invitrogen’s rnai - sirna & mirna...

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