preclinical microrna biomarker screening and functional analysis 2014

8/12/2019 Preclinical MicroRNA Biomarker Screening and Functional Analysis 2014

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Preclinical microRNA Biomarker Screeningand Functional Analysis using

LNA Technology

Dr. Michael Hansen

March 10th2014


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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

214 patents and patent applications (114 issued)

cover products, pipeline and miRNA biomarkers

Business divisions

Exiqon Life Sciences combines leading-edgescientific 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 providingtechnologies 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


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Exiqon Life Sciences: Established one-stop

shop for microRNA research products


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4

Agenda

Introduction microRNA and their potential as biomarkers

The choice of profiling platforms detecting microRNAs

Identification of microRNA biomarkers in FFPE samples

Case study: Identification of skin cancer

Identification of microRNA biomarkers in biofluid samples

Case studies: early detection of CRC in plasma

microRNA biomarkers of nephrotoxicity

Functional analysis tools


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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 inprotein synthesis

tRNATransfer RNA.

Interface betweenmRNA and amino acids

snRNASmall nuclear RNA.

Incl. RNAthat form part

of the spliceome

snoRNASmall nucleolar RNA.

Found in nucleolus, involvedin modification of rRNA

OtherIncluding large and small

RNAs with variousfunctions

RegulatoryRNAs

Includes small and largeregulatory RNAs

siRNASmall interfering RNA.

Active molecules inRNA interference

TASRTermini-associated

sRNAs

PASRPromoter-

associated sRNAs

TERRATelomeric repeatcontaining RNA

piRNASmall RNAs which

are enriched ingerm cells.

miRNAmicroRNA.

Involved in generegulation


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microRNA Biogenesis and Mode of Action

Introduction to microRNAs

1. miRNAs are short ~22nt long RNAs

2. Post-transcriptional regulators of mRNA

3. Regulate at least one third of all human

genes

4. 2387 annotated human microRNAs*5. Phylogenetically well conserved

6. Altered microRNA expression profiles are

associated with a variety of diseases, toxic

response, and injury.

Filipowicz et al. Nature Genetics 2008

*Sanger miRBase release 20.0, July 2013

http://www.mirbase.org


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Tumors of unknown primary origin


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Clinical plasma samples indicate high stability

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Feature Challenge

miRNAs are short Limited flexibility in e.g. capture probe or primer

designhard to achieve sensitivity

miRNAs are highly diverse GC content varies between 5-95%hard toachieve specificity

miRNA family members are

highly homologous

Requires single nucleotide discrimination

miRNAs are often rare

targets

Requires highly sensitive methods for detection

miRNAs are a challenging target for analysis


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LNA technology increases binding affinity

LNA is a bicyclic high affinity RNA mimic with thesugar ring locked in the 3-endo conformation

Stable A-helix with good base-stacking

Increased Tm (Tm increases by 2 - 8C per base) Improved mismatch discrimination

High sensitivity and specificity in hybridizationassays

Obeys Watson-Crick base-pairing rules

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

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

LNA technology is the solution

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LNA is used to adjust the melting temperature

(Tm) of oligonucleotides

Tm adjustments enables:

Increased affinity towards DNA and RNA

Increased specificity (shorter sequences)

Increased flexibility in oligo design

Sequence independence (GC content less important) 11


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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

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Agenda










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miRBase v. 20 statistics


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miRBase v. 20 statistics and

discoverage of microRNAs

2387 human miRNAs

1400 miRNAs detected with < 30 read counts

Sequencing

or profiling ?


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RNA Requirements

qPCR miRNA seq Array

Unknown small/novel miRNAs

miRNA seq qPCR/Array

Budget

Array qPCR miRNA seq

Low High

Yes No

IsomiR analysis

miRNA seq qPCR/Array

Yes No

Validation of limited #miRs

qPCR miRNA seq/Array

Yes No

Low High

Comparison the main metrics


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microRNA input quantity ABSOLUTELY

DICTATES platform to use

NATURE REVIEWS | GENETICS VOLUME 13 | MAY 2012 | 359

PCR or NGS or Array

PCR only

PCR and Array


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Exiqon Services scientists can advise on

validation of novel and known microRNAs

Take advantage of Exiqon Services 8+ years of experience withmicroRNA research and get their support for qPCR validation andfunctional analysis


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Agenda










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Lovn et al. PNAS, 2010 vol. 107 no. 4 1553-1558

Hundreds of publications using LNA microRNA Arrays

PLoS ONE 2011, 6(7): e22484.

Ralfkiaer et al. 2011 Nov 24;118(22):5891-900.

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Certain types of skin cancers can be very difficult to discriminate from non-cancerous inflammatory skin diseases such as

Eczema

Psoriasis

Allergic contact dermatitis

Some are rare conditions with many subtypes and many doctors have neverheard of them

Diagnosis may take months to years and require multiple skin biopsies

Developing a Skin Cancer Diagnostic Profile

Cutaneous T-cell lymphoma

(CTCL)Psoriasis Eczema

A diagnostic dilemma


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MicroRNAs are much more stable in archival FFPE material than

messenger RNAsmore likely to reflect the true nature of the

sample or effect of the drug (less influenced by differences in sample

collection).

microRNAs are stable in typical archival clinical

samples used in early stage biomarker discovery

Fresh Frozen FFPE


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LNA based arrays makes accurate microRNA

profiling form FFPE samples possible

Excellent correlation of

microRNA profiles from

FFPEand fresh frozen

samples for manydifferent tissues


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The classification study includes formalin-fixed and paraffin-embedded (FFPE)biopsies from;

63 patients with cutaneous T-cell lymphoma

85 patients/individuals with benign skin disease or normal skin

Biopsies from the lymphoma patients were sampled during the period 1979-2004, and the clinical characteristics of the cohort were reviewed to establish

the final diagnoses.

Biopsies from the patients with benign skin diseases and healthy controls werecollected after informed consent at Dermatology departments at three different

Hospitals and as part of clinical trials at LEO Pharma A/S.

Study design of the Skin Cancer Case

Ralfkiaer U, et. al., Blood. 2011 Aug 24 (PMID:21865341).

Selection of samples


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Initial statistical analysis of the training set identified 27 miRNAs showing strong (at least50% change) and highly significant (Bonferroni corrected P-values


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The five identified miRNA classifiers,two reference miRNAs and seven

candidate miRNAs reported from

other studies were included.

The nearest shrunken centroid

algorithm identified miR-155, miR-203, and miR-205 as the most

discriminative set of miRNAs.

Results of the validation step using Exiqon qPCR

miR-155 functions as an oncogene inother hematological malignancies.

miR-203 is important in keratinocytedevelopment and may act as a tumor

suppressor.

miR-205 is a tumor suppressorpossibly through targeting VEGF.

Identification of a qPCR-based miRNA classifier


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Agenda










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Estimated new cases in 2013 in the USA: 142,820

Estimated deaths in 2013 in the USA: 50,830

Early diagnosis results in resectable cancer with much improved prognosis

BUT around 50% of patients are diagnosed at Stage III / IV

Stage 5 yr relative

survival

Treatment

0-I 93% Surgery

II 80% Surgery/discretionary

adjuvant chemotherapy

III 58% Surgery/adjuvant

chemotherapy

IV 6.9% Chemotherapy

Unmet need for minimally invasive early

detection test of Colorectal Cancer

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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

analysisCandidate miRNA

discovery screen

4000 patients

Defined miRNA

signature

Pick & Mix panel Multiple controls

Validation Set

miRNA signature

DISCOVERY PHASE VALIDATION PHASE

Development of microRNA Early Detection Test of CRC in Blood

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miRCURY LNA Universal RT microRNA PCR enables

robust biofluid microRNA profiling

TwoLNA enhanced

microRNA specificprimers

SYBR Green detection

Polyadenylation

Universal Reverse

Transcription reaction

1 RT per sample

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The difference in miR-451 and miR-23a

levels can be used gauge hemolysis

Severely affected Moderately affected Not affected

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The degree of hemolysis can vary according to

sample source

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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

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325 samples

Focused

Serum/Plasma

panel Multiple controls

Candidate miRNA

discovery screen

4000 patients

Defined miRNA

signature

Pick & Mix panel Multiple controls

Validation Set

miRNA signature

DISCOVERY PHASE VALIDATION PHASE

Investigations before the final validation

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Serum samples

More homogeneous?

Less inter-hospitalvariation?

Serum/Plasma panel

Signal enhancement viaexosomes?

Serum/Plasma panel


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Exosomes are one of many types of compartment

within biofluids

Blood cells (various types)

Circulating cells (e.g tumour cells)

Cells shed from organs, dying cells, apoptotic bodies (1000-4000nm)

Platelets

Microparticles / microvesicles (1001000 nm)

Exosomes (20120 nm)

Protein complexes (e.g. Ago2 , NPM1)

HDL/LDL


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miRCURY Exosome Isolation Kit

successfully isolates exosomes from urine

Nanosight data comparing enriched exosomes (pellet)

with supernatant


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miRCURY Exosome Isolation Kit improves

microRNA call rate


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Agenda

Introduction microRNA and their potential as biomarkers The choice of profiling platforms detecting microRNAs








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Identifying microRNA biomarkers for kidney

damage

Experimental set up:

Rats injected i.p with either nephrotoxin (treated) or saline (control)

Urine samples kindly provided by Bayer AG, Germany

Time Day 3 Day 5

Treatment Control Treated Control Treated

Number per group N=4 N=4 N=4 N=4

Kidney Necrosis None Mild None Moderate


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Clear separation of sample groups in PCA


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Potential miRNA Biomarkers of Nephrotoxicity


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Summary

LNA provides a clear advantage in the exploration ofmicroRNA as biomarkers in biofluids and tissue samples

Of utmost importance for microRNA profiling in biofluids: High sensitivity

Controlling for inhibitors

Qualification of samples Assessment of data quality

Potential housekeeping genes in Biofluids incl plasma, serum and

urine (-425, -93, -103 and -191not U6U6 is for tissue and cell

samples)

Potential microRNA biomarkers of CRC have been identified,and the importance of monitoring hemolysis has beendemonstrated

Putative microRNA biomarkers of nephrotoxicity were identified


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Agenda

Introduction microRNA and their potential as biomarkers The choice of profiling platforms detecting microRNAs






Functional analysis tools using LNA gapmeRs


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Overview of LNA based tools for Functional

Analysis

X

miRCURY LNA

microRNA Inhibitors

X

X

X

LNA LongRNA

GapmeR

miRCURY LNA

microRNA Target

Site Blockers


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LNA spiking pattern determines mode of action of

Antisense Oligonucleotides (ASOs)

Large gap betweenLNAs (14-16mer)

Enzymaticdegradation of RNA

LNA GapmeRLNA Inhibitor

Short gaps between LNAs

Stable complex with miRNA Masks ribonucleoprotein binding

sites on mRNA and lncRNA

No degradation of RNA, notranslational attenuation


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Key features of LNA Antisense Oligonucleotides

(ASOs)

Cellular uptake is possible without use oftransfection reagent by gymnotic uptake

Typically incubation in micro-molar concentration

of LNA ASO

LNA ASOs will translocate to both nucleus andcytoplasm

In vivo, LNAASOs distribute to most organs

Stein CA, et al. 2010,

Nucleic Acids Res., 38

Zhang Y, et al., 2011

Gene Therapy 18

Straarup EM et al. 2010,Nucleic Acids Res., 38


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Single stranded DNA

DNA/RNA duplex recruits RNase H

RNase H digests the RNA in the

middle of the target sequence

The two generated fragments aredegraded by exonucleases

LNA GapmeR is released andcan go on and catalyze degradation

of another RNA target

LNA GapmeR technology:

An alternative to siRNA


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LNA longRNA GapmeRs have higher success rate

and potency compared to siRNAs

Effective KD of Target X

in BT474 cells using five

different LNA GapmeR

designs


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LNA longRNA GapmeRs have higher success rate

and potency compared to siRNAs

Effective KD of Target Y

in HN5 cells using five

different LNA GapmeR

designs


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Advantages of LNA GapmeRs over siRNA

Short single stranded DNA/LNA oligonucleotides not incorporated into RISC:

No microRNA like effect

No saturation of RISC

No passenger strand

Efficient KD of nuclear retained lncRNA

Unassisted (transfection free) delivery possible with many cell lines

- so called Gymnosis

Active in vivo with no formulation

Different and fewer

off-target effects


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Malat 1 case story

lncRNA ~8KB

Highly conserved in mammals

Ubiquitously expressed and extremely abundant

Expression dysregulated in cancer and associated with metastasisand poor patient prognosis in NSCLC (lung cancer)

RNA interference (RNAi) based Malat1 silencing suggested anessential role in cell cycle and important role in splicing

Three different mouse KD models show that Malat 1 is dispensablefor proliferation and normal development and RNA splicing

Recent results suggest a critical role in the regulation of metastasisof lung cancer cells


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From in vi t roscreening to in v ivoKD using

LNA GapmeRs: Successful KD of Malat1

mEC-H5V, Transfection 50nM, 48h


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mEC-H5V, Transfection 10nM, 48h




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Efficient KD 48h after gymnotic delivery (without transfection)into mEC-H5V



From in v i t ro screening to in v ivo KD using


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



LNA GapmeRs are ideal for studying lncRNA function in live animals

In vivo inhibition of Malat1

48h after single subcutaneous injection of 20mg/kg

Liver Aorta Lung Muscle Heart


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Thank you for your attention

preclinical microrna biomarker screening and functional analysis 2014

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