Targeted delivery of miRNA therapeutics for cardiovascular ... 2.pdf · muscle cells) leads to...

2Targeted delivery of miRNA

therapeutics for cardiovascular

diseases: opportunities and

challenges

Published in Clinical Science Vol. 126, No. 6, pp. 351-65, 2014Rick F. J. Kwekkeboom1,

Zhiyong Lei2,

Pieter A. Doevendans2,3,

René J. P. Musters1

Joost P. G. Slijter2,3

1 Department of Physiology, VU University Medical Center, Van der Boechorststraat 7,

1081 BT, Amsterdam, The Netherlands2 Experimental Cardiology Laboratory, Department of Cardiology,

Division of Heart and Lungs, University Medical Center Utrecht, P.O. Box 85500,

3508 GA, Utrecht, The Netherlands3 ICIN, Netherlands Heart Institute, Catharijnesingel 52, 3511 GC Utrecht,

The Netherlands

Chapter 2

24

Abstract

Dysregulation of miRNA expression has been associated with many cardiovascular diseases

in animal models, as well as in patients. In the present review, we summarize recent findings

on the role of miRNAs in cardiovascular diseases and discuss the opportunities, possibilities

and challenges of using miRNAs as future therapeutic targets. Furthermore, we focus on

the different approaches that can be used to deliver these newly developed miRNA thera-

peutics to their sites of action. Since siRNAs are structurally homologous with the miRNA

the rapeutics, important lessons learned from siRNA delivery strategies are discussed that

might be applicable to targeted delivery of miRNA therapeutics, thereby reducing costs and

potential side effects, and improving efficacy.

Key words: antimiR, cardiovascular disease, miRNA, siRNA, targeted delivery method

Abbreviations: ApoB, apolipoprotein B; AAV, adeno-associated virus; EC, endothelial cell,

i.v., intravenous; JNK1, c-Jun N-terminal kinase 1; LNA, locked nucleic acid; MB, microbubble;

PEI, polyethylenimine; PLGA, poly(lactic-co-glycolic acid); PLL, poly-L-lysine; RISC, RNA-

induced silencing complex; SHP-1, Src homology 2 domain-containing protein tyrosine

phosphatase-1; TLR, Toll-like receptor; US, ultrasound.

miRNA therapeutics for cardiovascular diseases

25

2

miRNA BASICS

miRNAs are small regulatory non-coding RNA molecules 19–22 nucleotides in length, which

bind to the 3’-UTR region of mRNAs and regulate mRNA expression via degradation or inhi-

bition of their translation [1,2]. miRNAs are transcribed in the nucleus by RNA polymerase II,

like mRNAs, and are subsequently processed into a pre-miRNA by a combination of proteins

including the nuclear-located endonucleases Pasha and Drosha [3]. After being transported

out of the nucleus into the cytosol by ATPconsuming exportin-5 [4,5], pre-miRNAs are

processed further into mature miRNAs by the RNA endonuclease Dicer [6,7]. This step is

followed by the binding of RISC (RNA-induced silencing complex) [8], after which miRNAs

are ready to fulfil their inhibitory roles. The biogenesis and function of miRNAs have been

extensively discussed in previous work [9–14].

miRNAs IN CARDIOVASCULAR DISEASES

Over the last decade, miRNAs have been investigated extensively. In mammalians, they

are involved in many biological processes, if not all, including ESC (embryonic stem cell)

pluripotency [15], cellular proliferation [16–22], driving and directing differentiation [23,24],

regulating apoptosis [25,26], and aging [27–29]. The overall importance of miRNAs in the

cardiovascular system was established by knocking out key miRNA-processing components.

For example, cardiac-specific knockout of Dicer leads to dilated cardiomyopathy, heart

failure and eventually lethality in mice [30]. These Dicer-knockout mice have deregulated

cardiac contractile proteins and profound sarcomere disarray [30]. Similar to Dicer-knockout

mice, post-natal inactivation of Pasha in the heart leads to left ventricular malfunction,

dilated cardiomyopathy and lethality in adult mice [31]. The importance of miRNAs is not

limited to cardiomyocytes, since specific knockout of Dicer or Pasha in SMCs (smooth

muscle cells) leads to reduced proliferation and impaired contractility [32,33], and in ECs

(endothelial cells) to impaired post-natal angiogenesis [34]. Furthermore, specific miRNAs

were found to have pivotal roles in cardiovascular diseases, as has been explored exten-

sively in different animal models and is summarized in Figure 1. For example, Wang and

co-workers [35] and van Rooij co-workers [36] have shown that inhibition of dysregulated

miR-208 in pressureoverloaded hearts preserved cardiac function and reduced mortality.

In addition, when the heart is under stress, cardiomyocytes produce more ‘rubbish’, which

needs to be removed via autophagy. Ucar et al. [37] showed that misregulated miR-132/212

in the pressure-overloaded heart suppressed cardiomyocyte autophagy, which turned out

to be detrimental to cardiac function. Pharmaceutical inhibition of miR-132/212 rescued

the autophagic response and preserved cardiac function [37]. In another study, systemic

inhibition of miR-92a enhanced vessel growth and functional recovery of damaged tissue

Chapter 2

26

in a mouse hindlimb ischaemia model and myocardial infarction [38]. Similar results have

been reported in a large animal study in which inhibition of miR-92a reduced infarct size

and resulted in improved ejection fraction [39]. Owing to limited space, we refer to Table 1

and recent reviews for the role of individual miRNAs [13,40–43].

Figure 1: miRNAs involved in cardiovascular diseases. miRNAs are reported to be involved in many dif-

ferent types of cardiovascular diseases and in different cell types such as in angiogenesis for endothelial cells,

in cardiac fibrotic remodeling for myofibroblasts, and in proliferation, cell death, conduction and arrhythmias

for cardiomyocytes.

POTENTIAL OF mRNA THERAPEUTICS FOR CARDIOVASCULAR DISEASES

miRNA gain-of-functionAn advantage of miRNAs as potential therapeutic targets is their synergistic effects. miRNAs

bind to their targets in a partial complementary manner and increasing evidence suggests

that a single miRNA can target several genes within a similar signalling pathway [44,45].

The effect of a miRNA on an individual mRNA target sometimes is low, but, due to the

simultaneous regulation of multiple targets within a pathway, the combined effect is more

pronounced [45]. For example, miR-29 regulates the level of fibrosis through the direct

targeting of different collagens, fibrillins and elastins, which are all components of the


27

2

Chapter 2

28

extracellular matrix and collectively determine tissue stiffness, whereas each component

individually is only partly inhibited [46]. Therefore one advantage of using miRNA mimics is

the inhibitory effect they have on a selected pathway, by blocking several targets and lead-

ing to pronounced effects, both in vitro and in vivo. Another advantage of using miRNAs as

a therapeutic target is that their gain-of-function is easy to achieve by using miRNA mimics

or viral approaches. Depending on the vendor, the synthesized structure of miRNA mimics

varies, but they all have similar structures. The structure either resembles endogenously

present pre-miRNAs with a stem–loop or consists of a doublestranded RNA to help the

mimic get incorporated into RISCs. Many miRNA mimics have been shown to be functional

in vitro by numerous laboratories, including our own [19,47–

50]. miRNAs are relatively stable in the cell when compared with other RNA species, with

a half-life ranging from 28 h to 5 days, which is approximately 10 times longer than that of

mRNA molecules [51]. The long half-life of miRNAs indicates that potential miRNA mimics

may have long-lasting effects. van Rooij et al. [46] were among the first researchers who

tried to overexpress miR-29 mimics by systematic injection of cholesteroltagged miRNA

mimics at a dose of 80 mg/kg of body weight. They showed that there was a modest down-

regulation of miR-29-targeted genes, but this was accompanied by less fibrotic remodelling

[46]. Only a few in vivo applications of miRNA mimics can be found in the literature; however,

viral approaches, especially AAV (adeno-associated virus)-mediatedmiRNA gain-of-function,

are more widely used [52,53]. Owing to its low immunogenicity, the transgene can remain

active for months [53]. Eulalio et al. [53] used AAV9, a cardiac tropism of AAV serotypes,

to overexpress miR-590-3p and miR-199a-3p and showed a marked cardiac regeneration

aftermyocardial infarction [53]. The long-lasting high level of transgene expression make

AAVs an ideal tool for research, with potential future clinical applications.

miRNA inhibitionmiRNAs can be inhibited by synthetic miRNA inhibitors in vitro as well as in vivo [54].

The synthesis of miRNA inhibitors is even easier comparedwith miRNAmimics as they are

single-stranded. AntimiRs, widely used for in vitro studies, are chemically engineered single-

stranded oligonucleotides completely complementary to the 20-nucleotide-long targeted

miRNAs. To facilitate entry into the cell, antimiRs can be modified with cholesterol into a

so-called antagomiR [55].Upon systemic intravenous injection, this cholesterol-tagged an-

tagomiR can efficiently and specifically silence endogenous miRNAs throughout the body,

except for the brain [55]. miRNA inhibitors do not require incorporation into RISC to become

functional, but bind to endogenously present miRNAs and block their activities. This makes

it possible to modify their structure and thereby improve resistance to nucleases and speci-

ficity by increasing their affinity for the targeted miRNA. Several miRNA inhibitors have been

designed with different modifications at the 2’ position of the sugar molecule [55,56]. Among

these, 2’-MOE (2’-O-methoxyethyl) and 2’-fluoro modifications are the most commonly used


29

2

[57–59]. Another type of modification, named LNA (locked nucleic acid) and using an O, 4’-C

methylene bridge to lock the furanose ring in the sugar-phosphate backbone, displayed

enhanced affinity for their targeted miRNA [60–65]. In fact, the LNA miRNA-binding affin-

ity is so high that tiny LNA molecules, complementary to only the 7-nucleotidelong seed

sequence of a family of miRNAs, can effectively block the wholemiRNA family [66]. The effect

of a single injection can last several months and this long-lasting inhibition of endogenous

miRNA targets probably originates from antimiRs having a different structure than natural

RNA species so that endogenous RNase cannot recognize them anymore.

CHALLENGES OF miRNA THERAPEUTICS FOR CARDIOVASCULAR DISEASES

miRNA gain-of-functionThe capacity to induce a long-lasting high level of transgene expression in combination with

low production cost makes the AAV an ideal tool for miRNA gain-of-function research [52,53].

However, the safety of AAVs in clinical applications is still under debate, and using viruses for

gene therapy still carries a negative connotation from failures in the past. It has been reported

that neutralizing antibodies against AAV virus can be detected in healthy populations [67],

and transgene products themselves can induce cell-mediated and humoral responses [68].

Besides, a definitive cardiac-specific serotype is not yet fully developed. For example, AAV9

is considered to be a cardiac tropism serotype [69–73]; however, AAV9 transgenes are also

expressed in other tissues, such as the liver and brain [68,74,75]. AAVs are promising and

further developments might well make them a successful clinical tool; however, the negative

public opinion regarding virus-based transgene delivery methods will most likely frustrate

developments. Even though miRNAmimics are powerful and widely used for in vitro studies,

in vivo delivery of miRNA mimics is challenging. Synthetic miRNA mimics, such as siRNA, have

a very short half-life in the blood [76]. As mentioned above, miRNA mimics have to be double-

stranded or need a stem–looped precursor structure so that they can be processed correctly

and functionally incorporated into RISC. This makes it difficult to modify their structure to

improve their stability, as with miRNA inhibitors, while maintaining their biological function.

Interestingly, endogenously expressed miRNAs are stable in serum for 4–6 years when stored

at −20 °C, whereas other RNA species are degraded [77]. These ‘circulating’ miRNAs may form

complexes with the miRNA effector protein Ago2 (Argonaute 2) [76], HDL (high-density

lipoprotein) [78], or can be incorporated into microvesicles, such as exosomes, microvesicular

bodies and apoptotic bodies, thereby being protected from RNase degradation [76,79]. On

the basis of these observations, it might be possible to improve the stability of miRNA mimics

in the circulation by forming complexes with protective proteins or polymers or loading them

into vesicles before injecting them into the body.

Chapter 2

30

miRNA inhibitionDifferent from miRNA mimics, miRNA inhibitors are already chemically modified RNA

molecules, which may be recognized by the body as foreign materials and thereby induce

inflammation, as is observed for siRNA [80]. It is well documented that foreign single- or

double-stranded synthetic siRNA can induce innate immune response through activation

of TLRs (Toll-like receptors) [80,81]. For instance, bacterial DNA or even mitochondrial DNA

fragments can induce inflammation through TLR9 signalling upon myocardial infarction

[82]. Although very useful, the high stability of miRNA therapeutics (antagomiRs and LNA)

is a double-edged sword. As we mentioned above, the effect of antagomiRs is very long-

lasting compared with conventional pharmaceuticals. With a single injection of antagomiR

at a dose of 80 mg/kg of body weight, inhibition of endogenous miRNAs can still be ob-

served after 1 month [37,52], probably due to a lack of nucleases that can recognize them in

mammalian cells. These long-lasting systemic effects of antagomiR or LNAwill increase the

chance of potential side effects in healthy regions or other organs of the body.

General challenges for miRNA mimics and inhibitorsBesides these specific challenges for miRNA mimics or inhibitors, there are some common

challenges for both of them. miRNA mimics and inhibitors are negatively charged. It is there-

fore difficult for them to leave the bloodstream and enter into the cell. Various delivery vehicles

have been used to help them enter into the cell, such as liposomes and polymers, which sig-

nificantly enhance cellular uptake and will be discussed below in more detail. However, 90%

of the mimics are still trapped in endolysosomal vesicles and cannot escape into the cytosol

of the cell [83]. Thus agents which can disrupt the endolysosomal compartments and help

them escape from endolysosome degradation, or new delivery techniques, are needed for

further development. A lack of effective organ-specific (e.g. cardiac-specific) delivery methods

is another problem for both miRNA mimics and inhibitors. In vivo administration of miRNA

mimics and inhibitors mainly relies on systemic injection, with related issues such as high

costs, potential side effects and low efficacy. It costs up to 250€ per mouse using antagomiR

with a dose of 80 mg/kg of body weight via systemic injection [84]. Given the body weight of

a human, it will cost more than 500000€ for a single treatment in humans. Even though lower

doses (<10 mg/kg of body weight) via repetitive injections [47] or using low-dose LNAs [66]

have been shown to be effective and cheaper, it will still cost approximately 100000€ for using

them in human trials. The cost may be reduced in the future with the development of synthe-

sis technology and more potent miRNA inhibitors. Another problem with systemic delivery of

miRNA therapeutics are the potential side effects. First, one miRNA can have multiple targets,

thus one miRNA may have different functions in different organs or cell types. For example,

miR-214 has been shown to play an inhibitory role in angiogenesis [59,85]; however, this

miRNA is also essential in regulating calcium homoeostasis in cardiomyocytes [86]. Therefore

systemic inhibition of miR-214 may improve neovascularization in the heart, but it may also


31

2

cause problems in myocardial calcium handling. In summary, successful implementation of

miRNA therapeutics relies on strategies to improve the stability in the circulation, to reach

high efficient and tissue/cell-specific delivery, to have effective cellular uptake and eventually

to escape from the endolysosomal compartment.

OVERCOMING CHALLENGES: LESSONS LEARNED FROM siRNA DELIVERY

Several of the previously mentioned challenges for using miRNAs as therapeutic modalities,

both mimics and inhibitors, also apply when using siRNA as a therapeutic molecule. siRNAs

are double-stranded RNA-molecules 21–23 nucleotides in length that can be synthetically

produced. After cytosolic delivery they are incorporated into RISC like miRNAs. siRNAs have a

completely complementary sequence to their target mRNAs and binding of the siRNA–RISC

to their targets results in mRNA degradation [87]. As with miRNA mimics, siRNAs have similar

problems, such as being susceptible to degradation in the blood, not leaving the blood ves-

sels readily, and not readily being taken up by cells. To overcome these challenges, several

siRNA delivery vehicles, such as liposomal delivery vehicles, polymeric delivery vehicles

and US (ultrasound) MBs (microbubbles), have been studied. In order to overcome these

challenges for miRNA-based treatment, we can learn important lessons from these siRNA

delivery vehicles. An overview of these commonly used delivery vehicles is shown in Table 2.

Liposomal delivery vehiclesLiposomal are the most-studied and oldest delivery vehicle for siRNA, as reviewed recently

by Buyens et al. [88]. In the circulation, it has been shown extensively that liposomes protect

siRNA from RNase degradation and remain stable in the blood [88,89]. Additionally, Elbashir

et al. [90] have shown that cationic liposomes can induce the cellular uptake of siRNA in cell

culture through endocytosis, overcoming the challenge of low uptake of naked siRNA by cells.

Subsequently, cationic liposomes have been used to successfully treat liver metastasis in mice

by increasing apoptosis via targeting the bcl-2 oncogene in cancer cells [91]. Specific delivery

of an siRNA for CD31 to endothelial cells inhibited angiogenesis and decreased tumour growth

[92], thereby demonstrating that liposomes not only facilitate cellular uptake, but also assist in

the exit of siRNA from blood vessels into the tissue where they become biologically active in

vivo at several doses of 1–4 mg/kg of body weight. However, after i.v. (intravenous) injection,

siRNA-loaded liposomes tend to end up in the liver, kidney, lung, tumours, spleen and bone

[89,93,94], but not in the heart. Thus liposomes can solve major challenges for miRNA, antimiR

and siRNA delivery, including stability, cellular uptake and targeting of blood vessels or specific

organs, but still there is a need to increase delivery to the myocardium for cardiac applications

in order to improve therapeutic effects and avoid off-target side-effects.

Chapter 2

32


33

2

Polymeric delivery vehiclesAnother strategy to overcome the challenges of siRNA delivery is the use of polymers, which

have been used with mixed successes [87,95,96]. There are many polymers that have been

used to improve siRNA delivery, and most commonly used are PEI (polyethylenimine), chitosan,

PLGA [poly(lactic-co-glycolic acid)], PLL (poly-L-lysine) and dendrimers. These polymers mostly

rely on their cationic charge to bind siRNA and subsequently protect siRNA from degradation

in the blood. Additionally, a cationic charge also induces endocytosis, thereby increasing cel-

lular uptake of the delivery vehicle. PEI was found to protect siRNA from RNase degradation in

the circulation and, after i.v. injection, siRNA ended up in tumours, liver, spleen, lung, kidney

and, to a low extent, in the heart of mice [97]. Indeed, tumour growth was decreased upon

i.v. treatment with a specific PEI–siRNA complex against VEGFR2 (vascular endothelial growth

factor receptor 2) [97]. Similarly, chitosan was found to protect siRNA from degradation for at

least 7 h, whereas naked siRNA is degraded within minutes upon systemic injection [98]. In

addition, after i.v. injection of an siRNA carrying chitosan in mice, the siRNA could be found

in the brain, liver, lungs, kidney, spleen, to a very low degree in the heart and abundantly

in tumours [99]. These delivered siRNA for POSTN (periostin), FAK (focal adhesion kinase) or

PLXDC1 (plexin domain containing 1) were subsequently capable of reducing tumour size

in mice [99]. Another polymer, PLGA, has been studied as an siRNA delivery vehicle because

it is FDA (Food and Drug Administration)-approved and provides a slow release of siRNA due

to the hydrolysis of PLGA [100], thereby making its therapeutic effects last longer. However,

PLGA is not cationic and is not readily taken up by cells, which makes loading PLGA with siRNA

more difficult and additional functionalities to improve cellular uptake have to be added [101].

If these additional functionalities are used, the resulting composite polymer is capable of

protecting an siRNA from degradation, enabling the exit of the siRNA out of the bloodstream

into tissue and subsequently inhibiting tumour growth [101]. Cardiac accumulation of these

particles, however, was not reported and, besides providing protection from degradation, the

challenges for siRNA delivery were not necessarily overcome by the characteristics of PLGA,

but rather by adding other polymers or moieties. PLL has been shown to deliver siRNA for

luciferase into cells, knocking down protein expression up to 80%in vitro [102]. However, when

trying to translate the use of PLL to in vivo models, several issues were encountered, such

as blood instability [103] and severe toxicity [102]. Additionally, PLL–siRNA delivery vehicles

mainly accumulate in the liver, spleen and tumours [104]. Dendrimers are branching polymers

and chemically challenging to produce [105]. For siRNA and oligonucleotide delivery, PPI

(polypropylenimine) or PAMAM [poly(amido amine)] have received most interest because of

their cationic nature [105]. Dendrimers have been proven to be able to cross cell membranes

and deliver siRNA intracellularly [106]. However, low dendrimer–siRNA stability in the blood

has been reported [107]. After i.v. injection, dendrimers are capable of transferring siRNA out

of the bloodstream after which they accumulate in the liver, kidney and spleen [106] or in

tumours after the addition of tumour-targeting functionalities such as a synthetic analogue of

Chapter 2

34

LHRH (luteinizing hormone-releasing hormone) [106], but no studies have reported specific

uptake and use in the heart. Several challenges have been conquered by using polymers to

deliver siRNA in vivo. This has resulted in successful therapeutic effects in laboratory animals

at several doses of siRNA, ranging from 1 to 3 mg/kg of body weight, whereas antagomiRs are

administered at 20–80 mg/kg of body weight. By using polymers such as chitosan, PEI and

PLGA it is possible to provide protection against degradation in the blood, and transfer the

siRNA out of the bloodstream and into cells to facilitate mRNA degradation. An overview of

the characteristics of discussed polymers is shown in Table 2. However, these siRNA–polymers

mostly end up in the liver, spleen, kidneys and tumours, and not in other organs such as the

heart, introducing the additional challenge of specifically targeted these delivery vehicles to

the myocardium.

Cardiovascular application of liposomal and polymeric delivery devicesAs mentioned above, liposomal and polymeric delivery vehicles for siRNA naturally accumulate

in tumours, the liver or spleen, and not in the vasculature and heart. Owing to this biodistribu-

tion, targeting the cardiovascular system with synthetic delivery vehicles is quite difficult. In

addition, the heart muscle is a difficult organ to transfect, and it is even believed that direct

injection of naked nucleotides results in higher cardiac uptake then nucleotides combined

with a delivery vehicle [108]. It has been hypothesized that the cationic nature of delivery

vehicles caused them to get attached to the densely packed anionic extracellular matrix of the

heart [109]. Even so, several approaches have been used to target the cardiovascular system

using siRNA delivery vehicles. To circumvent issues with biodistribution, delivery vehicles can

be directly injected into the myocardium [109–111] (Figure 2: delivery route 1). Kim et al. [109]

used a PEI-based delivery vehicle to deliver an siRNA against SHP-1 (Src homology 2 domain-

containing protein tyrosine phosphatase-1) to the myocardium of rats in an ischaemia/reper-

fusion model, resulting in a successful knockdown of SHP-1 and thereby reducing apoptotic

cell death and infarction size by 50% [109]. Alternatively, Somasuntharam et al. [111] used

pH-sensitive nanoparticles for intramyocardial siRNA delivery in a mouse modelwith a perma-

nent ligation in the left anterior descending coronary. As a result of Nox2 (NADPH oxidase 2)

knockdown, fractional shortening in the heart improved to non-infarcted myocardial levels

[111]. Additionally, direct injection of liposomes carrying miR-590 and miR-199 in the heart of

neonatal rats successfully increased cardiomyocyte proliferation within 3 days [53].

Even though biodistribution of cationic liposomes after i.v. injection does not favour the heart,

the i.v. approach to achieve cardiac effects has been reported (Figure 2: delivery route 2). For

instance, Santel et al. [112] successfully knocked down CD31 mRNA levels by 40%in the heart

after i.v. injection of liposomes loaded with siRNA in mice. CD31 is an EC-specific marker and

was used in that study to prove endothelial targeting of the delivery vehicle. Furthermore,

Betigeri et al. [113] used i.v. injection of liposomes, loaded with siRNA against JNK1 (c-Jun N-


35

2

terminal kinase 1) to decrease apoptosis in the heart of mice during hypoxia, knocking down

JNK1 by 80% and significantly reducing apoptosis. Both studies report, however, that uptake

of liposomes in organs other than the heart is much higher [112,113]. Another approach to af-

fect the cardiovascular system is by indirect targeting via organs which are easier to transfect,

such as the liver [114,115] or spleen [116,117], which naturally show high accumulation of

delivery vehicles (Figure 2: delivery routes 3 and 4). In the liver, both PEI-based delivery vehicles

and liposomes were successfully used to knockdown ApoB (apolipoprotein B), resulting in a

reduction in serum cholesterol levels and thereby preventing atherosclerosis [114]. In another

approach, an siRNA against CCR2 (C-C chemokine receptor 2) was intravenously injected after

complexing with cationic liposomes. These liposomes accumulated in the spleen and trans-

fected monocytes, macrophages and neutrophils, leading to a lower responsiveness of these

cells to MCP-1 (macrophage chemoattractant protein-1), a chemoattractant which is released

from the infarcted heart. As a result, monocyte numbers in infarcted hearts were reduced

by 70%, which led to a 34% reduction in infarct size (Figure 2: delivery route 5) [116]. In a

follow up study, the same group found that the reduction in infarct size also leads to increased

left ventricular ejection fraction [117]. This approach was also used to decrease the number

Figure 2: Targeting the heart with siRNA delivery vehicles. Cardiac application of siRNA delivery ve-

hicles can occur via different application routes. Delivery strategies either rely on (1) intramyocardial injection

of siRNA-carrying delivery vehicles, or intravenous injection routes (2, 3, 4). After intravenous injection, delivery

vehicles can directly enter the heart (2) or enter off-target organs like the liver (3) or the spleen (4). Subsequently,

secondary effects of those organs can have therapeutic effects on atherosclerosis by reducing ApoB levels (3)

or by reducing monocyte chemotaxis (4). Reduced chemotaxis of monocytes from the spleen can also reduce

infarct size after myocardial infarction (5).

Chapter 2

36

of macrophages and monocytes in atherosclerotic plaques, leading to an 82% reduction in

macrophages/monocytes in the plaque [116].

The main issue in treating the heart with siRNA remains the low accumulation of delivery

vehicles and siRNA. Although liposomal–siRNA formulations have entered the clinical arena

for treatment of cancer [118] and the liver [119], the heart still lags behind, creating a need

for improved cardiac homing of siRNA delivery vehicles. Interestingly, promising alternative

methods are being developed to treat myocardial infarction or atherosclerosis by targeting

organs such as the liver or spleen with subsequent effects on the heart, although these are

still in an early pre-clinical phase and mostly rely on a reduction in total cholesterol levels or

modulating the immune responses.

MicrobubblesMBs are micrometre-diameter gas-filled spheres with a flexible shell which can be modified

in order to bind nucleic acids such as DNA or siRNA [120–122]. The characteristic that makes

MBs really interesting as a drug delivery vehicle is their US responsiveness. Having a core of

gas, MBs react to US pressure waves. During the peak positive pressure phase, the gas-filled

MBs will be compressed and during the peak negative phase the MBs will expand, causing

the MBs to oscillate on the US wave. Increasing the peak-to-peak pressure of the US wave

to a certain point causes the MBs to collapse, thereby releasing the therapeutic payload

and permeabilizing cells [123,124] and blood vessels [125]. This results in an increased local

delivery and uptake of therapeutic payload if US is applied locally [126]. MBs have been used

for the local delivery of liposomes, nanoparticles, plasmid DNA and also siRNA [127].

MBs have been found to protect siRNA from RNase degradation in the circulation and for

transferring them out of the blood vessel into cells in tumour tissue, subsequently slow-

ing down tumour growth [120]. Similarly, Un et al. [128] used MBs to deliver siRNA against

ICAM-1 (intercellular adhesion molecule-1) to the liver and successfully delivered siRNA

in endothelial cells, Kupffer cells and hepatocytes, thereby providing protection against

ischaemia/reperfusion damage in the liver. MBs are therefore capable of protecting siRNA,

enable siRNA to exit the blood stream, and induce the entry of siRNA into cells.

Furthermore, the ability to use a physical force to induce local delivery makes the MB suit-

able to target tissues in which delivery vehicles do not naturally accumulate, and MB+US

has thereby been used to deliver siRNA or oligonucleotides to the heart [129,130]. In 2005,

Tsunoda et al. [130] successfully delivered siRNA against GFP in GFP transgenic mice. They

found that delivery was confined to the ECs in the heart [130] and was not observed in

cardiomyocytes, in contrast with delivery of plasmid DNA, which did enter cardiomyocytes

as well as ECs after MB+US treatment. Alter et al. [129] also showed that single-stranded

oligonucleotides could be delivered locally to the heart, inducing the transcription of

dystrophin in cardiomyocytes. Both studies showed successful delivery of small oligonucle-

otides to compartments of the heart while retaining biological activity without off-target


37

2

effects in other organs. Although Tsunoda et al. [130] only found tissue penetration to ECs

by using double stranded siRNA, Alter et al. [129] observed tissue penetration reaching the

cardiomyocytes by using single-stranded oligonucleotides, which is an interesting differ-

ence between these RNA analogues.

MB+US-induced local delivery techniques look a promising approach for local miRNA

delivery in the cardiovascular system. Small RNA molecules are only delivered to organs

where US is applied and therefore the heart can also be reached, in contrast with the other

synthetic delivery vehicles discussed above. Penetration of siRNA into tissue, however, only

extends to ECs. For miRNA mimics, similar results can be expected as these molecules are

double-stranded as well. For antimiR molecules, being single-stranded, affecting both EC

cells and cardiomyocytes seems feasible. Although promising, MB+US-induced delivery of

siRNA is still rather complex and many factors are known to influence the success of local

delivery, including MB type [129], US intensity [131], US frequency [132], US pulsing [127]

and MB dose [129]. Furthermore, cavitating MBs can cause stress to blood vessels and cells

[121]. However, when all of these factors are well balanced, the MB+US-induced delivery

of miRNA mimics is a very promising technique for the treatment of cardiovascular disease,

including the myocardium.

IMPROVING CARDIAC DELIVERY

Strategies to improve the homing of delivery vehicles to the heart include the use of

antibodies,EC- or cardiac-targeting peptides, or pH-sensitive-targeting moieties. These

cardiac-targeting agents take advantage of distinct properties of cardiac tissue, such as

cardiac-specific cell-surface markers which can be recognized by antibodies or targeting

peptides. Ko et al. [133] used modified liposomes with a cell-penetrating peptide TATp for

increased cellular uptake and an anti-myosin antibody for cardiac targeting. This approach

resulted in increased accumulation of DNA–liposome complexes in the ischaemicmyocar-

dium [133]. Another well-studied targeting peptide is RGD that specifically binds to integrin

αvβ3 and αvβ5,which is enriched on the surface of ECs. This RGD peptide has been used

for targeted delivery of siRNA to ECs in combination with delivery vehicles such as chitosan

nanoparticles [99], PEI [134] and liposomes [135]. Using another approach, Sosunov et al

[136] reported that a low-pH-sensitive insertion peptide accumulates only in the ischaemic-

myocardium, but not in the healthy regions, as the pH in the ischaemic myocardium is

lower. These strategies improve cardiac homing, but have not been used in combination

with siRNA or miRNA therapeutics yet. It will be interesting to see how these promising

targeting strategies by themselves, or as a combination, improve cardiac homing of delivery

vehicles for siRNA and miRNA therapeutics.

Chapter 2

38

IMPROVING ENDOSOMAL RELEASE AND CYTOSOLIC DELIVERY OF miRNA THERAPEUTICS

Cellular entry of siRNA or miRNA therapeutics using delivery vehicles is usually endocytosis-

mediated, thus resulting in endolysomal localization. The last challenge for small RNA

therapeutic delivery after its entry into cells is release from its carrier and escape from the

endolysosomal compartment. To this end, fusogenic peptides have been used to facilitate

endolysosomal escape and improve cytosolic delivery of therapeutic molecules after inter-

nalization of drug delivery vehicles [137]. Hatakeyama and co-workers [138,139] showed that

the introduction of a pH-responsive fusogenic GALA peptide into a drug delivery vehicle

improved gene silencing due to more efficient endosomal escape. By combining fusogenic

peptides and cell-penetrating peptides, even more efficient knockdown has been achieved

[140,141]. Many protein/peptides such as GALA have been identified and can enhance

endosomal release, which can be considered for incorporation into the miRNA-therapeutics

delivery complex [142].

FUTURE PERSPECTIVES

miRNAs have been shown to play important roles in many different aspects of cardiovascu-

lar disease. Some properties that make the miRNA an excellent therapeutic target are the

ability to influence whole cellular processes instead of a single gene, the easy manipulation

of miRNA activity in vivo using miRNA inhibitors, the high intracellular stability of miRNA

therapeutics, and their involvement in regenerative processes in cardiovascular disease. Ac-

tually, the first successful Phase II clinical trial with antimiR-122 [143], a hepatitis C-targeting

antimiR, has been carried out and should definitely encourage the introduction of more

miRNA therapeutics towards clinical practice, and also potentially in the cardiovascular

field. However, the intrinsic nature of miRNAs targeting multiple target mRNAs can achieve

synergistic effects, but may also cause unexpected off-target side effects, since no organ- or

tissue selectivity is achieved, especially as the heart is difficult to reach. Moreover, in vivo

delivery of miRNA-mimicking molecules to enhance miRNA function is limited and is mainly

achieved by viral overexpression. There is therefore a persistent need for methods to in-

crease miRNA therapeutic delivery, prevent off-target uptake and increase tissue-specific

uptake. Recent developments in drug delivery vehicles for siRNA have resulted in success-

ful protection of siRNA in the blood, successful exit from the circulation and intracellular

delivery of siRNA, and important lessons can be learnt from these siRNA delivery methods.

Three important challenges have been conquered using liposomal and polymeric delivery

vehicles. However, the big issue remains that these delivery vehicles end up in tumours,

the spleen, liver and lungs, but not readily in the heart. For a cardiac application, one would


39

2

need high systemic doses of siRNA to reach therapeutic levels in the myocardium, resulting

in large off-target effects and low therapeutic benefit. Strategies to circumvent this problem

include local injection or indirectly treating the heart by targeting other organs with known

effects on the heart. However, local injections are invasive and targeting the immune system

is not cardiac-specific and can result in major side effects. An alternative approach is the use

of MBs and US, which are known to have low off-target effects. However, cardiac delivery

of siRNA by MB has not been studied extensively and data so far point towards low-tissue

penetration of siRNA. For a cardiac application of siRNA (or miRNA) therapy, cardiac target-

ing remains an issue. However, several strategies to improve cardiac uptake and targeting

of delivery vehicles are being developed, and encouraging results are being obtained with

antibodies, cardiac-targeting peptides and pH-sensitive-targeting peptides (for an overview

see Figure 3). These results, however, still need to be translated to delivery vehicles for miRNA

therapeutics. A growing body of knowledge is available, but this needs to be combined

with experimental and translational efforts to open up the successful delivery of miRNAs,

which are, at this point in time, the most potent therapeutic small RNA available.

Figure 3: Delivery vehicles for siRNA, targeting options and improving cytosolic delivery. Deliv-ery vehicles that can be used for siRNA delivery are represented in the left column, the size of the circles represents the size of the delivery vehicle. In the middle column, tissue targeting options which can be combined with delivery vehicles from the left column are depicted. Additionally, the right column gives an overview for options to increase cellular entry and endosomal escape.

Chapter 2

40

REFERENCES

1. Guo, H., Ingolia, N. T., Weissman, J. S. and Bartel, D. P. (2010) Mammalian microRNAs predominantly act

to decrease target mRNA levels Nature 466, 835-840

2. Baek, D., Villen, J., Shin, C., Camargo, F. D., Gygi, S. P. and Bartel, D. P. (2008) The impact of microRNAs on

protein output Nature 455, 64-71

3. Tomari, Y. and Zamore, P. D. (2005) MicroRNA biogenesis: drosha can‘t cut it without a partner Curr Biol

15, R61-64

4. Yi, R., Qin, Y., Macara, I. G. and Cullen, B. R. (2003) Exportin-5 mediates the nuclear export of pre-

microRNAs and short hairpin RNAs Genes Dev 17, 3011-3016

5. Bohnsack, M. T., Czaplinski, K. and Gorlich, D. (2004) Exportin 5 is a RanGTP-dependent dsRNA-binding

protein that mediates nuclear export of pre-miRNAs Rna 10, 185-191

6. Jiang, F., Ye, X., Liu, X., Fincher, L., McKearin, D. and Liu, Q. (2005) Dicer-1 and R3D1-L catalyze microRNA

maturation in Drosophila Genes Dev 19, 1674-1679

7. Chendrimada, T. P., Gregory, R. I., Kumaraswamy, E., Norman, J., Cooch, N., Nishikura, K. and Shiekhattar,

R. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing Nature

436, 740-744

8. Forstemann, K., Horwich, M. D., Wee, L., Tomari, Y. and Zamore, P. D. (2007) Drosophila microRNAs are

sorted into functionally distinct argonaute complexes after production by dicer-1 Cell 130, 287-297

9. Barad, O., Mann, M., Chapnik, E., Shenoy, A., Blelloch, R., Barkai, N. and Hornstein, E. (2012) Efficiency

and specificity in microRNA biogenesis Nat Struct Mol Biol 19, 650-652

10. Gregory, R. I., Chendrimada, T. P., Cooch, N. and Shiekhattar, R. (2005) Human RISC couples microRNA

biogenesis and posttranscriptional gene silencing Cell 123, 631-640

11. Kim, V. N. (2005) MicroRNA biogenesis: coordinated cropping and dicing Nat Rev Mol Cell Biol 6,

376-385

12. Krol, J., Loedige, I. and Filipowicz, W. (2010) The widespread regulation of microRNA biogenesis, func-

tion and decay Nat Rev Genet 11, 597-610

13. Sluijter, J. P. (2013) MicroRNAs in Cardiovascular Regenerative Medicine: Directing Tissue Repair and

Cellular Differentiation ISRN Vascular Medicine 2013,

14. Winter, J., Jung, S., Keller, S., Gregory, R. I. and Diederichs, S. (2009) Many roads to maturity: microRNA

biogenesis pathways and their regulation Nat Cell Biol 11, 228-234

15. Heinrich, E. M. and Dimmeler, S. (2012) MicroRNAs and stem cells: control of pluripotency, reprogram-

ming, and lineage commitment Circ Res 110, 1014-1022

16. Jin, C., Zhao, Y., Yu, L., Xu, S. and Fu, G. (2013) MicroRNA-21 mediates the rapamycin-induced suppres-

sion of endothelial proliferation and migration FEBS Lett 587, 378-385

17. Sluijter, J. P., van Mil, A., van Vliet, P., Metz, C. H., Liu, J., Doevendans, P. A. and Goumans, M. J. (2010)

MicroRNA-1 and -499 regulate differentiation and proliferation in human-derived cardiomyocyte

progenitor cells Arterioscler Thromb Vasc Biol 30, 859-868


41

2

18. Wu, W. H., Hu, C. P., Chen, X. P., Zhang, W. F., Li, X. W., Xiong, X. M. and Li, Y. J. (2011) MicroRNA-130a

mediates proliferation of vascular smooth muscle cells in hypertension Am J Hypertens 24, 1087-

1093

19. Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E., Hammond, S. M., Conlon, F. L. and Wang, D.

Z. (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentia-

tion Nat Genet 38, 228-233

20. Choe, N., Kwon, J. S., Kim, J. R., Eom, G. H., Kim, Y., Nam, K. I., Ahn, Y., Kee, H. J. and Kook, H. (2013) The

microRNA miR-132 targets Lrrfip1 to block vascular smooth muscle cell proliferation and neointimal

hyperplasia Atherosclerosis 229, 348-355

21. Chen, J., Huang, Z. P., Seok, H. Y., Ding, J., Kataoka, M., Zhang, Z., Hu, X., Wang, G., Lin, Z., Wang, S.,

Pu, W. T., Liao, R. and Wang, D. Z. (2013) mir-17-92 cluster is required for and sufficient to induce

cardiomyocyte proliferation in postnatal and adult hearts Circ Res 112, 1557-1566

22. Porrello, E. R., Mahmoud, A. I., Simpson, E., Johnson, B. A., Grinsfelder, D., Canseco, D., Mammen, P. P.,

Rothermel, B. A., Olson, E. N. and Sadek, H. A. (2013) Regulation of neonatal and adult mammalian

heart regeneration by the miR-15 family Proc Natl Acad Sci U S A 110, 187-192

23. Marson, A., Levine, S. S., Cole, M. F., Frampton, G. M., Brambrink, T., Johnstone, S., Guenther, M. G.,

Johnston, W. K., Wernig, M., Newman, J., Calabrese, J. M., Dennis, L. M., Volkert, T. L., Gupta, S., Love, J.,

Hannett, N., Sharp, P. A., Bartel, D. P., Jaenisch, R. and Young, R. A. (2008) Connecting microRNA genes

to the core transcriptional regulatory circuitry of embryonic stem cells Cell 134, 521-533

24. Ohtani, K. and Dimmeler, S. (2011) Control of cardiovascular differentiation by microRNAs Basic Res

Cardiol 106, 5-11

25. Subramanian, S. and Steer, C. J. (2010) MicroRNAs as gatekeepers of apoptosis J Cell Physiol 223,

289-298

26. Li, P. (2010) MicroRNAs in cardiac apoptosis J Cardiovasc Transl Res 3, 219-224

27. Smith-Vikos, T. and Slack, F. J. (2012) MicroRNAs and their roles in aging J Cell Sci 125, 7-17

28. Jansen, F., Yang, X., Nickenig, G., Werner, N. and Vasa-Nicotera, M. (2013) Role, Function and Therapeu-

tic Potential of MicroRNAs in Vascular Aging Curr Vasc Pharmacol

29. Boon, R. A., Iekushi, K., Lechner, S., Seeger, T., Fischer, A., Heydt, S., Kaluza, D., Treguer, K., Carmona, G.,

Bonauer, A., Horrevoets, A. J., Didier, N., Girmatsion, Z., Biliczki, P., Ehrlich, J. R., Katus, H. A., Muller, O.

J., Potente, M., Zeiher, A. M., Hermeking, H. and Dimmeler, S. (2013) MicroRNA-34a regulates cardiac

ageing and function Nature 495, 107-110

30. Chen, J. F., Murchison, E. P., Tang, R., Callis, T. E., Tatsuguchi, M., Deng, Z., Rojas, M., Hammond, S. M., Sch-

neider, M. D., Selzman, C. H., Meissner, G., Patterson, C., Hannon, G. J. and Wang, D. Z. (2008) Targeted

deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure Proc Natl Acad Sci U

S A 105, 2111-2116

31. Rao, P. K., Toyama, Y., Chiang, H. R., Gupta, S., Bauer, M., Medvid, R., Reinhardt, F., Liao, R., Krieger, M.,

Jaenisch, R., Lodish, H. F. and Blelloch, R. (2009) Loss of cardiac microRNA-mediated regulation leads

to dilated cardiomyopathy and heart failure Circ Res 105, 585-594

Chapter 2

42

32. Albinsson, S., Suarez, Y., Skoura, A., Offermanns, S., Miano, J. M. and Sessa, W. C. (2010) MicroRNAs are

necessary for vascular smooth muscle growth, differentiation, and function Arterioscler Thromb Vasc

Biol 30, 1118-1126

33. Chen, Z., Wu, J., Yang, C., Fan, P., Balazs, L., Jiao, Y., Lu, M., Gu, W., Li, C., Pfeffer, L. M., Tigyi, G. and Yue,

J. (2012) DiGeorge syndrome critical region 8 (DGCR8) protein-mediated microRNA biogenesis is

essential for vascular smooth muscle cell development in mice J Biol Chem 287, 19018-19028

34. Suarez, Y., Fernandez-Hernando, C., Yu, J., Gerber, S. A., Harrison, K. D., Pober, J. S., Iruela-Arispe, M. L.,

Merkenschlager, M. and Sessa, W. C. (2008) Dicer-dependent endothelial microRNAs are necessary for

postnatal angiogenesis Proc Natl Acad Sci U S A 105, 14082-14087

35. Callis, T. E., Pandya, K., Seok, H. Y., Tang, R. H., Tatsuguchi, M., Huang, Z. P., Chen, J. F., Deng, Z., Gunn, B.,

Shumate, J., Willis, M. S., Selzman, C. H. and Wang, D. Z. (2009) MicroRNA-208a is a regulator of cardiac

hypertrophy and conduction in mice J Clin Invest 119, 2772-2786

36. Montgomery, R. L., Hullinger, T. G., Semus, H. M., Dickinson, B. A., Seto, A. G., Lynch, J. M., Stack, C.,

Latimer, P. A., Olson, E. N. and van Rooij, E. (2011) Therapeutic inhibition of miR-208a improves cardiac

function and survival during heart failure Circulation 124, 1537-1547

37. Ucar, A., Gupta, S. K., Fiedler, J., Erikci, E., Kardasinski, M., Batkai, S., Dangwal, S., Kumarswamy, R., Bang,

C., Holzmann, A., Remke, J., Caprio, M., Jentzsch, C., Engelhardt, S., Geisendorf, S., Glas, C., Hofmann, T.

G., Nessling, M., Richter, K., Schiffer, M., Carrier, L., Napp, L. C., Bauersachs, J., Chowdhury, K. and Thum, T.

(2012) The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy

Nat Commun 3, 1078

38. Bonauer, A., Carmona, G., Iwasaki, M., Mione, M., Koyanagi, M., Fischer, A., Burchfield, J., Fox, H., Doe-

bele, C., Ohtani, K., Chavakis, E., Potente, M., Tjwa, M., Urbich, C., Zeiher, A. M. and Dimmeler, S. (2009)

MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice Science

324, 1710-1713

39. Hinkel, R., Penzkofer, D., Zuhlke, S., Fischer, A., Husada, W., Xu, Q. F., Baloch, E., van Rooij, E., Zeiher, A. M.,

Kupatt, C. and Dimmeler, S. (2013) Inhibition of MicroRNA-92a Protects Against Ischemia-Reperfusion

Injury in a Large Animal Model Circulation

40. Small, E. M., Frost, R. J. and Olson, E. N. (2010) MicroRNAs add a new dimension to cardiovascular

disease Circulation 121, 1022-1032

41. van Rooij, E. and Olson, E. N. (2012) MicroRNA therapeutics for cardiovascular disease: opportunities

and obstacles Nat Rev Drug Discov 11, 860-872

42. Ono, K., Kuwabara, Y. and Han, J. (2011) MicroRNAs and cardiovascular diseases Febs J 278, 1619-1633

43. Lorenzen, J. M., Batkai, S. and Thum, T. (2013) Regulation of cardiac and renal ischemia-reperfusion

injury by microRNAs Free Radic Biol Med

44. Backes, C., Meese, E., Lenhof, H. P. and Keller, A. (2010) A dictionary on microRNAs and their putative

target pathways Nucleic Acids Res 38, 4476-4486

45. Cloonan, N., Wani, S., Xu, Q., Gu, J., Lea, K., Heater, S., Barbacioru, C., Steptoe, A. L., Martin, H. C.,

Nourbakhsh, E., Krishnan, K., Gardiner, B., Wang, X., Nones, K., Steen, J. A., Matigian, N. A., Wood, D.

L., Kassahn, K. S., Waddell, N., Shepherd, J., Lee, C., Ichikawa, J., McKernan, K., Bramlett, K., Kuersten, S.


43

2

and Grimmond, S. M. (2011) MicroRNAs and their isomiRs function cooperatively to target common

biological pathways Genome Biol 12, R126

46. van Rooij, E., Sutherland, L. B., Thatcher, J. E., DiMaio, J. M., Naseem, R. H., Marshall, W. S., Hill, J. A. and

Olson, E. N. (2008) Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in

cardiac fibrosis Proc Natl Acad Sci U S A 105, 13027-13032

47. Xiao, J., Liang, D., Zhang, H., Liu, Y., Zhang, D., Liu, Y., Pan, L., Chen, X., Doevendans, P. A., Sun, Y., Liang,

X., Sluijter, J. P. and Chen, Y. H. (2012) MicroRNA-204 is required for differentiation of human-derived

cardiomyocyte progenitor cells J Mol Cell Cardiol 53, 751-759

48. van Mil, A., Vrijsen, K. R., Goumans, M. J., Metz, C. H., Doevendans, P. A. and Sluijter, J. P. (2013) mi-

croRNA-1 enhances the angiogenic differentiation of human cardiomyocyte progenitor cells J Mol

Med (Berl) 91, 1001-1012

49. Liu, J., van Mil, A., Vrijsen, K., Zhao, J., Gao, L., Metz, C. H., Goumans, M. J., Doevendans, P. A. and Sluijter,

J. P. (2011) MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via

targeting RIP1 J Cell Mol Med 15, 1474-1482

50. Grundmann, S., Hans, F. P., Kinniry, S., Heinke, J., Helbing, T., Bluhm, F., Sluijter, J. P., Hoefer, I., Paster-

kamp, G., Bode, C. and Moser, M. (2011) MicroRNA-100 regulates neovascularization by suppression

of mammalian target of rapamycin in endothelial and vascular smooth muscle cells Circulation 123,

999-1009

51. Gantier, M. P., McCoy, C. E., Rusinova, I., Saulep, D., Wang, D., Xu, D., Irving, A. T., Behlke, M. A., Hertzog,

P. J., Mackay, F. and Williams, B. R. (2011) Analysis of microRNA turnover in mammalian cells following

Dicer1 ablation Nucleic Acids Res 39, 5692-5703

52. Eulalio, A., Mano, M., Dal Ferro, M., Zentilin, L., Sinagra, G., Zacchigna, S. and Giacca, M. (2012) Func-

tional screening identifies miRNAs inducing cardiac regeneration Nature 492, 376-381

53. Dirkx, E., Gladka, M. M., Philippen, L. E., Armand, A. S., Kinet, V., Leptidis, S., El Azzouzi, H., Salic, K.,

Bourajjaj, M., da Silva, G. J., Olieslagers, S., van der Nagel, R., de Weger, R., Bitsch, N., Kisters, N., Seyen, S.,

Morikawa, Y., Chanoine, C., Heymans, S., Volders, P. G., Thum, T., Dimmeler, S., Cserjesi, P., Eschenhagen,

T., da Costa Martins, P. A. and De Windt, L. J. (2013) Nfat and miR-25 cooperate to reactivate the

transcription factor Hand2 in heart failure Nat Cell Biol 15, 1282-1293

54. van Mil, A., Doevendans, P. A. and Sluijter, J. P. (2009) The potential of modulating small RNA activity in

vivo Mini Rev Med Chem 9, 235-248

55. Krutzfeldt, J., Rajewsky, N., Braich, R., Rajeev, K. G., Tuschl, T., Manoharan, M. and Stoffel, M. (2005)

Silencing of microRNAs in vivo with ‚antagomirs‘ Nature 438, 685-689

56. Krutzfeldt, J., Kuwajima, S., Braich, R., Rajeev, K. G., Pena, J., Tuschl, T., Manoharan, M. and Stoffel, M.

(2007) Specificity, duplex degradation and subcellular localization of antagomirs Nucleic Acids Res

35, 2885-2892

57. van Mil, A., Grundmann, S., Goumans, M. J., Lei, Z., Oerlemans, M. I., Jaksani, S., Doevendans, P. A. and

Sluijter, J. P. (2012) MicroRNA-214 inhibits angiogenesis by targeting Quaking and reducing angio-

genic growth factor release Cardiovasc Res 93, 655-665

Chapter 2

44

58. Meister, G., Landthaler, M., Dorsett, Y. and Tuschl, T. (2004) Sequence-specific inhibition of microRNA-

and siRNA-induced RNA silencing Rna 10, 544-550

59. Hutvagner, G., Simard, M. J., Mello, C. C. and Zamore, P. D. (2004) Sequence-specific inhibition of small

RNA function PLoS Biol 2, E98

60. Fabani, M. M. and Gait, M. J. (2008) miR-122 targeting with LNA/2‘-O-methyl oligonucleotide mixmers,

peptide nucleic acids (PNA), and PNA-peptide conjugates Rna 14, 336-346

61. Elmen, J., Lindow, M., Schutz, S., Lawrence, M., Petri, A., Obad, S., Lindholm, M., Hedtjarn, M., Hansen, H.

F., Berger, U., Gullans, S., Kearney, P., Sarnow, P., Straarup, E. M. and Kauppinen, S. (2008) LNA-mediated

microRNA silencing in non-human primates Nature 452, 896-899

62. Varallyay, E., Burgyan, J. and Havelda, Z. (2007) Detection of microRNAs by Northern blot analyses

using LNA probes Methods 43, 140-145

63. Orom, U. A., Kauppinen, S. and Lund, A. H. (2006) LNA-modified oligonucleotides mediate specific

inhibition of microRNA function Gene 372, 137-141

64. Naguibneva, I., Ameyar-Zazoua, M., Nonne, N., Polesskaya, A., Ait-Si-Ali, S., Groisman, R., Souidi, M.,

Pritchard, L. L. and Harel-Bellan, A. (2006) An LNA-based loss-of-function assay for micro-RNAs Biomed

Pharmacother 60, 633-638

65. Kloosterman, W. P., Wienholds, E., de Bruijn, E., Kauppinen, S. and Plasterk, R. H. (2006) In situ detection

of miRNAs in animal embryos using LNA-modified oligonucleotide probes Nat Methods 3, 27-29

66. Hullinger, T. G., Montgomery, R. L., Seto, A. G., Dickinson, B. A., Semus, H. M., Lynch, J. M., Dalby, C. M.,

Robinson, K., Stack, C., Latimer, P. A., Hare, J. M., Olson, E. N. and van Rooij, E. (2012) Inhibition of miR-15

protects against cardiac ischemic injury Circ Res 110, 71-81

67. Boutin, S., Monteilhet, V., Veron, P., Leborgne, C., Benveniste, O., Montus, M. F. and Masurier, C. (2010)

Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6,

8, and 9 in the healthy population: implications for gene therapy using AAV vectors Hum Gene Ther

21, 704-712

68. Ciesielska, A., Hadaczek, P., Mittermeyer, G., Zhou, S., Wright, J. F., Bankiewicz, K. S. and Forsayeth, J.

(2013) Cerebral infusion of AAV9 vector-encoding non-self proteins can elicit cell-mediated immune

responses Mol Ther 21, 158-166

69. Ying, Y., Muller, O. J., Goehringer, C., Leuchs, B., Trepel, M., Katus, H. A. and Kleinschmidt, J. A. (2010)

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral

display peptide library Gene Ther 17, 980-990

70. Sands, M. S. (2011) AAV-mediated liver-directed gene therapy Methods Mol Biol 807, 141-157

71. Pleger, S. T., Shan, C., Ksienzyk, J., Bekeredjian, R., Boekstegers, P., Hinkel, R., Schinkel, S., Leuchs, B.,

Ludwig, J., Qiu, G., Weber, C., Raake, P., Koch, W. J., Katus, H. A., Muller, O. J. and Most, P. (2011) Cardiac

AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model Sci

Transl Med 3, 92ra64

72. Katwal, A. B., Konkalmatt, P. R., Piras, B. A., Hazarika, S., Li, S. S., John Lye, R., Sanders, J. M., Ferrante, E.

A., Yan, Z., Annex, B. H. and French, B. A. (2013) Adeno-associated virus serotype 9 efficiently targets

ischemic skeletal muscle following systemic delivery Gene Ther 20, 930-938


45

2

73. Bish, L. T., Morine, K., Sleeper, M. M., Sanmiguel, J., Wu, D., Gao, G., Wilson, J. M. and Sweeney, H. L.

(2008) 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 19, 1359-1368

74. Geisler, A., Jungmann, A., Kurreck, J., Poller, W., Katus, H. A., Vetter, R., Fechner, H. and Muller, O. J. (2011)

microRNA122-regulated transgene expression increases specificity of cardiac gene transfer upon

intravenous delivery of AAV9 vectors Gene Ther 18, 199-209

75. Foust, K. D., Nurre, E., Montgomery, C. L., Hernandez, A., Chan, C. M. and Kaspar, B. K. (2009) Intravascu-

lar AAV9 preferentially targets neonatal neurons and adult astrocytes Nat Biotechnol 27, 59-65

76. Arroyo, J. D., Chevillet, J. R., Kroh, E. M., Ruf, I. K., Pritchard, C. C., Gibson, D. F., Mitchell, P. S., Bennett, C.

F., Pogosova-Agadjanyan, E. L., Stirewalt, D. L., Tait, J. F. and Tewari, M. (2011) Argonaute2 complexes

carry a population of circulating microRNAs independent of vesicles in human plasma Proc Natl Acad

Sci U S A 108, 5003-5008

77. Jung, M., Schaefer, A., Steiner, I., Kempkensteffen, C., Stephan, C., Erbersdobler, A. and Jung, K. (2010)

Robust microRNA stability in degraded RNA preparations from human tissue and cell samples Clin

Chem 56, 998-1006

78. Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D. and Remaley, A. T. (2011) MicroRNAs are

transported in plasma and delivered to recipient cells by high-density lipoproteins Nat Cell Biol 13,

423-433

79. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J. and Lotvall, J. O. (2007) Exosome-mediated

transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nat Cell

Biol 9, 654-659

80. Judge, A. D., Sood, V., Shaw, J. R., Fang, D., McClintock, K. and MacLachlan, I. (2005) Sequence-depen-

dent stimulation of the mammalian innate immune response by synthetic siRNA Nat Biotechnol 23,

457-462

81. Sioud, M. (2005) Induction of inflammatory cytokines and interferon responses by double-stranded

and single-stranded siRNAs is sequence-dependent and requires endosomal localization J Mol Biol

348, 1079-1090

82. Arslan, F., de Kleijn, D. P. and Pasterkamp, G. (2011) Innate immune signaling in cardiac ischemia Nat

Rev Cardiol 8, 292-300

83. Thomson, D. W., Bracken, C. P., Szubert, J. M. and Goodall, G. J. (2013) On measuring miRNAs after

transient transfection of mimics or antisense inhibitors PLoS One 8, e55214

84. Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz,

S., Castoldi, M., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M. A., Licht, J. D., Pena, J. T., Rou-

hanifard, S. H., Muckenthaler, M. U., Tuschl, T., Martin, G. R., Bauersachs, J. and Engelhardt, S. (2008)

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts

Nature 456, 980-984

85. Chan, L. S., Yue, P. Y., Mak, N. K. and Wong, R. N. (2009) Role of microRNA-214 in ginsenoside-Rg1-

induced angiogenesis Eur J Pharm Sci 38, 370-377

Chapter 2

46

86. Aurora, A. B., Mahmoud, A. I., Luo, X., Johnson, B. A., van Rooij, E., Matsuzaki, S., Humphries, K. M., Hill, J.

A., Bassel-Duby, R., Sadek, H. A. and Olson, E. N. (2012) MicroRNA-214 protects the mouse heart from

ischemic injury by controlling Ca(2)(+) overload and cell death J Clin Invest 122, 1222-1232

87. Whitehead, K. A., Langer, R. and Anderson, D. G. (2009) Knocking down barriers: advances in siRNA

delivery Nat Rev Drug Discov 8, 129-138

88. Buyens, K., De Smedt, S. C., Braeckmans, K., Demeester, J., Peeters, L., van Grunsven, L. A., de Mollerat

du Jeu, X., Sawant, R., Torchilin, V., Farkasova, K., Ogris, M. and Sanders, N. N. (2012) Liposome based

systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design

J Control Release 158, 362-370

89. Jiang, N., Zhang, X., Zheng, X., Chen, D., Siu, K., Wang, H., Ichim, T. E., Quan, D., McAlister, V., Chen, G. and

Min, W. P. (2012) A novel in vivo siRNA delivery system specifically targeting liver cells for protection

of ConA-induced fulminant hepatitis PLoS One 7, e44138

90. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of

21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 411, 494-498

91. Yano, J., Hirabayashi, K., Nakagawa, S., Yamaguchi, T., Nogawa, M., Kashimori, I., Naito, H., Kitagawa,

H., Ishiyama, K., Ohgi, T. and Irimura, T. (2004) Antitumor activity of small interfering RNA/cationic

liposome complex in mouse models of cancer Clin Cancer Res 10, 7721-7726

92. Santel, A., Aleku, M., Keil, O., Endruschat, J., Esche, V., Durieux, B., Loffler, K., Fechtner, M., Rohl, T., Fisch,

G., Dames, S., Arnold, W., Giese, K., Klippel, A. and Kaufmann, J. (2006) RNA interference in the mouse

vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy Gene Ther

13, 1360-1370

93. Zhang, G., Guo, B., Wu, H., Tang, T., Zhang, B. T., Zheng, L., He, Y., Yang, Z., Pan, X., Chow, H., To, K., Li, Y.,

Li, D., Wang, X., Wang, Y., Lee, K., Hou, Z., Dong, N., Li, G., Leung, K., Hung, L., He, F., Zhang, L. and Qin, L.

(2012) A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy

Nat Med 18, 307-314

94. Lin, S. Y., Zhao, W. Y., Tsai, H. C., Hsu, W. H., Lo, C. L. and Hsiue, G. H. (2012) Sterically polymer-based

liposomal complexes with dual-shell structure for enhancing the siRNA delivery Biomacromolecules

13, 664-675

95. Li, R. C., Tao, J., Guo, Y. B., Wu, H. D., Liu, R. F., Bai, Y., Lv, Z. Z., Luo, G. Z., Li, L. L., Wang, M., Yang, H. Q., Gao,

W., Han, Q. D., Zhang, Y. Y., Wang, X. J., Xu, M. and Wang, S. Q. (2013) In vivo suppression of microRNA-24

prevents the transition toward decompensated hypertrophy in aortic-constricted mice Circ Res 112,

601-605

96. Singha, K., Namgung, R. and Kim, W. J. (2011) Polymers in small-interfering RNA delivery Nucleic Acid

Ther 21, 133-147

97. Schiffelers, R. M., Ansari, A., Xu, J., Zhou, Q., Tang, Q., Storm, G., Molema, G., Lu, P. Y., Scaria, P. V. and

Woodle, M. C. (2004) Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically

stabilized nanoparticle Nucleic Acids Res 32, e149

98. Katas, H. and Alpar, H. O. (2006) Development and characterisation of chitosan nanoparticles for

siRNA delivery J Control Release 115, 216-225


47

2

99. Han, H. D., Mangala, L. S., Lee, J. W., Shahzad, M. M., Kim, H. S., Shen, D., Nam, E. J., Mora, E. M., Stone,

R. L., Lu, C., Lee, S. J., Roh, J. W., Nick, A. M., Lopez-Berestein, G. and Sood, A. K. (2010) Targeted gene

silencing using RGD-labeled chitosan nanoparticles Clin Cancer Res 16, 3910-3922

100. Alshamsan, A., Haddadi, A., Hamdy, S., Samuel, J., El-Kadi, A. O., Uludag, H. and Lavasanifar, A. (2010)

STAT3 silencing in dendritic cells by siRNA polyplexes encapsulated in PLGA nanoparticles for the

modulation of anticancer immune response Mol Pharm 7, 1643-1654

101. Zhou, J., Patel, T. R., Fu, M., Bertram, J. P. and Saltzman, W. M. (2012) Octa-functional PLGA nanoparticles

for targeted and efficient siRNA delivery to tumors Biomaterials 33, 583-591

102. Meyer, M., Dohmen, C., Philipp, A., Kiener, D., Maiwald, G., Scheu, C., Ogris, M. and Wagner, E. (2009)

Synthesis and biological evaluation of a bioresponsive and endosomolytic siRNA-polymer conjugate

Mol Pharm 6, 752-762

103. Buyens, K., Meyer, M., Wagner, E., Demeester, J., De Smedt, S. C. and Sanders, N. N. (2010) Monitor-

ing the disassembly of siRNA polyplexes in serum is crucial for predicting their biological efficacy J

Control Release 141, 38-41

104. Kano, A., Moriyama, K., Yamano, T., Nakamura, I., Shimada, N. and Maruyama, A. (2011) Grafting of

poly(ethylene glycol) to poly-lysine augments its lifetime in blood circulation and accumulation in

tumors without loss of the ability to associate with siRNA J Control Release 149, 2-7

105. Wang, Y. and Grayson, S. M. (2012) Approaches for the preparation of non-linear amphiphilic polymers

and their applications to drug delivery Adv Drug Deliv Rev 64, 852-865

106. Taratula, O., Garbuzenko, O. B., Kirkpatrick, P., Pandya, I., Savla, R., Pozharov, V. P., He, H. and Minko, T.

(2009) Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA

delivery J Control Release 140, 284-293

107. Merkel, O. M., Zheng, M., Mintzer, M. A., Pavan, G. M., Librizzi, D., Maly, M., Hoffken, H., Danani, A.,

Simanek, E. E. and Kissel, T. (2011) Molecular modeling and in vivo imaging can identify successful

flexible triazine dendrimer-based siRNA delivery systems J Control Release 153, 23-33

108. Wolff, J. A. and Budker, V. (2005) The mechanism of naked DNA uptake and expression Adv Genet 54,

3-20

109. Kim, D., Hong, J., Moon, H. H., Nam, H. Y., Mok, H., Jeong, J. H., Kim, S. W., Choi, D. and Kim, S. H. (2013)

Anti-apoptotic cardioprotective effects of SHP-1 gene silencing against ischemia-reperfusion injury:

use of deoxycholic acid-modified low molecular weight polyethyleneimine as a cardiac siRNA-carrier


110. Saliba, Y., Mougenot, N., Jacquet, A., Atassi, F., Hatem, S., Fares, N. and Lompre, A. M. (2012) A new

method of ultrasonic nonviral gene delivery to the adult myocardium J Mol Cell Cardiol 53, 801-808

111. Somasuntharam, I., Boopathy, A. V., Khan, R. S., Martinez, M. D., Brown, M. E., Murthy, N. and Davis, M.

E. (2013) Delivery of Nox2-NADPH oxidase siRNA with polyketal nanoparticles for improving cardiac

function following myocardial infarction Biomaterials 34, 7790-7798

112. Santel, A., Aleku, M., Keil, O., Endruschat, J., Esche, V., Fisch, G., Dames, S., Loffler, K., Fechtner, M., Arnold,

W., Giese, K., Klippel, A. and Kaufmann, J. (2006) A novel siRNA-lipoplex technology for RNA interfer-

ence in the mouse vascular endothelium Gene Ther 13, 1222-1234

Chapter 2

48

113. Betigeri, S., Zhang, M., Garbuzenko, O. and Minko, T. (2011) Non-viral systemic delivery of siRNA or

antisense oligonucleotides targeted to Jun N-terminal kinase 1 prevents cellular hypoxic damage

Drug Deliv Transl Res 1, 13-24

114. Kang, J. H., Tachibana, Y., Obika, S., Harada-Shiba, M. and Yamaoka, T. (2012) Efficient reduction of

serum cholesterol by combining a liver-targeted gene delivery system with chemically modified

apolipoprotein B siRNA J Control Release 163, 119-124

115. Tadin-Strapps, M., Peterson, L. B., Cumiskey, A. M., Rosa, R. L., Mendoza, V. H., Castro-Perez, J., Puig, O.,

Zhang, L., Strapps, W. R., Yendluri, S., Andrews, L., Pickering, V., Rice, J., Luo, L., Chen, Z., Tep, S., Ason,

B., Somers, E. P., Sachs, A. B., Bartz, S. R., Tian, J., Chin, J., Hubbard, B. K., Wong, K. K. and Mitnaul, L. J.

(2011) siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with

human-like serum lipids J Lipid Res 52, 1084-1097

116. Majmudar, M. D., Keliher, E. J., Heidt, T., Leuschner, F., Truelove, J., Sena, B. F., Gorbatov, R., Iwamoto, Y.,

Dutta, P., Wojtkiewicz, G., Courties, G., Sebas, M., Borodovsky, A., Fitzgerald, K., Nolte, M. W., Dickneite,

G., Chen, J. W., Anderson, D. G., Swirski, F. K., Weissleder, R. and Nahrendorf, M. (2013) Monocyte-

directed RNAi targeting CCR2 improves infarct healing in atherosclerosis-prone mice Circulation 127,

2038-2046

117. Leuschner, F., Dutta, P., Gorbatov, R., Novobrantseva, T. I., Donahoe, J. S., Courties, G., Lee, K. M., Kim, J. I.,

Markmann, J. F., Marinelli, B., Panizzi, P., Lee, W. W., Iwamoto, Y., Milstein, S., Epstein-Barash, H., Cantley,

W., Wong, J., Cortez-Retamozo, V., Newton, A., Love, K., Libby, P., Pittet, M. J., Swirski, F. K., Koteliansky,

V., Langer, R., Weissleder, R., Anderson, D. G. and Nahrendorf, M. (2011) Therapeutic siRNA silencing in

inflammatory monocytes in mice Nat Biotechnol 29, 1005-1010

118. Strumberg, D., Schultheis, B., Traugott, U., Vank, C., Santel, A., Keil, O., Giese, K., Kaufmann, J. and Drevs,

J. (2012) Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with

advanced solid tumors Int J Clin Pharmacol Ther 50, 76-78

119. Coelho, T., Adams, D., Silva, A., Lozeron, P., Hawkins, P. N., Mant, T., Perez, J., Chiesa, J., Warrington, S.,

Tranter, E., Munisamy, M., Falzone, R., Harrop, J., Cehelsky, J., Bettencourt, B. R., Geissler, M., Butler, J. S.,

Sehgal, A., Meyers, R. E., Chen, Q., Borland, T., Hutabarat, R. M., Clausen, V. A., Alvarez, R., Fitzgerald, K.,

Gamba-Vitalo, C., Nochur, S. V., Vaishnaw, A. K., Sah, D. W., Gollob, J. A. and Suhr, O. B. (2013) Safety and

efficacy of RNAi therapy for transthyretin amyloidosis N Engl J Med 369, 819-829

120. Carson, A. R., McTiernan, C. F., Lavery, L., Grata, M., Leng, X., Wang, J., Chen, X. and Villanueva, F. S.

(2012) Ultrasound-targeted microbubble destruction to deliver siRNA cancer therapy Cancer Res 72,

6191-6199

121. Christiansen, J. P., French, B. A., Klibanov, A. L., Kaul, S. and Lindner, J. R. (2003) Targeted tissue transfec-

tion with ultrasound destruction of plasmid-bearing cationic microbubbles Ultrasound Med Biol 29,

1759-1767

122. Hernot, S. and Klibanov, A. L. (2008) Microbubbles in ultrasound-triggered drug and gene delivery

Adv Drug Deliv Rev 60, 1153-1166


49

2

123. Tlaxca, J. L., Anderson, C. R., Klibanov, A. L., Lowrey, B., Hossack, J. A., Alexander, J. S., Lawrence, M. B.

and Rychak, J. J. (2010) Analysis of in vitro transfection by sonoporation using cationic and neutral

microbubbles Ultrasound Med Biol 36, 1907-1918

124. Meijering, B. D., Juffermans, L. J., van Wamel, A., Henning, R. H., Zuhorn, I. S., Emmer, M., Versteilen, A.

M., Paulus, W. J., van Gilst, W. H., Kooiman, K., de Jong, N., Musters, R. J., Deelman, L. E. and Kamp, O.

(2009) Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction

of endocytosis and pore formation Circ Res 104, 679-687

125. Choi, J. J., Selert, K., Gao, Z., Samiotaki, G., Baseri, B. and Konofagou, E. E. (2011) Noninvasive and local-

ized blood-brain barrier disruption using focused ultrasound can be achieved at short pulse lengths

and low pulse repetition frequencies J Cereb Blood Flow Metab 31, 725-737

126. Chappell, J. C., Song, J., Burke, C. W., Klibanov, A. L. and Price, R. J. (2008) Targeted delivery of nanopar-

ticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic

arteriogenesis Small 4, 1769-1777

127. Kinoshita, M. and Hynynen, K. (2005) A novel method for the intracellular delivery of siRNA using

microbubble-enhanced focused ultrasound Biochem Biophys Res Commun 335, 393-399

128. Un, K., Kawakami, S., Yoshida, M., Higuchi, Y., Suzuki, R., Maruyama, K., Yamashita, F. and Hashida, M.

(2012) Efficient suppression of murine intracellular adhesion molecule-1 using ultrasound-responsive

and mannose-modified lipoplexes inhibits acute hepatic inflammation Hepatology 56, 259-269

129. Tsunoda, S., Mazda, O., Oda, Y., Iida, Y., Akabame, S., Kishida, T., Shin-Ya, M., Asada, H., Gojo, S., Imanishi,

J., Matsubara, H. and Yoshikawa, T. (2005) Sonoporation using microbubble BR14 promotes pDNA/

siRNA transduction to murine heart Biochem Biophys Res Commun 336, 118-127

130. Alter, J., Sennoga, C. A., Lopes, D. M., Eckersley, R. J. and Wells, D. J. (2009) Microbubble stability is a

major determinant of the efficiency of ultrasound and microbubble mediated in vivo gene transfer

Ultrasound Med Biol 35, 976-984

131. Saito, M., Mazda, O., Takahashi, K. A., Arai, Y., Kishida, T., Shin-Ya, M., Inoue, A., Tonomura, H., Sakao,

K., Morihara, T., Imanishi, J., Kawata, M. and Kubo, T. (2007) Sonoporation mediated transduction of

pDNA/siRNA into joint synovium in vivo J Orthop Res 25, 1308-1316

132. Haag, P., Frauscher, F., Gradl, J., Seitz, A., Schafer, G., Lindner, J. R., Klibanov, A. L., Bartsch, G., Klocker, H.

and Eder, I. E. (2006) Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide

targeting the human androgen receptor into prostate tumours J Steroid Biochem Mol Biol 102, 103-

113

133. Ko, Y. T., Hartner, W. C., Kale, A. and Torchilin, V. P. (2009) Gene delivery into ischemic myocardium by

double-targeted lipoplexes with anti-myosin antibody and TAT peptide Gene Ther 16, 52-59

134. Kim, J., Kim, S. W. and Kim, W. J. (2011) PEI-g-PEG-RGD/small interference RNA polyplex-mediated

silencing of vascular endothelial growth factor receptor and its potential as an anti-angiogenic tumor

therapeutic strategy Oligonucleotides 21, 101-107

135. Holig, P., Bach, M., Volkel, T., Nahde, T., Hoffmann, S., Muller, R. and Kontermann, R. E. (2004) Novel RGD

lipopeptides for the targeting of liposomes to integrin-expressing endothelial and melanoma cells

Protein Eng Des Sel 17, 433-441

Chapter 2

50

136. Sosunov, E. A., Anyukhovsky, E. P., Sosunov, A. A., Moshnikova, A., Wijesinghe, D., Engelman, D. M.,

Reshetnyak, Y. K. and Andreev, O. A. (2013) pH (low) insertion peptide (pHLIP) targets ischemic myo-

cardium Proc Natl Acad Sci U S A 110, 82-86

137. Bongartz, J. P., Aubertin, A. M., Milhaud, P. G. and Lebleu, B. (1994) Improved biological activity of

antisense oligonucleotides conjugated to a fusogenic peptide Nucleic Acids Res 22, 4681-4688

138. Hatakeyama, H., Ito, E., Akita, H., Oishi, M., Nagasaki, Y., Futaki, S. and Harashima, H. (2009) A pH-

sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of

siRNA-containing nanoparticles in vitro and in vivo J Control Release 139, 127-132

139. Sakurai, Y., Hatakeyama, H., Sato, Y., Akita, H., Takayama, K., Kobayashi, S., Futaki, S. and Harashima,

H. (2011) Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic

peptide shGALA Biomaterials 32, 5733-5742

140. Ye, S. F., Tian, M. M., Wang, T. X., Ren, L., Wang, D., Shen, L. H. and Shang, T. (2012) Synergistic effects of

cell-penetrating peptide Tat and fusogenic peptide HA2-enhanced cellular internalization and gene

transduction of organosilica nanoparticles Nanomedicine (Lond) 8, 833-841

141. Choi, S. W., Lee, S. H., Mok, H. and Park, T. G. (2010) Multifunctional siRNA delivery system: poly-

electrolyte complex micelles of six-arm PEG conjugate of siRNA and cell penetrating peptide with

crosslinked fusogenic peptide Biotechnol Prog 26, 57-63

142. Varkouhi, A. K., Scholte, M., Storm, G. and Haisma, H. J. (2011) Endosomal escape pathways for delivery

of biologicals J Control Release 151, 220-228

143. Janssen, H. L., Reesink, H. W., Lawitz, E. J., Zeuzem, S., Rodriguez-Torres, M., Patel, K., van der Meer, A.

J., Patick, A. K., Chen, A., Zhou, Y., Persson, R., King, B. D., Kauppinen, S., Levin, A. A. and Hodges, M. R.

(2013) Treatment of HCV infection by targeting microRNA N Engl J Med 368, 1685-1694

144. Kuhnert, F., Mancuso, M. R., Hampton, J., Stankunas, K., Asano, T., Chen, C. Z. and Kuo, C. J. (2008)

Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126 Development

135, 3989-3993

145. Yin, K. J., Olsen, K., Hamblin, M., Zhang, J., Schwendeman, S. P. and Chen, Y. E. (2012) Vascular en-

dothelial cell-specific microRNA-15a inhibits angiogenesis in hindlimb ischemia J Biol Chem 287,

27055-27064

146. Spinetti, G., Fortunato, O., Caporali, A., Shantikumar, S., Marchetti, M., Meloni, M., Descamps, B., Floris, I.,

Sangalli, E., Vono, R., Faglia, E., Specchia, C., Pintus, G., Madeddu, P. and Emanueli, C. (2013) MicroRNA-

15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the

proangiogenic cells and serum of patients with critical limb ischemia Circ Res 112, 335-346

147. Zhou, Q., Gallagher, R., Ufret-Vincenty, R., Li, X., Olson, E. N. and Wang, S. (2011) Regulation of an-

giogenesis and choroidal neovascularization by members of microRNA-23~27~24 clusters Proc Natl

Acad Sci U S A 108, 8287-8292

148. Fiedler, J., Jazbutyte, V., Kirchmaier, B. C., Gupta, S. K., Lorenzen, J., Hartmann, D., Galuppo, P., Kneitz,

S., Pena, J. T., Sohn-Lee, C., Loyer, X., Soutschek, J., Brand, T., Tuschl, T., Heineke, J., Martin, U., Schulte-

Merker, S., Ertl, G., Engelhardt, S., Bauersachs, J. and Thum, T. (2011) MicroRNA-24 regulates vascularity

after myocardial infarction Circulation 124, 720-730


51

2

149. Meloni, M., Marchetti, M., Garner, K., Littlejohns, B., Sala-Newby, G., Xenophontos, N., Floris, I., Suleiman,

M. S., Madeddu, P., Caporali, A. and Emanueli, C. (2013) Local Inhibition of MicroRNA-24 Improves

Reparative Angiogenesis and Left Ventricle Remodeling and Function in Mice With Myocardial Infarc-

tion Mol Ther 21, 1390-1402

150. van Rooij, E., Sutherland, L. B., Qi, X., Richardson, J. A., Hill, J. and Olson, E. N. (2007) Control of stress-

dependent cardiac growth and gene expression by a microRNA Science 316, 575-579

151. Huang, Z. P., Chen, J., Seok, H. Y., Zhang, Z., Kataoka, M., Hu, X. and Wang, D. Z. (2013) MicroRNA-22

regulates cardiac hypertrophy and remodeling in response to stress Circ Res 112, 1234-1243

152. da Costa Martins, P. A., Salic, K., Gladka, M. M., Armand, A. S., Leptidis, S., el Azzouzi, H., Hansen,

A., Coenen-de Roo, C. J., Bierhuizen, M. F., van der Nagel, R., van Kuik, J., de Weger, R., de Bruin, A.,

Condorelli, G., Arbones, M. L., Eschenhagen, T. and De Windt, L. J. (2010) MicroRNA-199b targets the

nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling Nat Cell

Biol 12, 1220-1227

153. Ganesan, J., Ramanujam, D., Sassi, Y., Ahles, A., Jentzsch, C., Werfel, S., Leierseder, S., Loyer, X., Giacca, M.,

Zentilin, L., Thum, T., Laggerbauer, B. and Engelhardt, S. (2013) MiR-378 controls cardiac hypertrophy

by combined repression of mitogen-activated protein kinase pathway factors Circulation 127, 2097-

2106

154. Care, A., Catalucci, D., Felicetti, F., Bonci, D., Addario, A., Gallo, P., Bang, M. L., Segnalini, P., Gu, Y., Dalton,

N. D., Elia, L., Latronico, M. V., Hoydal, M., Autore, C., Russo, M. A., Dorn, G. W., 2nd, Ellingsen, O., Ruiz-

Lozano, P., Peterson, K. L., Croce, C. M., Peschle, C. and Condorelli, G. (2007) MicroRNA-133 controls

cardiac hypertrophy Nat Med 13, 613-618

155. Pan, Z., Sun, X., Shan, H., Wang, N., Wang, J., Ren, J., Feng, S., Xie, L., Lu, C., Yuan, Y., Zhang, Y., Wang, Y.,

Lu, Y. and Yang, B. (2012) MicroRNA-101 inhibited postinfarct cardiac fibrosis and improved left ven-

tricular compliance via the FBJ osteosarcoma oncogene/transforming growth factor-beta1 pathway

Circulation 126, 840-850

156. Duisters, R. F., Tijsen, A. J., Schroen, B., Leenders, J. J., Lentink, V., van der Made, I., Herias, V., van Leeuwen,

R. E., Schellings, M. W., Barenbrug, P., Maessen, J. G., Heymans, S., Pinto, Y. M. and Creemers, E. E. (2009)

miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in

myocardial matrix remodeling Circ Res 104, 170-178, 176p following 178

157. Ali, R., Huang, Y., Maher, S. E., Kim, R. W., Giordano, F. J., Tellides, G. and Geirsson, A. (2012) miR-1 medi-

ated suppression of Sorcin regulates myocardial contractility through modulation of Ca2+ signaling

J Mol Cell Cardiol 52, 1027-1037

158. Lu, Y., Zhang, Y., Wang, N., Pan, Z., Gao, X., Zhang, F., Zhang, Y., Shan, H., Luo, X., Bai, Y., Sun, L., Song,

W., Xu, C., Wang, Z. and Yang, B. (2010) MicroRNA-328 contributes to adverse electrical remodeling in

atrial fibrillation Circulation 122, 2378-2387

159. Grueter, C. E., van Rooij, E., Johnson, B. A., DeLeon, S. M., Sutherland, L. B., Qi, X., Gautron, L., Elmquist,

J. K., Bassel-Duby, R. and Olson, E. N. (2012) A cardiac microRNA governs systemic energy homeostasis

by regulation of MED13 Cell 149, 671-683

Chapter 2

52

160. Noh, S. M., Park, M. O., Shim, G., Han, S. E., Lee, H. Y., Huh, J. H., Kim, M. S., Choi, J. J., Kim, K., Kwon, I. C.,

Kim, J. S., Baek, K. H. and Oh, Y. K. (2010) Pegylated poly-l-arginine derivatives of chitosan for effective

delivery of siRNA J Control Release 145, 159-164

161. Ghosn, B., Singh, A., Li, M., Vlassov, A. V., Burnett, C., Puri, N. and Roy, K. (2010) Efficient gene silenc-

ing in lungs and liver using imidazole-modified chitosan as a nanocarrier for small interfering RNA

Oligonucleotides 20, 163-172

162. Thibault, M., Astolfi, M., Tran-Khanh, N., Lavertu, M., Darras, V., Merzouki, A. and Buschmann, M. D.

(2011) Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal

release of the polyplexes Biomaterials 32, 4639-4646

163. Holzerny, P., Ajdini, B., Heusermann, W., Bruno, K., Schuleit, M., Meinel, L. and Keller, M. (2012) Biophysi-

cal properties of chitosan/siRNA polyplexes: profiling the polymer/siRNA interactions and bioactivity


164. Yuan, X., Shah, B. A., Kotadia, N. K., Li, J., Gu, H. and Wu, Z. (2010) The development and mechanism

studies of cationic chitosan-modified biodegradable PLGA nanoparticles for efficient siRNA drug

delivery Pharm Res 27, 1285-1295

165. Kolhar, P., Doshi, N. and Mitragotri, S. (2011) Polymer nanoneedle-mediated intracellular drug delivery

Small 7, 2094-2100

166. Shen, M., Gong, F., Pang, P., Zhu, K., Meng, X., Wu, C., Wang, J., Shan, H. and Shuai, X. (2012) An MRI-

visible non-viral vector for targeted Bcl-2 siRNA delivery to neuroblastoma Int J Nanomedicine 7,

3319-3332

167. Zheng, M., Librizzi, D., Kilic, A., Liu, Y., Renz, H., Merkel, O. M. and Kissel, T. (2012) Enhancing in vivo

circulation and siRNA delivery with biodegradable polyethylenimine-graft-polycaprolactone-block-

poly(ethylene glycol) copolymers Biomaterials 33, 6551-6558

168. Gao, S., Dagnaes-Hansen, F., Nielsen, E. J., Wengel, J., Besenbacher, F., Howard, K. A. and Kjems, J. (2009)

The effect of chemical modification and nanoparticle formulation on stability and biodistribution of

siRNA in mice Mol Ther 17, 1225-1233

169. Lee, D., Kim, D., Mok, H., Jeong, J. H., Choi, D. and Kim, S. H. (2012) Bioreducible crosslinked polyelec-

trolyte complexes for MMP-2 siRNA delivery into human vascular smooth muscle cells Pharm Res 29,

2213-2224

170. Liu, P., Yu, H., Sun, Y., Zhu, M. and Duan, Y. (2012) A mPEG-PLGA-b-PLL copolymer carrier for adriamycin

and siRNA delivery Biomaterials 33, 4403-4412

171. Qi, R., Liu, S., Chen, J., Xiao, H., Yan, L., Huang, Y. and Jing, X. (2012) Biodegradable copolymers with

identical cationic segments and their performance in siRNA delivery J Control Release 159, 251-260

172. Sato, A., Choi, S. W., Hirai, M., Yamayoshi, A., Moriyama, R., Yamano, T., Takagi, M., Kano, A., Shimamoto,

A. and Maruyama, A. (2007) Polymer brush-stabilized polyplex for a siRNA carrier with long circulatory

half-life J Control Release 122, 209-216

173. Kang, H., DeLong, R., Fisher, M. H. and Juliano, R. L. (2005) Tat-conjugated PAMAM dendrimers as

delivery agents for antisense and siRNA oligonucleotides Pharm Res 22, 2099-2106


53

2

174. Arima, H., Yoshimatsu, A., Ikeda, H., Ohyama, A., Motoyama, K., Higashi, T., Tsuchiya, A., Niidome, T.,

Katayama, Y., Hattori, K. and Takeuchi, T. (2012) Folate-PEG-appended dendrimer conjugate with

alpha-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier Mol Pharm 9, 2591-2604

Targeted delivery of miRNA therapeutics for cardiovascular ... 2.pdf · muscle cells) leads to...

Documents

Transcript of Targeted delivery of miRNA therapeutics for cardiovascular ... 2.pdf · muscle cells) leads to...