Progress in Extrahepatic Silencing with siRNA Conjugates · Intrathecal Delivery of CNS Optimized...
Transcript of Progress in Extrahepatic Silencing with siRNA Conjugates · Intrathecal Delivery of CNS Optimized...
1© 2018 Alnylam Pharmaceuticals, Inc.
March 29, 2019
Progress in Extrahepatic Silencing with siRNA
Conjugates
Stuart Milstein
2
2004
2014
2018
STC
ESC
Advanced
ESC
Potency/duration/
specificity
Stability, optimal
ligand orientation
Efficient delivery
to target cells
ESC+
siRNA
design/chemistryLinker Ligand
0.01
0.1
1
10
100
2006 2008 2010 2012 2014 2016 2018
ED
50
(m
g/k
g)
Year
Partially
modified
Evolution of conjugate potency
(mouse, SD ED50)
siRNA designs with enhanced potency and stability may extend to extrahepatic tissues
Transformative Advancements in Conjugate-Based Delivery
3
Extensive Durability and Safety Demonstrated in Liver Programs
STC
ESC
Advanced
ESC
Potency/duration/
specificity
Stability, optimal
ligand orientation
Efficient delivery
to target cells
ESC+
siRNA
design/chemistryLinker Ligand
0.01
0.1
1
10
100
2006 2008 2010 2012 2014 2016 2018
ED
50
(m
g/k
g)
Year
Partially
modified
Evolution of conjugate potency
(mouse, SD ED50)
4
Expanding Alnylam opportunities beyond liver
Investigational RNAi Therapeutics for CNS and Ocular Diseases
Devastating diseases with enormous burden and unmet need
• Alzheimer’s disease
• Amyotrophic lateral
sclerosis (ALS)
• Cerebral amyloid
angiopathy
• Frontotemporal
dementia
• Huntington’s disease
• Multi-system atrophy
• Parkinson’s disease
• Spinocerebellar ataxia
• AMD, dry
• AMD, wet
• Birdshot
chorioretinopathy
• Dominant retinitis
pigmentosa 4
• Fuch’s dystrophy
• hATTR amyloidosis
• Hereditary and
sporadic glaucoma
• Stargardt’s disease
Number of genetically validated targets known but few disease modifying therapies for these devastating or
life threatening disorders.
RNAi therapeutics directed to disease-causing, CNS- or ocular-expressed genes represent a potential
opportunity to address diseases with some of the greatest unmet need.
5
Single dose and dose response in rat
Intrathecal Delivery of CNS Optimized siRNA Conjugates
siRNAs targeting b-catenin or SOD1 in single dose or dose response
• Single siRNA conjugate doses of 0.9 mg, 0.3 mg, 0.07 mg
• Multidose arm- 0.3 mg monthly x 5
• Time points through 1 month for b-catenin and 6 months for SOD1
Day 0 427 28
Tissue
collection
16856 70 84 98 126
Tissues: Spinal cord: Lumbar, thoracic and cervical
Brain: prefrontal cortex, cerebellum and remaining brain
Fluids: CSF and plasma
Assays: mRNA, tissue siRNA levels, Histology
Strand (5'-3') Sequence
b-cateninS UACUGUUGGAUUGAUUCGAAA
AS TUUCGAAUCAAUCCAACAGUAGC
SOD1S CAUUUUAAUCCUCACUCUAAA
AS UUUAGAGUGAGGAUUAAAAUGAG
6
Silencing of b-catenin following a single IT dose
Robust and Durable Silencing Demonstrated Following a Single IT Dose
Lumbar
Thoracic
Cervical
Cerebellum
Frontal Cortex
Remaining Brain
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
7
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.3 mg Multi Dose
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Lumbar Spinal Cord
Messag
e R
em
ain
ing
Control
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Control
Cervical Spinal Cord
Messag
e R
em
ain
ing
SOD1 VP - 0.3 mg Multi Dose
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Control
Cerebellum
Messag
e R
em
ain
ing
SOD1 VP - 0.3 mg Multi Dose
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Control
Frontal Cortex
Messag
e R
em
ain
ing
SOD1 VP - 0.3 mg Multi Dose
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Control
Thoracic Spinal Cord
Messag
e R
em
ain
ing
SOD1 VP - 0.3 mg Multi Dose
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00
0.25
0.50
0.75
1.00
1.25
1.50
Time (Days)
SOD1 Parent - 0.9 mg
SOD1 VP - 0.9 mg
SOD1 VP - 0.3 mg
SOD1 VP - 0.075 mg
Control
Temporal Cortex
Messag
e R
em
ain
ing
SOD1 VP - 0.3 mg Multi Dose
Silencing of SOD1 following a single or multiple IT doses
Robust and Durable Silencing Demonstrated Following a Single IT Dose
Lumbar
Thoracic
Cervical
Cerebellum
Frontal Cortex
Temporal Cortex
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
Messag
e R
em
ain
ing
8
Intrathecal delivery of siRNA provides durable knockdown throughout CNS
Robust Silencing Throughout the Brain
• Consistent lowering across animals in most regions of the brain
• PD comparison in liver across species together with extended duration seen in rodents expected to support
infrequent dosing in human
PD comparison in liver across species
9
Higher drug levels and robust silencing observed in brain with SOD1 siRNA conjugate
siRNA Conjugates Show Enhanced Uptake and Activity
• Rapid siRNA clearance from CSF
• Conjugate reveals superior uptake and
stability in brain over unconjugated
siRNA
• Increased uptake in brain results in
substantial improvement in mRNA
knockdown
Unconjugated siRNA
Conjugated siRNA
Unconjugated siRNA
Conjugated siRNA
Target silencing in cerebellum
Unconjugated siRNA
Conjugated siRNA
Control siRNA
10
CSF and Plasma PK in Rat Following IT dosing
0.9 mg SOD1, lumbar puncture
• Rapid disappearance from CSF
• Rapid appearance in plasma, tmax
5 min – 1 h
• Plasma concentrations parallel
CSF, but ~2 logs lower
0 6 1 2 1 8 2 4
0 . 0 0 0 1
0 . 0 0 1
0 . 0 1
0 . 1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
S O D 1 - C 1 6 - V P , P l a s m a a n d C S F
T i m e ( h )
[s
iRN
A],
g/m
L
P l a s m a
C S F
U r in e
n=2
n=1n=2
n=2 n=3
n=3
Intrathecal (IT) dosing, 0.9 mg
Dose 42 8 24 h
5, 15, 30 min
Plasma
CSF
61
Sampling timeline
11
SOD1 IT Dose Response Reveals Minimal Silencing in the Liver and Kidney
Liver
Kidney
Though rapidly cleared from the CSF to the systemic circulation,
conjugated siRNAs do not show robust silencing in the liver or kidney
12
Rat IHC probed for b-catenin
b-cateninControl
Neocortex
Cerebellum
b-cateninControl
Basal forebrain
b-cateninControl
Striatum
b-cateninControl
Silencing Is Seen In All Regions of the Brain
13
Single dose NHP study design
Evaluating Translation of CNS siRNA Conjugate Delivery to NHP
Day 0 31
• siRNA conjugate dosed at 72 mg – IT bolus
• Evaluated a single target – b-catenin
Tissues: Spinal cord: Lumbar, thoracic and cervical
Brain: Prefrontal cortex, temporal cortex, cerebellum, brain stem
hippocampus
Fluids: CSF and plasmaHistology
Assays: mRNA, siRNA levels
Tissue
collection
Strand (5'-3') Sequence
b-cateninS UACUGUUGGAUUGAUUCGAAA
AS TUUCGAAUCAAUCCAACAGUAGC
14
siRNA uptake varies between CNS regions
siR
NA
ng
/gsiRNA levels
IT Dosed b-catenin siRNA is Detected Throughout the Spinal Cord and Brain
Regions of NHP
The day 31 time point shows the presence of significant siRNA
levels across all tissues tested
Lu
mb
ar
Th
ora
cic
Cerv
ical
Bra
in S
tem
Cere
bellu
m
Pre
fro
nta
l C
or t
ex
Tem
po
ral C
or t
ex
Hip
po
cam
pu
s
Liv
er
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
5 0 0 0
6 0 0 0
7 0 0 0
8 0 0 0 s iR N A le v e ls - d a y 3 1
15
Robust Silencing Across CNS Demonstrating Successful Translation NHPb-catenin mRNA knockdown by tissue, Day 31
• The conjugate targeting b-catenin produces robust knockdown throughout the spinal cord
and brain at the 31 day time point.
b-catenin mRNA
Messag
e R
em
ain
ing
Lu
mb
ar
Sp
ine
Th
ora
cic
Sp
ine
Cerv
ical S
pin
e
Cere
bellu
m
Pre
fro
nta
l C
or t
ex
Tem
po
ral C
or t
ex
Hip
po
cam
pu
s
Bra
in S
tem
Liv
er
0 .0 0
0 .2 5
0 .5 0
0 .7 5
1 .0 0
1 .2 5
1 .5 0
P B S C o n tro l
C T N N B s iR N A
PBS
CTNNB
16
siRNAs Distribute Throughout the CNS in NHP Following IT Dosing
Lumbar cord Cervical cord Cerebellum
Neocortex Hippocampus Dentate Gyrus Basal Ganglia
17
Most neurons show siRNA uptake
siRNA Conjugates are Localized to Neurons Following IT Dosing
MAP2
siRNA
MAP2 is a neuronal marker
CTNNB siRNA probed with siRNA antibody
40X
100X
18
siRNA Conjugates are Localized to Microglia Following IT Dosing
Iba1 is a microglia marker
b-catenin siRNA probed with siRNA antibody
40X
100X
Iba1
siRNA40X
100X
19
siRNAs Conjugates are Localized to Astrocytes Following IT Dosing
GFAP is an Astrocyte marker
b-catenin siRNA probed with siRNA antibody
40X
100X
GFAP
siRNA40X
100X
40X
100X
20
siRNA vs ASO in hSOD1 (SOD1G93A) Rats
McCampbell et al, J Clin Invest. 2018;128(8):3558-3567
Study Purpose
• Head-to-head comparison of siRNA and ASO
– 3 siRNAs selected from mouse AAV-hsSOD1 screen
– ASO 1, based on McCampbell et al. (2018)◦ Demonstrated ~75% maximum silencing at 2 weeks in the same rat model
Study Design
• Dose: single IT injection of 0.9 mg in 30µl (Study Day 0)
– Same dose used for siRNA and ASO
• Dosed 13 week old hSOD1 rats (SOD1G93A)1
– Single Day 7 timepoint ◦ Single early time point was chosen to capture the highest expression of SOD1G93A before animals developed degenerative phenotype
Day 7 collection after single IT dose
Study Day
Animal Age (Weeks)13
Tissues collected
across spinal
cord and brain
0
14
7
IT injection
siRNA & ASO
SOD1G93A
Levels15
21
Improved hSOD1 mRNA Reduction with siRNA compared to ASO in CNS
siRNA vs ASO in hSOD1 (SOD1G93A) Rats
aC
SF
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
AS
O
siR
NA
0
20
40
60
80
100
120
% h
SO
D1 m
essage r
em
ain
ing
rela
tive to a
CS
F
Lumbar Thoracic CervicalBrain
StemCerebellum Hippo
Frontal
CortexTemporal
Cortex
An siRNA targeting hSOD1 showed superior silencing in all regions of the brain and spinal cord
22
Expanding Alnylam opportunities beyond liver
Investigational RNAi Therapeutics for CNS and Ocular Diseases
Devastating diseases with enormous burden and unmet need
• Alzheimer’s disease
• Amyotrophic lateral
sclerosis (ALS)
• Cerebral amyloid
angiopathy
• Frontotemporal
dementia
• Huntington’s disease
• Multi-system atrophy
• Parkinson’s disease
• Spinocerebellar ataxia
• AMD, dry
• AMD, wet
• Birdshot
chorioretinopathy
• Dominant retinitis
pigmentosa 4
• Fuch’s dystrophy
• hATTR amyloidosis
• Hereditary and
sporadic glaucoma
• Stargardt’s disease
Number of genetically validated targets known but few disease modifying therapies for these devastating or
life threatening disorders.
RNAi therapeutics directed to disease-causing, CNS- or ocular-expressed genes represent a potential
opportunity to address diseases with some of the greatest unmet need.
23
TTR is Produced in the Eye as Well as the Liver
Ocular manifestations of hATTR amyloidosis in patients
• Glaucoma (~12%-23%)
• Vitreous opacities (Wide range)
• Retinal abnormalities (~4% - 15%)
− retinal amyloid deposit
• Iris abnormalities (~14% - 38%)
− iris amyloid deposit
• Amyloid deposits on lens (~33%)
Pupillary deposits and abnormality predict onset of
glaucoma
• V30M and T114C study
− 62% eyes had high IOP
Liver transplant patients
• Study of Japanese liver transplant patients with V30M continue to
have ocular symptoms
24
Ocular TTR Silencing by Differentially Modified siRNA Conjugates in Rat
After Single Intravitreal InjectionOcular silencing of hTTR in transgenic mice
Specificity: No impact on expression of mTTR, Crx or Rhodopsin
PB
S
Par t
ially M
od
ifie
d
ES
C
EH
op
tim
ized
0 .2 5
0 .5
1
2
4
8
1 6
3 2
6 4
1 2 8
2 5 6
% m
es
sa
ge
re
ma
inin
g
Mouse TTR mRNA
Day 14, 50 g siRNA conjugate
25
Near complete reduction of TTR mRNA and protein 28 days following a single IVT dose
Robust Silencing of Ocular TTR by siRNA Conjugates in NHP
TTR protein analysis by IHC
TTR protein analysis by ELISA
TTR mRNA analysis by qPCR
Aqueous Humor
Vitreous Humor
0 0 .1 m g 0 .3 m g 1 .0 m g 3 .0 m g
0
5 0
1 0 0
1 5 0
D o s e
% R
em
ain
ing
TT
R P
ro
tein
0 0 .1 m g 0 .3 m g 1 .0 m g 3 .0 m g
0
5 0
1 0 0
1 5 0
D o s e
% R
em
ain
ing
TT
R P
ro
tein
Ciliary Body RPE
PB
S
0.1
mg
0.3
mg
1.0
mg
3.0
mg
PB
S
0.1
mg
0.3
mg
1.0
mg
3.0
mg
0
5 0
1 0 0
1 5 0
% T
TR
Me
ss
ag
e R
em
ain
ing
0.1
mg
/eye
PB
S
Dose
Dose
26
Expanding a pipeline of potentially transformative medicines
Alnylam CNS and Ocular Pipeline Strategy
Genetically validated
target gene
Biomarker for
POC in
Phase 1
Definable path
to approval and
patient access
27
• Durable silencing of target mRNA observed
across the CNS of rat and NHP following IT
administration
• Tissue uptake observed in all CNS tissues
examined with drug levels in the ng/g to g/g
range
• In both rat and NHP studies, intrathecal
administration of the novel siRNA conjugates
was found to be generally well tolerated
• Robust silencing throughout the brain observed
for an siRNA targeting SOD1
Successful delivery of siRNA conjugate to the CNS and eye
Consistent Silencing Observed Across Pre-clinical Species
• TTR silencing demonstrated in rodents and
NHP
• Target silencing seen in the CE and RPE
• Target silencing is specific
• Target knockdown demonstrated in NHP
following a single IVT dose of siRNA
• Equivalent silencing demonstrated for mRNA
and protein across the eye