MicroRNA-based therapeutics in cardiovascular disease: screening and delivery to the target

David Mellis and Andrea Caporali

University/BHF Centre for Cardiovascular Science, the QMRI, University of

Edinburgh, Edinburgh EH16 4TJ, UK.

Corresponding Author:

Dr Andrea Caporali, PhD

University/British Heart Foundation Centre for Cardiovascular Science,

The Queen's Medical Research Institute,

University of Edinburgh, Edinburgh, EH16 4TJ, UK

Tel: +44 131 2426760

Email: a.caporali@ed.ac.uk

Words: 3,665

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22-nucleotides,

which have increasingly been recognized as potent post-transcriptional regulators of

gene expression. MiRNA targeting is defined by the complementarities between

positions 2 to 8 of miRNA 5′-end with generally the 3′ untranslated region of target

mRNAs. The capacity of miRNAs to simultaneously inhibit many different mRNAs

allows for an amplification of biological responses. Hence, miRNAs are extremely

attractive targets for therapeutic regulation in several diseases, including

cardiovascular. Novel approaches are emerging to identify the miRNA functions in

cardiovascular biology processes and to improve miRNA delivery in the heart and

vasculature. In this article, we provide an overview of current studies of miRNA

functions in cardiovascular cells by the use of high-content screening. We also

discuss the challenge to achieve a safe and targeted delivery of miRNA therapeutics

in cardiovascular cells.

Abbreviations: microRNAs (miRNAs), long non-coding RNAs (lncRNA), high-

content screening (HCS); untranslated region (UTR); cardiac myocytes (CMs);

smooth muscle cells (SMC), endothelial Cells (ECs); adeno associated virus (AAV);

myocardial infarction (MI)

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IntroductionThe foundation of molecular biology states that DNA is copied to RNA by the

process of transcription. The transcribed RNA then translates the information to

produce proteins. This process is one of the most well characterised areas of

modern day biology. Although clearly a pivotal role for RNA, the human genome

encodes many RNAs that do not code for proteins, these are referred to as the non-

coding RNAs (ncRNAs). Although ncRNAs lack the ability to encode proteins, recent

insights over many years have revealed an important role for ncRNAs in the

regulation of gene expression in health and disease (1). Different classes of ncRNA

have been identified, differing from each other by nucleotide sequence length,

folding and function (2). The most well-known ncRNAs are structural RNA belonging

to ribosomal RNA (rRNA) and transfer RNA (tRNA), both involved in translation

events (3). Other ncRNA classes are small nuclear RNAs (snRNA), piwi-interacting

RNA (piRNA) and long non-coding RNAs (lncRNA) (4). The lncRNAs are ncRNA

longer than 200 nucleotides which have recently attracted the interest of many

research groups attempting to understand their role in human biology (5) lncRNAs

are involved in numerous important biological phenomena such as imprinting

genomic loci, shaping chromosome conformation and allosterically regulating

enzymatic activity. Specific patterns of lncRNA expression coordinate cell state,

differentiation, development and disease (6). Another interesting class of ncRNA are

microRNAs (miRNAs), From the discovery in C.elegans (7), miRNAs have been

identified in animals, plants and viruses where they are characterized as

endogenous single stranded small ncRNAs of 20-22 nucleotides in length that play

key roles in RNA silencing and in the post-transcriptional regulation of gene

expression (8). They are highly conserved between mammals and are thought to be

a vital evolutionary component of gene regulation (9, 10). The vast number of

different miRNAs currently identified can target and degrade between 50-60% of all

mammalian genes by binding to target sites found within the 3′ untranslated region

(3’ UTR) of the targeted messenger RNA (mRNA) (11). Most target sites on mRNA

only share a partial complementarity with their corresponding miRNAs. This gives a

single miRNA the ability to target many different mRNA within a genetic network,

thus contributing to biological processes and disease (12).

Interestingly, miRNAs that belong to miR-16 family and contain the AGCAGC motif

at the 5’ end, have many more complementary sites in the CDS compared with

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3’UTR and they inhibit the protein translation (13, 14). Moreover, miRNAs can bind

5’-UTR of the target gene and regulate gene transcription as it has been reported for

hsa-miR-34-5p (15).

These features of miRNAs make them a highly potent regulator of post-

transcriptional gene expression and begins to explain their involvement in such a

wide range of different biological and pathological processes. Studies over recent

years have revealed that miRNAs effect a diverse range of biological processes

including development, cell differentiation, proliferation and apoptosis, linking

miRNAs to multi-disease processes including cancer, neurodegenerative and

cardiovascular disease (16-18).

This diverse range of biological effects have made them a tempting target for drug

discovery (19). By exploiting miRNAs ability to effect multi-gene complexes in a wide

range of biological systems we have an excellent opportunity to study the

mechanisms behind these processes and their resultant phenotypes. Before we can

begin to understand the genetic networks, we need to identify the miRNAs involved

and their contributions. The most effective way to achieve this is to modulate the

intracellular levels of miRNAs by transfecting cells with miRNA mimics or inhibitors.

There are several commercially available mimics that allow the overexpression of

miRNAs in vitro by introducing synthetic miRNA hairpin precursors or duplex miRNA

mimics. Inhibitors are also available that allow us to decrease miRNA expression.

These predominately function by sequestering miRNAs away from their targets and

are based around modified nucleotides including the locked nucleic acids technology

(LNA) (20). We can use these tools to gain better insight into the role of miRNAs

and identify their functions and develop a miRNA-based therapeutic (Figure 1).

Looking for miRNA-derived phenotype in cardiovascular cellsIn the past decade, the focus on the identification of novel therapeutic targets for

clinical applications in cardiovascular disease has shifted from gene-expression

analysis, to identification of genes based on their function (21). Disease-target genes

can now be identified in a high-throughput fashion based on functional properties

that are directly related to the disease phenotype (high content screening; HCS)(22-

24). Several research teams have adopted the functional genomic approach and

HCS technique to identify the primary function of each miRNA in normal biological

functions and in human disease (25, 26) . Over the last decade, this technique has

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helped identify and elucidate miRNAs roles and functions in cell signalling (27), stem

cell homeostasis (28), human infection (29) and cancer biology (30, 31). However, in

recent years, HCS characterized the phenotype of miRNAs involved in

cardiovascular cell function (32-34).

A functional screening study by Ana Eulalio and colleagues in 2012 investigated the

role of miRNAs in cardiomyocytes (CMs) proliferation and heart regeneration (33).

Once damaged by ischemic injury, the human heart has a very poor regenerative

ability. CMs have some potential to repair damaged tissue, however, this remains

limited and finding miRNAs that could increase cardiac regeneration would have

huge therapeutic benefit. In this study, they set out to systematically identify any

miRNAs that can increase cardiomyocyte proliferation. To identify any novel miRNAs

involved in CM proliferation, they use a library of 875 miRNA mimics in neonatal rat

CMs in conjunction with HCS to identify potential targets. Further functional testing

identified miRNA hsa-miR-590-5p and hsa-miR-199-5p as the top two candidates for

promoting cell cycle re-entry of adult cardiomyocyte ex vivo and in promoting CM

proliferation in both neonatal and adult animals. For these miRNAs to be a useful

therapeutic target, they need to retain their function in vivo. To investigate hsa-miR-

199-5p and hsa-miR-590-5p mimics in vivo, Eulalio et al used two different delivery

methods. They used the readily available miRNA mimics in a complex with a lipid

based transfection reagent to study short-term expression and for longer-term

studies, cloned the miRNAs into adeno-associated viruses (AAVs). Both were

injected into the neonatal rat heart and proliferation of CMs were studied. As

predicted, in mice subjected to myocardial infarction (MI), hsa-miR-199-5p and hsa-

miR-590-5p significantly increased CM proliferation in vivo with a reduction in the

size of the infarction and improved cardiac function. Their adenoviral delivery

methods produced a high rate of expression in the heart overcoming the notoriously

difficult challenges with delivery miRNAs. The poor delivery to the human heart has

been a major limitation of miRNAs therapeutic benefit; we discuss further in the next

section. This study nicely demonstrates that using HCS allows you to screen

hundreds of miRNAs quickly and through well thought out functional analysis,

specific miRNAs can be identified and validated in your model of interest. Although

this study shows hsa-miR-199-5p and hsa-miR-590-5p as having a benefit post-

infarction in the mouse heart, the translation from mouse or rat to the much larger

human heart has still proven difficult.

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A tremendous benefit of high-content screening with high throughput microscopy is

the diversity and flexibility of the technique. By changing the cell type, the

immunostaining or the functional output, it can reveal a completely new set of miRNA

candidates. Jentzsch et al used a similar high throughput technique to the author

Eulalio but rather than finding miRNA candidates that regulate cardiomyocyte

proliferation, they investigated cardiomyocyte hypertrophy relating to heart failure

(32). This study investigated the role miRNAs have in effecting CMs size in vitro.

Jentzech and colleagues identified miRNAs that increased cell hypertrophy and were

also highly expressed in CMs (35). These studies demonstrate the huge potential

HCS has in identifying novel miRNAs and the multiple roles they have in human

health and disease. This technique is highly adaptable and works with a variety of

different biological assays to address specific research question.

To understand whether miRNAs could control heart contractility, Wahlquist and

collaborators performed a high-throughput functional screening to identify miRNAs

that target the sarcoplasmic reticulum calcium ATPase SERCA2a (34). The authors

identified and characterized hsa-miR-25-5p as a strong regulator of SERCA2a

expression by a reporter construct composed of the 3′UTR of SERCA2a fused with

EGFP. Inhibition of hsa-miR-25-5p by the administration of an antagomiR specific to

hsa-miR-25-5p improved cardiac function and survival of a mouse model of

established heart failure.

To identify miRNAs that alter human aortic smooth muscle cells (SMC), Fiedler et al

performed a high-throughput miRNA mimic screening (36). Applying a miRNA library

of 250 miRNA mimics, the authors identified several miRNAs that altered cellular

proliferation 72 h after miRNA mimic transfection. Hsa-miR-24-3p was identified as a

master regulator of SMC proliferation. Proteome profiling showed that hsa-miR-24-

3p dependent impact on cellular stress-associated factors was due to the regulation

of heme oxygenase 1. In vitro and ex vivo models showed that hsa-miR-24-3p was

inhibiting the development of vasculature in a model of engineered heart tissue.

In our lab, we have taken advantage of functional HCS to identify, for the first time,

miRNAs that regulate EC proliferation, whereby each miRNA represents a potential

therapeutic angiogenesis target. Human vein umbilical endothelial cells were

transiently transfected with 1500 unique miRNA Mimics and proliferation was

analysed. We have identified 124 miRNAs that have significantly enhanced EC

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growth; among them 24 were both evolutionarily conserved and accounted for high

expression in four different ECs sources (unpublished data).

Finally, high-content phenotypic screening can be used for miRNA target

identification, pinpointing the targets that are relevant for a specific phenotype

caused by a specific miRNA. This is particularly important, because one miRNA can

have hundreds of putative targets, but only few of these might be relevant for the

phenotype under study. In line with this, knockdown or genetic ablation of the

candidate target gene should recapitulate the phenotype observed with the miRNA

under study. Importantly, this can be performed by combining gene expression

analysis upon miRNA modulation, followed by RNAi screening to discover genes

whose knockdown phenocopies the modulation of the selected miRNA. For example,

Eulalio et al. have also used this method to discover relevant targets of hsa-miR-

199a-3p and hsa-miR-590-3p for their role in increasing cardiomyocyte proliferation,

through screening of a siRNA library against genes downregulated by these miRNAs

(33).

MiRNA-based therapy in cardiovascular diseaseMicroRNAs make extremely attractive targets for drug intervention therapies (37,

38). As reported in the introduction of this review, a single miRNA can affect several

different genes simultaneously to alter complex genetic networks. This gives

miRNAs an advantage over conventional drug therapies that traditionally target a

single site within a cellular pathway (39) (Figure 2).

To date, there are few miRNA-based trials that are in Phase I showing significant

clinical promise. This includes, antimiR-103/107 (RG-125/AZD4076; Regulus

Therapeutics) for type 2 diabetes (40), miRNA mimics based on the miR-15 family

consensus sequence, packaged in EDVs that are targeted with an anti-EGFR-

specific antibody (TargomiR) for small cell lung cancer and mesothelioma (41), hsa-

miR-29a-3p mimic (MRG-201; miRagen Therapeutics) for scleroderma (42), hsa-

miR-34a-5p mimic (MRX34; Mirna Therapeutics) in patients with advanced solid

tumours (43), antimiR-155 (MRG-106; miRagen Therapeutics) for cutaneous T cell

lymphoma and mycosis fungoides (44) and antimiR-21 (RG-012; Regulus

Therapeutics) for Alport syndrome (45) (Table 1).

Only Mirvirasen (Santaris Pharma A/S) and RG-101 (Regulus Therapeutics),

antisense designed against hsa-miR-122-5p for the treatment of chronic hepatitis C

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are advanced to Phase II clinical trials. The on-going clinical trials have concluded

that patients receiving these drugs have a significant reduction in their viral titres with

no rebound effect after finishing treatment (Regulus Therapeutics, press release

dated 7 June 2016). However, the clinical development of RG-101 was discontinued

upon completion of the one remaining clinical study in July 2017. Evaluation of the

clinical data from RG-101 has led to the identification of a bilirubin transport

mechanism as the likely cause for the cases of hyperbilirubinemia in the RG-101

program (see Regulus Therapeutics, press release dated 12 June 2017).

Studies have highlighted that miRNAs could be a potential molecular therapeutic

strategy for cardiovascular disease (46, 47). Several miRNAs have key roles in

different aspects of the progression of cardiovascular diseases and have been

analysed in pre-clinical model of MI(48). miR-15 family, including hsa-miR-15a-5p,

hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-195-5p, and hsa-miR-497-5p, could serve

as a therapeutic target for the manipulation of cardiac remodelling and function in the

settings of MI. The miR-15 family is consistently found to be upregulated in cardiac

diseases and during postnatal development of the heart.

Knockdown of the miR-15 family with LNA-modified anti-miRNAs was associated

with reduced infarct size after ischaemia–reperfusion injury (49) and an increased

number of mitotic cardiomyocytes in neonatal mice (50).

Inhibition of miR-24-3p improved neovascularization in two studies. AntagomiRs

directed against miR-24-3p or local adenovirus-mediated miR-24-3p decoy delivery

improved the recovery after MI in mice (51, 52); however, the proapoptotic effect on

cardiomyocytes observed after miR-24-3p inhibition suggests that cell-specific

activity of this miRNA (53).

MiRNAs have also an important role in regulating therapeutic angiogenesis,

particularly through the regulation of EC function. MiR-92a-3p, miR-100-5p, miR-

424-5p and miR-93-5p control perfusion recovery and angiogenesis during MI and

critical limb ischemia (reviewed in (54, 55). In particular, antimiR-92a (MRG-110) is

in the pipeline to proceed to phase I clinical trial for Miragen and it could offer a

potential therapeutic to accelerate the healing process and revascularization in

chronic ischemic disease. Interestingly, inhibition of miRNAs belonging to the 14q32

locus has led to improvements in post-ischaemic blood flow in the limb (56).

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The delivery of miRNA therapies into the myocardium and vessels is a major

challenge in order to translate the pre-clinical discovery to clinical applications

(Figure 3).

One of the major problem to solve for widespread development of miRNA

therapeutics is to overcome the lipid bilayer in the cell membrane to deliver miRNA

into cells. Lipid bilayers allow small neutral, slightly hydrophobic molecules to

passively diffuse across them, while preventing large, charged molecules, like RNAs,

from crossing them (57).

Viral vectors, including the AAVs, are adaptable for miRNA delivery into the

myocardium (58) (Figure 3a). However, the safety and efficacy of AAVs in clinical

applications are still under debate. Widespread natural exposure to AAVs has

resulted in a large fraction of the population harbouring neutralizing anti-capsid

antibodies in blood and other bodily fluids (59). Furthermore, following cellular

transduction, AAV capsid epitopes can become cross-presented on MHC I

complexes, leading to the elimination of transduced cells by T lymphocytes and

corresponding loss of gene expression (60). Although AAV9 is considered a cardiac

tropism serotype; AAV9 transgenes can also be present in other tissues (61).

Moreover, the AAVs bind tightly to heparan sulphate proteoglycans and they do not

easily escape from the intraluminal space when injected into the myocardium (62).

Although most of these trials are in their infancy, non-viral delivery vectors for

miRNAs have been developed based on experimental experience on siRNA delivery

(63) (Figure 3a). The approach includes the use of liposomes or cationic polymers

that have been modified with specific ligands for receptors on target tissues to

enhance nanoparticles ability to bypass the plasma membrane and enter the target

cells (64). These modifications increase delivery of the RNA cargo by increasing

cellular uptake of genes via receptor mediated endocytosis (Figure 3b).

Lipid-based approaches utilize the lipid/nucleic acid complexes, named liposome as

delivery carriers. Liposomes are composed of the membrane-like surface, and

nucleic acids encapsulated inside. Giacca lab analysed the efficacy of different lipid

formulations in delivering miRNA mimics in a mouse model of MI (65). They showed

that single intracardiac injection of miR-199a-3p and miR-590-3p mimics complexed

with RNAi max (Thermo Fishers Scientific), a commonly available transfection

reagent, has prolonged effects on the downregulation of their target genes and is

sufficient to promote stable cardiac repair after MI in mice (65).

8

Polymer-based approaches utilize polyethylenimine (PEI), poly (lactide-co-glycolide)

(PLGA) or poly-amidoamine dendrimers(PAMAMs), as delivery carriers. PLGA and

PANAM show lower levels of damage to cell membranes and less cytotoxic to cells

compare to PEI. PEI and PLGA nanoparticles have been previously used to deliver

VEGF to stimulate neovascularization in the heart post MI (66).

We proposed a novel strategy for delivering miRNAs to ECs to regulate

angiogenesis, using polymer functionalized carbon nanotubes (CNTs) (67). CNTs

were coated with two different polymers PEI or PAMAM, followed by conjugation of

hsa-miR-503-5p oligonucleotides as recognized regulators of angiogenesis. We

demonstrated that polymer-coated CNT conferred resistance to miRNA mimics

degradation, allowed their efficient delivery to ECs and regulated angiogenic

sprouting in vitro and in vivo (67). Notably, the recent development of polymeric

nanoparticle comprising a mix of low-molecular-weight polyamines and lipids opens

the possibility to deliver siRNAs to ECs with high efficiency, thereby facilitating the

simultaneous silencing of multiple endothelial genes in vivo (68).These nanoparticles

have also been successfully used to deliver miRNA mimics to ECs in vivo (69).

An extremely novel technique called ultrasound mediated sonoporation has been

considered for miRNA delivery in the myocardium (70) (Figure 3b). It uses albumin

shelled microbubbles, which carry genetic material to target sites. The microbubbles

are gas filled acoustic microspheres that burst with ultrasound and deliver their

contents to the target site (71). Recently, this approach has been used to deliver

miR-21-5p in pig myocardium by ultrasound-targeted microbubble and prevented

coronary microembolization-induced cardiac dysfunction (72).

Most therapies were designed and developed in animal models that have seen low

success rates when translated into human clinical trials with gene and cell transfer

efficiency remaining very low (73). The size of the human heart is one reason for the

poor translation from animal to human models (74).

Many trials have attempted to use direct intramyocardial or intracoronary injection of

viral and non-viral vectors (75) and cells (76) during heart surgeries (Figure 3c).

Local injection overcome the issues with injecting the vectors systemically and lead

to better transduction efficiencies but diffusion of the therapeutic in the myocardium

remains challenging. The development of new techniques using electromechanical

mapping (77) or the use of positron emission tomography to study blood flow have

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improved the situation (78), allowing clinicians to better target site of myocardial

ischemia thus, achieving high efficiency around the site of injection.

It is hoped that by exploiting the delivery techniques discussed, miRNAs will become

an extremely effective tool in the field of vascular and cardiovascular biology. Single

gene therapy has had limited success, a single miRNA has the far greater

therapeutic potential with its unique ability to alter complex genetic network. Over the

next several years, we will begin to see the results of ongoing human clinic trials and

it is hoped that miRNAs will become a new line of treatment in multiple human

diseases.

However, at the moment, limitations are still overcome the therapeutic benefits.

Limitations in miRNA-based therapyIn vivo administration of miRNA mimics and inhibitors which relies on systemic

injection is expensive and has low efficacy. Another problem with systemic delivery

of miRNA therapeutics are the potential side effects. Since one miRNA can have

multiple targets, it may have different functions in different organs or cell types. Cell-

type-specific delivery tools might need to be developed for some miRNAs, such as

hsa-miR-24-3p for example, which has divergent effects in vascular (52) versus

cardiac cells (53).

On the other side, several miRNA family members are located on multiple different

chromosomes, with multiple miRNAs recognizing the same target. Considering this

functional redundancy, the often-modest effects after deletion or inhibition of one

miRNA of the family on individual target genes is not entirely surprising (79).

Understanding the potential for redundancy in the regulatory hierarchy of miRNA

interactions will be important to design future miRNA-based therapies.

Gain- and loss-of-function by synthesized oligonucleotides are the most frequently

employed strategies to study a target gene and miRNA function in vitro. This

approach induces very high abundance of miRNA into the cells in comparison to the

physiological miRNA expression level and it can lead to irreproducible research and

misguided interpretation of results.

Finally, most of the miRNA oligonucleotides are still trapped in endolysosomal

vesicles and cannot escape into the cytosol of the cell. Thus, agents which can

disrupt the endolysosomal compartments and help them escape from endolysosome

degradation are of great benefit. The classic approach has been to use chloroquine

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that disrupts or lyses endosomes (80). An alternative endosomal escape approach is

to conjugate endosomolytic peptides directly to the miRNA (57).

Conclusion and prospectsSuccessful implementation in miRNA-based therapeutics relies on identifying the

best miRNA candidates or miRNA targets for each disease type and the design of

delivery vehicles that confer higher stability to the therapeutic candidate and enable

tissue-specific targeting.

On the identification of the targets, advances in gene-library generation, transfection

methods, miniaturization of assays, and screening techniques have provided the

opportunity for target validation to keep up with the pace of target identification at a

genomic scale. Searching for targets by high- content analysis, which generates

associative data rather than causative data, is being caught up by novel methods

that enable direct functional linkage between gene-expression levels and phenotype.

Although technical challenges remain, recent progress has been made in the

generation of libraries, and high-content data analysis will enable a broader scientific

community to use these tools and to generate a more complete understanding of

cellular processes and associated gene-expression changes in relation to

cardiovascular phenotype. The application of genome-editing technologies based on

CRISPR/Cas9 for screening and/or validation studies (81) will further improve

miRNA characterization as recently happened for long noncoding RNA (82).

Delivery is a key point to consider in planning miRNA-based therapeutic approach in

cardiovascular disease. Local injection methods could provide a better control for

the delivery procedure and decrease costs in comparison to systemic delivery.

Tissue specific delivery would be desirable; however, at present it is very difficult to

achieve. Beside miRNA delivery using viral vectors, non-viral gene transfer, has

become a possible option for transgene delivery.

In summary, miRNA-based therapeutics in cardiovascular disease will benefit from

the development of future innovative platforms and tools, which combined with a

comprehensive analysis of mechanisms and limitations during pre-clinical

experiments, should enable miRNA therapeutics to translate to clinical practice.

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Funding information

AC and DM are supported by British Heart Foundation (PG/16/58/32275)

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Disease Therapeutic miRNA /Drug Name

Clinical Trial Status Delivery Method

Hepatitis C (including chronic

infections)

AntimiR-122 / Mirvirasen/RG-101

Completed Phase I and Phase II single

and multicentre trials LNA modified anti-sense

inhibitor

Type 2 diabetes

AntimiR-103/107 /RG-125/AZD4076

Ongoing single centre Phase I/IIa trials

N-Acetylgalactosamine -conjugated antimiR

Mesothelioma, non-small cell lung cancer

miRNA mimics based on the miR-15/107 consensus

sequence

Ongoing multicentre Phase I trial

EnGeneIC delivery vehicle: nanocells with an anti-EGFR-specific

antibody

Scleroderma miR-29 mimic / MRG-201

Ongoing single centre Phase I trial

Cholesterol conjugated miRNA duplex

Solid tumours

miR-34 mimic/ MRX34

Multicentre Phase I Lipid nanoparticles

Cutaneous T cell lymphoma and mycosis

fungoides

AntimiR-155/ MRG-106 Multicentre Phase I LNA-modified antisense

inhibitor

Alport syndrome antimiR-21/RG-012 Phase II trial

chemically modified oligonucleotide

Table 1: Selection of current miRNA-based clinical trials

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Figure Legends

Figure 1: Schematic illustrating the use of high content screening to identify miRNA

targets and the steps required to validate the targets as a treatment in disease

Figure 2: A single miRNA can affect several different genes simultaneously to alter

complex genetic networks, unlike current conventional therapeutics.

Figure 3: (A) Selection of novel methods for delivering and increasing the

effectiveness of microRNAs as a therapeutic reagent in human disease. (B)

Systemic delivery: liposomes modified with the addition of a tissue specific ligand to

enhance delivery to target tissue and subsequent uptake by receptor mediated

endocytosis. Technique of ultrasound mediated sonoporation. MiRNAs are contained

within albumin-shelled microbubbles that burst and release their contents when

exposed to ultrasonic waves. (C) Local delivery: miRNAs delivered into the heart by

intracoronary injection and intramyocardial injection. The accuracy and effectiveness

of local injections in the heart are improved by using state of the art imaging

techniques such as electromechanical mapping and positron emission tomography

(images modified from ref 77 and 78)

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