Towards detection of diagnostic microRNA biomarkers in...

Towards detection of diagnostic microRNA

biomarkers in biofluids and the functional analysis of

long non-coding RNA using LNA technology

Michael Hansen, PhD

Technical Specialist, Senior Scientist

Technical Seminar

The University of Hong Kong – October 4th 2016

2

Agenda

● Introduction

● The choice of platform detecting microRNAs in biofluids

● Identification of microRNA biomarkers in biofluid samples

● Case study I: Early detection of CRC in plasma

● Case study II: Identifying microRNAs in ‘exosomes’

● Silencing long RNA using Antisense Gapmer

● LNA™ longRNA GapmeRs – an efficient tool for RNA

knockdown

● Case study: From in vitro screening to in vivo KD using LNA™

GapmeRs: successful KD of Malat1

Exiqon at a glance

Life Sciences Diagnostics

Highlights Locations

• Based on proprietary LNA™ detection technology

• Established one-stop shop for miRNA products

• Promising diagnostic pipeline based on miRNA

• 256 patents and patent applications (205 issued)

cover products, pipeline and miRNA biomarkers

Business divisions

• Exiqon Life Sciences combines leading-edge

scientific expertise in gene expression with our

proprietary LNA™ technology. Our products,

services and scientific staff enable life science

researchers to make groundbreaking discoveries.

Exiqon Diagnostics is the leader in providing

technologies for miRNA biomarker detection. Exiqon

Diagnostics is dedicated in collaboration with partners

to develop novel molecular diagnostic tests for early

detection of diseases and knowledge based treatment

selection.

Exiqon facilities

Distributors

Biostation

A Diverse World of RNA Molecules

Transcriptome

ncRNANon-coding RNA. Transcribed RNA with a structural,

functional or catalytic role

mRNAMessenger RNA.Encodes protein

rRNARibosomal RNA.

Participate in protein synthesis

tRNATransfer RNA.

Interface between mRNA and amino acids

snRNASmall nuclear RNA.

Incl. RNA that form part

of the spliceome

snoRNASmall nucleolar RNA.

Found in nucleolus, involved in modification of rRNA

OtherIncluding large and small

RNA’s with variousfunctions

Regulatory RNAs

Includes small and large regulatory RNAs

siRNASmall interfering RNA.

Active molecules in RNA interference

TASRTermini-associated

sRNAs

PASRPromoter-

associated sRNAs

TERRATelomeric repeat containing RNA

piRNASmall RNAs which

are enriched in germ cells.

miRNAmicroRNA.

Involved in gene regulation

Exiqon Diagnostics – examples of

microRNA biomarker applications

MicroRNAs in biofluids make excellent biomarkers

Adapted from Guire et al. Clinical Biochemistry 2013

● Highly stable in a

range of biofluids

● Minimally invasive

● Biomarkers for:

● Diagnosis

● Prognosis

● Treatment response

● Safety

6

Clinical plasma samples indicate high stability

7

8

Broad product and service portfolio for RNA analysis

Clinical test based on Exiqon qPCR system

The test uses Exiqon cDNA, Master mix and PnM panels.

http://www.interpacediagnostics.com/thygenx-thyramir/

http://www.interpacediagnostics.com/thygenx-thyramir/

10

Agenda

● Introduction to Exiqon







knockdown

● Introduction to long non-coding RNA



Translation to the real world ?

?

miRBase statistics and

discoverage of microRNAs

● ~2500 human miRNAs

● 1400 miRNAs detected with < 30 read counts

Sequencing

or profiling ?

miRBase v. 21 statistics

microRNA input quantity ABSOLUTELY

DICTATES which platform to use

NATURE REVIEWS | GENETICS VOLUME 13 | MAY 2012 | 359

When to use PCR or NGS or Array ?

qPCR

qPCR

qPCR and Exiqon NGS services

microRNA Array, qPCR or NGS

LNA™ technology increases binding affinity

• LNA is a bicyclic high affinity RNA mimic with the

sugar ring locked in the 3’-endo conformation

• Stable A-helix with good base-stacking

• Increased Tm (Tm increases by 2 - 8ºC per base)

• Improved mismatch discrimination

• High sensitivity and specificity in hybridization

assays

• Obeys Watson-Crick base-pairing rules

• Easy to implement in standard oligo synthesis K. Bondensgaard et al., Chem. Eur. J. 2000, 6, 2687

M. Petersen et al., J. Am. Chem. Soc. 2002, 124, 5974

LNA™ technology overview

15

Skeletal muscle Brain/CNS Liver

Images kindly provided by Dr. Ronald Plasterk,

Hubrecht Laboratory, The Netherlands

High specificity and clear differentiation is obtained with miRCURY™ LNA DIG labeled detection probes

Superior specificity with single nucleotide discrimination

16

17

Agenda




● Case study I: Early detection of Colorectal Cancer in plasma




knockdown



Challenges of working with cell-free biofluids

for screening Colorectal Cancer patients

19

Use carrier RNA to improve reproducibility

Carrier RNA (e.g. MS2)

● minimizes sample loss

● increases reproducibility

of RNA isolation

● minimizes selective loss

of structured low GC

content miRNA

(observed in Trizol only

isolations)*

* Kim et al.2012. Mol Cell 46:893 - 895

22

A unique system for microRNA profiling23

Polyadenylation

Reverse transcription

Two LNA™ enhanced microRNA specific primers

SYBR Green detection

Individual assays

Universal RT Complex setup

Exiqon vs TaqMan: Reverse transcription

AAAAAAAAAAAAAAA

AAAAAAAAAAAAAAA

Poly-A tailing Binding of loop primer

TTTTTTTTT

Panels/cards

Multiplex: compromised performance

Exiqon vs TaqMan: PCR reaction

PCR with one specific DNA primerPCR with two specific LNA primers

?

SYBR Green detection Detection with TaqMan probe

170 pages of supplementary data!

Specificity

Quote from paper:

One platform (miRCURY (EX); Exiqon) showed absolute specificity for both miRNA families.

Perfect

specificity!

Exiqon qPCR best platform for

serum/plasma samples

These platforms have

problems with specificity,

so high call rates are

most likely due to false

positives

Quality control of samples by PCR (RNA PCR QC)

● Monitor RNA isolation efficiency

(add RNA spike-ins to lysis buffer)

● Monitor RT and PCR inhibitors (add

RNA spike-ins to cDNA synthesis)

● qPCR monitoring (inter-plate

calibrator, IPC)

● Unique hemolysis indicator

∆Cq(miR-23a – miR-451)

● Biologically relevant endogenous

microRNAs

miRCURY LNA™ Universal RT microRNA

QC PCR Panels are available as ready-to-

use PCR plates (96-well or 384 well).

30

• 50 controls

• 50 CRC patients

• 742 miRNA

screen

• Genome wide

• Multiple QC

check

• Data flagging

• Normalization

• Data analysis

• Quality control

• ROC curve

• miRNA selection

• 325 samples

• Focused

Serum/Plasma

panel

• Multiple controls

Genome wide

screening

Normalization, QC,

processing

Bioinformatics, data

analysis

Candidate miRNA

discovery screen

• 3000 patients

• Defined miRNA

signature

• Pick & Mix panel

• Multiple controls

Validation Set

miRNA signature

DISCOVERY PHASE VALIDATION PHASE

Development of microRNA Early Detection Test of

Colorectal Cancer in Blood

33

Hemolyzed samples have a peak in the

absorbance spectrum

● Monitor hemolysis of serum

and plasma samples by

measuring absorbance on

nanodrop

● observe peak at A414 nm

34

The difference in miR-451 and miR-23a

levels can be used gauge hemolysis

Severely affected Moderately affected Not affected

35

The degree of hemolysis can vary according to

sample source

36

Early Detection Test of CRC in blood plasma

- monitoring pre-analytical variables is crucial

All samples Hospital 2

(discovery)

Remaining Hospitals

(validation)

AUC 0.68 0.81 0.87

Sensitivity (%) 67 75 82

Specificity (%) 65 79 89

37

NGS vs qPCR miRNome profiling

miRNome panel I+II

mic

roR

NA

exp

ressio

n

microRNAs

qPCR is a hypothesis-driven experimental set-up.

Require previous knowledge

Very robust (accurate) detection of even low copy

number of microRNAs

NGS vs qPCR miRNome profiling

– theoretical correlation

All microRNA

mic

roR

NA

exp

ressio

n

microRNAs

Platform sensitivity

NGS is a hypothesis-free experimental set-up.

Possibility to detect ”all” microRNAs.

Bevare of introduction of technical mutations in library

prep and sequencing steps

Library preparation of Biofluid samples

● NEBNext Small RNA Library Kit

● Proprietary optimization of the protocol to

meet challenges of biofluid samples:

● microRNA from serum and plasma

● pg -low concentration of starting material

● Size selection to maximize microRNA reads

● miRNA [15-30 nt], piRNA [15-50 nt]

● Automated XCalibur Gel Cutter

Robust call rate using 10 mill. raw reads per sample

Call

rate

10 M

60 M

High mapping percentage from biofluids

● > 80 % of reads can be mapped in a typical biofluids microRNA

sequencing project

Typical distribution of mapable reads

Exiqon LNA™ microRNA PCR system demonstrates

excellent concordance with NGS results in serum

64

Analysis performed by Exiqon Services in collaboration with

Dr. Ferruccio Bonino, University Hospital of Pisa, Italy.

Global mean normalization is

recommended in screening studies

65

Reference gene normalization is

recommended in validation studies

66

*

* Tip: See Biofluids Guidelines for

selection of standard normalizers

67

Agenda





● Case study II: Identifying microRNAs in ‘exosomes’ of urine



knockdown

● Introduction to long non-coding RNA



Prostate cancer - Unmet need for new biomarkers in the clinic

68

microRNA signals in urine are low

Exiqon developed the miRCURY™ Exosome Isolation kit

to concentrate dilute biofluid samples

Urine (control)

Urine (treated)

69

miRCURY™ Exosome Isolation Kit enables

detection of more microRNAs in urine

0.2 0.2 1.5 10

Whole Exosomes

Urine

Urine starting

volume (ml)70

miRCURY™ Exosome Isolation Kit

successfully isolates exosomes from urine

Nanosight data comparing enriched exosomes (pellet)

with supernatant

Nanosight data kindly provided by

Dr. Ken Howard

Aarhus University

miRCURY™ Exosome Isolation Kit provides

greater yield than ultracentrifugation

● Similar size distribution of pelleted vesicles

● Greater yield of exosomes compared to ultracentrifugation

73

miRCURY™ Exosome Isolation Kit Ultracentrifugation Yield comparison

Storage of cell free urine samples

0

5

10

15

20

25

30

35

40Storage 3°C

AvT0

Av4days

Av7days

Av14days

Av28days

0

5

10

15

20

25

30

35

40

Storage -20°C

AvT0

AvT14

Av/28

Store at

- 80°C

Genome wide microRNA profiling in urine

Samples:

● 50 individuals (healthy and with disease)

● No dietary control

● 3 mL of cell-free urine

Sample collection

● Danish hospitals

● Urine collected during the morning before prostatectomy

● Cryo tubes (DNase / RNase free)

● Stabilizer not applied

● Stored briefly at -20°C, then transferred to -80°C for long term storage 78

A 3-microRNA signature performs in all three cohorts

The urine microRNA signature can identify individuals with prostate

cancer in all three independent cohorts

79

DISCOVERY VALIDATION

What are the recommended starting volumes?

93

Urine samples: 2-5 mL

CSF samples: 1 mL

Cell conditioned media: 1 - 10 mL

Serum/plasma samples: 0.5 - 1 mL

Summary

● Precipitation based exosome isolation provides an efficient

method for biomarker discovery in exosomes

● microRNA signal is increased through exosome isolation in

urine.

● microRNA from exosomes are promising biomarkers of cancer

● LNA™ provides clear advantage in the exploration of microRNA

as biomarkers in biofluids.

94

Overview of LNA™ based tools for Functional Analysis

X

miRCURY LNA™

microRNA Inhibitors

miRCURY LNA™

microRNA Target

Site Blockers

95

XmiRCURY LNA™

mimics

97

Agenda






● Silencing RNA targets using Antisense Gapmer


knockdown



Non-coding RNA: the next great frontier in biology

● Only ~2% of the transcriptome is translated

● 60,500 human genes in GENCODE:

● 19,800 protein coding

● 15,900 lncRNA

● 9,900 small ncRNA

● 14,500 pseudogenes

● lncRNAs perform a range of functions in

both nucleus and cytoplasm

● Lots of interesting biology and drug targets

to be discovered

~ 40,000 ncRNA

“Long non-coding RNAs

may be for transcription

what microRNAs are for

translation”

Central dogma revisited

99

Central dogma revisited

100

Factors that complicate KD of lncRNAs

● Many lncRNA are nuclear retained or have long residence time in

the nucleus – inefficient KD with siRNA!

● lncRNA are often expressed from complex loci with overlapping

sense and antisense transcription – strand specificity of double

strand siRNA a concern!

● Many lncRNA are folded in very tight secondary structures

Overview of LNA™ based tools for Functional

Analysis targeting mRNA and lncRNA

X

X

X

LNA™ LongRNA

GapmeR

102

● 14-16mer LNA™ /DNA ASO

● LNA™ at each extremity enhances

affinity for the target.

● RNase H activity requires a certain

distance (”gap”) between LNAs

LNA™ longRNA GapmeRs

103

● DNA/RNA duplex recruits RNase H

● RNase H digests the RNA in the

middle of the target sequence

● The two generated fragments are

degraded by exonucleases

● LNA™ GapmeR is released and

can go on and catalyze degradation

of another RNA target

LNA™ GapmeR technology:

An alternative to siRNA

Efficient in vitro silencing of nuclear retained lncRNA

by unassisted uptake

Gymnosis - unassisted uptake in vitro

Gymnotic silencing

● Intracellular delivery without using delivery technology e.g.

lipofection

● Gymnotic delivery does not require the use of any

transfection reagent

● Works in most cells lines, both adherent and suspension

cells

● Better predictive power from in vitro to in vivo setting

Bcl-2

Tubulin

C 2.5 5 mM

3 days

SPC2996

5 days

7 days

10 days

Bcl-2

Tubulin

Bcl-2

Tubulin

Bcl-2

Tubulin

Stein CA, et al. 2010, Nucleic Acids Res., 38(1): e3

106

Efficient knockdown with LNA™ GapmeRs irrespective

of type of RNA target and subcellular localization

mRNA

109

Efficient knockdown with LNA™ GapmeRs irrespective

of type of RNA target and subcellular localization

lncRNA (nuclear retained)

111

Antisense LNA™ GapmeRs are far superior

to siRNA with nuclear retained lncRNA

siRNA LNA™ GapmeRs

KD achieved using 1000x less LNA™ GapmeR compared to siRNA

Malat 1 case story

● lncRNA ~8KB

● Highly conserved in mammals and Ubiquitously expressed

● Expression dysregulated in cancer and associated with metastasis

and poor patient prognosis in NSCLC (lung cancer)

● Three different mouse KD models show that Malat 1 is dispensable

for proliferation and normal development and RNA splicing

● Recent results suggest a critical role in the regulation of metastasis

of lung cancer cells

mEC-H5V, Transfection 10nM, 48h

From in vitro screening to in vivo KD using

LNA™ GapmeRs: Successful KD of Malat1

Efficient KD 48h after gymnotic delivery (without transfection)

into mEC-H5V



In vivo inhibition of Malat148h after single subcutaneous injection of 20mg/kg



LNA™ GapmeRs are ideal for studying lncRNA function in live animals

Liver Lung Muscle HeartAorta

First paper with new generation of LNA gapmers

“Yes, the GapmeRs showed similar effects to our KO mice, or even better in the adults

(since there is likely some compensation by other mechanisms). This is actually very similar

to our experience with miRNAs, where we also see better effects with LNAs than in the KO

mice.”

Malat1 knockdown – animal experimental setup

Week 1: 2x10mg/kg

Week 2-5: 2x15mg/kg

Subcutaneous administration

Group size: 6 animals

2 days 5 weeks 15 weeks

Euthanasia and collection of tissue samples after last dose

118

Malat 1 KD in vivo study

12 different tissue samples Biopsies taken from:

• Liver

• Kidney (cross section)

• Stomach

• Jejunum

• Muscle (musculus gastrocnemius)

• Heart

• Lung

• Spleen

• Fat pad

• Ovary

• Skin

• Brain

Total RNA extracted from all samples and

analyzed by qPCR for content of Malat1

119

Effective Malat1 knockdown with LNA™

GapmeR in a broad range of tissues

Long lasting knockdown of Malat1 with LNA™ GapmeR

Malat1 KD – 2 days, 5 & 15 weeks after last dose

Long lasting KD of Malat1 with LNA gapmer

– especially in muscle!

Summary - Functional analysis of non-coding

RNA species

● Multiple types of antisense activity has been achieved with LNATM

oligonucleotides

● microRNA

● mRNA

● lncRNA

● LNATM GapmeR Technology is superior in knocking down mRNA

and lncRNA targets in both cytoplasm and nucleus

● LNATM GapmeR Online Design Tool is freely available for design

of potent ASOs with high hit rates and superior hit quality.

● LNATM antisense technology demonstrates potent activity both in

vitro and in vivo - considered a promising drug development

platform.

130

Aknowledgements

Reinier A. Boon

Katharina M. Michalik

Stefanie Dimmeler

FF Institute for Cardiovascular Regeneration

Goethe University

Frankfurt, Germany

Bodo Brunner

Mike Helms

Sanofi-Aventis Deutschland GmbH

R&D Biologics / Nucleic Acid Therap

Frankfurt, Germany

mRNA

Thank you for your attention

www.exiqon.com

miRNA

lncRNA

circRNA

pseudogenes

GapmeR and

qPCR starter kit

promotion

20% discount

Towards detection of diagnostic microRNA biomarkers in...

Documents

Transcript of Towards detection of diagnostic microRNA biomarkers in...