >50% LDL-C lowering maintained for over 3 months in 10 mg/kg group
25 Fitzgerald, ATVB, May 2014
Comparing STC- to ESC-GalNAc-siRNA Conjugates Key Data in NHP
26
STC
(ALN-TTRsc)
ESC
(ALN-PCSsc)
Potency
ED50
SD 10.0 mg/kg 1.0 mg/kg
MD (qw) ca. 2.0 mg/kg 0.2 mg/kg
Stability
t1/2
Liver ~24 h ~120 h
Duration ~40 days ~120 days
Safety
Cytokine/Complement
Activation None None
Adverse ISRs None None
NOAEL (MD) >300 mg/kg >300 mg/kg
Agenda
siRNA-GalNAc Conjugates: Background
Standard Template Chemistry (STC)
» Human POC of ALN-TTRsc
Enhanced Stabilization Chemistry (ESC)
» mTTRsc
» ALN-AT3
» ALN-PCSsc
Summary
27
Summary
28
Alnylam has developed an effective, well-tolerated, SC-administered
GalNAc-conjugate platform with proven human clinical translation
Enhanced Stabilization Chemistry (ESC) of siRNA-GalNAc conjugates
has led to increased metabolic stability resulting in:
» Increased liver exposure
» Improved potency: Up to 10-fold SD potency improvement and less
injection volume
» Increased duration of effect, which further increases from rodents to
NHP
» No compromise in safety
ESC-conjugate design translates well across multiple siRNAs and
targets
Expect favorable translation of potency and duration in humans for
ESC-GalNAc-conjugates