Nonviral gene therapy targeting cardiovascular system - AJP

Am J Physiol Heart Circ Physiol 303: H629 –H638, 2012.
First published July 20, 2012; doi:10.1152/ajpheart.00126.2012.
Nonviral gene therapy targeting cardiovascular system
Cheng-Huang Su,1,2 Yih-Jer Wu,1,3 Hsueh-Hsiao Wang,3 and Hung-I Yeh1,3
Departments of Internal Medicine and Medical Research, Mackay Memorial Hospital, New Taipei City, Taiwan; 2Mackay
Medicine, Nursing and Management College, New Taipei City, Taiwan; 3Mackay Medical College, New Taipei City, Taiwan
Submitted 15 February 2012; accepted in final form 17 July 2012
gene therapy; vector; ultrasound; cavitation; microbubble
is part of a collection on Physiological
Basis of Cardiovascular and Gene Therapies. Other articles
appearing in this collection, as well as a full archive of all
Review collections, can be found online at http://ajpheart.
Overview of Applications of Gene Therapy
Gene therapy is a novel approach that can be applied through
a variety of clinical therapeutic procedures in which nucleic
acids are introduced into human cells for the purpose of
treating, curing, or ultimately preventing disease. The development of gene vectors for effectively carrying genes into cells
has made a great deal of progress in recent years. Nonviral
vectors should have a better chance to circumvent some of the
problems possibly occurring with viral vectors, such as unwanted immune response and oncogenesis. In general, although nonviral vectors, such as plasmid DNA, allow remarkable organ specificity, they are often limited by low transfection efficiency (TE). In contrast, virus-based vectors deliver the
transgene more efficiently, but organ specificity may be reduced and immunogenic properties can limit their practical
Address for reprint requests and other correspondence: H.-I Yeh, Depts. of
Internal Medicine and Medical Research, Mackay Memorial Hospital; Mackay
Medical College, No. 46, Sec. 3, Jhong-Jheng Rd, San-Jhih District, New
Taipei City 252, Taiwan (e-mail: [email protected]).
applications. Both systems need dose optimization and development of strategies to improve vector technology. Thus far,
the main categories of methods that have been used to deliver
genes into cells or tissues in gene therapy protocols are as follows:
viral vectors (68.8%), nonviral chemical vectors (6.4%), naked/
plasmid DNA (18.6%), and physical delivery systems (0.3%; data
adapted from
clinical/, clinical trials website of The Journal of Gene Medicine, updated June 2011). In the present review, we summarized the progress made in the past few years regarding
techniques developed in the following two areas: 1) chemical
carrier-mediated gene delivery, such as cationic polymer and
lipid, and 2) naked DNA transfection by a physical method,
such as ultrasound (US) along with microbubbles. In addition,
the nonviral carrier and technology of cardiovascular gene
delivery are briefly overviewed, while the applications to the
biomedical researches including regenerative medicine are
Candidate Diseases for Gene Therapy
There are several promising areas for gene therapy in genetic
and acquired diseases. Thus far, more than 1,500 clinical trials
have taken place worldwide. The diseases most commonly
treated with gene therapy are cancer (64.6%), cardiovascular
diseases (8.5%), monogenic diseases (8.3%), and infectious
0363-6135/12 Copyright © 2012 the American Physiological Society
Downloaded from by on June 15, 2017
Su C, Wu Y, Wang H, Yeh H. Nonviral gene therapy targeting cardiovascular
system. Am J Physiol Heart Circ Physiol 303: H629 –H638, 2012. First published
July 20, 2012; doi:10.1152/ajpheart.00126.2012.—The goal of gene therapy is
either to introduce a therapeutic gene into or replace a defective gene in an
individual’s cells and tissues. Gene therapy has been urged as a potential method
to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues
after extensive investigation in recent preclinical and clinical studies. A successful
gene therapy mainly relies on the development of the gene delivery vector.
Developments in viral and nonviral vector technology including cell-based gene
transfer will further improve transgene delivery and expression efficiency. Nonviral
approaches as alternative gene delivery vehicles to viral vectors have received
significant attention. Recently, a simple and safe approach of gene delivery into
target cells using naked DNA has been improved by combining several techniques.
Among the physical approaches, ultrasonic microbubble gene delivery, with its
high safety profile, low costs, and repeatable applicability, can increase the
permeability of cell membrane to macromolecules such as plasmid DNA by its
bioeffects and can provide as a feasible tool in gene delivery. On the other hand,
among the promising areas for gene therapy in acquired diseases, ischemic
cardiovascular diseases have been widely studied. As a result, gene therapy using
advanced technology may play an important role in this regard. The aims of this
review focus on understanding the cellular and in vivo barriers in gene transfer and
provide an overview of currently used chemical vectors and physical tools that are
applied in nonviral cardiovascular gene transfer.
Among the target genes that encode angiogenic factors used in
clinical trials for CAD and PAD, VEGF (121 and 165 aminoacid isoforms of VEGF1 and VEGF2) and fibroblast growth
factor (FGF, FGF1, and FGF4) families are relatively well
investigated. VEGF and FGF in gene delivery have been
shown to induce functionally significant angiogenesis in preclinical (19, 41) and clinical studies (53, 79, 91) for ischemic
diseases. Although some studies showed no benefit or inconsistent results of growth factor gene therapy, designs may need
to be further optimized with proper patient selection and
growth factor delivery (40, 93). Another strategy investigated in
angiogenic therapy is based on the transcription factor hypoxiainducible factor-1␣ (82) by which the expression of several
angiogenic genes, including VEGF and the VEGF receptors
KDR/FLT-1, are regulated. Additionally, a factor associated with
regulation of angiogenesis is the peptide PR-39, which is a
proline- and arginine-rich peptide and increases the cellular levels
of hypoxia-inducible factor-1␣ by inhibiting its proteosomal degradation (2). Although it is possible that nonspecific action is
related to charge (55), PR-39 has been shown to enhance the
expression of VEGF, its receptors KDR and FLT-1, and the FGF
receptor 1.
For treatment of cardiovascular diseases, in addition to
angiogenic genes, there are important nonangiogenic genes or
nucleotides related to cell survival, such as genes associated
with reduction-oxidation (redox) reactions and microRNAs
(miRNAs). Redox regulation is an essential physiological process in the cell survival of almost all types of cells, including
cardiomyocytes. Loss of cellular redox balance results in the
development of oxidative stress in the cells, causing cellular
dysfunction (12). There are critical antioxidant systems in cardiomyocytes to balance the oxidative stress: thioredoxin (Trx),
glutaredoxin (Grx), and peroxiredoxin (Prx), all of which participate in redox regulation to protect the cells from oxidative stress
and to antagonize apoptotic signaling (6). Overexpression of
mitochondrial Prx-3 in mouse model showed resistance to
ventricular remodeling and failure after myocardial infarction
(68). Therapeutic strategies designed to attenuate mitochondrial oxidative stress such as enhanced expression of the
antioxidant Prx-3 might be beneficial in preventing cardiac
failure. Overexpression of both Grx-1 and Grx-2 rendered the
heart resistant to ischemia-reperfusion injury and implicates a
role in cardioprotection and redox signaling of the ischemic
myocardium (66, 75). The above findings may suggest that
Trx, Grx, and Prx are potential treatment targets for myocardial
salvage in conditions associated with overwhelming oxidative
stress. miRNAs consist of noncoding single-stranded RNAs of
⬃22 nucleotides that negatively regulate gene expression via
degradation or translational inhibition of their target mRNAs.
They have been implicated in the regulation of a diverse
spectrum of cardiac functions, including cardiovascular cell
differentiation, growth, proliferation, and apoptosis (50, 101).
Among human miRNA genes, miRNA-21 was reported to be a
mediator of extracellular signal-regulated kinase/mitogen-activated protein kinase signaling and is crucial for fibroblast
survival and activation (43, 102). Further analysis showed that
miRNA-21 is predominantly overexpressed in cardiac fibroblasts, especially those existing in the failing heart (83, 102).
Moreover, in 2011, functional recovery and engraftment of
transplanted cardiac progenitor cells that were transduced with
lentivirus carrying a miRNA prosurvival cocktail (miRNA-21,
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
diseases (8.1%) (data adapted from clinical trials website of
The Journal of Gene Medicine).
Cardiovascular disease is among the leading cause of morbidity and mortality in the developed world. The first investigation of vascular gene transfer was demonstrated in 1989 by
Nabel et al. (73), who transfected porcine endothelial cells ex
vivo with a retrovirus carrying the ␤-galactosidase gene and
reintroduced the cells onto the denuded iliofemoral artery of a
syngeneic pig. Since then, recombinant viruses have been used
for gene transfer in most clinical studies. However, the death of
a 18-year-old boy (54), who died of complications from a
massive inflammatory response shortly after receiving a dose
of adenovirus carrying a corrective gene in a phase I trial in
1999, illustrates the challenge in gene therapy. The mechanisms for this catastrophic reaction have never been completely
understood, but a dose-specific adverse effect or a previous
immunization due to a viral infection has been hypothesized as
a potential cause. The future choice of viral or nonviral vectors
will critically depend on specific purposes in gene therapy,
potential adverse effects, and further improvements in vector
design, though this review will focus on nonviral methods by
which clinical or preclinical trials have shown feasibility into
cardiovascular diseases.
Among those diseases related to aging process, gene therapy
may provide an alternative treatment option in peripheral artery
disease (PAD) and coronary artery disease (CAD). Neovascularization that plays a critical role in PAD and CAD involves
complex processes that include the coordination of cytokines to
induce new conduits of blood supply. Among these events,
angiogenesis involves enhanced vascular permeability, vascular cell migration and proliferation, and collateral vessel formation (34). In patients with ischemic cardiovascular diseases,
progressive occlusion of arteries often results in the formation
of collateral vessels that supply the ischemic tissues. However,
the natural compensatory development of neovascularization is
often incomplete partly due to insufficient availability of angiogenic factors. Therapeutic angiogenesis is a method based
on direct administration of angiogenic agents such as recombinant protein, indirect delivery of target genes that encode
relevant proteins, or use of stem cells that have been shown to
secret angiogenic factors and enhance angiogenesis in preclinical and clinical studies (26, 37). Clinical trials of the vascular
endothelial growth factor (VEGF) gene was started in 1994 by
Isner et al. (37). Since then, several angiogenic cytokines have
been investigated in clinical trials. Administration of proper
angiogenic proteins with controllable therapeutic levels and
pharmacokinetics are required in this regard. Since the limited
half-life of angiogenic proteins restricts the action duration,
development of slow-release delivery systems to prolong the
action may be more effective. In addition, multifactor therapy
may be necessary to achieve adequate angiogenesis and provide functionally significant improvements in human tissue
perfusion because of the sophisticated angiogenic process
(103). Regarding angiogenic gene therapy, induction of inflammatory and immune responses, low efficiency of gene expression, and nonspecific gene transfer to other cell types with
uncontrolled levels of growth factor are limiting factors (51).
Advantages of gene therapy in angiogenesis included persistent
expression of the angiogenic factor with prolonged, local
action. So far, several genes of growth factors and transcription
factors have been tried to achieve therapeutic angiogenesis.
-24, and -221) can be improved in a murine model of myocardial infarction (36). Therefore, miRNAs may be a gene therapy
target to promote cell survival in heart failure by regulating
other genes.
Current Nonviral Gene-transfer Systems
their key mechanisms, diseases/status, limitations, and advantages.
Nonviral vectors. The safety concerns associated with viral
vectors have encouraged the development of nonviral vectors.
pDNA delivered by nonviral methods is maintained in an
extrachromosomal site, not integrated into the cellular genome.
The most popular materials used in current nonviral applications include purified pDNA, chemical vectors such as lipids
and synthetic polymers. Nonviral methods of gene delivery
used in current clinical trials include pDNA and liposomal
safest nonviral gene delivery system currently in use in vivo is
the direct gene transfer with naked pDNA. However, as the
rapid degradation by nucleases and the clearance by the mononuclear phagocyte system in the systemic circulation, expression levels after the injection of naked DNA are generally
limited. In 1990, Wolff et al. (108) first reported that intramuscularly injected naked pDNA can be expressed in myofibers.
One of the promising approaches in this field is the combined
use of naked DNA and physical approaches (such as electroporation) to enhance plasmid-mediated gene expression in
muscle (20, 87). In the past 10 years, intramuscular injection of
pDNA has been applied in a strategy of therapeutic angiogenesis (96). Isner and colleagues have used VEGF to enhance the
formation of collateral blood vessels around occluded native
arteries. Two catheter-based trials of myocardial gene delivery
using pDNA encoding VEGF-2 have been reported (63, 105).
In 2011, an open-label clinical study investigated the efficacy
and safety of intramuscular injection of naked pDNA encoding
the human hepatocyte growth factor (HGF) gene in Japanese
patients with Buerger’s disease (89). The size of ischemic
ulcers decreased in 6/9 (66.7%) patients, and the ulcers healed
completely in 5/9 (55.6%) patients after gene therapy. The
investigators concluded that HGF gene therapy is safe and
effective for treating critical limb ischemia.
LIPID-BASED GENE DELIVERY SYSTEMS. First reported by Felgner et al. in 1987 (23), lipid-based gene delivery is still one of
the major systems to increase the TE of naked DNA (15).
Liposomes or lipoplexes are formed by DNA with positively
charged lipids and detergents (16, 60). In lipoplex structure,
DNA backbones are surrounded with positively charged lipids
to form condensed complexes that possibly protect them
Fig. 1. Entry of plasmid into nucleus using nonviral gene
delivery can be achieved either by physical methods, such
as ultrasound (US) or electroporation, to increase cell
permeability or via chemical vectors through endocytosis.
Main steps impeding the nuclear accumulation of DNAvector complexes that need to be overcome include 1) cell
binding, 2) cell entry, 3) endosomal escape, 4) degradation
by endonucleases, 5) nuclear entry, and 6) decomplexation. In contrast, entry of plasmid DNA into nucleus by
physical methods mainly encounters steps 2, 4, and 5. Note
that both approaches required cytoplasmic transport between steps 2 and 5.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
Extra- and intracellular barriers for a successful gene
delivery. A feasible gene therapy vector needs to meet three
criteria: safety, adequate gene transfer efficiency, as well as
stable expression of the transgene for a duration appropriate for
treating the disease. Nonviral methods may only cause transient transfection and lack the ability to integrate into the
genome that limit their use in conditions requiring long-term
gene expression such as chronic heart failure. However, it is
possible that transient expression of therapeutic genes can be
favorable in certain applications. One such example is in bone
healing, in which bone morphogenetic protein-2 just needs to
express only for a few days because it triggers a cascade of
activity that does not end even if it is withdrawn (56, 57).
Moreover, nonviral vectors, due to anatomical and cellular
barriers, limit the overall efficiency in gene therapy. There are
biological obstacles impeding the nuclear accumulation of
pDNA or DNA-vector complexes that need to be overcome:
cell binding, cell entry/endocytosis, endosome escape, cytoplasmic transport, and nuclear entry (Fig. 1). Most of the
chemical vectors compact DNA into particles by neutralizing
the anionic DNA phosphate backbone and balancing charge
repulsion force from the anionic cell surface (67). Considering
a normal intracellular trafficking, after DNA-vector complexes
interact with the cell membrane, they are endocytosed (97).
This step may be nonspecific (mediated via electrostatic interactions) or may involve a specific target (using an antibody
conjugates) (98). The vector should be designed to allow the
DNA-vector complex to effectively escape from the endosome
into the cytoplasm to protect it from the subsequent lysosomal
degradation, and the vector should be capable of transporting
its cargo to the nucleus. Additionally, there are many factors
influencing the efficacy and safety of nonviral vector-mediated
gene transfer, such as the charge of the vector, formulation of
DNA/vector complexes (42), and interaction with serum and
blood cells (85). Unfortunately, the ideal gene delivery systems
are still under investigation. The potential nonviral vectors and
physical approaches are summarized in Table 1 in terms of
Table 1. A summary of nonviral gene delivery systems for treatment of cardiovascular diseases
Delivery Methods
Naked plasmid (VEGF, HGF, FGF)
Chemical vectors
Central Mechanism
PAD and CAD (phase I/II)*
Safety and simplicity
Low transfection efficiency
PAD and CAD (phase I/II)*
Low to medium high-efficiency,
In vitro and in vivo
Low cytotoxicity
Low immunogenicity
Fair efficiency
Complement activation, low
Low efficiency
Safety and flexibility
Low efficiency
Good efficiency and
Tissue damage, limited
accessibility of electrodes to
internal organs
Shallow penetration of DNA
into target tissues
Physical methods
Ultrasound ⫾ microbubble
In vitro and in vivo
Enhancement of cell
membrane permeability
Enhancement of cell
membrane permeability
Gene gun
High-pressure helium
Magnetic force plus
Good efficiency, transfer
genes to nondividing
Flexibility and low
Transient transfection
See text for abbreviations. *Clinical trials completed or currently conducted.
against extracellular or intracellular nucleases. The resulting
net-positive charge of lipid-DNA complexes may facilitate
fusion with the negatively charged molecules of the cell membrane (glycoproteins and proteoglycans) that may subsequently
facilitate their cellular uptake by endocytosis (112). Many
cationic molecules cannot form liposomes alone and are normally
accompanied by a neutral lipid (colipid or helper lipids), such as
dioleoylphosphatidylethanolamine (DOPE) (22). DOPE promotes
hexagonal-phase lipid polymorphism that is related to membrane
fusion and increase in TE in vitro (47). In addition, most of the
cationic lipids are more or less toxic to cells, and the presence of
DOPE could lead to reduced charge ratio and less toxicity.
Cationic lipids generally have the advantages of being inexpensive and can be engineered to have targeted specificity. However,
because of the excessive surface charge, the half-life of cationic
lipids in circulation is very short. Elimination of cationic lipids
in systemic circulation occurs upon the formation of larger
aggregates because of their interactions with the negatively
charged serum molecules. Lipoplexes tend to initially accumulate in the pulmonary vasculature because of the first passage
effect (58). As a result, cells that are transfected are mainly
pulmonary vascular and endothelial cells. The drawback of a
fast clearance of cationic lipids from the circulation limits their
use to transfer gene to cells located beyond vascular endothelial cells. Surface shielding or modification of DNA-vector
complex through the use of hydrophilic and charge neutral
polymers such as polyethylene glycol (PEG) to reduce excessive charge-charge interaction appears very effective in prolonging the circulation half-life of lipoplexes (32). However, it
is noted that the presence of the PEG moiety on the surface of
lipoplexes reduces an interaction between lipoplexes and cell
membrane and, as a result, reduces the TE. Several strategies
have been used to make the PEG shielding conditional and
nonpermanent, such as PEG-lipid conjugates. Other drawbacks
of lipoplex-mediated gene delivery are short duration of gene
expression and acute toxicity. When combined with unmethylated cytosine-phosphate guanosine-containing DNA, cat-
ionic lipids can stimulate potent inflammatory response in the
hosts. Surface shielding with PEG-lipid and systematic deletions and mutations of cytosine-phosphate-guanosine-containing DNA sequences from plasmid sequences have resulted in
some promising data in suppressing cytokine production (8).
Delivery of growth factors by liposomes has been reported to
be effective in animal models of angiogenesis (3, 65). In 2009,
Hedman et al. (33) studied the long-term effects (a total
follow-up time of 8.1 years) and safety of the local VEGF-A
catheter-based gene transfer in 103 patients with CAD between
three groups (adenoviral vector, liposome vector, or control)
(33). The results showed that local intracoronary VEGF gene
transfer is safe and does not cause an increased risk of major
adverse cardiovascular events, cancer, or diabetes. More recently, in 2011, Muona et al. (71) examined the effects of
VEGF on 25 patients having received VEGF-A gene transfer
for the treatment of symptomatic PAD (71). There were no
significant differences between study groups in the causes of
death or in the incidence of cancer or diabetic retinopathy.
Polymer-based gene delivery systems. The principle of cationic polymers as nonviral DNA vehicles is based on the
concept of forming condensed DNA particles by complex
formation with cationic polymers-polyplexes. The use of polycationic polymers leads to electrostatic neutralization of anionic charges of DNA and condenses the polynucleotide structure of DNA into nanosized complexes, thereby protecting it
from nuclease digestion and facilitating the cellular uptake
through endocytosis.
Many polycationic molecules are used, including poly-Llysine (PLL), dendrimers, polyornithine, and polyethyleneimine (PEI). PEI and PLL are the commonest and most important ones used as nonviral vectors. PLL has been used to
condense pDNA under various salt conditions (59). The PLLDNA particles have been shown to be protected against DNA
degradation (28). Electron microscopic studies had demonstrated that PLL-DNA complexes assumes a rod-like appearance with a diameter of 15 nm and a length of 109 ⫾ 36 nm,
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
DNA condensation
Protein sponge effect
DNA-gold beads are cheap and easy to prepare. The gene gun
delivery into the skin is a promising alternative to the injection
of naked pDNA into muscle for genetic vaccinations (13).
Several animal studies have reported that gene gun transfection
of skin tissues can elicit immune responses to protect the
animals from infection (27, 64). These immune responses have
also been employed as a therapeutic approach for the treatment
of cancer in human clinical trials (107). As a method to treat
other disorders, Goudy et al. (30) have shown that vaccination
in an animal model of type 1 diabetes with glutamic acid
decarboxylase gene has induced type 2 immunity that resulted
in blocking ␤-cell autoimmunity (30).
ELECTROPORATION. The application of controlled electric
fields to facilitate cell permeabilization (electroporation) was
first described in the 1960s (14). Extracellular molecules can
pass through the cell membrane via the pores transiently
generated by the electrical pulses. Since 1982, the use of
electric pulses for cell electroporation has been used to introduce foreign DNA into cells in vitro and in vivo (77, 104).
Skeletal muscle is a good candidate for electroporation (7). In
general, DNA injection followed by electroporation showed
10- to 1,000-fold enhancements of expression compared with
naked DNA injection alone but are accompanied by an elevation of serum creatine kinase levels (31). Indeed, one key
limitation of electroporation is that a substantial procedurerelated damage may occur and, as a result, may limit the
efficiency of transfection and its use in cardiovascular tissues
(18, 69). In addition to electroporation followed by local
injection, in vivo electroporation performed in a localized
manner after a systemic injection of pDNA has been reported
(84). Although in vivo electroporation technique is generally
efficient when parameters are optimized and can produce good
reproducibility, the key limitation is the accessibility of the
electrodes to the internal organs and its clinical applications
need further optimization.
US-BASED TECHNOLOGY IN GENE DELIVERY. US waves are defined as mechanical sound waves that have a frequency above
the audible sound of humans, generally about 20 kHz.
Bioeffects of US. Among the physical effects of US, acoustic
cavitation (4) is thought to be the most important bioeffect.
Briefly, as the US waves propagate through the medium, the
characteristic compression (Fig. 2) and rarefaction (Fig. 2)
cause microscopic gas bubbles in the tissue fluid to contract
and expand. Two types of cavitation are recognized. Gas body
activation (61) or stable cavitation is the term used to describe
bubbles that oscillate in diameter with the passing pressure
Fig. 2. Dynamic size change of microbubbles (inertial
cavitation) within an acoustic field of a continuouswave US. The rapid growth in size (rarefaction cycle)
and the collapse of microbubbles (compression cycle)
lead to the implosion of the microbubbles and local
generation of heat, shock wave, free radical formation,
and light, some of which provide the energy for sonoporation of cell membrane.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
much smaller than lipoplexes. The poor circulatory half-lives
of PLL-DNA complexes, typically shorter than 3 min, also
limit their use in vivo (110). In the recent years, PLL-PEG
conjugates with defined chemical composition have been
shown to improve delivery to different organs (113) and have
been applied in phase I/II clinical trials for treatment of ocular
degenerative diseases and cystic fibrosis (48). Generally, PLL
or PLL-DNA complexes were reported to have low immunogenicity (92).
Among cationic polymers, PEI, which is used in both linear
and branched forms with molecular masses (MMs) between
0.7 and 800 kDa (29), has been one of the most effective
polymer-based transfection agents. The polycationic PEI is
receiving much attention because of its characteristic of condensing DNA with an intrinsic endosomolytic activity and has
the ability to capture protons that are pumped into endolysosomes—“proton sponge” effect (9). This permits the escape of
endocytosed PEI-DNA complexes. However, it is highly cytotoxic. Factors influencing cytotoxicity, TE, and the use for
in vivo transfection include MM, incubation time, and the
charge ratio of polymer to DNA used (25, 39). In general, high
MM PEI (⬎25 kDa) is toxic to cells, whereas polymers with
medium to low MM (5–25 kDa) are more efficient and less
toxic (25). The toxic effect of PEI on cells can be partially
reduced by conjugation with other polymers such as PEG (80).
Upon systemic administration, these polyplexes of small particle size tend to aggregate to form larger complexes and
accumulate in major tissues including lung and liver. So far,
there have been no major clinical trials of cardiovascular
diseases conducted by using cationic polymers alone.
Physical approaches. To date, there are three major physical
approaches of gene delivery-gene gun, electroporation and US
by creating transient membrane holes using mechanical forces.
Additionally, magnetofection, which uses magnetic force to
concentrate particles containing nucleic acid into target cells,
has been recently developed as a feasible nonviral method to
combine current chemical and physical methods in gene delivery (35, 76, 81).
high-pressure helium stream to deliver DNA, coated onto gold
particles of varying sizes (1–3 ␮m), directly into the cytoplasm. The efficiency of the gene gun is variable, and the
duration of the expression is transient. A drawback of this tool
is the shallow penetration of DNA into target tissue. The
advantages of the gene gun, relative to some viral vectors, are
that it can be used to transfer genes to nondividing cells and the
transfection up to 18-fold and the combination of US and PEI
synergistically increased transfection up to 200-fold, resulting
in TF 34%. In 2010, our results showed that ultrasonic microbubble transfection causes phenotypic changes of primary
vascular endothelial cells by enhancing proliferation and upregulating kinase insert domain-containing receptor and finslike tyrosine kinase-1, while possesses no obvious adverse
effect on viable transfected cells (94).
Although these are promising in vitro findings, US-based
gene delivery is still in its infancy. Since 1996, there have been
several in vivo investigations concerning US-assisted gene
delivery. Most importantly, in 2002, Taniyama et al. (100), by
using intramuscular injection of rat hindlimbs, reported a
10-fold increase in luciferase activity after injection of plasmid
and Optison, followed by topical external US compared with
plasmid alone. Subsequently, using HGF plasmid in a rabbit
ischemic hindlimb model, they demonstrated that transfection
with plasmid and Optison followed by US resulted in increased
capillary formation in the treatment group after 5 wk. Therefore, ultrasonic microbubble-mediated HGF plasmid delivery
could be feasible for safe clinical gene therapy to treat PAD
without a viral vector system. Using intravenously infusion of
microbubbles loaded with plasmids encoding the human
VEGF165 gene into rat myocardial microcirculation, Korpanty
et al. (49) performed US exposure along with microbubbles
(49). The data showed that the capillary density increased to
18% at 5 days and 33% at 10 days before returning to control
levels at 30 days. Theoretically, it is feasible to apply the US
energy to deliver the growth factor gene into the ischemic
tissue in clinical setting. The molecular mechanism of USassisted gene delivery in therapeutic angiogenesis remains to
be determined. In 2011, Chen et al. (10), by using tail vein
injections in mice, investigated the efficiency of in vivo gene
transfer to the myocardium mediated by US-targeted liposome
microbubble destruction accompanied by PEI. The significantly enhanced transgene expression was noted related to
US-targeted microbubble destruction in the presence of PEI,
and the results showed the feasibility of US-based method for
primary myocardial disease.
At present, there are two main hurdles as to why gene
therapy has not globally succeeded in the clinical setting: first,
inefficient delivery of gene of interest to their correct sites of
action and, second, safety concern of some viral-based vectors
that are one to three orders of magnitude more efficient than
nonviral techniques in gene delivery in vivo. Many gene
delivery methods are much less efficient in vivo than in vitro
(such as liposome-mediated transfection). US is a versatile
modality and has several potential advantages over other techniques, especially that it can be focused and in turn targeted to
specific and, if necessary, to deep locations within the body
along with its high safety profile, low costs, and repeatable
applicability. Last but not least, in efforts to further improve
the level of transgene expression, targeted gene delivery may
be one of the promising methods that will work through US. In
this regard, it would be feasible to design a target-specific
microbubble that can selectively bind to the areas of interest in
the tissue/body. These active targeting strategies can be
achieved by the development of targeted microbubbles by
attaching antibodies or peptides to microbubble shells (45, 95).
Therefore, the targeted microbubbles with a specific ligand to
the target receptors that are expressed in the diseased area can
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
variations of the sound wave. As the ultrasonic intensity
increases, inertial cavitation (70) (Fig. 2) occurs when the
amplitude of bubble oscillations enhances to a point where the
inward moving wall of fluid has sufficient inertia that it cannot
reverse direction when the acoustic pressure reverses and
subsequently persists to compress the gas in the bubble. Finally, the bubble implodes violently, producing pressure discontinuities (shock waves), free radicals, microjets, extremely
high-localized temperatures (at least 5,000 K), pressures (up to
1,200 bars), and light (Fig. 2). Formation of shock waves,
bubble wall motion, and microjets may affect membrane permeability (62). Both stable and inertial cavitation may induce
membrane permeabilization (100).
Applications of US in gene delivery. US-mediated transient
pore formation in the cell membranes (sonoporation) can be
regarded to be the same as the promotion of membrane permeability induced by acoustic cavitation. Generally, the efficiency of sonoporation-mediated gene delivery depends on
DNA concentration, the frequency and intensity of US exposure, the duration of exposure, and the presence of contrast
agent. Most gene delivery studies use the therapeutic US at
driving frequency of 1–3 MHz with an intensity of 0.5–2.5
W/cm2 (44).
Microbubble echo contrast agents and their applications in
gene delivery. The concept of US contrast imaging was introduced in the 1960s, and gene delivery using microbubbles has
been intensively studied since 2000 (90).
The ideal microbubble contrast agents should be nontoxic,
injectable intravenously, capable of crossing the pulmonary
capillary bed after a peripheral injection, and stable enough to
achieve enhancement for the duration of the examination. They
are typically gas-encapsulated microbubbles around 1–10 ␮m
in diameter (1). Contrast agents have a gas core that is filled
with air or a higher MM substance such as perfluoropropane
with lower aqueous solubility. The surrounding shell can be
stiff (e.g., denatured albumin) or more flexible (lipid or phospholipids), and the shell thickness can vary from 10 to 200 nm.
A critical improvement in US-assisted gene delivery was the
combination of US irradiation with microbubbles. Most importantly, microbubbles have been shown to lower the energy
threshold for cavitation by US energy and to have the potential
of enhancing cavitation (24). When US interacts with the
microbubbles leading to cavitation, pDNA are driven across
cell membranes into the target cells (106).
US-assisted gene delivery in vitro and in vivo. Naked pDNA
is the simplest nonviral vector. However, disadvantages in
systemic gene delivery with naked DNA have also been found,
since pDNA vector can be rapidly degraded and neutralized by
endogenous DNases. Therefore, it is reasonable to combine
naked pDNA delivery with another method to improve the
transgene efficiency. In 1987, Fechheimer et al. (21) first
demonstrated that US had potential as a tool of pDNA delivery
into murine fibroblasts. The first major research in this field
came in 1996. Kim et al. (44) investigated the potential use of
US exposure as a novel transfection method for experimental
use. In 2000, Lawrie et al. (52), sonicating vascular smooth
muscle cells in the presence of Optison and naked luciferase
pGL3 plasmid (52), achieved transgene expression levels 300fold higher than with naked pDNA alone. In 2007, Deshpande
and Prausnitz (17) performed US-assisted in vitro reporter gene
transfection and found that US sonication alone increased
be applied either for the purpose of attaining US imaging or for
a potentially therapeutic purpose via US-induced cavitation.
Furthermore, since most of the major progress in US-based gene
delivery involves microbubble cavitation by which a synergistic
effect in enhancing US-assisted gene transfer occurs, further
understanding of cavitation physics and US-microbubble-cell interactions cannot be overemphasized.
Integrated Nonviral Gene Delivery into Cell Therapy:
Cell-Based Gene Delivery
Future Perspectives
Gene therapy by growth factor gene delivery in therapeutic
angiogenesis or nonangiogenic gene delivery for myocardial
salvage is emerging as a potential strategy for the treatment of
Fig. 3. Schematic presentation of the application of cell-based nonviral gene
delivery technology in regenerative medicine. Therapeutic angiogenesis is
achieved via paracrine effects and/or incorporation of transfected cells into
tissues by implanting the cells using different methods.
cardiovascular diseases. Viral-based gene delivery is efficient
but safety aspects have usually limited its clinical applications
(78). Various nonviral transfer approaches were developed to
facilitate the delivery of transgenes into target cells without
concern of insertion mutation, though dose optimization of
nonviral vectors needs to be performed because of potential
cytotoxicity. In the past few years, the work continued in
improving delivery modalities with increased local distribution
and reduced systemic circulation of chemical vectors. Naked
DNA remains the preferred method of DNA transfer to cardiovascular ischemic tissues and has been explored extensively
in clinical trials because of its simplicity and safety. However,
insufficient gene delivery is still the major hurdle of plasmidmediated gene therapy. Improving plasmid-mediated gene
transfer either by physical methods or chemical carriers to
enhance cellular uptake and reduce susceptibility to circulating
nucleases has shown successful applications for in vivo gene
delivery. Further improvements to increase the efficiency and
reduce the cytoxicity of nonviral vectors are required before
their clinical implication. The potential interest will rely on
developing new vector system that are more target-specific and
a better understanding of the limiting steps that nonviral vector
must overcome. Last but not least, although only short- and
mid-term follow-up data are available, the methods and tech-
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
Gene transfer can be performed either by isolating stem cells
from the patient, delivering the gene(s) into the cells in vitro
and then transplanting the genetically modified stem cells back
into the patient, or by directly introducing the delivery vector
into the target sites in vivo. The in vivo approach is limited by
nonspecific cellular targeting and inefficient gene delivery.
This may explain that several clinical trials failed to show the
benefit of in vivo growth factor gene therapy. With the recent
rapid progress in molecular biology together with the steady
advance of genome projects, gene and cell therapy has become
an exciting new hope for the treatment of cardiovascular
diseases. Cell-based gene delivery is a novel ex vivo strategy
that uses autologous cells (such as endogenous stem cells and
progenitor cells) as natural carrier vehicles of gene to the target
site after in vitro transfection with a functional gene of interest
(11) (Fig. 3). The use of adult progenitor cells, for example,
endothelial progenitor cells (EPCs), as angiogenic substrate that is
capable of synthesizing multiple cytokines is an attractive strategy
for neovascularization of ischemic cardiovascular diseases (86,
99, 109). Genetic manipulation of EPCs to overexpress angiogenic genes may enhance their biological functions, such as
migration, proliferation, and secretion of angiogenic growth factors (5, 46). The advantage of the ex vivo approach is to provide
specific genetically modified cells that may directly participate in
tissue regeneration and, most importantly, allow for both paracrine (mainly) and autocrine effects from the expressed cytokine
(72, 88). In cell-based gene delivery, the EPCs serve not only as
a vehicle for gene delivery to target sites but also as a tissueengineering tool in restoring normal tissues (74). By means of
delivering autologous cells, cell-based gene delivery is capable of
circumventing the unwanted inflammatory response, eliminating
the possibility of insertional mutagenesis of other genes caused by
viral vectors, and possibly achieving prolonged expression by
transfection concomitantly using various nonviral methods including ultrasonic microbubble transfection, electroporation, and
in vitro chemical vector transfection. However, in the long term,
this approach needs to develop further steps to avoid contamination, to isolate, manipulate, and expand cells, as well as to develop
less invasive strategies to enhance endogenous stem cell function
and survival ex vivo. In the recent years, insights have been
focused on gene transfer by means of cellular transplantation to
induce neovascularization in ischemic tissue (38, 46). Genetically
manipulated stem cells, in which a VEGF gene can be transferred
by US or other physical methods ex vivo, may yield a promising
future in therapeutic angiogenesis.
nology that merge nonviral gene and cell therapy such as
delivery of proangiogenic plasmids with EPCs may give rise to
several attractive opportunities to achieve more efficient and
nontoxic strategies for gene therapy in regenerative medicine.
This study was supported by National Science Council (Taiwan) Grant
99-2314-B-195-010-MY2 and Medical Research Department of the Mackay
Memorial Hospital (Taiwan) Grant MMH-E 100003.
No conflicts of interest, financial or otherwise, are declared by the author(s).
1. Allen JS, May DJ, Ferrara KW. Dynamics of therapeutic ultrasound
contrast agents. Ultrasound Med Biol 28: 805–816, 2002.
2. Anbanandam A, Albarado DC, Tirziu DC, Simons M, Veeraraghavan S. Molecular basis for proline- and arginine-rich peptide inhibition
of proteasome. J Mol Biol 384: 219 –227, 2008.
3. Aoki M, Morishita R, Taniyama Y, Kida I, Moriguchi A, Matsumoto
K, Nakamura T, Kaneda Y, Higaki J, Ogihara T. Angiogenesis
induced by hepatocyte growth factor in non-infarcted myocardium and
infarcted myocardium: up-regulation of essential transcription factor for
angiogenesis, ets. Gene Ther 7: 417–427, 2000.
4. Apfel RE. Acoustic cavitation: a possible consequence of biomedical
uses of ultrasound. Br J Cancer Suppl 45: 140 –146, 1982.
5. Asahara T, Kalka C, Isner JM. Stem cell therapy and gene transfer for
regeneration. Gene Ther 7: 451–457, 2000.
6. Berndt C, Lillig CH, Holmgren A. Thiol-based mechanisms of the
thioredoxin and glutaredoxin systems: implications for diseases in the
cardiovascular system. Am J Physiol Heart Circ Physiol 292: H1227–
H1236, 2007.
7. Bettan M, Emmanuel F, Darteil R, Caillaud JM, Soubrier F, Delaere
P, Branelec D, Mahfoudi A, Duverger N, Scherman D. High-level
protein secretion into blood circulation after electric pulse-mediated gene
transfer into skeletal muscle. Mol Ther 2: 204 –210, 2000.
8. Blume G, Cevc G. Liposomes for the sustained drug release in vivo.
Biochim Biophys Acta 1029: 91–97, 1990.
9. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D,
Demeneix B, Behr JP. A versatile vector for gene and oligonucleotide
transfer into cells in culture and in vivo: polyethylenimine. Proc Natl
Acad Sci USA 92: 7297–7301, 1995.
10. Chen ZY, Liang K, Qiu RX, Luo LP. Ultrasound- and liposome
microbubble-mediated targeted gene transfer to cardiomyocytes in vivo
accompanied by polyethylenimine. J Ultrasound Med 30: 1247–1258,
11. Cho HC, Marban E. Biological therapies for cardiac arrhythmias: can
genes and cells replace drugs and devices? Circ Res 106: 674 –685, 2010.
12. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems,
and apoptosis. Free Radic Biol Med 48: 749 –762, 2010.
13. Condon C, Watkins SC, Celluzzi CM, Thompson K, Falo LD Jr.
DNA-based immunization by in vivo transfection of dendritic cells. Nat
Med 2: 1122–1128, 1996.
14. Coster HG. A quantitative analysis of the voltage-current relationships
of fixed charge membranes and the associated property of “punchthrough”. Biophys J 5: 669 –686, 1965.
15. Dass CR. Lipoplex-mediated delivery of nucleic acids: factors affecting
in vivo transfection. J Mol Med (Berl) 82: 579 –591, 2004.
16. Dauty E, Remy JS, Blessing T, Behr JP. Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles
and transfect cells in culture. J Am Chem Soc 123: 9227–9234, 2001.
17. Deshpande MC, Prausnitz MR. Synergistic effect of ultrasound and
PEI on DNA transfection in vitro. J Control Release 118: 126 –135,
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
C.-H.S. and H.-I.Y. conception and design of research; C.-H.S. performed
experiments; C.-H.S., Y.-J.W., H.-H.W., and H.-I.Y. interpreted results of
experiments; C.-H.S. drafted manuscript; C.-H.S. and H.-I.Y. edited and
revised manuscript; H.-I.Y. approved final version of manuscript.
18. Durieux AC, Bonnefoy R, Busso T, Freyssenet D. In vivo gene
electrotransfer into skeletal muscle: effects of plasmid DNA on the
occurrence and extent of muscle damage. J Gene Med 6: 809 –816, 2004.
19. Faries PL, Pomposelli FB Jr, Quist WC, LoGerfo FW. Assessing the
role of gene therapy in the treatment of vascular disease. Ann Vasc Surg
14: 181–188, 2000.
20. Fattori E, La Monica N, Ciliberto G, Toniatti C. Electro-genetransfer: a new approach for muscle gene delivery. Somat Cell Mol Genet
27: 75–83, 2002.
21. Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer
SG. Transfection of mammalian cells with plasmid DNA by scrape
loading and sonication loading. Proc Natl Acad Sci USA 84: 8463–8467,
22. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R,
Ramsey P, Martin M, Felgner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol
Chem 269: 2550 –2561, 1994.
23. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M,
Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci
USA 84: 7413–7417, 1987.
24. Feril LB Jr, Kondo T, Zhao QL, Ogawa R, Tachibana K, Kudo N,
Fujimoto S, Nakamura S. Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents. Ultrasound Med Biol 29:
331–337, 2003.
25. Fischer D, Bieber T, Li Y, Elsasser HP, Kissel T. A novel non-viral
vector for DNA delivery based on low molecular weight, branched
polyethylenimine: effect of molecular weight on transfection efficiency
and cytotoxicity. Pharm Res 16: 1273–1279, 1999.
26. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston.
Clinical applications of research on angiogenesis. N Engl J Med 333:
1757–1763, 1995.
27. Fuller DH, Murphey-Corb M, Clements J, Barnett S, Haynes JR.
Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination. J Med
Primatol 25: 236 –241, 1996.
28. Gao X, Huang L. Potentiation of cationic liposome-mediated gene
delivery by polycations. Biochemistry (Mosc) 35: 1027–1036, 1996.
29. Godbey WT, Wu KK, Mikos AG. Size matters: molecular weight
affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J
Biomed Mater Res 45: 268 –275, 1999.
30. Goudy KS, Wang B, Tisch R. Gene gun-mediated DNA vaccination
enhances antigen-specific immunotherapy at a late preclinical stage of
type 1 diabetes in nonobese diabetic mice. Clin Immunol 129: 49 –57,
31. Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M, Nishioka WK, Wheeler CJ, Manthorp M, Sawdey M. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle:
plasmid dependence of muscle damage and effect of poloxamer 188. Mol
Ther 4: 407–415, 2001.
32. Harvie P, Wong FM, Bally MB. Use of poly(ethylene glycol)-lipid
conjugates to regulate the surface attributes and transfection activity of
lipid-DNA particles. J Pharm Sci 89: 652–663, 2000.
33. Hedman M, Muona K, Hedman A, Kivela A, Syvanne M, Eranen J,
Rantala A, Stjernvall J, Nieminen MS, Hartikainen J, Yla-Herttuala
S. Eight-year safety follow-up of coronary artery disease patients after
local intracoronary VEGF gene transfer. Gene Ther 16: 629 –634, 2009.
34. Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis
versus angiogenesis: similarities and differences. J Cell Mol Med 10:
45–55, 2006.
35. Holzbach T, Vlaskou D, Neshkova I, Konerding MA, Wortler K,
Mykhaylyk O, Gansbacher B, Machens HG, Plank C, Giunta RE.
Non-viral VEGF(165) gene therapy—magnetofection of acoustically
active magnetic lipospheres (’magnetobubbles’) increases tissue survival
in an oversized skin flap model. J Cell Mol Med 14: 587–599, 2010.
36. Hu S, Huang M, Nguyen PK, Gong Y, Li Z, Jia F, Lan F, Liu J, Nag
D, Robbins RC, Wu JC. Novel microRNA prosurvival cocktail for
improving engraftment and function of cardiac progenitor cell transplantation. Circulation 124: S27–S34, 2011.
37. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T,
Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of
angiogenesis after arterial gene transfer of phVEGF165 in patient with
ischaemic limb. Lancet 348: 370 –374, 1996.
59. Liu G, Molas M, Grossmann GA, Pasumarthy M, Perales JC,
Cooper MJ, Hanson RW. Biological properties of poly-L-lysine-DNA
complexes generated by cooperative binding of the polycation. J Biol
Chem 276: 34379 –34387, 2001.
60. Lleres D, Dauty E, Behr JP, Mely Y, Duportail G. DNA condensation
by an oxidizable cationic detergent. Interactions with lipid vesicles.
Chem Phys Lipids 111: 59 –71, 2001.
61. Lohse D. Sonoluminescence: cavitation hots up. Nature 434: 33–34,
62. Lokhandwalla M, McAteer JA, Williams JC Jr, Sturtevant B. Mechanical haemolysis in shock wave lithotripsy (SWL): II. In vitro cell
lysis due to shear. Phys Med Biol 46: 1245–1264, 2001.
63. Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD,
Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2
placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in
patients with chronic myocardial ischemia. Circulation 105: 2012–2018,
64. Lunn DP, Soboll G, Schram BR, Quass J, McGregor MW, Drape RJ,
Macklin MD, McCabe DE, Swain WF, Olsen CW. Antibody responses
to DNA vaccination of horses using the influenza virus hemagglutinin
gene. Vaccine 17: 2245–2258, 1999.
65. Makinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava
E, Yla-Herttuala S. Increased vascularity detected by digital subtraction
angiography after VEGF gene transfer to human lower limb artery: a
randomized, placebo-controlled, double-blinded phase II study. Mol Ther
6: 127–133, 2002.
66. Malik G, Nagy N, Ho YS, Maulik N, Das DK. Role of glutaredoxin-1
in cardioprotection: an insight with Glrx1 transgenic and knockout
animals. J Mol Cell Cardiol 44: 261–269, 2008.
67. Manning GS. The molecular theory of polyelectrolyte solutions with
applications to the electrostatic properties of polynucleotides. Q Rev
Biophys 11: 179 –246, 1978.
68. Matsushima S, Ide T, Yamato M, Matsusaka H, Hattori F, Ikeuchi
M, Kubota T, Sunagawa K, Hasegawa Y, Kurihara T, Oikawa S,
Kinugawa S, Tsutsui H. Overexpression of mitochondrial peroxiredoxin-3 prevents left ventricular remodeling and failure after myocardial
infarction in mice. Circulation 113: 1779 –1786, 2006.
69. McMahon JM, Signori E, Wells KE, Fazio VM, Wells DJ. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment
with hyaluronidase—increased expression with reduced muscle damage.
Gene Ther 8: 1264 –1270, 2001.
70. Miller MW, Miller DL, Brayman AA. A review of in vitro bioeffects
of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 22: 1131–1154, 1996.
71. Muona K, Makinen K, Hedman M, Manninen H, Yla-Herttuala.
10-year safety follow-up in patients with local VEGF gene transfer to
ischemic lower limb. Gene Ther 19: 392–395, 2012.
72. Murayama T, Tepper OM, Silver M, Ma H, Losordo DW, Isner JM,
Asahara T, Kalka C. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced
neovascularization in vivo. Exp Hematol 30: 967–972, 2002.
73. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ. Recombinant
gene expression in vivo within endothelial cells of the arterial wall.
Science 244: 1342–1344, 1989.
74. Nagaya N, Kangawa K, Kanda M, Uematsu M, Horio T, Fukuyama
N, Hino J, Harada-Shiba M, Okumura H, Tabata Y, Mochizuki N,
Chiba Y, Nishioka K, Miyatake K, Asahara T, Hara H, Mori H.
Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 108: 889 –
895, 2003.
75. Nagy N, Malik G, Tosaki A, Ho YS, Maulik N, Das DK. Overexpression of glutaredoxin-2 reduces myocardial cell death by preventing both
apoptosis and necrosis. J Mol Cell Cardiol 44: 252–260, 2008.
76. Namgung R, Singha K, Yu MK, Jon S, Kim YS, Ahn Y, Park IK,
Kim WJ. Hybrid superparamagnetic iron oxide nanoparticle-branched
polyethylenimine magnetoplexes for gene transfection of vascular endothelial cells. Biomaterials 31: 4204 –4213, 2010.
77. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene
transfer into mouse lyoma cells by electroporation in high electric fields.
EMBO J 1: 841–845, 1982.
78. Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R,
Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer
into normal rabbit arteries results in prolonged vascular cell activation,
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017
38. Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H,
Hayashi S, Silver M, Li T, Isner JM, Asahara T. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular
regeneration. Circulation 105: 732–738, 2002.
39. Jeong JH, Song SH, Lim DW, Lee H, Park TG. DNA transfection
using linear poly(ethylenimine) prepared by controlled acid hydrolysis of
poly(2-ethyl-2-oxazoline). J Control Release 73: 391–399, 2001.
40. Kastrup J, Jorgensen E, Ruck A, Tagil K, Glogar D, Ruzyllo W,
Botker HE, Dudek D, Drvota V, Hesse B, Thuesen L, Blomberg P,
Gyongyosi M, Sylven C. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe
angina pectoris A randomized double-blind placebo-controlled study: the
Euroinject One trial. J Am Coll Cardiol 45: 982–988, 2005.
41. Khan TA, Sellke FW, Laham RJ. Therapeutic angiogenesis for coronary artery disease. Curr Treat Options Cardiovasc Med 4: 65–74, 2002.
42. Kichler A, Zauner W, Ogris M, Wagner E. Influence of the DNA
complexation medium on the transfection efficiency of lipospermine/
DNA particles. Gene Ther 5: 855–860, 1998.
43. Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in
human diseases. Biochim Biophys Acta 1802: 396 –405, 2010.
44. Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME.
Ultrasound-mediated transfection of mammalian cells. Hum Gene Ther
7: 1339 –1346, 1996.
45. Klibanov AL. Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem 16: 9 –17,
46. Kobayashi K, Kondo T, Inoue N, Aoki M, Mizuno M, Komori K,
Yoshida J, Murohara T. Combination of in vivo angiopoietin-1 gene
transfer and autologous bone marrow cell implantation for functional
therapeutic angiogenesis. Arterioscler Thromb Vasc Biol 26: 1465–1472,
47. Koltover I, Salditt T, Radler JO, Safinya CR. An inverted hexagonal
phase of cationic liposome-DNA complexes related to DNA release and
delivery. Science 281: 78 –81, 1998.
48. Konstan MW, Davis PB, Wagener JS, Hilliard KA, Stern RC,
Milgram LJ, Kowalczyk TH, Hyatt SL, Fink TL, Gedeon CR, Oette
SM, Payne JM, Muhammad O, Ziady AG, Moen RC, Cooper MJ.
Compacted DNA nanoparticles administered to the nasal mucosa of
cystic fibrosis subjects are safe and demonstrate partial to complete cystic
fibrosis transmembrane regulator reconstitution. Hum Gene Ther 15:
1255–1269, 2004.
49. Korpanty G, Chen S, Shohet RV, Ding J, Yang B, Frenkel PA,
Grayburn PA. Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles. Gene Ther 12:
1305–1312, 2005.
50. Kumarswamy R, Volkmann I, Thum T. Regulation and function of
miRNA-21 in health and disease. RNA Biol 8: 706 –713, 2011.
51. Laham RJ, Simons M, Sellke F. Gene transfer for angiogenesis in
coronary artery disease. Annu Rev Med 52: 485–502, 2001.
52. Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC,
Newman CM. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 7: 2023–2027, 2000.
53. Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia
AN, Hermiller JB, Hillegass WB, Rocha-Singh K, Moon TE, Whitehouse MJ, Annex BH. Therapeutic angiogenesis with recombinant
fibroblast growth factor-2 for intermittent claudication (the TRAFFIC
study): a randomised trial. Lancet 359: 2053–2058, 2002.
54. Lehrman S. Virus treatment questioned after gene therapy death. Nature
401: 517–518, 1999.
55. Li J, Post M, Volk R, Gao Y, Li M, Metais C, Sato K, Tsai J, Aird
W, Rosenberg RD, Hampton TG, Sellke F, Carmeliet P, Simons M.
PR39, a peptide regulator of angiogenesis. Nat Med 6: 49 –55, 2000.
56. Lin CY, Chang YH, Kao CY, Lu CH, Sung LY, Yen TC, Lin KJ, Hu
YC. Augmented healing of critical-size calvarial defects by baculovirusengineered MSCs that persistently express growth factors. Biomaterials
33: 3682–3692, 2012.
57. Lin CY, Lin KJ, Kao CY, Chen MC, Lo WH, Yen TC, Chang YH,
Hu YC. The role of adipose-derived stem cells engineered with the
persistently expressing hybrid baculovirus in the healing of massive bone
defects. Biomaterials 32: 6505–6514, 2011.
58. Liu F, Qi H, Huang L, Liu D. Factors controlling the efficiency of
cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther 4: 517–523, 1997.
inflammation, and neointimal hyperplasia. J Clin Invest 96: 2955–2965,
Nikol S, Baumgartner I, Van Belle E, Diehm C, Visona A, Capogrossi MC, Ferreira-Maldent N, Gallino A, Wyatt MG, Wijesinghe
LD, Fusari M, Stephan D, Emmerich J, Pompilio G, Vermassen F,
Pham E, Grek V, Coleman M, Meyer F. Therapeutic angiogenesis with
intramuscular NV1FGF improves amputation-free survival in patients
with critical limb ischemia. Mol Ther 16: 972–978, 2008.
Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E. PEGylated
DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene
delivery. Gene Ther 6: 595–605, 1999.
Plank C, Zelphati O, Mykhaylyk O. Magnetically enhanced nucleic
acid delivery. Ten years of magnetofection-progress and prospects. Adv
Drug Deliv Rev 63: 1300 –1331, 2011.
Rajagopalan S, Olin J, Deitcher S, Pieczek A, Laird J, Grossman
PM, Goldman CK, McEllin K, Kelly R, Chronos N. Use of a
constitutively active hypoxia-inducible factor-1alpha transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase I
dose-escalation experience. Circulation 115: 1234 –1243, 2007.
Roy S, Khanna S, Hussain SR, Biswas S, Azad A, Rink C, Gnyawali
S, Shilo S, Nuovo GJ, Sen CK. MicroRNA expression in response to
murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc Res 82:
21–29, 2009.
Sakai M, Nishikawa M, Thanaketpaisarn O, Yamashita F, Hashida
M. Hepatocyte-targeted gene transfer by combination of vascularly
delivered plasmid DNA and in vivo electroporation. Gene Ther 12:
607–616, 2005.
Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O,
Taga T, Yamashita F, Takakura Y, Hashida M. Interaction between
DNA-cationic liposome complexes and erythrocytes is an important
factor in systemic gene transfer via the intravenous route in mice: the role
of the neutral helper lipid. Gene Ther 8: 677–686, 2001.
Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe
C, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Dimmeler S,
Zeiher AM. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the
TOPCARE-AMI Trial. J Am Coll Cardiol 44: 1690 –1699, 2004.
Scherman D, Bigey P, Bureau MF. Applications of plasmid electrotransfer. Technol Cancer Res Treat 1: 351–354, 2002.
Shi Q, Wu MH, Fujita Y, Ishida A, Wijelath ES, Hammond WP,
Wechezak AR, Yu C, Storb RF, Sauvage LR. Genetic tracing of
arterial graft flow surface endothelialization in allogeneic marrow transplanted dogs. Cardiovasc Surg 7: 98 –105, 1999.
Shigematsu H, Yasuda K, Sasajima T, Takano T, Miyata T, Ohta T,
Tanemoto K, Obitsu Y, Iwai T, Ozaki S, Ogihara T, Morishita R.
Transfection of human HGF plasmid DNA improves limb salvage in
Buerger’s disease patients with critical limb ischemia. Int Angiol 30:
140 –149, 2011.
Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH,
Grayburn PA. Echocardiographic destruction of albumin microbubbles
directs gene delivery to the myocardium. Circulation 101: 2554 –2556,
Shyu KG, Chang H, Wang BW, Kuan P. Intramuscular vascular
endothelial growth factor gene therapy in patients with chronic critical
leg ischemia. Am J Med 114: 85–92, 2003.
Stankovics J, Crane AM, Andrews E, Wu CH, Wu GY, Ledley FD.
Overexpression of human methylmalonyl CoA mutase in mice after in
vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes.
Hum Gene Ther 5: 1095–1104, 1994.
Stewart DJ, Kutryk MJ, Fitchett D, Freeman M, Camack N, Su Y,
Della Siega A, Bilodeau L, Burton JR, Proulx G, Radhakrishnan S.
VEGF gene therapy fails to improve perfusion of ischemic myocardium
in patients with advanced coronary disease: results of the NORTHERN
trial. Mol Ther 17: 1109 –1115, 2009.
Su CH, Chang CY, Wang HH, Wu YJ, Bettinger T, Tsai CH, Yeh
HI. Ultrasonic microbubble-mediated gene delivery causes phenotypic
changes of human aortic endothelial cells. Ultrasound Med Biol 36:
449 –458, 2010.
Su CH, Yeh HI, Hou JY, Tsai CH. Nonviral technologies for gene
therapy in cardiovascular research. Int J Gerontol 2: 35–47, 2008.
Takeshita S, Isner JM. Peripheral angiogenesis: therapeutic angiogenesis for peripheral vascular occlusive disease. Curr Interv Cardiol Rep 1:
147–156, 1999.
Tan PH, Chan CL, George AJ. Strategies to improve non-viral vectors—potential applications in clinical transplantation. Expert Opin Biol
Ther 6: 619 –630, 2006.
Tan PH, Manunta M, Ardjomand N, Xue SA, Larkin DF, Haskard
DO, Taylor KM, George AJ. Antibody targeted gene transfer to
endothelium. J Gene Med 5: 311–323, 2003.
Tanaka K, Sata M. Therapeutic application of bone marrow-derived
progenitor cells for vascular diseases: magic bullets having the good
without the bad? Int J Gerontol 1: 10 –21, 2007.
Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S,
Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R.
Development of safe and efficient novel nonviral gene transfer using
ultrasound: enhancement of transfection efficiency of naked plasmid
DNA in skeletal muscle. Gene Ther 9: 372–380, 2002.
Thum T, Catalucci D, Bauersachs J. MicroRNAs: novel regulators in
cardiac development and disease. Cardiovasc Res 79: 562–570, 2008.
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 MA, Licht JD, Pena JT, Rouhanifard SH, Muckenthaler MU, Tuschl T, Martin GR, Bauersachs J,
Engelhardt S. MicroRNA-21 contributes to myocardial disease by
stimulating MAP kinase signalling in fibroblasts. Nature 456: 980 –984,
Tirziu D, Simons M. Angiogenesis in the human heart: gene and cell
therapy. Angiogenesis 8: 241–251, 2005.
Titomirov AV, Sukharev S, Kistanova E. In vivo electroporation and
stable transformation of skin cells of newborn mice by plasmid DNA.
Biochim Biophys Acta 1088: 131–134, 1991.
Vale PR, Losordo DW, Milliken CE, McDonald MC, Gravelin LM,
Curry CM, Esakof DD, Maysky M, Symes JF, Isner JM. Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular
electromechanical mapping in patients with chronic myocardial ischemia. Circulation 103: 2138 –2143, 2001.
Vannan M, McCreery T, Li P, Han Z, Unger E, Kuersten B, Nabel
E, Rajagopalan S. Ultrasound-mediated transfection of canine myocardium by intravenous administration of cationic microbubble-linked plasmid DNA. J Am Soc Echocardiogr 15: 214 –218, 2002.
Weiner DB, Kennedy RC. Genetic vaccines. Sci Am 281: 50 –57, 1999.
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A,
Felgner PL. Direct gene transfer into mouse muscle in vivo. Science
247: 1465–1468, 1990.
Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P,
Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous
bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364: 141–148, 2004.
Xu B, Wiehle S, Roth JA, Cristiano RJ. The contribution of poly-Llysine, epidermal growth factor and streptavidin to EGF/PLL/DNA
polyplex formation. Gene Ther 5: 1235–1243, 1998.
Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D. In vivo
and in vitro gene transfer to mammalian somatic cells by particle
bombardment. Proc Natl Acad Sci USA 87: 9568 –9572, 1990.
Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ.
Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol
Chem 270: 18997–19007, 1995.
Ziady AG, Gedeon CR, Miller T, Quan W, Payne JM, Hyatt SL,
Fink TL, Muhammad O, Oette S, Kowalczyk T, Pasumarthy MK,
Moen RC, Cooper MJ, Davis PB. Transfection of airway epithelium by
stable PEGylated poly-L-lysine DNA nanoparticles in vivo. Mol Ther 8:
936 –947, 2003.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 •
Downloaded from by on June 15, 2017