The world leader in serving science Proprietary & Confidential May 2016 Xingwang Fang PhD Invitrogen’s RNAi - siRNA & miRNA Solutions
The world leader in serving science Proprietary & Confidential
May 2016
Xingwang Fang PhD
Invitrogen’s RNAi - siRNA & miRNA Solutions
The world leader in serving science Proprietary & Confidential
Combining design algorithms and LNA™ modifications to enhance siRNA specificity without compromising potency
3 Proprietary & Confidential
Functional Assay: Apoptosis
Silencer ™ Select siRNA
Western-Star™ kit
Ambion MagMAX™-96
Total RNA Isolation or
Cells-to-CT™ kits Lipofectamine™
RNAiMax
TaqMan™:
AB 7900
48h Eg5 siRNA 48h Cdc siRNA
8200 Cellular
Detection System
mRNA Knockdown
18s neg1 surv
Protein Knockdown
GAPDH
Survivin
Biology
IF
0
1000
2000
3000
4000
5000
6000
An
ti-miR
NC
An
ti-miR
23
An
ti-miR
NC
An
ti-miR
21
An
ti-miR
NC
An
ti-miR
15
a
An
ti-miR
NC
An
ti-miR
16
An
ti-miR
NC
An
ti-miR
19
a
No
Inh
ibito
r
No
rmali
zed
LU
Dual-
Light
Reagent
Functional analysis using siRNA
4 Proprietary & Confidential
short-
interfering
~21 bp
dsRNA RNA Induced
Silencing
Complex
Mechanism of RNAi
5 Proprietary & Confidential
Consistent Knockdown
Better Potency
Reduced Toxicity
Better Specificity
Silencer™ Select siRNAs
Increased Confidence in Results
Design
Algorithm
Bioinformatic
Filters
Chemical
Modifications
Phenotype
from
siRNA 3
Phenotype
from
siRNA 1
Phenotype
from
siRNA 2
Silencer™ Select siRNA
6 Proprietary & Confidential
Developing a New siRNA Design Algorithm
Biological features
Data for SVM training
SVM classifier
Experimental data
Feature extraction
Data transformation and scaling
Validation with
independent data siRNA prediction
SVM training and optimization
7 Proprietary & Confidential
Base Count Statistical Significance (P Value)
Base P
osit
ion
s (
1–
21
)
>90 Sequence
and
Biophysical
Parameters in
New Algorithm
Class Separation
Developing a New siRNA Design Algorithm
8 Proprietary & Confidential
Design Algorithm Improves siRNA Prediction Accuracy
9 Proprietary & Confidential
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.03nM 0.3nM 3nM 30nM
Av
era
ge
Re
ma
inin
g G
en
e E
xp
res
sio
n (%
)
Concentration
Supplier A - Modified (n=40)
Supplier B - Unmodified (n=30)
Silencer™ Select (n=30)
SVM Silencer™ Select siRNAs outperform siRNAs
10 Proprietary & Confidential
Mismatch Filter
Silencer™ Select
Toxicity Classifier
Natural miRNA
Seed Region Filter
Antiviral Response
Motif Filter
siRNA Seed
Region Ranking
Selection of hyperfunctional siRNA with
improved potency and specificity.
Wang, X, et al., NAR, Oct. 21, 2009
Five BioInformatic Filters for Enhancing Specificity
11 Proprietary & Confidential
Consistent Knockdown
Better Potency
Reduced Toxicity
Better Specificity
Silencer™ Select siRNAs
Increased Confidence in Results
Design
Algorithm
Bioinformatic
Filters
Chemical
Modifications
Phenotype
from
siRNA 3
Phenotype
from
siRNA 1
Phenotype
from
siRNA 2
Silencer™ Select siRNA Silencer™ Select siRNA
12 Proprietary & Confidential
LNA
2’OMe
RNA
NNNNNNNNNNNNNNNNNNNnn
nnNNNNNNNNNNNNNNNNNNN Potency
Efficacy
Strand Bias
Cell Assays
siRNA Testing
Array
>30 formats
48 siRNA
Optimal siRNA Modifications and Patterns Optimal siRNA Modifications and Patterns
13 Proprietary & Confidential
Modification & formats – siRNA potency Uncompromised
14 Proprietary & Confidential
5’ cDNA 3’ fLuciferase
3’ cDNA 5’ fLuciferase
Target for Guide Strand
Target for Passenger Strand
Passenger Guide
+
OR
Luciferase Assay
siRNA
Strandedness Assay to Test Chemical Modifications
15 Proprietary & Confidential
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
A B C D E F G H I J K L M N O P Luc
siRNA
No
siRNA
Lu
cif
era
se
Sig
na
l
Poor formats – compromise silencing
Of the guide strand
Passenger
Guide
Chemical modification patterns impact of the siRNA
16 Proprietary & Confidential
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
A B C D E F G H I J K L M N O P Luc
siRNA
No
siRNA
Lu
cif
era
se
Sig
na
l
Lead formats – maintain silencing
potency of the guide strand
Passenger
Guide
Chemical modification patterns impact of the siRNA Chemical modification patterns impact of the siRNA
17 Proprietary & Confidential
• Oncology assay
• Apoptosis – cleaved lamin A
• Mitosis – Phospho-histone H3
• Cell size/shape – tubulin
• Nucleus size/shape – Hoechst
• U-2 OS osteosarcoma cells
• WEE1, PLK1, AURKA
ImageXpressmicro
Cenix Bioscience GmBH
• Endocytosis assay − LDL uptake
− Transferrin uptake
− Dextran uptake
− Cell number- Hoechst
− HUH7 hepatoma cells
− HMGCR, FDFT1, LDLR, TFRC
Specificity of chemically modified siRNA: cell assays
18 Proprietary & Confidential
HUH7 cells
Optimal formats reduce off-target effects in cell assays
19 Proprietary & Confidential
Optimal format reduces apoptosis off-target effects
20 Proprietary & Confidential
Unmodified LNA
↓50%
2’ OMe
↓6%
Unmodified LNA
↓70%
2’OMe
0%
Affymetrix U133 2.0 Plus
2-fold DEGs (P<0.001)
HeLa Cells 24 hours post-transfection
FDFT1 siRNA 1 FDFT1 siRNA 2
Optimal format LNA® modified siRNA reduced off-targets
21 Proprietary & Confidential
2- fold changers p<0.001
siRNA ID s477 siRNA ID s475 siRNA ID s476
3 siRNA to
CLTC
30 nM
Hela
Optimal format LNA™ modified siRNA reduced off-targets
22 Proprietary & Confidential
[siRNA]
conc
# of
DEGs
30 nM 24
3 nM 4
2-fold changers
p<0.001
Off-target reduction without decrease in knock-down
23 Proprietary & Confidential
85% reduction in 2-fold
DEG p<0.001
Affy U133 2.0 plus
2-fold p>0.001
Negative control siRNA
Functional analysis using siRNA Optimal format LNA™ modified siRNA footprint
24 Proprietary & Confidential
Modified siRNAs Produce Coherent Phenotypes
% mitotic/apoptotic nuclei (3nM)
Off-target
On-t
arg
et
HMGCR Silencer ™ Select
HMGCR
siRNA Neg control
HMGCR Unmodified
25 Proprietary & Confidential
Better Knockdown
Higher Potency
Better Specificity
Design
Algorithm
Bioinformatic
Filters
Chemical
Modifications
Phenotype
from
siRNA 3
Phenotype
from
siRNA 1
Phenotype
from
siRNA 2
Silencer™ Select siRNAs
• Superior knockdown in side-by-side tests
• Up to 100X more potency
• Off-target effects reduced by up to 90%
• 100% guaranteed knockdown
(see www.ambion.com/siRNA/guarantee)
Silencer™ Select siRNAs give • Fewer false positives
• Fewer false negatives
• Verified results with less follow up
Silencer™ Select siRNA
26 Proprietary & Confidential
Human Genome Collection (21,585)
Extended Druggable Genome (10,415)
Druggable Genome (9,032)
GPCR (380) Protease
(494)
Kinases
(710)
Phosphotase
(298)
Nuclear
Hormones
(47)
Ion Channels
(338)
Silencer™ Select siRNA Library Modularity
27 Proprietary & Confidential
RNAi ordering made easy – Thermofisher.com
The world leader in serving science Proprietary & Confidential
microRNA functional analysis
29 Proprietary & Confidential
• microRNAs (miRNAs) are short 18-25 nt RNA molecules, found in abundance in plants and
animals
• miRNAs are unique & ubiquitous post-transcriptional regulators that bind to complementary
sequences on target mRNAs, resulting in translational repression and gene silencing
• miRNAs are well conserved in eukaryotic organisms and are thought to be a vital and
evolutionarily ancient component of genetic regulation
• the human genome encodes about 2000 miRNAs, which may target and regulate >50% of
mammalian genes
• each miRNA typically regulates multiple mRNAs, and many mRNAs are regulated by several
miRNAs.
• roles in many human diseases
Overview: what are microRNAs?
30 Proprietary & Confidential
mirVana™ mimics and inhibitors
Antisense oligonucleotides
bind and inhibit miRNAs
Mature miRNAs enter the
miRNA pathway when
transfected into cultured
mammalian cells
mirVana™ miRNA inhibitors
mirVana™ miRNA mimics
Novel tools for microRNA functional analysis
mirVana™ mimics and inhibitors
31 Proprietary & Confidential
Screening by TaqMan® assays & Validating by miRNA mimics/inhibitors
Example: miRNA mimic Example: miRNA inhibitor
Normal
cell line
Disease
cell line
miRNA TaqMan®
expression profile
Results: Down-regulated
miRNA in Disease cell line
Transfect Disease cell line with Mimic
See if phenotype reverts to normal
Normal
cell line
Disease
cell line
miRNA TaqMan®
expression profile
Results: Up-regulated
miRNA in Disease cell line
Transfect Disease cell line with Inhibitor
See if phenotype reverts to normal
Re-Validate by measuring mRNA, miRNA by TaqMan®
Reporter assays pLuc; Cell Based Assays, Protein Assays
Functional Analysis
MicroRNA Functional Analysis Process
32 Proprietary & Confidential
Select miRNA
mirVana™ inhibitors
mirVana™ mimics
Controls
Transfect
RNAiMAX™
Invivofectamine2.0
Reporter Assays
mRNA/miRNA
TaqMan® Assays
Cells-to-Ct™ kits
Protein/Phenotype
Western
Immunoassay
MicroRNA Functional Analysis Workflow
33 Proprietary & Confidential
mirVana™ miRNA mimics Enhanced Specificity
34 Proprietary & Confidential
Star (Passenger) strand Mature miRNA (Guide) strand miRNA
Luc miRNA Target
Luc Reverse Target
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
miRNAmimic 1
miRNAmimic 2
miRNAmimic 3
miRNAmimic 4
miRNAmimic 5
miRNAmimic 6
Neg
Fo
ld C
ha
ng
e
0.000
0.200
0.400
0.600
0.800
1.000
1.200
miRNAmimic 1
miRNAmimic 2
miRNAmimic 3
miRNAmimic 4
miRNAmimic 5
miRNAmimic 6
Neg
Fo
ld C
ha
ng
e
Mimic mature strand is highly potent while “star” strand is inactivated
mirVana™ miRNA
35 Proprietary & Confidential
0
0.2
0.4
0.6
0.8
1
1.2
mimic neg mimic neg mimic neg mimic neg mimic neg
miR19a miR23a let7c let7a miR21
Fold
ch
ange
3 nM
0.3 nM
Are highly functional in reporter assays
mirVana™ miRNA mimics
36 Proprietary & Confidential
0
20
40
60
80
100
120
miR1 mimic Neg
Re
mai
nin
g P
TK9
exp
ress
ion
(%
)
30nM
3nM
mirVana™ miR1 mimic induces significant down
37 Proprietary & Confidential
Cancer Research
38 Proprietary & Confidential
mirVana™ miRNA Inhibitors Enhanced Potency
39 Proprietary & Confidential
0
1
2
3
4
5
6
7
8
9
10
inh
ibit
or
ne
g
inh
ibit
or
ne
g
inh
ibit
or
ne
g
inh
ibit
or
ne
g
inh
ibit
or
ne
g
mir19a miR23a Let7c Let7a miR21
Fold
ch
ange
3 nM
0.3 nM
Display high functionality in reporter assays
mirVana™ miRNA inhibitors
40 Proprietary & Confidential
Let-7 endogenous assay, 10 nM inhibitors, HMGA2 TaqMan
0
2
4
6
8
10
12
14
16
18
Neg ctrl E D mirVana
Rela
tive H
MG
A2 e
xp
ressio
n (a-l
et7
/neg
)Are more potent than lead competitors products
mirVana™ miRNA inhibitors
41 Proprietary & Confidential
0
1
2
3
4
5
6
pM
IR-3
1
pM
IR-2
1
pM
IR-let7
a
pM
IR-1
06a
pM
IR-2
3a
pM
IR-1
9a
pM
IR-1
7
pM
IR-2
4
pM
IR-3
1
pM
IR-2
1
pM
IR-let7
a
pM
IR-1
06a
pM
IR-2
3a
pM
IR-1
9a
pM
IR-1
7
pM
IR-2
4
let7a Inhibitor neg
Avg
. F
old
Ch
an
ge
0
2
4
6
8
10
12pM
IR-3
1
pM
IR-2
1
pM
IR-let7
a
pM
IR-1
06a
pM
IR-2
3a
pM
IR-1
9a
pM
IR-1
7
pM
IR-2
4
pM
IR-3
1
pM
IR-2
1
pM
IR-let7
a
pM
IR-1
06a
pM
IR-2
3a
pM
IR-1
9a
pM
IR-1
7
pM
IR-2
4
miR21 Inhibitor neg
Avg
. F
old
Ch
an
ge Are specific to a given
miRNA target
mirVana™ miRNA inhibitors
42 Proprietary & Confidential
0
1
2
3
4
5
6
7
8
inhibitor 1 year inhibitor 2 months inhibitor 1 week Neg
Fo
ld C
han
ge
3nM
0.3nM
Real-time stability data. miR-23a inhibitors, 3 batches: synthesized
1 yr, 2 mo and 1 wk before test. Storage conditions: 20 M water stocks, +4ºC
• In addition to functional test, integrity of mirVana inhibitors was confirmed by HPLC
• Inhibitors were also stable upon storage for 1 mo at +37C and 40 freeze/thaw cycles
mirVana™ miRNA inhibitors stable for 1 year at +4ºC
43 Proprietary & Confidential
• Lipid nanoparticle reagent
• Designed for systemic liver delivery
• Potent at low dose (<5mg/kg)
• Easy to use
• Robust
• Non toxic
Phalloidin
siRNA
Hoechst
Delivery Reagent — Invivofectamine® 2.0
44 Proprietary & Confidential
Upregulation of miR122 four target genes after miR122 inhibition in the mouse liver
0
50
100
150
200
250
300
350
miR122 inhibitor neg
Hfe
2 p
rote
in l
evels
(%
)
0
20
40
60
80
100
120
miR122 inhibitor neg
Pla
sm
a C
ho
leste
rol
(mg
/dL
)
mirVana miR122 inhibitors + Invivofectamine 2.0 are highly potent in vivo-
similar to the drug candidates from Santaris and Regulus
Invivofectamine 2.0 and suppress microRNA targets
mirVana™ miRNA inhibitors efficiently delivered to liver
45 Proprietary & Confidential
mirVana™ miRNA inhibitors (in vitro, in vivo)
mirVana™ miRNA mimics (in vitro, in vivo) In vitro: 5 and 20 nmols, standard purity, desalted
In vivo: 250 nmols, HPLC purified, in vivo ready (IVR)
Human & Mouse Libraries at 0.25 nmol & 1.0 nmol
RNAiMax® reagent (in vitro)
Invivofectamine® 2.0 reagent (in vivo)
in vitro & in vivo miRNA functional analysis
The Complete Solution