Klinikum der

Johann Wolfgang Goethe Universität

Frankfurt am Main

The microRNAs: Novel therapeutic

targets in cardiovascular disease

Stefanie Dimmeler

Conflict of interest: Miragen, Exiqon

Non-coding DNA & RNA and

microRNAs Human

Genome

Project:

Graig

Venter,

2003

Source: Wikipedia

Human Genome: ca. 25.000 Genes

(only 2 x more than worms or flies)

Proteins

% non-coding DNA

Human 97 %

Fungi and Plants 7-75 %

Mammals 75-93 %

Eurkaryotes 34-53 %

„Junk“ DNA

3x109 bp

>200nt

transcribed by RNA

Polymerase II

mostly 5‘-cap, polyadenylated

(in part), spliced

poorly conserved

Genome

Transcriptome

Protein-

coding

<200nt

transcribed by RNA

Polymerase II

endogenously processed

well conserved

Small non-coding RNAs

„microRNAs“

72 %

2 %

Long non-coding RNAs

„LncRNAs“

mRNA Protein

Non-coding RNAs

> 2.000 microRNAs > 30.000 lncRNAs

Non-coding sequences

microRNAs: Processing and functions

Drosha

Primary-

miRNA

Pre-miRNA

Dicer

miRNA

Duplex

RISC

Complex Translational

Repression

mRNA

Degradation

Nucleus

microRNA

Gene Gene

microRNA

Exon

Intergenic miRNAs

Exon Exon

Intronic miRNAs

Cluster Single

miRNA

siRNA:

microRNA:

up to

hundreds of

mRNAs

One target mRNA

microRNAs: therapeutic targets and

biomarkers

Pharmacological/

Gene therapy microRNA

therapy

Therapeutic option

Target

Micro RNA

Target

Target Target

Target

One

pathway Networks of genes

Cardiac-specific

miRNA:

miR-208a

miR-

499 miR-208

Muscle enriched

miRNAs:

miR-1

miR-133a/b

miR-499

Release of

miRNAs

Biomarker

(Dimmeler EHJ 2010)

MicroRNAs and postinfarction

repair & regeneration

Seeger et al, ATVB 2013

miR-34a

AAAAAA

Heart function

Apoptosis

AMI Aging

Fibrosis

Vessel growth

Inhibition of age-induced miR-34a improves cardiac

function

Confirmed by:

Bernardo et al PNAS 2012 109:17615-20; Huang et

al Expert Opin Ther Targets. 2014 18:1355-1365.

Fan et al Curr Pharm Des. 2013;19:4865-73.

Ant-Control Ant-34a0

5

10

15

20

25

30

35

40

Eje

ctio

nfr

act

ion

(%)

*

Ant-Control Ant-34a LNA-Control LNA-34a

Day 0 (after myocardial infarction)

Day 14

Eje

ction fra

ction (

%)

Eje

ction fra

ction (

%)

Antagomir-34a LNA-antimiR-34

Boon et al

Nature 2013

miR-34a

AAAAAA

Heart function

Apoptosis

AMI Aging

Fibrosis

Vessel growth

Inhibition of age-induced miR-34a improves cardiac

function

Boon et al

Nature 2013

weeks

Improvement of age-associated cardiac function

MicroRNAs and postinfarction

repair & regeneration

Seeger et al, ATVB 2013

Circ Res 2012; in vivo Circ Res 2014

Circ Res 2012

Direct reprogramming by microRNAs

Regulating cardiomyocyte proliferation by miRs

MicroRNAs to enhance regeneration

Nature 2012

Eulalio et al Nature 2012

MicroRNAs and postinfarction

repair & regeneration

Seeger et al, ATVB 2013

Targeting the

vascular niche

for repair

(and

regeneration?)

miR-92a regulates angiogenesis and

vessel patterning

Angiogenic sprouting &

Vessel formation

Pre-miR-92

miR-92

Pre-miR-Co

Pre-miR-92a

Pre-miR-Co

Pre-miR-92a

Pre-miR-Co

Pre-miR-92a

Pre-miR-Co

Pre-miR-92a

Spheroid model Network formation Matrigel plug model Zebra fish

Bonauer et al

Science 2009

miR-92a inhibition by antimiRs

LNA –modified antimiRs have been tested in non-human primates and were successful and safe in a phase II study (NEJM 2013)

Antagomir-92a

0

20

40

60

80

100

120

PBS 1 mg/kg 8 mg/kg 40 mg/kg

miR

-92

a e

xp

res

sio

n

(%

of

PB

S)

Heart of mice

(van Rooij et al, Circ Res, 2008)

L

N

A

R

N

A

LNA-92a

Heart of mice

DNA LNA

0

20

40

60

80

100

120

miR

-92

a e

xp

res

sio

n

(% o

f L

NA

-Co

)

LNA- LNA- LNA- LNA- LNA- LNA - Co 92a Co 92a Co 92a

Inhibition of miR-92a enhances

neovascularization & recovery after ischemia

0

50

100

150

200

250

300

Ve

sse

ls (

% A

nta

go

mir

-Co

)

Doebele et al, Blood 2010

* *

Bonauer et al, Science 2009

Antagomir-Co

Antagomir-92a

miR-92a

Heart

function

0

2000

4000

6000

8000

10000

12000

dP

/dt

ma

x (

mm

Hg

/se

c)

Angiogenesis

(Matrigel-Model)

Recovery after myocardial

infarction

Inhibition of miR-92a improves cardiac

function after AMI

anterograde retrograde

L

N

A

R

N

A

LNA-92a

DNA

LNA

Dosing: 5 mg/kg heart weight

Intravenous vs catheter-based delivery

of LNA-92a

Expression of miR-92a

Ischemia/reperfusion in

pigs

Inhibition of miR-92a improves cardiac

function after AMI

• Infarct size

% l

eft

ven

tric

le

Anti-miR-92a

* * *

Hinkel et al, Circulation 2013

0

20

40

60

Controls LNA-92a Controls LNA-92a Controls LNA-92a

**

**

****

***** *

Regional myocardial function

Baseline 120 bpm 150 bpm

SE

S [

% c

on

tro

l a

rea

]

• Regional function

Local delivery of LNA-92a

- Reduced infarct size

- Improved global and regional cardiac function

- Reduced inflammation & augmented neovascularization

in a large animal I/R pig model

Pig model of reperfused AMI

miR-92a

eNOS

AntimiR-92a

miR-92a effects in the cardiovascular system

Tumor growth

Hind limb ischemia

Acute myocardial

infarction

Vasculoprotection/

Atheroprotection Metabolism

0

50

100

150

200

250

Blo

od

flo

w (

% v

s.

PB

S) *

PBS

Antagomir 92a

Ischemic leg

Ischemic leg

0

2000

4000

6000

8000

10000

12000

dP

/dt

ma

x (

mm

Hg

/se

c)

(Bonauer et al,

Science 2009)

Re

en

doth

elia

lisatio

n (

%)

0

10

20

30

40

50

60

70

80

NaClcontrol

LNAcontrol

LNA 92a

*

Klf2 Integrin a5

Hmox1

SIRT1 0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

Tu

mo

r a

rea

(cm

²)

Antagomir-Co

AntagomiR-92a *

Endothelial repair and

atheroprotective effect of

miR-92a inhibition:

Ciaconetti et al.

Bas Res Cardiol 2012

Loyer et al. Circ Res 2014

Cardioprotective effect

confirmed in

miR-92a-/- mice: Hinkel et al Circ 2013

untreat.

LNA-Co

LNA-92a

AntimiR-92a reduces body weight and

pericardial fat in db/db mice

• 4.5 month old db/db

mice

• Weekly injection of

LNA-92a

(0.5 mg/kg) for

4 months

db/db

WAT miR-92a_ohne outlier

y WT untr LNA-Co LNA-92a0

100

200

300

rel. e

xp

ressio

n m

iR-92a

vs.

U6 [

% u

ntreated

]

miR-92a

expression

Adipose

tissue

LNA-92a

Pharmacological inhibition of miR-92a:

- was well tolerated, no side effects (mice, pigs) (so far!)

- reduces atherosclerosis

- improved re-endothelialisation after denudation

- improves the recovery of cardiac function after ischemia

in mice and pigs

36 patients, double-blind,

placebo controlled

AntimiR-122 (Miravirsen)

LNA-based antimiRs have been tested in clinical phase IIa trials:

AntimiR-92a- the path to the clinic

Systemic inhibition of some miRNAs may have

adverse effects

e.g. pro-regenerative antimiR-15 or cardioprotectiv

antimiR-34 may support tumor growth

(both miRNAs are tumor suppressors)

Challenges for the development of

miRNA therapeutics

Selective delivery?

Selective delivery of miRNA therapeutics

Aptamers Viral Vectors Light-induced AntimiRs

Cell-specific

Aptamer

Pre-

miRNA

Stick

GFP U6 miRNA/antimiR SFFV

mCD105-LV mCD105-AAV

Aptamer-AAV

l=365 nm

5-6 mW

Local delivery improves the silencing in the heart, but also acts systemically

Catheter-based delivery Antegrade and retrograde infusion in pigs Retro

Ante

Ante high

Development of light-induced antimiRs

In vitro Angiogenesis

A6c + Licht

l=365 nm

5-6 mW

(Schäfer/Wagner et al, Angew. Chemie 2013)

miR-92a Expression

miRNA

Control

Light

Light-induced activation of antimiR-92

enhances sprouting angiogenesis

miR-92a Target gene-Expression

In vitro Angiogenesis

A6c A6c + Light

miR-92a

ITGa5

l=365 nm

5-6 mW

(Schäfer/Wagner et al, Angew. Chemie 2013)

Target gene

de-repression

Angiogenesis

Effect on wound healing?

Light-induced activation of antimiR-92

in skin tissue

The RNA world

Aim: understand the biology of RNAs and develop novel RNA

therapeutics for the treatment of cardiovascular disease

Long non-coding

RNAs:

Reinier A. Boon

Katharina Michalik

Nicolas Jae

Niels Boeckel

Andreas Heumüller

Yosif Manavski

Teresa Hartung

Phillip Neumann

Technical support:

Ariane Fischer

Marion Reinholz

Natalja Lerch

Denise Berghäuser

Bioinformatics:

Shizuka Uchida

David John

Yuliya Ponomareva

MicroRNAs:

Angelika Bonauer

Carmen Doebele

Daniela Penzkofer

Shemsi Demolli

Anne Maron

DZHK

TR-SFB23

Jasmin Wagner

Tina Lucas

Cong Zhao

Timon Seeger

Klinikum der

Johann Wolfgang Goethe Universität

Frankfurt am Main

Andreas Zeiher

Light-induced antimiRs

F. Schäfer, A. Heckel,

Frankfurt

Large animal studies

E. van Rooij, Miragen

R. Hinkel &

Christian Kupatt,

Munich

Stephan Fichtlscherer

Birgit Assmus

Florian Seeger

Till Keller

Kostas Stellos

Christoph Zehendner

Summary

Cholesterol

lowering

White adipose tissue

„Browing“

UCP-1 Cidea PGC1a

Reduced

Inflammation

Prdmt16 Klf2 SIRT1

Putative direct

miR-92a targets:

PCSK9

export eNOS

miR-92a Inhibition or

Deletion

Endothelium

in WAT White

Adipose

Tissue

Liver

Potential pathways that may contribute to miR-92a effects in metabolism