Bioinformatics in the

development and

understanding of

oligonucleotide drugsMorten Lindow

Group leader, Bioinformatics

Santaris Pharma A/S

Aalborg University November 2010

Therapeutic antagonism of microRNAs

Most drugs work on proteins

…GGGCGACACUCCACCAUGAAU……

translation

CGCUGUGAGGUG

GUA

|||||||||||||||

Chemically diverse

3D

Many types of interactions with ligands

Diverse cellular compartments

Hard to develop regulator

Chemically simple

1D

Fewer cellular compartments

Easy to develop regulator

RNAseHRISC

RISC

Enzyme-directing oligonucleotides

ASOs siRNA miRNA

Designing oligos as RNA regulators

miRNAssiRNAs

Natural selection

ASOs

Intelligent design

(oligoinformatics)

TargetSurveyor.pl

7/10 have IC50<5nM

Conserved across species

Minimal off-targets

…

Lindow et al.,

PLoS Comput Biol. 2007

Plant genomes have

>2000 genomic pre-miR

structures with

complementarity to

mRNA. Just waiting for

the right niche to show up.

• Vast compound libraries

• Combinatorial chemistry

• High through put screening on

primary target

• Specificity screen on related

receptors

• Tolerability screen

Lead optimization

Clinical development

CGCUGUGAGGUGGUA

|||||||||||||||

CCCCCUGAUGGGGGCGACACUCCACCAUGAAUCACUCCCCUG

~2 m

onth

s

TargetSurveyor.pl

Automated oligosynthesis

qPCR

23 optimized leads

2 in pre-clinical dev

2 in phase 1

1 in phase 2

~2 y

ears

Both Nature and Business make

use of oligonucleotides

LNA-antimiR – miravirsen

- an oligo to regulate an oligo

RISC

miR-122

miravirsen

Inhibits expression

8 LNA,

7 DNA

Wienholds et al, Science (2005)

Antagonism of miR-122 leads to

reduced plasma cholesterol

Elmen&Lindow et al, Nature 2008

Esau et al, Cell Metab 2006

Kreutzfeldt et al. Nature 2005

Single i.v. injection of

miravirsen in mice

Three i.v. injections of miravirsen

in African green monkeys

miR-122 and hepatitis C virus

HCV is a single stranded RNA virus

HCV genome resembles an mRNA

170 million infected worldwide

Current treatment often ineffective and

with serious side effects

2x miR-122 binding

sites in 5‟NTR Viral replication

Jopling et al, Science 2005

Elmen&Lindow et al., Nature 2008

HCV is the target indication for

miravirsen

?

?

?

?

Can miravirsen reduce HCV-load in vivo?

Can HCV mutate to escape miravirsen treatment?

What is the physiological role of miR-122?

Does miravirsen have any off-targets?

miravirsen reduces HCV in

chimpanzees

Lanford et al, Science 2010

Can HCV mutate to escape

miravirsen treatment?

Cooper et al, J Hepat, 2009

Direct-acting small molecule inhibitor

of viral RNA polymerase

Period of treatment

Rebound during

treatment

Lanford et al, Science 2010

LNA-antimiR targeting the host factor

miR-122

Rebound 2 weeks after

end of treatment

Deep sequencing of virus from

treated animalsHCV specific primers to amplify miR-122 binding region

454 deep sequencing

73,000 to 214,000 reads at 4 time points

Does frequency of variants change?

No evidence of viral escape from

miravirsen!

What is the physiological role of miR-122?

Are miR-122 targets upregulated after miravirsen

treatment? Can this be used as an efficacy

endpoint?

Is there a non-sequence specific effect of treatment

with LNA-oligos?

Is there a sequence specific effect of treatmentwith

miravirsen? (off-target effect on mRNAs?)

Antagonism of miR-122:

Effects on gene expression

Distance between transcriptomes

Data from Elmen & Lindow et al, Nature 2008

5 fat mice treated with miravirsen

5 fat mice treated with 2 mismatch control

5 fat mice treated with saline

Are miR-122 targets upregulated

after miravirsen treatment?

RISC

miR-122

Inhibits expression

On the level of the individual gene only a few targets are significantly upregulated

(n=5) and only with about 25%

Sequence and expression analysis

combined yields a miR-signature

All ~20 000 genes

mRNA changes

antimiR

Control

predicted miR-122 targets

log2(antimiR/control)0 means no change

for each gene:

Expression analysis Sequence analysis

Null-hypothesis:Are the distributions of

background and

predicted targets

identical?

Test: two-sided Kolmogorov-

Smirnov

Response to miravirsen treatment

log2(miravirsen/saline)

Density

14503

mRNAs with

no site

879 mRNA

with

miR-122 site

0 0

miR-signature is an efficacy endpoint

p=6.60E-27

Microarray data from four different experiments

Model Liver Northern Serum cholesterol levels

Monkey

Normal diet

Monkey

High-fat diet

Mouse

Normal diet

Mouse

High-fat diet

On gene level: only little consistency

between mice and primates

Pathway level changesUnbiased pathway analysis

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;"*&)5/.('%,

5"8-.#%)%<-#%,,+"()*%:%*,

!"#$%&

'%(%)*+,$

monkey mouse

high fat mRNA changes mRNA changes

normal mRNA changes mRNA changesHagedorn et al, unpublished

Pathways responding consistently

across diets and species

Cholesterol synthesis enzymes are

downregulated across diets and species

Hagedorn, et. al, unpublished

Systems biology successfully

explains pharmacology!

Off-target effects?

RISCmiR

antimiR

Yes, the antimiR binds and

derepress targets of the miR

Direct effect on (partially)

complementary targets?

cells or animals

treated with oligo

or siRNA

Untreated or

mock treated

animals

1. Calculate fold

change in the

concentration of

each mRNA

2. Rank mRNAs by fold change

up in treated down in treated

3. Sequence analysis: Mark presence of all „words‟ of length k

4. Test if a word is over/under represented in one end of the ranked list

Sylamer, Enright lab, 2009

Obad et al., Nature Genetics, in press

6 nt words 7 nt words

Sites complementary

to miR-122

Sites complementary

to oligo

Noise level

No evidence of direct regulation of

mRNAs by LNA-antimiRs!

★Preclinical tox: “SPC3649 (miravirsen) was tolerated at doses that far

exceed those intended for human clinical use”

★Phase Ia study completed: Single dose, dose-escalationin healthy

volunteers

★Phase Ib completed: Multiple ascending doses in healthy volunteers

★Phase II ongoing: Hepatitis C patients

miravirsen is in clinical trials

Phase 1a. Dose dependent reduction of plasma

cholesterol in humans

Summary

Bioinformatics: Sequence analysis facilitates

oligonucleotide drug development

Design of specific, potent and tolerable oligonucleotide drugs

Methods for expression data analysis on efficacy and specificity

Systems biology: miR-122 coordinates expression level of

cholesterol biosynthesis enzymes

HCV-treatment: Miravirsen appears to be a promising new

HCV treatment

First time a microRNA is a drug target

No escape mutants in treated chimpanzees

Awaiting phase II data

Acknowledgements

Santaris microRNA research group

Sakari Kauppinen

Susanna Obad

Joacim Elmen

Santaris Bioinformatics group

Andreas Petri

Lena Hansson (now Intomics)

Elfar Torarinson (sysadm consult)

Peter Hagedorn (post doc, now at LEOPharma)

Center for biological sequence analysis, DTU

Henrik Bjørn Nielsen

More….. mirmaid.org microRNA information for computers, open

source API to miR data resources

bio-geeks.com, blog with other geeks about bioinformatics

RNA.dk – homepage for new big project about Enabling RNA

Therapeutics

oligoinformatics.org, New. Specialized blog on oligos and

informatics

Student project inquiries: mol@santaris.com