Selection of Well-Tolerated GalNAc-siRNAs
by Screening for RNAi-Mediated Off-Target
Effects in Rodent Toxicity Studies
Maja Janas, PhD, DABT
Senior Scientist, Investigative Toxicology
TIDES, May 2nd 2017
2
Agenda
• Nonclinical safety profile of GalNAc-siRNAs
• Mechanisms of GalNAc-siRNA hepatotoxicity in rodents
3
GalNAc-siRNA Conjugates
SC-Administered Platform for Targeted Delivery to Hepatocytes
siRNA Metabolic stability
Duration of effect
Intrinsic potency
Safety
CMC
Ligand Receptor affinity & specificity
Metabolic stability
Safety
CMC
Asialoglycoprotein Receptor (ASGPR)
Highly expressed in hepatocytes
High turnover (recycling time ~15 min)
Conserved across species
4
GalNAc-siRNA Conjugates
Nonclinical Considerations
• Highly specific distribution to liver via
ASGPR
• Limited uptake/distribution in other organs
• Liver to kidney ratio >30 at PD doses
• Short plasma half-life; long tissue half-life;
long PD effects
• Metabolized largely by intracellular exo-
and endo-nucleases
• Maximum liver drug concentration limits
relevant toxicological doses
◦ Loss of dose-proportionality, approaching
saturation of liver uptake via ASGPR
◦ Cannot achieve MTDs
– Excess circulating siRNA levels not taken up
by liver; excreted through kidneys
5
GalNAc-siRNA Conjugates
Platform-Wide Responses at Toxicological Doses
Rat
◦ Liver
– Hepatocellular vacuolation: increased number and size of normal rat hepatocellular
vacuoles, most contain lipid
– Increased single cell necrosis
– Increased mitosis and regeneration
◦ Kidney
– Basophilic granules in proximal tubular epithelium; represents drug accumulation
NHP
◦ Liver
– Basophilic granules in Kupffer cells and hepatocytes; represents drug accumulation
◦ Lymph nodes
– Vacuolated macrophages (with basophilic stippling); represents phagocytosis of drug
Primarily dose-dependent and the result of drug accumulation in tissue
6
In silico prediction & In vitro efficacy
In vitro screen for predicted off-targets
Rodent Knockdown
Rat Tox @ >100x PD dose
NHP Knockdown DC
Bad Actors (40%):
Show hepatotoxicity
Good Actors (60%):
No hepatotoxicity
Path Towards a Development Candidate (DC)
Single cell necrosis and/or
hepatocellular degeneration with
↑LFT 2x upper limit of normal
7
Potential Causes of Hepatotoxicity in Rodent Toxicity Studies
1. Non-RNAi drug
effects e.g. protein binding
2. Competition for Ago
binding with miRNAs
3’-UTR
Off-target binding Partial sequence match
3. RNAi-mediated
off-target activity
RISC loading
mRNA
RISC
On-target binding Full sequence match
mRNA cleavage
Desired
on-target activity
Illustrative design
8
5’ Modifications on the Antisense Strand Can Block
RISC Loading With No Impact on Liver Exposure
RISC loading
mRNA
Sense strand removal
Target RNA cleavage
Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
- + - +
1
1 0
1 0 0
R a t L iv e r E x p o s u re
ug
AS
/ g
liv
er
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ck- + - +
0 .0
0 .2
0 .4
0 .6
R a t R IS C L o a d in g
ng
AS
/ g
liv
er
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ck
9
Blocking Antisense RISC Loading Without Altering
the 2’F/2’OMe/PS Content Mitigates Hepatotoxicity
*
* ^
#
- + RISC loading block
Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
S a lin e - + - +
0
1 0 0
2 0 0
3 0 0
R a t A L T
U/L
R e fe re n c e ra n g e
s iR N A -1 s iR N A -2
R IS C lo a d in g b lo ck
10
ESC
Advanced ESC
Changing siRNA Chemical Modification Pattern
Does Not Reduce Liver Exposure or RISC Loading Dose: 100 mg/kg
Regimen: qw x 9
Necropsy: Day 58
Altered modification pattern for
improved metabolic stability
E S C Ad v a n c e d E S C
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
R a t R IS C L o a d in g
ng
AS
/ g
liv
er
E S C Ad v a n c e d E S C
1
1 0
1 0 0
1 0 0 0
R a t L iv e r E x p o s u re
ug
AS
/ g
liv
er
Illustrative design
11
Changing siRNA Chemical Modification Pattern
Does Not Mitigate Hepatotoxicity Dose: 100 mg/kg
Regimen: qw x 9
Necropsy: Day 58
S a lin e E S C Ad v a n c e d E S C
0
1 0 0
2 0 0
3 0 0
R a t A L T
U/L
R e fe re n c e ra n g e
ESC Advanced ESC
#
* # *
12
Potential Causes of Hepatotoxicity in Rodent Toxicity Studies
1. Non-RNAi drug
effects e.g. protein binding
2. Competition for Ago
binding with miRNAs
3’-UTR
Off-target binding Partial sequence match
3. RNAi-mediated
off-target activity
RISC loading
mRNA
RISC
On-target binding Full sequence match
mRNA cleavage
Desired
on-target activity
Illustrative design
13
Reversir™ Platform Achieves Tailored Control of RNAi Pharmacology
0%
20%
40%
60%
80%
100%
120%
0 3 6 9 12 15 18 21 24
Rel
ativ
e Ta
rget
Pro
tein
Lev
el
No Reversir
Time (Days)
3 mg/kg
GalNAc-siRNA
0.1 mg/kg
GalNAc-Reversir
Reversir
mRNA cleavage
mRNA target
ASGPR
Inactive RISC
Functional
RISC
14
Prevention
Treatment
Reversir
siRNA
Reversir
siRNA
( )
Blocking RISC-Loaded Antisense Strand with Reversir
Does Not Reduce Liver Exposure or RISC Loading
3-10 mg/kg
30 mg/kg
3-10 mg/kg
30 mg/kg
1
1 0
1 0 0
1 0 0 0
R a t L iv e r E x p o s u re
ug
A
S /
g l
ive
r
- Targeting Ctr - Targeting Ctr Targeting Ctr RVR
siRNA1 2 3
1
1 0
1 0 0
1 0 0 0
R a t R IS C L o a d in g
ng
A
S /
g l
ive
r
- Targeting Ctr - Targeting Ctr Targeting Ctr RVR
siRNA1 2 3
15
Reversing Activity of the RISC-Loaded Antisense
Strand Mitigates Hepatotoxicity
Pre
ve
nti
on
+ Ctr RVR + Targeting RVR
# *
#
+
*
* #
* *
*
^
Tre
atm
en
t
0
5 0
1 0 0
1 5 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 1
0
1 0
2 0
3 0
4 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 2
0
1 0
2 0
3 0
4 0
R a t G L D H
U/L
R e fe re n c e ra n g e
- Targeting - Targeting Ctr RVR
siRNA- 3
16
Swapping Seed Regions Does Not Reduce Liver
Exposure or RISC Loading Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
1
1 0
1 0 0
R a t L iv e r E x p o s u re
ug
AS
/ g
liv
er
Toxic Non-toxic Non-toxic Toxic Seed
Toxic Non-toxic Toxic Non-toxic Non-seed
Parent Seed swap
0
5 0
1 0 0
1 5 0
2 0 0
R a t R IS C L o a d in g
ug
AS
/ g
liv
er
Toxic Non-toxic Non-toxic Toxic Seed
Toxic Non-toxic Toxic Non-toxic Non-seed
Parent Seed swap
Illustrative design
17
Swapping Seed Regions Mitigates Hepatotoxicity
* #
*
#
* *
To
xic
Toxic Non-toxic
No
n-t
ox
ic
SEED
NO
N-S
EE
D
Dose: 30 mg/kg
Regimen: q2d x 6
Necropsy: Day 15
S a lin e b a d C 5 g o o d C 5g o o d s e e db a d s e e d
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
R a t A L T
U/L
R e fe re n c e ra n g e
Toxic Non-toxic Non-toxic Toxic Seed
Toxic Non-toxic Toxic Non-toxic Non-seed
Parent Seed swap
18
GalNAc-siRNA Conjugates Can Cause Seed-Mediated Off-Targets
at High Doses
RNAseq in Rat Hepatocytes (24 hrs, 10 nM)
siRNA-2 siRNA-1
Downregulated genes Upregulated genes
Antisense seed
enrichment
Sense seed
enrichment
Antisense seed
enrichment
Sense seed
enrichment
siRNA-1 p < 2.2e-16 p = 0.05286 p = 0.667 0.2174
siRNA-2 p < 2.2e-16 p = 0.9679 p = 0.6345 p = 0.3364
65
199
160
190
19
RNAi Activity is Driving Gene Dysregulation In Vivo at High Doses RNAseq in Rat Liver (24 hrs, 50 mg/kg)
siR
NA
-1
siR
NA
-2
- RISC loading block
16
44
8
1
-50%
-98%
196
311
7
18
-96%
-94%
+
RISC
Loading
Block
Downregulated genes Upregulated genes
Antisense seed
enrichment
Sense seed
enrichment
Antisense seed
enrichment
Sense seed
enrichment
siRNA-1 - p = 1.25e-03 p = 0.3521 p = 1 p = 0.7103
+ p = 1 (NA) p = 1 (NA) p = 1 p = 0.5548
siRNA-2 - p = 2.06e-10 p = 0.1843 p = 1 p = 0.9872
+ p = 1 p = 1 p = 1 p = 1
20
Conclusions
• Antisense strand-driven RNAi-mediated hybridization-based off-target
effects, not chemical modifications, are a major driver of hepatotoxicity
of a subset of GalNAc-siRNA conjugates that fail in rodent toxicity
studies at >100x PD dose
• Careful selection of "good actor" siRNAs is an important part of
identifying optimal siRNA molecules for clinical development
3’-UTR
Off-target binding Partial sequence match
RNAi-mediated off-
target activity
21
Nonclinical Safety and Bioanalysis
Carole Harbison
Yongfeng Jiang
Onur Yilmaz
Michael Placke
Natalie Keirstead
Kirk Brown
Brenda Carito
Samantha Chigas
Sean Dennin
Kristin Fong
Paul Gedman
Toni Hayes
Emma Henchy
Lauren Moran
Elena Ooms
Rachel Peters
Kristina Perry
Kellie Sawyer
Catrina Wong
Research
Adam Castoreno
Rubina Parmar
Mark Schlegel
Svetlana Shulga-Morskaya
Huilei Xu
Ivan Zlatev
Martin Maier
Vasant Jadhav
Terence Cawley
Klaus Charisse
Anna Bisbe
Rajeev Kallanthottathil
Ryan Malone
Mano Manoharan
Jonathan O'Shea
Nate Taneja
Christopher Theile
Jeff Rollins
Stacy Seide
Thank You!