the micrornas: novel therapeutic targets in cardiovascular...
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![Page 1: The microRNAs: Novel therapeutic targets in cardiovascular .../media/Non-Clinical/Files-PDFs...targets in cardiovascular disease Stefanie Dimmeler Conflict of interest: Miragen, Exiqon](https://reader034.vdocuments.mx/reader034/viewer/2022050515/5f9f6bd78fe4c231dd4d5b97/html5/thumbnails/1.jpg)
Klinikum der
Johann Wolfgang Goethe Universität
Frankfurt am Main
The microRNAs: Novel therapeutic
targets in cardiovascular disease
Stefanie Dimmeler
Conflict of interest: Miragen, Exiqon
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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
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>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
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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
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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)
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MicroRNAs and postinfarction
repair & regeneration
Seeger et al, ATVB 2013
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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
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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
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MicroRNAs and postinfarction
repair & regeneration
Seeger et al, ATVB 2013
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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
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MicroRNAs and postinfarction
repair & regeneration
Seeger et al, ATVB 2013
Targeting the
vascular niche
for repair
(and
regeneration?)
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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
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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
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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
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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
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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
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Pig model of reperfused AMI
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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
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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
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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:
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AntimiR-92a- the path to the clinic
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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?
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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
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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
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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?
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Light-induced activation of antimiR-92
in skin tissue
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The RNA world
Aim: understand the biology of RNAs and develop novel RNA
therapeutics for the treatment of cardiovascular disease
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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
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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
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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