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
% non-coding DNA Human 97 % Mammals 75-93 %
3x109 bp Proteins
Human Genome: ca. 25.000 Genes (only 2 x more than worms or flies)
Fungi and Plants 7-75 %
Eurkaryotes 34-53 %
„Junk“ DNA Source: Wikipedia
Non-coding RNAs Non-coding sequences Genome
Transcriptome
72 % Proteincoding
2%
Small non-coding RNAs „microRNAs“
mRNA
Protein
Long non-coding RNAs „LncRNAs“
<200nt
>200nt
transcribed by RNA Polymerase II
transcribed by RNA Polymerase II
endogenously processed
mostly 5‘-cap, polyadenylated (in part), spliced
well conserved
poorly conserved
> 2.000 microRNAs
> 30.000 lncRNAs
microRNAs: Processing and functions Intronic miRNAs
Intergenic miRNAs microRNA
microRNA Exon
Exon
Exon
Single miRNA
PrimarymiRNA
Gene
Gene
Cluster
Drosha
Nucleus
miRNA Duplex
One target mRNA
microRNA:
up to hundreds of mRNAs
Pre-miRNA
Dicer
siRNA:
RISC Complex
Translational Repression
mRNA Degradation
microRNAs: therapeutic targets and biomarkers Biomarker
Muscle enriched miRNAs: miR-1 miR-133a/b miR-499
Therapeutic option Pharmacological/ Gene therapy
miR499
microRNA therapy
Micro RNA
Target
Target
miR-208
Release of miRNAs
Target
Target Target One pathway
Cardiac-specific miRNA: miR-208a
Networks of genes
(Dimmeler EHJ 2010)
MicroRNAs and postinfarction repair & regeneration
Seeger et al, ATVB 2013
Inhibition of age-induced miR-34a improves cardiac function Antagomir-34a 40
LNA-antimiR-34
*
Aging
miR-34a AAAAAA
30 25 20 15 10 5 0
Ant-Control Ant-34a Ant-Control Ant-34a
LNA-Control LNA-34a
Day 0 (after myocardial infarction) Day 14
Boon et al Nature 2013
Apoptosis
Ejection fraction (%)
AMI
fraction Ejection Ejection fraction(%) (%)
35
Vessel growth Fibrosis
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.
Heart function
Inhibition of age-induced miR-34a improves cardiac function Improvement of age-associated cardiac function AMI
Aging
weeks
miR-34a AAAAAA
Boon et al Nature 2013
Apoptosis
Vessel growth Fibrosis
Heart function
MicroRNAs and postinfarction repair & regeneration
Seeger et al, ATVB 2013
MicroRNAs to enhance regeneration Direct reprogramming by microRNAs
Circ Res 2012; in vivo Circ Res 2014
Regulating cardiomyocyte proliferation by miRs
Circ Res 2012
Nature 2012 Eulalio et al Nature 2012
MicroRNAs and postinfarction repair & regeneration Targeting the vascular niche for repair
(and regeneration?)
Seeger et al, ATVB 2013
miR-92a regulates angiogenesis and vessel patterning Pre-miR-92 miR-92 Bonauer et al Science 2009
Spheroid model
Pre-miR-Co
Pre-miR-92a
Angiogenic sprouting & Vessel formation Network formation
Pre-miR-Co
Pre-miR-92a
Matrigel plug model
Zebra fish
Pre-miR-Co
Pre-miR-Co
Pre-miR-92a
Pre-miR-92a
miR-92a inhibition by antimiRs Antagomir-92a
LNA-92a DNA LNA R N A
Heart of mice
(van Rooij et al, Circ Res, 2008)
100 80 60 40 20
100 80 60 40 20 0
0 PBS
1 mg/kg
8 mg/kg 40 mg/kg
Heart of mice
120 miR-92a expression (% of LNA-Co)
miR-92a expression (% of PBS)
120
L N A
LNACo
LNA92a
LNACo
LNA92a
LNACo
LNA 92a
LNA –modified antimiRs have been tested in non-human primates and were successful and safe in a phase II study (NEJM 2013)
Inhibition of miR-92a enhances neovascularization & recovery after ischemia Recovery after myocardial infarction
Angiogenesis (Matrigel-Model)
Antagomir-Co
250
miR-92a
*
*
200
Heart function
150
dP/dt max (mmHg/sec)
Vessels ( % Antagomir-Co)
300
100
50
0
Doebele et al, Blood 2010
Antagomir-92a
12000 10000 8000
6000 4000 2000 0
Bonauer et al, Science 2009
Inhibition of miR-92a improves cardiac function after AMI Ischemia/reperfusion in pigs
Intravenous vs catheter-based delivery of LNA-92a anterograde
Expression of miR-92a
LNA-92a DNA LNA R N A
retrograde
L N A
Dosing: 5 mg/kg heart weight
Inhibition of miR-92a improves cardiac function after AMI • Infarct size
• Regional function Regional myocardial function 60
*
*
Controls
*
SES [% control area]
% left ventricle
Anti-miR-92a
LNA-92a
Controls
LNA-92a
Controls
LNA-92a
**
40 **
**
*
*** ** **
20
0 Baseline
120 bpm
150 bpm
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 Hinkel et al, Circulation 2013
Pig model of reperfused AMI
miR-92a effects in the cardiovascular system
12000
0.800
10000
0.700
*
8000
Cardioprotective effect 6000 confirmed in 4000 miR-92a-/- mice: 2000 Hinkel et al Circ 2013 0
Hmox1 SIRT1
(Bonauer et al, Science 2009)
200
* PBS
150 100 Ischemic leg
50
Integrin a5
Vasculoprotection/ Atheroprotection
Antagomir 92a
0
Reendothelialisation (%)
250
miR-92a Klf2
Antagomir-Co AntagomiR-92a
0.600 0.500
0.400 0.300 0.200 0.100 0.000
eNOS
Hind limb ischemia Blood flow (% vs. PBS)
Tumor growth
AntimiR-92a Tumor area (cm²)
dP/dt max (mmHg/sec)
Acute myocardial infarction
*
80
Endothelial repair and 70 60 atheroprotective effect of 50 miR-92a inhibition: 40
30 Ciaconetti et al. 20 Bas Res Cardiol 2012 10
Loyer0 et al. Circ Res 2014 Ischemic leg
NaCl LNA LNA 92a control control
Metabolism
AntimiR-92a reduces body weight and pericardial fat in db/db mice • •
db/db
4.5 month old db/db mice Weekly injection of LNA-92a (0.5 mg/kg) for 4 months
untreat. LNA-Co LNA-92a rel. expression miR-92a vs. U6 [% untreated]
WAT miR-92a_ohne outlier 300
200
LNA-92a miR-92a expression Adipose tissue
100
0 y WT
untr
LNA-Co 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
LNA-based antimiRs have been tested in clinical phase IIa trials:
36 patients, double-blind, placebo controlled AntimiR-122 (Miravirsen)
AntimiR-92a- the path to the clinic
Challenges for the development of miRNA therapeutics
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) Selective delivery?
Selective delivery of miRNA therapeutics Antegrade and retrograde infusion in pigs
Catheter-based delivery
Retro Ante Ante high
Local delivery improves the silencing in the heart, but also acts systemically
Aptamers
Viral Vectors SFFV
GFP
U6
Light-induced AntimiRs
miRNA/antimiR
PremiRNA l=365 nm 5-6 mW
Stick Cell-specific Aptamer
mCD105-LV
mCD105-AAV Aptamer-AAV
Development of light-induced antimiRs
l=365 nm 5-6 mW
miRNA
miR-92a Expression Control
Light
(Schäfer/Wagner et al, Angew. Chemie 2013)
Light-induced activation of antimiR-92 enhances sprouting angiogenesis miR-92a Target gene-Expression
miR-92a ITGa5 l=365 nm 5-6 mW
In vitro Angiogenesis A6c
A6c + Light
Target gene de-repression Angiogenesis
Effect on wound healing? (Schäfer/Wagner et al, Angew. Chemie 2013)
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
MicroRNAs: Angelika Bonauer Carmen Doebele Daniela Penzkofer Shemsi Demolli Technical support: Anne Maron Ariane Fischer Jasmin Wagner Marion Reinholz Tina Lucas Natalja Lerch Cong Zhao Denise Berghäuser Timon Seeger
DZHK
TR-SFB23
Bioinformatics: Shizuka Uchida David John Yuliya Ponomareva
Klinikum der Johann Wolfgang Goethe Universität Frankfurt am Main
Andreas Zeiher Stephan Fichtlscherer Birgit Assmus Florian Seeger Till Keller Kostas Stellos Christoph Zehendner Large animal studies E. van Rooij, Miragen R. Hinkel & Christian Kupatt, Munich
Light-induced antimiRs F. Schäfer, A. Heckel, Frankfurt
Summary Potential pathways that may contribute to miR-92a effects in metabolism miR-92a Inhibition or Deletion Putative direct miR-92a targets:
SIRT1
Liver
PCSK9 export
Cholesterol lowering
Prdmt16
UCP-1
Cidea
White Adipose Tissue
PGC1a
White adipose tissue „Browing“
Klf2
Endothelium in WAT
eNOS
Reduced Inflammation
