Review
Cardiovascular RNA Interference Therapy
The Broadening Tool and Target Spectrum
Wolfgang Poller, Juliane Tank, Carsten Skurk, Martina Gast
Abstract: Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding
disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the
human genome is insufficient because ncRNA variants have been associated with important human diseases.
Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic
targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they
are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic
strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing.
RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up
to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and
key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as
a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of
RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which
underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of
the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference
against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under
certain restrictions. (Circ Res. 2013;113:588-602.)
Key Words: cardiovascular diseases ■ genetic therapy
■ RNA, long untranslated
■
■
C
lassical studies several decades ago have already shown
the presence of large amounts of RNAs that are transcribed
but do not encode proteins. Only part of these noncoding
RNAs (ncRNAs) could later be explained by mRNA splicing,
or was specifically involved in the translation machinery and
its regulation (eg, transfer RNAs [tRNAs], ribosomal RNAs),
whereas the overwhelming majority remained functionally
cryptic for a long time. Rather recent studies then led to the
discovery of entire new classes of small RNAs, generated not
only by novel biosynthetic pathways and mediating regulatory functions (eg, miRs [microRNA]), but also of unusually
large long ncRNAs (lncRNAs) of still uncertain functional
significance. Cumulative evidence derived from multiple genetic, biochemical, and other experimental and clinical studies during the past decade strongly suggests a central role of
ncRNAs in the genetic programming of complex organisms,
during their development and in health and disease. The observation of a steeply increasing fraction of ncRNAs in the
immunity, innate ■ nanoparticles
RNA, short untranslated
■
RNA interference
genome during evolution, from simple to highly complex organisms, in contrast to an only modest increase in the number
of protein-coding genes, is consistent with the assumption of
an overwhelming role of the ncRNA species1 in higher organisms. Regarding disease pathogenesis, it has become evident
from the Encyclopedia of DNA Elements (ENCODE) project2 that confinement to the analysis of protein-coding regions
of the human genome is inadequate because many noncoding variants are associated with important human diseases.
Inclusion of the noncoding genomic elements in pathogenetic
studies seems mandatory and 1 approach is comprehensive
transcriptome mapping encompassing protein-coding genes,
as well as small and large ncRNAs.
Importantly, ncRNA can be both tools (eg, short interfering
RNAs [siRNAs]) and targets (eg, lncRNAs) of novel therapeutic strategies. Thus, RNA interference (RNAi) may be
considered as a highly versatile general-purpose tool for the
silencing of not only specific single protein-encoding genes
Original received May 5, 2013; revision received July 16, 2013; accepted July 19, 2013. In June 2013, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 13.67 days.
From the Department of Cardiology and Pneumology, Campus Benjamin Franklin, Charité–Universitätsmedizin Berlin, Berlin, Germany.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
113.301056/-/DC1.
Correspondence to Wolfgang Poller, MD, Department of Cardiology and Pneumology, Charité Centrum 11 (Cardiovascular Medicine), Charité–
Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail [email protected]
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.113.301056
588
Poller et al Cardiovascular RNA Interference 589
Nonstandard Abbreviations and Acronyms
AAV
ASO
dsRNA
lncRNA
MALAT1
MI
MIAT
miR
ncRNA
RNAi
RNA-seq
shRNA
siRNA
tRNA
adeno-associated virus
antisense oligonucleotide
double-stranded RNA
long noncoding RNA
metastasis-associated lung adenocarcinoma transcript 1
(synonymous: NEAT2)
myocardial infarction
myocardial infarction-associated transcript
microRNA
noncoding RNA
RNA interference
RNA sequencing
short hairpin RNA
short interfering RNA
transfer RNA
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
via mRNA, but also against non–protein-coding RNA targets.
Recent studies have demonstrated therapeutic potential of organ-targeted RNAi on the basis of viral vectors3 or synthetic
RNA,4 and developed therapeutic strategies on the basis of
modulation of miR functions.5–9 Both RNAi-mediating molecules and miRs are of small size, but only parts of a far larger
spectrum of ncRNAs arising from the human genome up to
thousands of base pairs in size. lncRNAs range from ≈200 nt
to >100 kb in length, excluding infrastructural or regulatory
RNAs (tRNAs, miRs, siRNAs, etc). Transcriptional surveys
in mammals suggest that lncRNAs may outnumber proteincoding genes,1,10 but biological functions have been assigned
only to a minute fraction of them. Overall, only a small part
of the noncoding human genome has been investigated and
explored with regard to possible therapeutic implications for
cardiovascular medicine.
RNAi: The Broadening Target Spectrum
RNA has been the subject of landmark discoveries with catalytic RNA in 1989, splicing in 1993, RNAi in 2006, ribosomal
structure in 2009, and others, which have led to a radical reassessment of RNA functions during evolution and in human
diseases. Only ≈1.2% of the human genome encodes for proteins and the 20 000 recognized human proteins are similar
in number with orthologous functional overlaps to those in
animals down to the simple nematode worm. This revolutionary insight has triggered extensive research into the functions of the noncoding genome and also led to an extended
theory of evolution.11 Certain very ancient ncRNA systems
are well conserved,12 whereas others have diversified during
evolution13–20 (Figure 1). The evolution of miRs has already
been traced, allowing to distinguish ancient from recent miR
families. Regarding the use of animal models of human diseases for therapeutic proof-of-principle studies, it should be
noted that for many miRs17,18 and lncRNAs there is high sequence conservation across species, whereas some lncRNAs
found in other mammalians do not seem to exist in humans.
As certain capacities, for example, cardiac regeneration has
been lost during evolution, one might speculate this is related
to permanent loss of ncRNAs with complex morphogenic
functions. In searches for ncRNA with therapeutic potential,
conservation across species can be a selection criterion, but
important candidates may exist only in humans and thus be
detectable only by comprehensive transcriptome mapping in
patients. Truly novel therapeutic thinking has been triggered
by “Learning from Nature” and this has started with RNAi and
miRs but will certainly continue with other RNA species, including lncRNAs. A fascinating aspect of current therapeutic
exploration of the noncoding genome is the fact that peculiar
new RNA players and totally unexpected mechanisms of cellular function are continuing to arise.
It should be emphasized that large groups of essentially important small RNAs (miRs, RNAi triggers) went undetected
for long times also for technical limitations. Only a small fraction of these small RNAs was functionally explained (eg, regarding the translation machinery and its regulation, such as
ribosomal RNA, tRNA, RNase P). When miRs and RNAi triggers were discovered in the 1990s, this revolutionized thinking
about RNA functions and introduced a huge number of novel
players involved in cellular functions and disease pathogenesis. Although this first expansion in the number of biologically
and pathogenetically important RNA species already triggered
a huge number of important and highly innovative studies during the past 2 decades, it seems clear that a second expansion
arrived with the discovery of a huge number of lncRNAs (and
processing products) about which we do not know much more
than about miRs when they were discovered. Again, technological progress plays a key role in the further elucidation of
highly complex new systems. Knowledge of the mechanisms
by which lncRNAs act is sparse,19 and only few well-characterized lncRNAs have to date given clues about their cellular
roles. Current evidence strongly supports pervasive transcription of mammalian genomes, but the functional biological
significance of many “Dark Matter” transcripts (novel RNAs
of unknown function) remains enigmatic.20 Notwithstanding
these limitations, first functional and mechanistic insights have
begun to emerge and warrant their investigation regarding
pathogenetic and therapeutic implications, if they are found to
be dysregulated in certain diseases. As discussed by important
recent reviews and below, there is sufficient evidence to investigate them also in certain cardiovascular disorders.
Technological Progress as Key Driving Force
Novel technologies were importantly involved in the discovery of multiple classes of small RNAs, including miRs,
snRNAs, snoRNAs, siRNAs, piRNAs, tiRNAs, spliRNAs,
sdRNAs, and others, and of lncRNAs thousands of which
have been identified in intergenic regions alone. On the one
hand, this immense expansion of regulatory RNAs not encoding for proteins already requires a huge amount of research to
identify biological function of individual RNAs and to characterize their molecular mechanisms of action. On the other
hand, there are arguments to suggest that traditional extraction of isolated functions, for example, gain/loss-of-function
models or biochemical studies in vitro, may not be suitable
to clarify their overall biological role because highly complex interaction networks are involved. An interesting recent
theory aims at unified understanding of the extremely broad
590 Circulation Research August 16, 2013
A
Simple
Organisms
Highly Complex
Organisms
“Basic Molecules“
“Advanced Molecules“
de novo arising from the noncoding
parts of the genome
Protein-Coding Genes
Structural RNAs
Noncoding Transcripts
(mRNAs, rRNAs, tRNAs)
encompassing a broad and diverse
range of noncoding RNA
First organisms arising from
(siRNAs, miRNAs, lncRNAs …)
“RNA World“
B
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
B
Noncoding genome
evolution has reached
≈ 99% of total genome
in humans
C
miRNA family evolution
reaches 63 in mammals
(1424 separate miRNA genes
in humans)
Figure 1. RNA interference: the broadening target spectrum. A, The basic molecules of life already found in the earliest and most
simple organisms are increasingly supplemented, during the evolution to complex species, by molecules needed for correct embryonic
development and homeostatic stability of their morphology and functions. B, Whereas the number of protein-coding genes remains similar
from simple to complex species, it is the noncoding part of the genome that increases dramatically with morphological complexity to ≤99%
in humans. C, Few types of noncoding RNAs arising from the noncoding genome have been phylogenetically mapped in depth. Thus,
investigation of microRNA (miR) family evolution revealed impressive increases with the advent of vertebrates, and ancient miRs families
can well be distinguished from those more recently arising.13–21 Reprinted from Heimberg et al18 with permission of the publisher. Copyright
©2008, National Academy of Sciences, USA. lncRNA indicates long noncoding RNA; smRNA, microRNA; rRNA, ribosomal RNA; iRNA, short
interfering RNA; and tRNA, transfer RNA.
spectrum of RNA species. This hypothesis attempts to explain
how mRNAs, transcribed pseudogenes, and lncRNAs talk to
each other using miR response elements as letters of a new
language.21 The authors propose that this competing endogenous RNA activity forms a large regulatory network across
the transcriptome, fundamentally expanding the functional
genetic information in the human genome and playing important roles in pathological conditions.22,23
Toward Comprehensive Human
Transcriptome Mapping
When considering therapeutic targets for RNAi, the obvious
candidates are well-characterized protein-encoding genes
with already established cardiovascular pathogenic relevance,
in particular if no conventional inhibitory drug is available
for the coded protein. As reviewed below, however, there is
already sufficient evidence to warrant also the evaluation of
several lncRNAs dysregulated in cardiovascular diseases as
novel targets for RNAi. We discuss which selection criteria
one might use to identify lncRNAs of therapeutic relevance
in the cardiovascular system. Selection criteria are needed, indeed, because of the vast number of noncoding transcripts,10,24
which are identified by state-of-the-art technologies in normal and diseased hearts.25 Until recently, transcriptome mapping based on microarrays failed to capture the full catalogue
of transcripts and their variations.26 Advances in sequencing
technology now allow, however, the reconstruction of truly
complete transcriptomes by deep RNA sequencing (RNA-seq)
in the absence of a reference genome.27 Furthermore, comprehensive RNA quantification is now possible from very small
amounts of cellular material28 (eg, from cardiovascular bioptic
material taken from patients during diagnostic procedures).
Whereas microarrays mainly detect transcripts with more or
less well-defined functions; RNA-seq includes vast numbers
of transcript sequences of unknown functional relevance and
context. In the cardiovascular field, RNA-seq has revealed cardiac transcriptomic signatures for physiological and pathological hypertrophy,29 including not only overall gene expression
analysis, but also individual transcript isoforms, novel spliced
Poller et al Cardiovascular RNA Interference 591
exons, novel alternative terminal exons, novel transcript clusters, and lncRNAs.30
Broad Versatility of RNA Structures
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Different from DNA, RNAs are carrying information not only
in their linear sequences of nucleotides (primary structure),
but also local nucleotide pairing creates secondary structures,
for example, hairpins, and interaction among distantly located
sequences create tertiary structures. In fact, this extreme structural versatility needs to be considered for RNAs as therapeutic tools (short hairpin RNAs [shRNAs], siRNAs, antisense
oligonucleotides [ASOs], aptamers), as well as therapeutic
targets (eg, lncRNAs). The use of computational prediction
methods and recent experimental techniques to probe RNA
structure by high-throughput sequencing has enabled genome-wide measurements of RNA structure and may in the
future reveal a picture of the RNA structurome.31 The plethora
of RNA types, sequences, and structures, which were created
by evolution, is a treasure trove of potential therapeutic tools
and targets. However, criteria need to be developed so that
promising candidates for further in-depth mechanistic, pathogenetic, and therapeutic studies can be selected.
RNAi: Versatile Principle for Silencing of
Protein-Coding Genes and ncRNA Systems
Classification of Therapeutic RNAs
Regarding the plethora of possible therapeutic ncRNAs, one
should distinguish RNAs addressing a single gene encoding a protein with well-characterized function, for example,
shRNAs mediating classicalRNAi, others addressing multiple
protein-encoding genes, for example, miRs targeting multiple
3′-untranslated regions, lncRNAs interacting with multiple
RNAs and RNA–protein complexes, and processing products from lncRNAs. Whereas all types may have therapeutic
potential; current technologies do not permit therapeutic delivery of large RNA molecules in vivo, whereas the delivery
of small RNAs mediating posttranscriptional gene silencing
(PTGS) has made significant progress toward the clinic. In
this context, it is of interest that recent work on lncRNA processing has revealed new biosynthetic pathways generating
small RNAs from lncRNA precursors.32–34 It seems entirely
possible that hitherto unknown common biosynthetic pathways generate novel classes of small RNAs from lncRNAs,
in a similar way as the large spectrum of miRs is generated
from prepro-miRs via Dicer and Drosha. Such novel classes
of small ncRNAs could then be targeted by already available
PTGS technologies, including RNAi.
Similarities and Differences Between RNAi
and miRs
Regarding the mechanisms of action of ncRNAs, we first
review mechanisms of PTGS dependent on small RNAs, including siRNAs, shRNAs, and miRs.35,36 Second, we discuss
mechanisms beginning to emerge for lncRNAs, as well as first
data on functions of lncRNA processing products. When the
mechanisms of ncRNA actions are considered with the perspective of possible therapeutic application, one may distinguish different categories defined by their levels of specificity.
When an RNAi trigger is chosen to silence a single target gene
and its encoded protein specifically, the situation resembles
traditional drug therapies with well-defined single targets,
and also the concept of traditional gene therapy overexpressing single proteins. A second, less well-defined situation
arises when miRs are either enhanced or suppressed because
1 single modulated miR influences the function of multiple
genes and thus multiple encoded proteins. If the function of
a single miR within a specific cell or organ has been well defined, the consequences of its modulation should nevertheless
be predictable. As exemplified below, however, a single miR
may exert grossly different actions dependent on cell type and
pathogenic situation, so that miR modulation therapies require
precise cellular targeting. lncRNA systems have even more
complex and context-dependent mechanisms of action, so that
therapeutic translation will certainly take time and probably
the development of novel therapeutic tools.
Classical RNAi
Regarding RNAi, its original discovery had revealed that introduction of double-stranded RNA (dsRNA) into a cell can
trigger a target sequence-specific gene silencing mechanism.
Within the cell, the dsRNA is processed into ≈22-nt fragments
by an evolutionary ancient, endogenous machinery. This endogenous RNAi was first discovered and analyzed in plants
and nematodes, but later was found to occur also in higher
organisms, including mammals.35 It was also observed that the
silencing effect of dsRNA may be mimicked by chemically
synthesized siRNA, and one may designate this as exogenous
RNAi. The task to produce siRNAs in vivo was solved by designing vectors expressing shRNAs from RNA polymerase
III promotors (U6, H1, 7SK), which are then endogenously
processed to active siRNAs. In parallel with the study of
RNAi, progress in understanding the cellular processing and
function of miRs was achieved. RNAi and miR systems share
most components of the silencing pathway (Figure 2). In the
nucleus, the initial precursors of miRs designated as primary
miRs are transcribed from their own genes, or from introns,
by polymerase II or III, and processed by an enzyme complex (Microprocessor) consisting of Drosha, a polymerase
III–type dsRNA-specific endonuclease, and its cofactor microprocessor complex subunit DGCR8, to premiRs. In contrast, shRNAs are transcribed in the nucleus via polymerase
III, from plasmids of viral vectors transfected/transduced into
the cells. Both premiRs and shRNAs are exported from the
nucleus to the cytoplasm by exportin-5, a nuclear membrane
protein recognizing the 2-nucleotide overhang at the 3′-end
of the premiRs or shRNAs, respectively. In the cytoplasm,
both premiRs and shRNA are cleaved by Dicer, a polymerase
III–type dsRNA endonuclease which associates with its cofactors transactivation response RNA-binding element protein
and protein activator of PKR. The small RNA duplexes thus
generated are then processed to the mature double-stranded
miRs or siRNAs. One strand of the products represents the
mature guide strand miR or siRNA bringing the Dicer–
transactivation response RNA-binding protein-protein activator of PKR complex into contact with argonaute proteins,
whereas the opposite passenger strand is discarded. The mature miRs and siRNAs then guide the resulting RNA-induced
silencing complex complex to their respective target sites on
592 Circulation Research August 16, 2013
RNAi
Multipurpose gene silencing tool
ASOs
Antisense Oligonucleotides
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Protein-coding mRNAs
“Classical“ RNAi
Evaluation of suspected targets
by RNAi-based silencing
Figure 2. RNA interference (RNAi): versatile
principle for silencing of protein-coding genes
and noncoding RNA systems. The broad spectrum
of RNAs with therapeutic potential ranges from
RNAi triggers to antisense oligonucleotides (ASOs),
ribozymes, aptamers, and others. Among these
RNA tools, we consider here those mediating RNAi,
and as targets for RNAi we discuss protein-coding
mRNAs and long noncoding RNAs (lncRNAs).
Target silencing may be achieved using synthetic
RNA molecules modified to achieve biostability
and delivered by synthetic vehicles, for example,
nanoparticles, or using viral vectors (preferentially
organ-targeted and regulatable) with the capacity for
long-term production of therapeutic RNA. It should
be noted that all these RNA drugs act on delicately
balanced endogenous systems where target genes
have promotors and feedback mechanisms to
ensure homeostasis, and all endogenous RNAs
are immunologically inert, whereas synthetic and
recombinant RNA drugs and their vectors may arise
immune reactions, and dosage of RNA therapeutics
needs to be adapted to requirements. Reprinted
from Poller and Fechner77 with permission of
the publisher. Ago indicates argonaute; DGCR8,
microprocessor complex subunit DGCR8; POL,
polymerase; and shRNA, short hairpin RNA.
Long noncoding RNA
(lncRNA) systems
Silencing of confirmed targets by
RNAi or other RNA therapeutics
mRNAs. Perfect complementarity between a miR/shRNA and
its mRNA target sites leads to endonuclease cleavage of this
mRNA by argonaute-2, whereas imperfect complementarity
between a miR and its target mRNA results in translational
stop. Recent studies36,37 have elucidated subtle differences
between the mechanism by which siRNAs and miRs lead to
target gene silencing, by assigning distinct biochemical properties to different domains established by Argonaute: anchor,
seed, central, 3′-supplementary, and tail region.
Largely Unexplored Spectrum lncRNA Mechanisms
The molecular mechanisms of lncRNA action are highly diverse and complex (Figure 3), and they operate not only in
cis, but also in trans. Cis-acting lncRNAs are confined to their
sites of synthesis and act locally on spatially linked genes on
the same chromosome. Trans-acting lncRNAs may migrate
from their site of synthesis and act genome wide, similar to
transcription factors and small ncRNAs. First, lncRNAs function as molecular scaffolds by forming interaction interfaces
with other RNAs, as well as proteins (eg, polycomb group
complexes, chromatin modifiers). Thus, metastasis-associated lung adenocarcinoma transcript 1 (synonymous: NEAT2;
MALAT1) is involved in the ncRNA-dependent and Pc2 methylation–dependent gene relocation between nuclear structures and thus mediates gene activation programs.38 Second,
lncRNAs generate regulatory RNA–protein complexes in the
nucleus, and alternative lncRNA splicing change the interaction domains of the RNA altering the structure and resulting
function of the protein complex. Thus, NEAT1 is important
for a highly ordered spatial organization of paraspeckles and
other nuclear bodies.39 Third, lncRNAs may act as a guide
targeting a RNA–protein complex to specific genome regions
or through RNA–RNA interactions linking otherwise not
interacting protein complexes.40 Fourth, dynamic conformational changes of lncRNAs may act as biological switch in response to diverse cellular stimuli. lncRNAs can influence the
transcriptional landscape in a comprehensive manner, from
epigenetic regulation and chromatin remodeling to transcriptional and post-transcriptional control to protein metabolism.
Beyond these functions of the long primary transcript, there
may be a plethora of specific functions exerted by lncRNA
processing products, some of which are rather small and likely
to migrate between cell compartments possibly serving signaling functions. Our knowledge in this regard is very limited,
however, and must await the outcome of (technically demanding) kinetic studies.
The recent discovery of tens of thousands of lncRNAs has
temporarily overwhelmed the capacities to assign their functions and mechanisms of action.41 Nevertheless, important
Poller et al Cardiovascular RNA Interference 593
Complex functions
Dynamic structures
lncRNA primary transcripts
multiple shRNAs or ASOs
for complete blockade
very long primary transcripts
e.g. MALAT1 7kb, NEAT1
23kb
Guides
targets RNA – protein
complex to specific
genomic regions
Scaffolds
creating novel RNA –
protein interaction
surfaces
Novel links
Switches
between protein
complexes
mediates dynamic
conformational
changes
specific lncRNA processing
Novel processing pathways
distinct from RNAi and miRs
Analytic
RNAi
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
lncRNA processing products
Therapeutic
RNAi
RNAi or ASO to inhibit
specific lncRNA sub-functions
Figure 3. RNA interference (RNAi): toward therapeutic exploration of long noncoding RNA systems. One particular aspect of long
noncoding RNAs (lncRNAs) as therapeutic targets is their often extensive processing which has been (partially) characterized in only a
few cases to date. Therapeutic modulation of entire lncRNAs seems beyond reach for technical reasons, and because side effects arising
from these complex systems could probably not be controlled. Targeted ablation or overexpression of specific small processing products
by RNAi or antisense oligonucleotides (ASOs) is feasible and therapeutically interesting once specific functions have been assigned to
defined lncRNA products. Ago indicates argonaute; miR, micro RNA; and shRNA, short hairpin RNA.
pioneering work has led to functional insights for a distinct
group of vertebrate-specific lncRNAs, MALAT1/NEAT2,
NEAT1/MENε/β, and MI-associated transcript (MIAT)/
Gomafu, which accumulate abundantly in the nucleus as RNA
components of specific nuclear bodies.42 Animal models for
MALAT1 and for NEAT1 have been developed and although
they in the first place yielded apparently normal mice, impressive differences between normal and knockout animals were
revealed under pathological conditions. As in other traditional
gene knockout models, only pathogenic stress may reveal the
true functional spectrum of lncRNAs, and studies of this type
are to date very rare in the field. Functional investigation of
lncRNA systems may also require nonclassical knockout technologies by which the lncRNA gene is not germ line deleted
but silenced in the adult organism by RNAi.
RNAi: Classical Therapeutic Silencing of
Protein-Coding Genes
Progress by Independent Synergistic Technological
Developments
The therapeutic potential of strategies based on ncRNAs is
critically dependent on a combination of technological developments which have convened from different areas of research. Pioneering work in the first era of gene therapy has
focused on inherited diseases (eg, the hemophilias,43–45 viral
diseases,46–50 and cancer). For a long time, no delivery systems
for cardiovascular gene therapy were available, but recently
breakthrough and successful clinical translational was also
achieved in this area.3,51–55 One may consider the novel therapeutic approaches discussed in this review as a second era of
gene therapy where gene silencing is intended, whereas in the
first era the goal was the addition or substitution of proteincoding genes.43,51,56,57 Below we discuss analogies and differences between ncRNA therapy and classical gene therapy
because recent breakthroughs in vector technology are of importance for both.55,58
Recent Developments in Cardiovascular RNAi
One example of the second era in the cardiovascular field is
the combination of a highly efficient cardiotropic RNAi vector
to silence the cardiac-expressed regulatory protein phospholamban, with a cardiotropic vector system based on adeno-associated virus (AAV), which has for the first time shown high
efficacy of an RNAi therapeutic strategy in a cardiac disease.3
The AAV system enables long-term RNAi therapy by the continuous production of shRNAs from the long-term stable vector. Recently, nanoparticle-encapsulated synthetic siRNA was
used for silencing of C-C chemokine receptor type 2, the chemokine receptor that governs inflammatory Ly-6C monocyte
subset traffic. This approach reduced recruitment of Ly-6C
monocytes, attenuated infarct inflammation and post–myocardial infarction (MI) left ventricular remodeling.4 Another
important recent study introduced a strategy for in vivo RNAi
imaging.59 RNAi targeted prolyl hydroxylase domain protein
2 to treat myocardial ischemia, and the shRNA sequence was
594 Circulation Research August 16, 2013
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
followed by a separate hypoxia response element-containing
promoter driving a firefly luciferase reporter gene. This construct allowed noninvasive monitoring of gene expression and
was used to demonstrate that inhibition of prolyl hydroxylase domain protein 2 by RNAi improved angiogenesis and
contractility in vitro and in vivo. RNAi has also been used in
conjunction with cardiovascular stem cell therapy.60 One of
the limitations of this approach is poor stem cell survival in
the diseased microenvironment, and RNAi-based prolyl hydroxylase domain protein 2 silencing enhanced survival and
paracrine function of transplanted adipose-derived stem cells
in infarcted myocardium. Allele-specific RNAi as novel approach for the treatment of many autosomal-dominant–negative disorders rescued the long-QT syndrome phenotype in
human-induced pluripotency stem cell cardiomyocytes.61 In
the context of heart transplantation, RNAi has been used to
induce alloimmune tolerance through silencing of toll-like
receptor adaptors MyD88 and TIR-domain-containing adapter-inducing interferon-β.62 In vivo treatment of recipients
with anti-MyD88 and anti-TIR-domain-containing adapterinducing interferon-β siRNAs prolonged allograft survival. It
should emphasized that in addition to its direct use to improve
the treatment of cardiovascular diseases (by delivery of therapeutic RNAi triggers in vivo), RNAi has also proven highly
valuable for reverse genetics. It may first serve to identify novel functions of specific protein-coding genes and ncRNAs,63
and second to evaluate their potential as therapeutic targets.
Cardiovascular examples include myotrophin,64 myocardin,65
activator protein-2α,66 Na+/Ca2+ exchanger,67 malic-enzyme 1,68
cyclophilin-D,69 or chymase in the context of plaque stability
in atherosclerosis.70 These studies exemplify valuable indirect
applications of RNAI to improve therapy via the identification and validation of putative therapeutic targets for which no
conventional inhibitory drugs are available. A large number of
important studies have used RNAi system for in vitro and in
vivo ablation of specific protein-coding, as well as noncoding
gene functions, and this approach has also been systemically
used in genome-wide functional scans.
Table. Characteristic Properties of RNA Therapeutics
Although our ability to understand the impact of small ncRNA
in disease states is preliminary, and our ability to target them
in vivo is limited; several companies have already been formed
with the goal of developing RNA therapeutics.71 Similar to
other recent fields in human therapy research, early enthusiasm has been followed by a realistic assessment of the key
steps that are prerequisite before successful clinical translation. In the field of RNA therapeutics these are (Figure 4): (1)
specificity of the therapeutic RNA for the target, (2) stability
or continuous in situ production of the therapeutic RNA or the
ability to reapply if needed, (3) targeting to the correct cells
by the use of appropriate vectors and other methods, (4) side
effects induced by the therapeutic RNA itself or the delivery
system need to be minimized. In this context, innate immune
response to synthetic RNA is an important novel aspect,72
(5) regulatability of vector systems for therapeutic RNAs.
Because of the peculiar biochemical properties of small RNA
molecules, clinical translation is more demanding than with
small molecule drugs or proteins. Overall, RNA therapeutics
(Figure 4) may be chemically synthesized nucleic acids (ranging from Ribozymes to Aptamers/Spiegel mers) delivered using
synthetic chemicals, or produced by recombinant viral vectors
thus taking advantage of their biological targeting properties.
Viral vectors have the capacity to act as long-term productive
and, ideally, also regulatable57 RNA drug factories, whereas
chemical synthesis allows introduction of advantageous RNA
modifications that cannot be generated biologically.
Specificity
Systematic comparison of a broad spectrum of synthetic ASOs
with modified backbones (Figure 4) revealed that high-affinity modifications, for example, the incorporation of locked
nucleic acid, 2′-fluoro (2′-F), or 2′-O-methyl bases, increase
potency but maintain specificity. 2′-O-methyl/locked nucleic
acid chimeric ASOs with phosphorothioate linkages exchanging a nonbridging oxygen of a phosphodiester bond with sulfur (conferring nuclease resistance at this site) were the most
Future Directions in Cardiovascular RNA Interference
Investigation
Novel Strategies
Future Perspectives
References
Cardiac-targeted RNAi
Cardiotropic AAV9-based silencing of Ca2+ cycle regulator
phospholamban for the treatment of severe heart failure via
intravenous route
Cardiac-specific gene knockout for therapeutic
purposes, and as an alternative for cardiacspecific inducible knockout models
3
Monocyte-targeted RNAi
Nanoparticle-encapsulated synthetic siRNA for silencing of
monocytic chemokine receptor CCR2 in myocardial infarction
Nonviral RNAi delivery system targeting monocytes
in vivo, with high translational potential
4
RNAi imaging in vivo
PHD2-shRNA sequence followed by a hypoxia response elementcontaining promoter driving a firefly luciferase reporter gene
Imaging of RNAi distribution in space and time in
experimental animal models
59
Functional improvement
of stem cells by RNAi
To ameliorate a limitation of cardiovascular stem cell therapy, RNAi
was used to enhance survival and function of transplanted cells
Possible enhancement of the efficacy of
cardiovascular stem cell therapies
60
Allele-specific RNAi
Allele-specific RNAi for treatment of human-induced pluripotency
stem cell cardiomyocytes
Rescue of autosomal-dominant–negative
disorders eg, long-QT syndrome
61
Induction of alloimmune
tolerance by RNAi
In heart transplantation models, RNAi induced alloimmune
tolerance through silencing of TLR adaptors MyD88 and TRIF
In vivo treatment of recipients with siRNAs to
MyD88 and TRIF prolonged allograft survival.
62
Plaque stabilization
by RNAi
Lentivirus-based RNAi to silence chymase increased plaque
stability in atherosclerosis in vivo
Chymase as a target for plaque stabilization in
atherosclerosis, vasculature as an RNAi target
70
AAV indicates adeno-associated virus; CCR2, C-C chemokine receptor type 2; PHD, prolyl hydroxylase domain protein 2; shRNA, short hairpin RNA; siRNA, short
interfering RNA ; TLR, toll-like receptor; and TRIF, TIR-domain-containing adapter-inducing interferon-β.
Poller et al Cardiovascular RNA Interference 595
Synthetic RNA
Recombinant RNA
in vitro synthesis with RNA modifications
in vivo delivery requires synthetic vehicles
in vivo synthesis and in vivo delivery are
based on viral vector systems
Double-stranded RNAi triggers
Single-stranded oligonucleotides
mi-shRNA
Synthetic
ASOs
shRNA
siRNA
Erasers
Sponges
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Characteristics
Viral vectors
Specificity
Cellular entry
of target interaction
Stability
in blood and cells
Nanoparticles
Nuclear entry
shRNA
transcription
AAV
Targeting
Figure 4. RNA interference (RNAi): classical
therapeutic silencing of protein-coding
genes. The currently most important therapeutic
application of RNAi in cardiovascular medicine is
post-transcriptional silencing of genes involved in
disease pathogenesis. Two fundamentally distinct
approaches use synthetic RNAs, or recombinant
noncoding RNAs (ncRNAs) continuously produced
from viral vectors. RNA is inherently unstable and
must be modified to achieve sufficient biostability,
and delivered via synthetic carriers, to become
therapeutically useful. Viral vectors, which may be
organ-targeted and regulatable, may circumvent
targeting issues by their inherent biological
properties, and the RNA stability problem by
continuous synthesis in the host cells. Apart from
these differences, the same characteristics will
be considered when the therapeutic potential of
synthetic or recombinant RNA drugs is assessed.
AAV indicates adeno-associated virus; ASO,
antisense oligonucleotide; LNA, locked nucleic acid;
shRNA, short hairpin RNA; siRNA, short interfering
RNA; and TS, target site.
through circulation
and cell surface
Side Effects
immune activation
miR competition
Regulatability
conventional drugs
siRNA/shRNA
Argonaute
interactions
potent constructs tested for miR inhibition in vitro.73 Such in
vitro studies are of course only 1, but an important, step toward
development of in vivo therapies because whatever synthetic
targeting system is used it will need to encompass such modifications. As long as nonviral ncRNA therapy is attempted,
these findings may well direct the choice of the therapeutic
RNA type. Unfortunately, combination of these high specificity modifications with vector systems is impossible. Of current
analytic and possible future therapeutic interest is the discovery that seed-targeting tiny locked nucleic acids may be deliberately used to elucidating the functions of entire miR families
in vivo,74 as opposed to the commonly attempted modulation
of single miRs.
Stability
RNAi may be triggered by introducing chemically synthesized siRNAs into a cell system. A more stable knockdown,
however, requires the use of plasmids or viral vectors encoding shRNAs as precursors of siRNA. This requirement
likewise applies to miR modulation therapies, which may
have complex consequences in the recipient dependent on
biochemical stability and distribution of the therapeutic
RNA. The challenge to produce siRNAs in vivo continuously was met by designing vectors expressing shRNAs
or premiRs from RNA polymerase III promotors (U6, H1,
7SK), which are processed into active siRNAs or miRs in
target cells. On the contrary, miR inhibition can be achieved
by chemically synthesized Antagomirs (cholesteryl-conjugated 2′-O-methyl ASOs), peptide nucleic acids,75 or backbone-modified small RNAs to improve biostability, target
RNA affinity, or cellular uptake (Figure 4), or by vectorbased recombinant expression of miR Sponges76 or Erasers
which act by miR scavenging, or Target Masks blocking
miR binding. Antagomirs seem to exert rather long-lived effects and may for certain applications circumvent the need
for viral vectors.77
Targeting
The originally developed AAV vector prototypes have been
derived from AAV serotype 2 (AAV2). AAV2 vectors had low
transduction efficiencies in vivo and displayed no cardiotropism.78 This problem has been overcome. First, because vector genomes with intact AAV2-inverted terminal repeat can be
packaged into the capsids of other AAV serotypes by pseudotyping. Second, the identification of a large number of new
AAV serotypes79,80 with diverse properties allowed the development of AAV vectors with tropism for different organs in
vivo. In the first pioneering clinical studies, an AAV1 vector
has been successfully used for the treatment of severe heart
failure by cardiac overexpression of sarcoplasmic reticulum
596 Circulation Research August 16, 2013
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Ca2+-ATPase 2a.51,53 Availability of multiple AAV serotypes for
cardiac transduction is highly desirable because some patients
may have preexisting antibodies against certain AAV serotypes, or develop them after AAV vector application.81 In these
cases, switching to another serotype would allow continuation
of therapy. Importantly, targeting of therapeutic genes or regulatory RNAs to an organ of choice may be achieved in some
cases by simple intravenous vector injection. AAV9 pseudotyped vectors are highly efficient for cardiac-targeted gene
or RNAi transfer in mice and rats, with ≈80% cardiomyocyte
transduction after intravenous application. In rodents, AAV9
showed low expression in other organs.82–84 Cardiac specificity
is further improved by liver detargeting by incorporation of
target sites for liver-specific miRs in the 3′-untranslated region
of the transgene or shRNA or miR expression cassette.85 Liver
detargeting shall avoid oversaturation of cellular miR/shRNA
pathways which may result in liver toxicity.46 Capsid-modified
AAVs have been generated by DNA shuffling of different
AAV serotype capsid genes to improve targeting properties.86
Because of species-differences it not yet entirely clear which
AAV serotype is best suitable for ncRNA therapy in humans,
although AAV9 has already been shown highly efficient in
nonhuman primates.84,87
Transcriptional targeting is useful to further confine synthesis of ncRNAs to the target. Improved cardiac specificity
was achieved using modified myosin-light-chain promotors
for AAV vector-mediated gene therapy,88,89 but these polymerase II promotors are unsuitable to express conventional
shRNAs which are efficiently transcribed by polymerase
III (U6, H1, 7SK) promotors only. Importantly, naturally
occurring miR sequence may be replaced by an artificial
shRNA sequence embedded into the original miR environment, to achieve classical transcriptional targeting of
the therapeutic shRNA sequence to an organ of interest.90
This discovery has enabled expression of shRNAs from
tissue-specific promotors, as previously used in classical
gene therapy. Recently, post-transcriptional targeting was
introduced which is based on miR target site incorporation
into the expression cassette. Thus, a genetically engineered
miR-155–based shRNA has been shown to have similar silencing efficacy but less immunogenicity than a previously
successfully used3 AAV9-RNAi vector targeting the cardiac
regulator protein phospholamban (H. Fechner et al, unpublished, 2013). Multiplex microRNA strategies with several
miR-based shRNAs expressed from a common polycistronic miR structure have also been used,91 and may become
useful for the treatment of viral infections, where cosilencing of multiple virus-encoded genes significantly improves
antiviral efficacy.
Whereas biological vectors are characterized by complex and largely preformed targeting properties (Figure 4),
synthetic RNAi delivery systems must be build up step by
step. If both the therapeutic RNA and delivery vehicle, for
example, nanoparticle, are sufficiently stable in vivo (blood
plasma, cytoplasm), their traffic in vivo can be modulated.
Thus, in situ siRNA–albumin conjugation enhances extravasation, stability, and cardiac gene silencing in vivo.92 A
Gal-PEG-Et nanoparticle carrying angiotensinogen shRNA
nanoparticle shRNA transfection system mediated RNAi
in early atherosclerotic lesions.93 Aptamers and other ligands were used to address receptors on target cells.94
Importantly, however, such ligand–receptor interactions are
only efficient in vivo if the delivery vehicle is able to cross
endothelial barriers. A strictly local delivery system of high
interest to cardiovascular medicine are siRNA-eluting surfaces using technologies related to drug-eluting stents.95
Because of the high technological standard in this field,
further investigation of opportunities for RNAi delivery is
warranted.
Side Effects
Several potential application problems and side effects of
both synthetic therapeutic small ncRNAs and vector-based
ncRNA delivery systems arise from immune system stimulation. First, AAV vectors cannot be used in patients with
preexisting antibodies96 or lose their efficacy on development of such antibodies in treated patients. Second, even
subtle structural differences between synthetic or recombinant small RNA molecules and endogenous counterparts,
for example, miRs, may activate the innate immune system
through both toll-like receptor-dependent and toll-like receptor-independent mechanisms,97,98 and considerable efforts
have been made to define structural requirements to avoid this
problem. Only in noncardiovascular diseases, for example,
cancer, immunostimulation by ncRNAs may be an acceptable or even useful side effect.99 Third, RNAi triggers, for
example, shRNAs, may critically compete with endogenous
miR biosynthetic pathways leading to severe toxicity.46 Here
again detailed understanding of specific sequence determinants may help to avoid toxicity.
Regulatability
Major efforts have been undertaken to make vector-based
gene and also ncRNA delivery system regulatable, with the
prospect to adjust their levels to possible changing requirements. A desirable feature of ncRNA vectors would also be
a complete switch-off function. Several systems of possible
clinical relevance have been reviewed elsewhere.77
Clinical Translation of Findings From
Proof-of-Principle Studies
First, given the enormous amount of work and money required
to develop ncRNA therapeutics,100 it may be advisable to focus
efforts on targets of potential interest in large patient cohorts.
Second, targets should be dysregulated in human cardiovascular diseases as documented by patient-derived data. Third,
their relevance should be documented by proof-of-principle
studies in animals. Unfortunately, for several important human cardiovascular diseases no adequate animal models exist.
Fourth, there should be assessment of the chance for development of conventional small molecule drugs mimicking
the desired effect of the ncRNA sequence in vivo. In many
instances, however, the development of receptor or pathway
blocking agents was unsuccessful (eg, for phospholamban).
Fifth, there should be a stable system available for ncRNA
sequence delivery to the desired tissue. Such systems may be
tailor-made synthetic small RNAs in isolation, coupled to proteins or nanoparticles, or AAV-based vectors targeting cardiovascular structures.
Poller et al Cardiovascular RNA Interference 597
Genomewide Transcriptome Mapping
mRNAs, miRs, lncRNAs, other ncRNA
RNA seq, microarrays
Heuristic
strategies
to select for
ncRNAs with
likely clinical
therapeutic
potential
long noncoding RNA Processing, miR Transport
mechanisms, regulation, (extra)cellular kinetics of products
ncRNA expression profiling of human diseases
First hints to biological functions and possible pathogenic roles of ncRNA in humans
Evaluation of Suspected Therapeutic Targets using RNA Interference
Analytical RNAi as important complement to classical and ZFN knockouts of protein-coding genes,
long noncoding RNA systems, or other noncoding transcripts
Assignment of biological functions in context
Novel small or long ncRNAs and products
Phase of therapeutic clinical translation
• Therapeutic proof-of-principle studies in animals
• GMP strategies for clinical translation
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
RNAi: Search Tool for Novel Therapeutic
Targets in the Human Genome
Search Strategies to Identify ncRNAs of Likely
Clinical Relevance
Against the background of the complexities on the noncoding genome, one may adopt a pragmatic heuristic approach
(Figure 5) directed at the identification of noncoding therapeutic targets in human cardiovascular diseases. Data and
insights from genome-wide phylogenetic studies, comprehensive sequencing work, and animal models of human diseases
constitute an indispensible first step toward target identification. However, the functions of the same ncRNA may differ
between species, and most animal models differ significantly
from the respective human disorder. Moreover, some ncRNAs
do not exist in humans, such as the lncRNA Braveheart,101 or
have no close homologues (several miRs), so that their functions and possible pathogenic relevance cannot be adequately
investigated in animal models. It, therefore, seems suitable
and necessary to conduct transcriptome mapping also directly
in humans, and in the specific diseases of interest. Because
of the advancement of technologies, comprehensive transcriptional mapping is possible even from the minute quantities of
RNA obtained from cardiac or other human biopsy samples.
It is entirely possible that therapeutic proof-of-concept studies
addressing ncRNAs deregulated in animal models cannot be
efficient in humans because of such interspecies differences.
Functional Spectrum and Therapeutic Potential of
Classical RNAi
The mechanism of RNAi is conserved across species and involves small RNAs inhibiting translation or degrading mRNA
by PTGS, which may protect against viral infection and regulate
endogenous genes. Several RNA classes exert PTGS: siRNAs
with perfect complementarity to targets cause transcript degradation; miRs with imperfect complementarity cause translational
repression; and piRNAs target transposon transcripts in animal
germ lines.102 siRNA-mediated RNAi is an efficient broad spectrum antiviral mechanism, whereas immunologic systems are
rather virus specific.103 RNAi has potential for the treatment
Figure 5. RNA interference (RNAi):
search tool for novel therapeutic
targets in the human genome. Genomewide genomic and transcriptomic
mapping has revealed a huge number of
novel noncoding transcripts and provided
structural information on their gene loci,
but for the vast majority there is as yet
no functional assignment. Functional
characterization is complicated by their
often extensive processing. Prospecting
of this plethora of noncoding RNA
(ncRNA) for possible clinical significance
may be critically facilitated by stateof-the-art transcriptional mapping of
cardiovascular patients, to obtain first
functional hints to guide subsequent
in-depth experimental investigations,
including therapeutic proof-of-principle
studies. GMP, good manufacturing
practice; lncRNA, long noncoding
RNA; RNA-seq, RNA sequencing; miR,
microRNA; and ZFN, zinc finger nuclease.
of viral cardiomyopathies48,50 in conjunction with antiviral
drugs.49,57 Interestingly, several viruses generate small RNAs
capable to modulate host gene expression to their advantage.104
miR evolution has reached 63 families in mammals18 and
1424 separate miR genes in humans17,105 (Figure 1). Genomewide assessment of miR targets suggests that in humans ≈50%
of mRNAs are influenced by miR regulation.106 Regarding the
functional assignment of miRs in the cardiovascular system,
an interesting approach is high-throughput functional screening in cell cultures using a whole-genome miR library. Using
this screening and further selection steps, a recent investigation has identified miRs promoting cell cycle reentry of adult
cardiomyocytes. After MI, these miRs stimulated cardiac regeneration and recovery of cardiac functional parameters in
mice.107,108 For the cardiovascular system, several recent reviews have discussed the differential regulation of miRs, their
functions during development, roles in disease pathogenesis,
and therapeutic perspectives,5–9 and these small ncRNAs are,
therefore, not covered here. One difference between RNAi
and miR-modulating therapies should be emphasized, however, that is, deliberate modulation of 1 single target by RNAi,
whereas miR modulation influences multiple targets as integral
part of miR action. Endogenous miR genes are also subject
to regulation via their promotor, similar to protein-encoding
genes, and miR modulators antagonists or mimics overdrive
this physiological regulatory network with possible implications for the therapeutic setting.109 Complexity is added to the
endogenous miR systems by extracellular miR trafficking via
vesicles,110 the existence of genetic miR variants,111 and strong
disease-context dependency of miR actions.112 Despite several
similarities between RNAi and miR systems, their functional
biology is grossly distinct.
Functional Assignment and Therapeutic Perspective
of lncRNAs
After the discovery of RNAi, miRs, and other small regulatory
RNAs had already revolutionized our perception of gene regulation in higher organisms, a huge number of other cellular
players was added when a major fraction of the transcriptome
598 Circulation Research August 16, 2013
was found to encompass not small RNAs of the known types,
but far larger lncRNA transcripts expressed in a cell type
and development stage-specific manner. This closely regulated manner of expression, in conjunction with biochemical,
mechanistic, and functional findings for an as yet small number of paradigmatic lncRNAs, indicates high importance of
this novel class of ncRNA in cardiovascular health and disease.113 Clinical interest was triggered by their involvement
in viral infectious diseases,114 immune disorders,115 and stem
cell differentiation and function.116 Regarding cardiovascular
disorders, recent studies suggest involvement of a number of
lncRNAs in cardiac development and important cardiac diseases. As excellent recent reviews have addressed this topic,117
we focus here on the discovery path of the lncRNA data currently available, issues that need to be further investigated by
inclusion of cardiac transcriptome data from patients, and first
evidence suggesting therapeutic potential of lncRNA systems.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Assignment by Genome-Wide Association Studies
A first type of data stems from genome-wide association studies analyses.118 Human chromosome 9p21.3 region is a strong
genetic susceptibility locus for coronary artery disease, but
no association with traditional risk factors, common proteincoding variation, variants in splice donor/acceptor sites, or
copy number was observed. However, strong association with
an isoform of lncRNA ANRIL and cyclin-dependent kinase
inhibitor 2B was found and led to the hypothesis that complex
regulatory variation affecting ANRIL and cyclin-dependent kinase inhibitor 2B gene expression explains the increased MI
risk linked to this locus.119–121 ANRIL modulates atherogenic
pathway expression in vascular smooth muscle cells in vitro,
different ANRIL isoforms had differential effects,122,123 and
ANRIL was induced by the ataxia telangiectasia mutated kinase-E2F1 transcription factor signaling pathway.124 Another
lncRNA of cardiovascular relevance identified by genomewide association studies analysis is MIAT/Gomafu. Single
nucleotide polymorphisms in this locus in complete linkage
disequilibrium were associated with MI,125 but the underlying
mechanism is unknown to date.126
Assignment by Experimental Targeted Search
Braveheart, an lncRNA required for cardiovascular lineage
commitment, was identified by a systematic targeted search
for lncRNAs restricted to specific cell types during embryonic stem cell differentiation. lncRNA expression profiling by
RNA-seq in mouse embryonic stem cells and differentiated
tissues led to identification of a number of candidates which
were then individually further evaluated. Braveheart interacts
with a component of polycomb-repressive complex 2 during
cardiomyocyte differentiation and is thought to regulate cardiac
commitment epigenetically.101 One remarkable aspect of this
study, with regard to the identification of lncRNAs of potential
clinical relevance, is the observation that the murine Braveheart
has obviously no human homologue. The interesting question
arises whether this lncRNA locus has been lost during human
evolution, with a resulting loss of certain capabilities on cardiac
biology, or whether it was substituted in humans by a structurally distinct (and thus not recognizable by traditional genome
sequence comparison) transcriptional system.
Assignment by Transcriptome Mapping in Patients
This discovery paths led to cardiovascular interest in the evolutionary highly conserved lncRNAs MALAT1 and NEAT1.
MALAT1 and NEAT1 are located <70 kb apart on human chromosome 11.127 MALAT1 is post-transcriptionally processed
by a novel biosynthetic pathway32,128 involving RNAses P and
Z, to yield a small tRNA-like product designated MALAT1associated small cytoplasmic RNA (mascRNA; Online Figure
I). To identify functions of the MALAT1 system, murine knockout models have been used129–132 which showed that loss is
compatible with normal development and life under normal
laboratory conditions, and that its function becomes apparent
only in specific cell types and under particular conditions. A
pioneering recent study revealed that MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells.133 By
endomyocardial biopsy-based cardiac transcriptome mapping,
we recently found MALAT1 dysregulated in human cardiomyopathies. Modulation of the small lncRNA-product mascRNA
by recombinant overexpression or PTGS strongly influenced
intrinsic cellular immunity134–136 and may have therapeutic
potential (Poller et al, unpublished data, 2013). Cardiac transcriptome maps of lncRNAs here yielded first evidence of
their involvement in cardiac diseases, exemplifying the search
strategy for lncRNA functions of clinical interest as outlined
in Figure 5.
Processing of lncRNAs
It will be preferable to use RNA-seq instead of microarray
technology to achieve a comprehensive overview, including
not only the primary transcripts, but also their specific processing products, which are likely to be carriers of defined
cellular functions. Different transcripts (MENβ, MENε) also
originate from the NEAT1 locus, and the nonpolyadenylated
3′-end of MENβ is noncanonically processed by RNases and
yield a tRNA-like product designated as menRNA (Online
Figure II). This complex processing seems to be integral part
of the NEAT1 system.34 Live-cell imaging system allowing
inducible transcription of NEAT1 revealed that it serves as a
platform to recruit proteins to assemble paraspeckles,137 which
are assumed to control several biological processes, including
stress responses and cellular differentiation.131 It was recently
shown that NEAT1 influences virus infections.138 Regarding
the general aspect of differential cellular functions of lncRNA
processing products it is of interest that another NEAT1related sequence designated as trophoblast ncRNA inhibits
alloresponses by suppressing class II transactivator promotor
III and major histocompatibility complex class II expression
in murine B-lymphocytes.139–141 An important genome-wide
analysis of lncRNA stability142 has determined the half-lives
of ≈800 lncRNAs and ≈12 000 mRNAs, as a further step toward kinetic characterization of lncRNA systems.
RNAi: Toward Therapeutic Exploration of
lncRNA Systems
RNAi for the Functional Assignment of
lncRNA Systems
RNAi recently emerged as an important novel tool to identify
the physiological cardiovascular functions, and hence possible
Poller et al Cardiovascular RNA Interference 599
therapeutic potential, of newly identified lncRNAs.143 RNAibased functional profiling was applied to 56 loci found by genome-wide association studies to be associated with coronary
artery disease and MI. RNAi was used to analyze 133 candidate genes in these genomic regions showed that individual
genome-wide association studies loci may contain >1 gene
with cholesterol-regulatory functions. Several genes without
previously known lipid-regulatory roles were functionally
assigned using silencing by RNAi. This study demonstrates
that quantitative, cell-based RNAi is an important strategy for
identification of possible novel therapeutic targets.
RNAi for the Therapeutic Modulation of
lncRNA Systems
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Several of the basic principles and methods with clinical
translational potential as discussed in the previous article on
RNAi are also applicable to lncRNA modulation. Thus, it
is possible to block even very long precursor transcripts by
targeting different regions of it with a panel of ASOs, independent of methods such as homologous recombination or
zinc finger nucleases.130 To date, however, limitations arise
from our incomplete knowledge of lncRNA processing and
kinetics. Furthermore, primary lncRNA transcripts generally
seem to have such a large number of nucleic acid and proteintype interaction partners, and highly complex functions that
adequate control of possible side effects of lncRNA blockade seems impossible to achieve. It should be kept in mind,
however, that blockade or overexpression of an entire lncRNA
would be analogous to blockade or overexpression of a primary miR which is not the principle of miR therapeutics. To
the contrary, only mature miRs as highly specific processing
products were ever considered for clinical application, and
this would be analogous to the targeting of single processing
products of a lncRNA of interest (eg, mascRNA or menRNA;
Online Figures I and II). From a therapeutic perspective, research into the functions of lncRNAs in the cardiovascular
system, several of which apparently have impact in cardiovascular pathogenesis, should pay attention to these aspects
in both experimental studies and transcriptome analyses of
cardiovascular patients.
Concluding Remarks—Therapeutic
Exploration of the Human Genome
Because of the now apparent extreme complexity of the human genome, one may consider a biological analogy to
Heisenberg Uncertainty Principle in physics: Intact biological
systems encompassing all molecular components, including
the plethora of newly discovered ncRNAs, can be dissected
into small subsystems accessible to reductionistic molecular
characterization. By doing so, however, the original biological context and functions are lost because in the intact living
organism the subsystem studied is in fact intricately linked to
multiple neglected but essential parts. This might be a fundamental limitation, to be only partially overcome by combining data from different study systems, including thorough
genomic and transcriptomic investigations in patients using
state-of-the-art tools.
Apart from this fundamental limitation, it is currently obviously impossible to simulate the behavior of a normal or
diseased cell without knowledge of all relevant players. Until
2 decades ago, one might have hoped that this could change
in the foreseeable future, with a comprehensive data set on the
then known players. Even that would have been a tremendous
endeavor, followed under the headlines of Systems Biology
and Network Medicine,144 but with the recent discovery of a
huge number of previously unknown and even unsuspected
players from the noncoding human genome145–149 this goal has
certainly shifted far away. It is already entirely clear that it
will take a very long time, if ever possible, to arrive at a full
understanding of the workings of the human genome with its
multiple and exceedingly complex layers of regulation and
dynamic adaptation to changing requirements.
Whereas comprehensive understanding of the workings
of the genome is beyond reach now, current knowledge will
nevertheless become increasingly valuable for cardiovascular
research and medicine. If the complementary insights from
evolutionary biology, genomic and transcriptomic studies in
well-selected cardiovascular patient cohorts, and experimental evaluation of possible ncRNA therapeutic candidates in
cell and animal models are brought together; currently available technologies are likely to reveal a continuously growing panel of novel targets for cardiovascular therapy. From a
pragmatic perspective, a broad spectrum of most valuable discovery tools for target prospecting in the noncoding genome
is already at hand, as well as various delivery tools suitable
for clinical translation. Because of the complexity of the task,
clinical translation in this area continues to be a most interesting and suitable area for close interdisciplinary research.
Sources of Funding
This work has been supported by Deutsche Forschungsgemeinschaft
through Collaborative Research Center Transregio 19 by grants
W. Poller and C. Skurk. The other authors report no conflicts.
Disclosures
None.
References
1. Mattick JS, Taft RJ, Faulkner GJ. A global view of genomic information–moving beyond the gene and the master regulator. Trends Genet.
2010;26:21–28.
2. Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M. An
integrated encyclopedia of DNA elements in the human genome. Nature.
2012;489:57–74.
3. Suckau L, Fechner H, Chemaly E, et al. Long-term cardiac-targeted RNA
interference for the treatment of heart failure restores cardiac function and
reduces pathological hypertrophy. Circulation. 2009;119:1241–1252.
4.Majmudar MD, Keliher EJ, Heidt T, et al. Monocyte-directed RNAi
targeting CCR2 improves infarct healing in atherosclerosis-prone mice.
Circulation. 2013;127:2038–2046.
5. van Rooij E, Purcell AL, Levin AA. Developing microRNA therapeutics.
Circ Res. 2012;110:496–507.
6. Abdellatif M. Differential expression of microRNAs in different disease
states. Circ Res. 2012;110:638–650.
7. Zampetaki A, Mayr M. MicroRNAs in vascular and metabolic disease.
Circ Res. 2012;110:508–522.
8. Boettger T, Braun T. A new level of complexity: the role of microRNAs in
cardiovascular development. Circ Res. 2012;110:1000–1013.
9. Mayr M, Zampetaki A, Willeit P, Willeit J, Kiechl S. MicroRNAs within
the continuum of postgenomics biomarker discovery. Arterioscler Thromb
Vasc Biol. 2013;33:206–214.
10.Volders PJ, Helsens K, Wang X, Menten B, Martens L, Gevaert K,
Vandesompele J, Mestdagh P. LNCipedia: a database for annotated
600 Circulation Research August 16, 2013
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
human lncRNA transcript sequences and structures. Nucleic Acids Res.
2013;41:D246–D251.
11. Sun J, Gao B, Zhou M, Wang ZZ, Zhang F, Deng JE, Li X. Comparative
genomic analysis reveals evolutionary characteristics and patterns of microRNA clusters in vertebrates. Gene. 2013;512:383–391.
12. Wang QH, Zhou M, Sun J, Ning SW, Li Y, Chen L, Zheng Y, Li X, Lv
SL, Li X. Systematic analysis of human microRNA divergence based on
evolutionary emergence. FEBS Lett. 2011;585:240–248.
13.Meunier J, Lemoine F, Soumillon M, Liechti A, Weier M, Guschanski
K, Hu H, Khaitovich P, Kaessmann H. Birth and expression evolution of
mammalian microRNA genes. Genome Res. 2013;23:34–45.
14.Hertel J, Bartschat S, Wintsche A, Otto C, Stadler PF; Students of the
Bioinformatics Computer Lab. Evolution of the let-7 microRNA family.
RNA Biol. 2012;9:231–241.
15. Zhang X, Rossi JJ. Phylogenetic comparison of small RNA-triggered transcriptional gene silencing. J Biol Chem. 2011;286:29443–29448.
16. Sun J, Zhou M, Mao ZT, Hao DP, Wang ZZ, Li CX. Systematic analysis
of genomic organization and structure of long non-coding RNAs in the
human genome. FEBS Lett. 2013;587:976–982.
17. Wheeler BM, Heimberg AM, Moy VN, Sperling EA, Holstein TW, Heber
S, Peterson KJ. The deep evolution of metazoan microRNAs. Evol Dev.
2009;11:50–68.
18.Heimberg AM, Sempere LF, Moy VN, Donoghue PC, Peterson KJ.
MicroRNAs and the advent of vertebrate morphological complexity. Proc
Natl Acad Sci U S A. 2008;105:2946–2950.
19. Kung JT, Lee JT. RNA in the loop. Dev Cell. 2013;24:565–567.
20. Clark MB, Amaral PP, Schlesinger FJ, et al. The reality of pervasive transcription. PLoS Biol. 2011;9:e1000625; discussion e1001102.
21. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP. A ceRNA hypothesis:
the Rosetta Stone of a hidden RNA language? Cell. 2011;146:353–358.
22. Tay Y, Kats L, Salmena L, Weiss D, Tan SM, Ala U, Karreth F, Poliseno
L, Provero P, Di Cunto F, Lieberman J, Rigoutsos I, Pandolfi PP. Codingindependent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell. 2011;147:344–357.
23. Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP.
A coding-independent function of gene and pseudogene mRNAs regulates
tumour biology. Nature. 2010;465:1033–1038.
24. Chen G, Wang Z, Wang D, Qiu C, Liu M, Chen X, Zhang Q, Yan G, Cui
Q. LncRNADisease: a database for long-non-coding RNA-associated diseases. Nucleic Acids Res. 2013;41:D983–D986.
25. Yang JH, Li JH, Jiang S, Zhou H, Qu LH. ChIPBase: a database for decoding the transcriptional regulation of long non-coding RNA and microRNA
genes from ChIP-Seq data. Nucleic Acids Res. 2013;41:D177–D187.
26.Martin JA, Wang Z. Next-generation transcriptome assembly. Nat Rev
Genet. 2011;12:671–682.
27.Metzker ML. Sequencing technologies—the next generation. Nat Rev
Genet. 2010;11:31–46.
28. Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011;12:87–98.
29. Lee JH, Gao C, Peng G, Greer C, Ren S, Wang Y, Xiao X. Analysis of
transcriptome complexity through RNA sequencing in normal and failing
murine hearts. Circ Res. 2011;109:1332–1341.
30. Song HK, Hong SE, Kim T, Kim do H. Deep RNA sequencing reveals
novel cardiac transcriptomic signatures for physiological and pathological
hypertrophy. PLoS One. 2012;7:e35552.
31. Wan Y, Kertesz M, Spitale RC, Segal E, Chang HY. Understanding the
transcriptome through RNA structure. Nat Rev Genet. 2011;12:641–655.
32. Wilusz JE, Freier SM, Spector DL. 3’ end processing of a long nuclearretained noncoding RNA yields a tRNA-like cytoplasmic RNA. Cell.
2008;135:919–932.
33. Naganuma T, Nakagawa S, Tanigawa A, Sasaki YF, Goshima N, Hirose T.
Alternative 3’-end processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J. 2012;31:4020–4034.
34. Naganuma T, Hirose T. Paraspeckle formation during the biogenesis of
lncRNAs. RNA Biol. 2013;10:456–461.
35. Babiarz JE, Ruby JG, Wang Y, Bartel DP, Blelloch R. Mouse ES cells express
endogenous shRNAs, siRNAs, and other Microprocessor-independent,
Dicer-dependent small RNAs. Genes Dev. 2008;22:2773–2785.
36. Wee LM, Flores-Jasso CF, Salomon WE, Zamore PD. Argonaute divides
its RNA guide into domains with distinct functions and RNA-binding
properties. Cell. 2012;151:1055–1067.
37. De N, Young L, Lau PW, Meisner NC, Morrissey DV, MacRae IJ. Highly
complementary target RNAs promote release of guide RNAs from human
Argonaute2. Mol Cell. 2013;50:344–355.
38. Yang L, Lin C, Liu W, Zhang J, Ohgi KA, Grinstein JD, Dorrestein PC,
Rosenfeld MG. ncRNA- and Pc2 methylation-dependent gene relocation between nuclear structures mediates gene activation programs. Cell.
2011;147:773–788.
39. Souquere S, Beauclair G, Harper F, Fox A, Pierron G. Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within
paraspeckle nuclear bodies. Mol Biol Cell. 2010;21:4020–4027.
40. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annu
Rev Biochem. 2012;81:145–166.
41.Lee JT. Epigenetic regulation by long noncoding RNAs. Science.
2012;338:1435–1439.
42.Ip JY, Nakagawa S. Long non-coding RNAs in nuclear bodies. Dev
Growth Differ. 2012;54:44–54.
43. Kay MA. State-of-the-art gene-based therapies: the road ahead. Nat Rev
Genet. 2011;12:316–328.
44. Goonasekera SA, Lam CK, Millay DP, Sargent MA, Hajjar RJ, Kranias
EG, Molkentin JD. Mitigation of muscular dystrophy in mice by SERCA
overexpression in skeletal muscle. J Clin Invest. 2011;121:1044–1052.
45. Nathwani AC, Rosales C, McIntosh J, et al. Long-term safety and efficacy
following systemic administration of a self-complementary AAV vector
encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.
Mol Ther. 2011;19:876–885.
46.Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis
CR, Marion P, Salazar F, Kay MA. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature.
2006;441:537–541.
47. Eckstein A, Grössl T, Geisler A, Wang X, Pinkert S, Pozzuto T, Schwer C,
Kurreck J, Weger S, Vetter R, Poller W, Fechner H. Inhibition of adenovirus infections by siRNA-mediated silencing of early and late adenoviral
gene functions. Antiviral Res. 2010;88:86–94.
48. Fechner H, Sipo I, Westermann D, Pinkert S, Wang X, Suckau L, Kurreck
J, Zeichhardt H, Müller O, Vetter R, Erdmann V, Tschope C, Poller W.
Cardiac-targeted RNA interference mediated by an AAV9 vector improves
cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med (Berl).
2008;86:987–997.
49. Werk D, Pinkert S, Heim A, Zeichhardt H, Grunert HP, Poller W, Erdmann
VA, Fechner H, Kurreck J. Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral
activity against coxsackievirus B3. Antiviral Res. 2009;83:298–306.
50. Fechner H, Pinkert S, Wang X, Sipo I, Suckau L, Kurreck J, Dörner A,
Sollerbrant K, Zeichhardt H, Grunert HP, Vetter R, Schultheiss HP, Poller
W. Coxsackievirus B3 and adenovirus infections of cardiac cells are efficiently inhibited by vector-mediated RNA interference targeting their
common receptor. Gene Ther. 2007;14:960–971.
51. Hajjar RJ. Potential of gene therapy as a treatment for heart failure. J Clin
Invest. 2013;123:53–61.
52.Lompré AM, Hajjar RJ, Harding SE, Kranias EG, Lohse MJ, Marks
AR. Ca2+ cycling and new therapeutic approaches for heart failure.
Circulation. 2010;121:822–830.
53.Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B,
Yaroshinsky A, Zsebo KM, Dittrich H, Hajjar RJ; Calcium Upregulation
by Percutaneous Administration of Gene Therapy in Cardiac Disease
(CUPID) Investigators. Calcium Upregulation by Percutaneous
Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2
trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase
in patients with advanced heart failure. Circulation. 2011;124:304–313.
54. Lipskaia L, Chemaly ER, Hadri L, Lompre AM, Hajjar RJ. Sarcoplasmic
reticulum Ca(2+) ATPase as a therapeutic target for heart failure. Expert
Opin Biol Ther. 2010;10:29–41.
55. Poller W, Hajjar R, Schultheiss HP, Fechner H. Cardiac-targeted delivery
of regulatory RNA molecules and genes for the treatment of heart failure.
Cardiovasc Res. 2010;86:353–364.
56. Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease
using AAV: progress and challenges. Nat Rev Genet. 2011;12:341–355.
57. Pinkert S, Westermann D, Wang X, et al. Prevention of cardiac dysfunction in
acute coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirus-adenovirus receptor. Circulation. 2009;120:2358–2366.
58. Castanotto D, Rossi JJ. The promises and pitfalls of RNA-interferencebased therapeutics. Nature. 2009;457:426–433.
59. Huang M, Wu JC. Molecular imaging of RNA interference therapy targeting PHD2 for treatment of myocardial ischemia. Methods Mol Biol.
2011;709:211–221.
60. Wang WE, Yang D, Li L, Wang W, Peng Y, Chen C, Peng C, Xia X, Wang
H, Jiang J, Liao Q, Li Y, Xie G, Huang H, Guo Y, et al. Phd2 silencing enhances the survival and paracrine function of transplanted adipose-derived
Poller et al Cardiovascular RNA Interference 601
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
stem cells in infarcted myocardium. Circ Res. 2013;113:288–300. doi:
10.1161/CIRCRESAHA.113.300929.
61. Matsa E, Dixon JE, Medway C, Georgiou O, Patel MJ, Morgan K, Kemp PJ,
Staniforth A, Mellor I, Denning C. Allele-specific RNAi rescues the long-qt
syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes. Eur Heart J. March 6, 2013. doi: 10.1093/eurheartj/eht067. http://
eurheartj.oxfordjournals.org/content/early/2013/03/06/eurheartj.eht067.
long. Accessed August 2, 2013.
62. Zhang X, Beduhn M, Zheng X, Lian D, Chen D, Li R, Siu LK, Marleau
A, French PW, Ichim TE, Min WP. Induction of alloimmune tolerance
in heart transplantation through gene silencing of TLR adaptors. Am J
Transplant. 2012;12:2675–2688.
63. Ramachandran PV, Ignacimuthu S. RNA interference–a silent but an efficient therapeutic tool. Appl Biochem Biotechnol. 2013;169:1774–1789.
64. Gupta S, Maitra R, Young D, Gupta A, Sen S. Silencing the myotrophin
gene by RNA interference leads to the regression of cardiac hypertrophy.
Am J Physiol Heart Circ Physiol. 2009;297:H627–H636.
65. Torrado M, Iglesias R, Centeno A, López E, Mikhailov AT. Targeted genesilencing reveals the functional significance of myocardin signaling in the
failing heart. PLoS One. 2011;6:e26392.
66. Lin HH, Chen YH, Chiang MT, Huang PL, Chau LY. Activator protein-2α
mediates carbon monoxide-induced stromal cell-derived factor-1α expression and vascularization in ischemic heart. Arterioscler Thromb Vasc Biol.
2013;33:785–794.
67.Maddaford TG, Dibrov E, Hurtado C, Pierce GN. Reduced expression
of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury. Am J
Physiol Heart Circ Physiol. 2010;298:H360–H366.
68.O’Donnell JM, Kalichira A, Bi J, Lewandowski ED. In vivo, cardiacspecific knockdown of a target protein, malic enzyme-1, in rat via adenoviral delivery of DNA for non-native miRNA. Curr Gene Ther.
2012;12:454–462.
69. Kato M, Akao M, Matsumoto-Ida M, Makiyama T, Iguchi M, Takeda T,
Shimizu S, Kita T. The targeting of cyclophilin D by RNAi as a novel
cardioprotective therapy: evidence from two-photon imaging. Cardiovasc
Res. 2009;83:335–344.
70.Guo T, Wang J, Yang J, Chen W, An G, Fan L, Peng Q. Lentivirusmediated RNAi of chymase increases the plaque stability in atherosclerosis in vivo [published online ahead of print May 24, 2013]. Exp Mol
Pathol. 2013;95:51–56. doi: 10.1016/j.yexmp.2013.05.005.
71.Holley CL, Topkara VK. An introduction to small non-coding RNAs:
miRNA and snoRNA. Cardiovasc Drugs Ther. 2011;25:151–159.
72.Davidson BL, McCray PB Jr. Current prospects for RNA interferencebased therapies. Nat Rev Genet. 2011;12:329–340.
73. Lennox KA, Behlke MA. A direct comparison of anti-microRNA oligonucleotide potency. Pharm Res. 2010;27:1788–1799.
74. Obad S, dos Santos CO, Petri A, Heidenblad M, Broom O, Ruse C, Fu
C, Lindow M, Stenvang J, Straarup EM, Hansen HF, Koch T, Pappin D,
Hannon GJ, Kauppinen S. Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet. 2011;43:371–378.
75.Gambari R, Fabbri E, Borgatti M, Lampronti I, Finotti A, Brognara
E, Bianchi N, Manicardi A, Marchelli R, Corradini R. Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development. Biochem Pharmacol.
2011;82:1416–1429.
76.Kluiver J, Slezak-Prochazka I, Smigielska-Czepiel K, Halsema N,
Kroesen BJ, van den Berg A. Generation of miRNA sponge constructs.
Methods. 2012;58:113–117.
77. Poller W, Fechner H. Development of novel cardiovascular therapeutics
from small regulatory RNA molecules–an outline of key requirements.
Curr Pharm Des. 2010;16:2252–2268.
78. Nakai H, Thomas CE, Storm TA, Fuess S, Powell S, Wright JF, Kay MA.
A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated
liver transduction. J Virol. 2002;76:11343–11349.
79. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel
adeno-associated viruses from rhesus monkeys as vectors for human gene
therapy. Proc Natl Acad Sci U S A. 2002;99:11854–11859.
80. Gao GP, Lu Y, Sun X, Johnston J, Calcedo R, Grant R, Wilson JM. Highlevel transgene expression in nonhuman primate liver with novel adenoassociated virus serotypes containing self-complementary genomes.
J Virol. 2006;80:6192–6194.
81.Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg
B, Borow K, Dittrich H, Zsebo KM, Hajjar RJ; Calcium Up-Regulation
by Percutaneous Administration of Gene Therapy In Cardiac Disease
(CUPID) Trial Investigators. Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-inhuman phase ½ clinical trial. J Card Fail. 2009;15:171–181.
82. Bish LT, Morine K, Sleeper MM, Sanmiguel J, Wu D, Gao G, Wilson JM,
Sweeney HL. Adeno-associated virus (AAV) serotype 9 provides global
cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the
mouse and rat. Hum Gene Ther. 2008;19:1359–1368.
83.Inagaki K, Fuess S, Storm TA, Gibson GA, Mctiernan CF, Kay MA,
Nakai H. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther.
2006;14:45–53.
84.Pacak CA, Mah CS, Thattaliyath BD, Conlon TJ, Lewis MA, Cloutier
DE, Zolotukhin I, Tarantal AF, Byrne BJ. Recombinant adeno-associated
virus serotype 9 leads to preferential cardiac transduction in vivo. Circ
Res. 2006;99:e3–e9.
85. Geisler A, Jungmann A, Kurreck J, Poller W, Katus HA, Vetter R, Fechner
H, Müller OJ. microRNA122-regulated transgene expression increases
specificity of cardiac gene transfer upon intravenous delivery of AAV9
vectors. Gene Ther. 2011;18:199–209.
86. Yang L, Jiang J, Drouin LM, Agbandje-McKenna M, Chen C, Qiao C, Pu
D, Hu X, Wang DZ, Li J, Xiao X. A myocardium tropic adeno-associated
virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl
Acad Sci U S A. 2009;106:3946–3951.
87. Okada H, Ishibashi H, Hayashita-Kinoh H, Chiyo T, Nitahara-Kasahara Y,
Baba Y, Watanabe S, Takeda S, Okada T. Robust Long-term Transduction
of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9.
Mol Ther Nucleic Acids. 2013;2:e95.
88.Goehringer C, Rutschow D, Bauer R, Schinkel S, Weichenhan D,
Bekeredjian R, Straub V, Kleinschmidt JA, Katus HA, Müller OJ.
Prevention of cardiomyopathy in delta-sarcoglycan knockout mice after
systemic transfer of targeted adeno-associated viral vectors. Cardiovasc
Res. 2009;82:404–410.
89. Müller OJ, Leuchs B, Pleger ST, Grimm D, Franz WM, Katus HA,
Kleinschmidt JA. Improved cardiac gene transfer by transcriptional and
transductional targeting of adeno-associated viral vectors. Cardiovasc
Res. 2006;70:70–78.
90. Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs
can inhibit the expression of cognate mRNAs when expressed in human
cells. Mol Cell. 2002;9:1327–1333.
91. Liu YP, Haasnoot J, ter Brake O, Berkhout B, Konstantinova P. Inhibition
of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res. 2008;36:2811–2824.
92. Lau S, Graham B, Cao N, Boyd BJ, Pouton CW, White PJ. Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNAalbumin conjugation. Mol Pharm. 2012;9:71–80.
93. Lu P, Yuan L, Wang Y, Du Q, Sheng J. Effect of GPE-AGT nanoparticle
shRNA transfection system mediated RNAi on early atherosclerotic lesion. Int J Clin Exp Pathol. 2012;5:698–706.
94.Zhou J, Rossi JJ. Aptamer-targeted cell-specific RNA interference.
Silence. 2010;1:4.
95. Nolte A, Walker T, Schneider M, Kray O, Avci-Adali M, Ziemer G,
Wendel HP. Small-interfering RNA-eluting surfaces as a novel concept
for intravascular local gene silencing. Mol Med. 2011;17:1213–1222.
96.Rapti K, Louis-Jeune V, Kohlbrenner E, Ishikawa K, Ladage D,
Zolotukhin S, Hajjar RJ, Weber T. Neutralizing antibodies against AAV
serotypes 1, 2, 6, and 9 in sera of commonly used animal models. Mol
Ther. 2012;20:73–83.
97. Sioud M. Recent advances in small interfering RNA sensing by the immune system. N Biotechnol. 2010;27:236–242.
98. Toroney R, Hull CM, Sokoloski JE, Bevilacqua PC. Mechanistic characterization of the 5’-triphosphate-dependent activation of PKR: lack of
5’-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD. RNA. 2012;18:1862–1874.
99. Mao CP, Wu TC. Inhibitory RNA molecules in immunotherapy for cancer. Methods Mol Biol. 2010;623:325–339.
100. Büning H. Gene therapy enters the pharma market: the short story of a
long journey. EMBO Mol Med. 2013;5:1–3.
101.Klattenhoff CA, Scheuermann JC, Surface LE, Bradley RK, Fields
PA, Steinhauser ML, Ding H, Butty VL, Torrey L, Haas S, Abo R,
Tabebordbar M, Lee RT, Burge CB, Boyer LA. Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell.
2013;152:570–583.
102. Castel SE, Martienssen RA. RNA interference in the nucleus: roles for
small RNAs in transcription, epigenetics and beyond. Nat Rev Genet.
2013;14:100–112.
602 Circulation Research August 16, 2013
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
103. Kemp C, Mueller S, Goto A, Barbier V, Paro S, Bonnay F, Dostert C,
Troxler L, Hetru C, Meignin C, Pfeffer S, Hoffmann JA, Imler JL. Broad
RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila. J Immunol. 2013;190:650–658.
104. Parameswaran P, Sklan E, Wilkins C, et al. Six RNA viruses and fortyone hosts: viral small RNAs and modulation of small RNA repertoires in
vertebrate and invertebrate systems. PLoS Pathog. 2010;6:e1000764.
105. Berezikov E. Evolution of microRNA diversity and regulation in animals. Nat Rev Genet. 2011;12:846–860.
106. Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet.
2011;12:99–110.
107. Eulalio A, Mano M, Dal Ferro M, Zentilin L, Sinagra G, Zacchigna S,
Giacca M. Functional screening identifies miRNAs inducing cardiac regeneration. Nature. 2012;492:376–381.
108.Mercola M. Cardiovascular biology: a boost for heart regeneration.
Nature. 2012;492:360–362.
109. Pasquinelli AE. MicroRNAs and their targets: recognition, regulation and
an emerging reciprocal relationship. Nat Rev Genet. 2012;13:271–282.
110. Roberts CT Jr, Kurre P. Vesicle trafficking and RNA transfer add complexity and connectivity to cell-cell communication. Cancer Res.
2013;73:3200–3205.
111. Ohanian M, Humphreys DT, Anderson E, Preiss T, Fatkin D. A heterozygous variant in the human cardiac miR-133 gene, MIR133A2, alters miRNA duplex processing and strand abundance. BMC Genet. 2013;14:18.
112.Elton TS, Selemon H, Elton SM, Parinandi NL. Regulation of the
mir155 host gene in physiological and pathological processes. Gene.
2012;112:01512-0.
113. Sati S, Ghosh S, Jain V, Scaria V, Sengupta S. Genome-wide analysis
reveals distinct patterns of epigenetic features in long non-coding RNA
loci. Nucleic Acids Res. 2012;40:10018–10031.
114. Peng X, Gralinski L, Armour CD, et al. Unique signatures of long noncoding RNA expression in response to virus infection and altered innate
immune signaling. MBio. 2010;1:e00206–e00210.
115. Tsitsiou E, Williams AE, Moschos SA, Patel K, Rossios C, Jiang X,
Adams OD, Macedo P, Booton R, Gibeon D, Chung KF, Lindsay MA.
Transcriptome analysis shows activation of circulating CD8+ T cells in
patients with severe asthma. J Allergy Clin Immunol. 2012;129:95–103.
116. Guttman M, Donaghey J, Carey BW, et al. lincRNAs act in the circuitry
controlling pluripotency and differentiation. Nature. 2011;477:295–300.
117. Schonrock N, Harvey RP, Mattick JS. Long noncoding RNAs in cardiac
development and pathophysiology. Circ Res. 2012;111:1349–1362.
118. Marian AJ, Belmont J. Strategic approaches to unraveling genetic causes
of cardiovascular diseases. Circ Res. 2011;108:1252–1269.
119. Johnson AD, Hwang SJ, Voorman A, et al. Resequencing and clinical
associations of the 9p21.3 region: a comprehensive investigation in the
Framingham heart study. Circulation. 2013;127:799–810.
120. Congrains A, Kamide K, Oguro R, et al. Genetic variants at the 9p21
locus contribute to atherosclerosis through modulation of ANRIL and
CDKN2A/B. Atherosclerosis. 2012;220:449–455.
121.Pasmant E, Sabbagh A, Vidaud M, Bièche I. ANRIL, a long, noncoding RNA, is an unexpected major hotspot in GWAS. FASEB J.
2011;25:444–448.
122. Congrains A, Kamide K, Katsuya T, Yasuda O, Oguro R, Yamamoto K,
Ohishi M, Rakugi H. CVD-associated non-coding RNA, ANRIL, modulates expression of atherogenic pathways in VSMC. Biochem Biophys
Res Commun. 2012;419:612–616.
123.Congrains A, Kamide K, Ohishi M, Rakugi H. ANRIL: Molecular
Mechanisms and Implications in Human Health. Int J Mol Sci.
2013;14:1278–1292.
124. Wan G, Mathur R, Hu X, Liu Y, Zhang X, Peng G, Lu X. Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling
pathway. Cell Signal. 2013;25:1086–1095.
125. Ishii N, Ozaki K, Sato H, Mizuno H, Saito S, Takahashi A, Miyamoto
Y, Ikegawa S, Kamatani N, Hori M, Saito S, Nakamura Y, Tanaka T.
Identification of a novel non-coding RNA, MIAT, that confers risk of
myocardial infarction. J Hum Genet. 2006;51:1087–1099.
126. Tsuiji H, Yoshimoto R, Hasegawa Y, Furuno M, Yoshida M, Nakagawa
S. Competition between a noncoding exon and introns: Gomafu contains
tandem UACUAAC repeats and associates with splicing factor-1. Genes
Cells. 2011;16:479–490.
127.Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence
JB, Chess A. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics.
2007;8:39.
128. Wilusz JE, Sunwoo H, Spector DL. Long noncoding RNAs: functional
surprises from the RNA world. Genes Dev. 2009;23:1494–1504.
129. Eißmann M, Gutschner T, Hämmerle M, Günther S, Caudron-Herger M,
Groß M, Schirmacher P, Rippe K, Braun T, Zörnig M, Diederichs S. Loss
of the abundant nuclear non-coding RNA MALAT1 is compatible with
life and development. RNA Biol. 2012;9:1076–1087.
130.Gutschner T, Baas M, Diederichs S. Noncoding RNA gene silencing
through genomic integration of RNA destabilizing elements using zinc
finger nucleases. Genome Res. 2011;21:1944–1954.
131. Nakagawa S, Ip JY, Shioi G, Tripathi V, Zong X, Hirose T, Prasanth KV.
Malat1 is not an essential component of nuclear speckles in mice. RNA.
2012;18:1487–1499.
132. Zhang B, Arun G, Mao YS, Lazar Z, Hung G, Bhattacharjee G, Xiao X,
Booth CJ, Wu J, Zhang C, Spector DL. The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory
role in the adult. Cell Rep. 2012;2:111–123.
133.Gutschner T, Hämmerle M, Eissmann M, Hsu J, Kim Y, Hung G,
Revenko A, Arun G, Stentrup M, Gross M, Zörnig M, MacLeod AR,
Spector DL, Diederichs S. The noncoding RNA MALAT1 is a critical
regulator of the metastasis phenotype of lung cancer cells. Cancer Res.
2013;73:1180–1189.
134. Pichlmair A, Lassnig C, Eberle CA, Górna MW, Baumann CL, Burkard
TR, Bürckstümmer T, Stefanovic A, Krieger S, Bennett KL, Rülicke
T, Weber F, Colinge J, Müller M, Superti-Furga G. IFIT1 is an antiviral protein that recognizes 5’-triphosphate RNA. Nat Immunol.
2011;12:624–630.
135.Raychoudhuri A, Shrivastava S, Steele R, Kim H, Ray R, Ray RB.
ISG56 and IFITM1 proteins inhibit hepatitis C virus replication. J Virol.
2011;85:12881–12889.
136.Yan N, Chen ZJ. Intrinsic antiviral immunity. Nat Immunol.
2012;13:214–222.
137. Mao YS, Sunwoo H, Zhang B, Spector DL. Direct visualization of the
co-transcriptional assembly of a nuclear body by noncoding RNAs. Nat
Cell Biol. 2011;13:95–101.
138. Zhang Q, Chen CY, Yedavalli VS, Jeang KT. NEAT1 long noncoding
RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression. MBio. 2013;4:e00596–e00512.
139. Geirsson A, Bothwell AL, Hammond GL. Inhibition of alloresponse by
a human trophoblast non-coding RNA suppressing class II transactivator
promoter III and major histocompatibility class II expression in murine
B-lymphocytes. J Heart Lung Transplant. 2004;23:1077–1081.
140. Geirsson A, Paliwal I, Lynch RJ, Bothwell AL, Hammond GL. Class
II transactivator promoter activity is suppressed through regulation by a
trophoblast noncoding RNA. Transplantation. 2003;76:387–394.
141.Geirsson A, Lynch RJ, Paliwal I, Bothwell AL, Hammond GL.
Human trophoblast noncoding RNA suppresses CIITA promoter III
activity in murine B-lymphocytes. Biochem Biophys Res Commun.
2003;301:718–724.
142. Clark MB, Johnston RL, Inostroza-Ponta M, Fox AH, Fortini E, Moscato
P, Dinger ME, Mattick JS. Genome-wide analysis of long noncoding
RNA stability. Genome Res. 2012;22:885–898.
143. Blattmann P, Schuberth C, Pepperkok R, Runz H. RNAi-based functional
profiling of loci from blood lipid genome-wide association studies identifies
genes with cholesterol-regulatory function. PLoS Genet. 2013;9:e1003338.
144. Barabási AL, Gulbahce N, Loscalzo J. Network medicine: a networkbased approach to human disease. Nat Rev Genet. 2011;12:56–68.
145.Danchin É, Charmantier A, Champagne FA, Mesoudi A, Pujol B,
Blanchet S. Beyond DNA: integrating inclusive inheritance into an extended theory of evolution. Nat Rev Genet. 2011;12:475–486.
146.Lehman N. RNA in evolution. Wiley Interdiscip Rev RNA.
2010;1:202–213.
147. Shabalina SA, Koonin EV. Origins and evolution of eukaryotic RNA interference. Trends Ecol Evol. 2008;23:578–587.
148. Koonin EV, Senkevich TG, Dolja VV. The ancient Virus World and evolution of cells. Biol Direct. 2006;1:29.
149.Mukherjee K, Campos H, Kolaczkowski B. Evolution of animal and
plant dicers: early parallel duplications and recurrent adaptation of antiviral RNA binding in plants. Mol Biol Evol. 2013;30:627–641.
Cardiovascular RNA Interference Therapy: The Broadening Tool and Target Spectrum
Wolfgang Poller, Juliane Tank, Carsten Skurk and Martina Gast
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 2013;113:588-602
doi: 10.1161/CIRCRESAHA.113.301056
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/113/5/588
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2013/08/07/CIRCRESAHA.113.301056.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
SUPPLEMENTAL MATERIAL
1
2
3
4
5
Cardiovascular RNA Interference Therapy: The Broadening Tool and Target
Spectrum
6
7
Supplemental Figures I and II
8
Primary Transcription and Processing of the Long Noncoding RNAs MALAT1 and NEAT1
9
10
11
Refering to Fig. 5 which outlines therapeutic perspectives of lncRNAs and their processing products,
12
13
14
15
16
17
18
19
20
21
22
23
24
Figure I: Whereas the large 7 kb primary MALAT1 transcript sequence is overall not well conserved
25
26
Figure II: Different primary transcripts (23 kb MEN, 3.7 kb MEN) arise from the NEAT1 locus near
27
28
29
30
31
32
33
34
35
36
37
38
across species (Panel A). Similar to MALAT1, MEN is also processed by RNAase P and RNAse Z to
39
the graphs below show details regarding the long primary transcripts from the MALAT1 (Figure I) and
NEAT 1 (Figure II) genomic locus and their peculiar posttranscriptional processing by RNAses.
across species (sequence comparison, Panel A), a 61-nt tRNA-like processing product generated by
RNAase P and RNAse Z and designated as mascRNA, is highly conserved (Panel B)
37,133
. MALAT1
was recently observed to be deregulated in human viral cardiomyopathies, and recombinant
mascRNA modulates the antiviral immune response (Poller et al., unpublished), suggesting possible
pathogenic relevance of the MALAT1 system. From a therapeutic perspective, it is of interest that
these findings were triggered by systematic cardiac transcriptome mapping of cardiomyopathy
patients, whereas this system had not been linked to cardiac diseases before. This example also
illustrate difficulties which may arise in searches for lncRNAs of therapeutic interest. Sequence
conservation is low when the whole primary transcripts are considered, and without further information
as available for MALAT1 and NEAT1 it is nearly impossible to identify any domains of particular
functional importance. Further, measurement of both precursor and products expression in diseased
organs is required to obtain a complete picture of the lncRNA system.
MALAT1 on human chromosome 11q13.1. As MALAT1, NEAT1 is also overall not well conserved
yield a small tRNA-like product designated as menRNA (Panel B)
38,39,47,136,142
. From the biochemical
view it is remarkable that mascRNA and menRNA are generated by similar biosynthetic pathways, and
most probably these small RNA products have functions distinct from their large precursors, and
different intracellular kinetics and trafficking. Whereas MALAT1 is intensively investigated in oncology,
however, there is only fragmentary knowledge regarding MALAT1 and NEAT1 in the cardiovascular
system despite evidence that they are deregulated in human cardiac diseases. As outlined in Fig. 4,
the sequential steps of genomic discovery, structural characterization, functional genomic knockout of
the whole lncRNA systems, and transcriptome mapping in various diseases, further tasks are the
blockade or overexpression not only of the whole system, but also of single processing products, both
in various cardiovascular cell types and animal models, using ablative or ncRNA transfer technologies
as discussed above. Reference numbers in this legend refer to the main manuscript.
1
2
3
4
SUPPLEMENTAL FIGURE I
1
2
3
4
SUPPLEMENTAL FIGURE II