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Journal of the American College of Cardiology
© 2010 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Nonviral Intercellular Adhesion
Molecule-1 Small Interfering
Ribonucleic Acid Sequences
Transfection In Vivo
How Ultrasound Bursts Into Therapy*
Christian Kupatt, MD
Munich, Germany
A decade ago, gene therapy of atherosclerosis was thought
to have a bright future for its proven capacity to force
expression of a protein that is either lacking but necessary
(such as the low-density lipoprotein receptor in hyperlipoproteinemia type IIa) or a disease-modifying molecular
See page 904
switch (such as hypoxia-inducible factor 1␣ in the case of
chronic ischemia). Both approaches have been successfully
realized experimentally (1,2), but still await clinical approval
because of side effects or lack of efficacy. Conceptually, an
even more complex attempt is to get rid of an unwanted
protein, for example, a pro-inflammatory protein that is
leading to intima proliferation and vascular stenosis. In
addition to overexpression of dominant negative mutants
competing with the functional target protein, which is still
an arduous task in vivo, silencing of protein translation is an
attractive alternative. This approach was initially performed
by antisense oligonucleotides, but recently gained more
steam by the discovery of small interfering ribonucleic acid
sequences (siRNAs) (for review, see de Fougerolles et al.
[3]). After ribonucleic acid oligonucleotides align with
messenger ribonucleic acid (mRNA) of the target gene, they
induce its cleavage by the Ago2-Risc-complex. The discovery of this mechanism provided life sciences with a frequently used tool for the study of gene regulation in vitro
and in vivo, with even orally applied formulas providing
efficacy lately (4).
*Editorials published in the Journal of the American College of Cardiology reflect the
views of the authors and do not necessarily represent the views of JACC or the
American College of Cardiology.
From the Medizinische Klinik und Poliklinik I, Klinikum Grosshadern, Munich,
Germany. Dr. Kupatt is supported by the Deutsche Forschungsgemeinschaft (KU
1019/10-1, Transregio For 535), by the German Ministry for Research and
Education (Grant 01GU0525), and by the Dr. Helmut Legerlotz Foundation.
Vol. 55, No. 9, 2010
ISSN 0735-1097/10/$36.00
Conversely, in vivo transfection of the endothelium with
siRNAs still poses a distinct hurdle. Construction of sense
and antisense siRNA sequences (5) or short hairpin ribonucleic acids as precursors (6) allows for plasmid transfection by appropriate vectors. However, adenoviral transfection, although capable of transducing endothelium (7), lasts
for only a limited expression time, without a fair chance of
retransfection (8). In contrast, adeno-associated virus vectors are capable of long-term expression, but all known
strains lack endothelial tropism in vivo (9). Of note,
lentiviral endothelial transfection has recently been accomplished by coupling lentiviral particles to paramagnetic
nanoparticles and with subsequent application of magnetic
force (10). However, lentiviral-based gene therapy has the
capacity to integrate into the host genome at unwanted
sites, although the pro-oncogenic potential is less substantial than that in other retroviruses (11).
Therefore, an alternative source is sought. Liposomal
transfection has been used for the endothelium with considerable success, even in vivo in large animal experiments
(12). Liposomal formulas appear to be of use for local drug
delivery in oncologic patients (13) and are examined as
vehicles for siRNA transfection in tumors (14). However,
transfection efficacy is clearly a limiting factor.
A short regional pulse of ultrasound may help deliver the
payload. Since its first description in this setting (15),
ultrasound energy has been repetitively used to burst microbubbles of various origins (16) at the endothelial lining and
enhance uptake of genetic material into the target cells (17).
Ultrasound waves apparently do the trick by punching
transient pores into the cell wall simultaneously to induce
collapse of the microbubble carriers in the near vicinity
(16,18). A second mechanism of uptake of genetic material
may be endocytosis (19). These principles make the endothelium a privileged transfection target, which is in closest
contact with the liposome carrier (Fig. 1), although deeper
cell layers may also be affected (20). As the field advances,
and apoptosis induction by sonoporation has become apparent in vitro (21), a closer look to cell detriment by
ultrasound in vivo will be required.
Utilizing ultrasound-microbubble mediated transfection, Suzuki et al. (22), in this issue of the Journal,
provide proof of the concept that anti-inflammatory
siRNA delivery to injured endothelium is efficiently
preventing inflammatory cell invasion and subsequent
intimal hyperplasia in an injury model of the femoral
artery. Green fluorescent protein (GFP)-expressing mice
were used to demonstrate a loss of fluorescence in the
vicinity of arterial lumina after GFP-siRNA treatment.
The authors then target the expression of the adhesion
molecules intercellular adhesion molecule (ICAM)-1 and
vascular cell adhesion molecule (VCAM)-1, preventing
adhesion of leukocytes expressing ␤2-integrin or very late
antigen-4 on their surface. As a result, neointima formation was significantly inhibited in this murine model. A
Nonviral ICAM-1 siRNA Transfection In Vivo
JACC Vol. 55, No. 9, 2010
March 2, 2010:914–6
Endothelial cell
mRNA strand
Figure 1
Mechanism of Ultrasound-Based Transfection of siRNA
Liposomal small interfering ribonucleic acid (siRNA) oligonucleotides are released from their carrier by ultrasound energy, the same mechanism enabling entry into
endothelial cells through putative transient pores. Once in the cytosol, siRNA aligns with messenger RNA (mRNA) strands, inviting attachment of the Ago-Risc complex
and subsequent mRNA cleavage. DOTAP ⫽ 1,2 dioleoyl-3-trimethylammonium-propane.
complementary approach, application of an antibody
against the ␤2-integrin Mac-1, had resulted in neointima
reduction in a carotid injury model earlier, but relied on
daily antibody infusion (23). Suzuki et al. (22) demonstrate that 1-time siRNA application yielded a significant
decrease of intimal hyperplasia for as long as 7 days of the
therapeutic intervention. This interval provides a starting
point, but requires long-term follow-up, exclusion of late
catch-up of the intimal hyperplasia, and feasibility studies
in large animals before clinical trials can be appropriately
Applying siRNAs to ICAM-1 and VCAM-1, the
authors demonstrate the pinpoint precision of the inhibitory siRNA approach in vivo. Although stent implantation was not assessed, a novel option may evolve taking
advantage of this routine: siRNA treatment might be
eluting from appropriate stents—although superiority
versus the current gold standard of antimitotic drugeluting stent treatment would have to be proved. Conversely, stents eluting siRNAs are an appealing vision of
individualized lesion treatment, as pinpoint molecular
signal interruption becomes feasible, taking emerging
differences in the molecular basis of restenosis in, for
example, heart transplant vasculopathy (24) versus diabetic angiopathy (25) into account. Moreover, targeting
microbubbles to a particular subset of activated endothelial cells displaying distinct surface molecules (26) is a
further step of limiting inhibitory gene therapy to the loci
of intense inflammation, for example, to the atherosclerotic plaques, where stabilization and prevention of
clinical manifestation is of utter clinical relevance.
Reprint requests and correspondence: Dr. Christian Kupatt,
Medizinische Klinik und Poliklinik I, Klinikum Grosshadern,
LMU, Marchioninistrasse 15, 81377 Munich, Germany. E-mail:
[email protected]
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JACC Vol. 55, No. 9, 2010
March 2, 2010:914–6
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Key Words: gene therapy y siRNA y adhesion molecules y ultrasound
y artery.

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