Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents

Abstract

A synergistic impact of research in the fields of post-angioplasty restenosis, drug-eluting stents and vascular gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the field of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based gene delivery and discusses the main optimization strategies required to move the field of gene-eluting stents to clinical translation.

Full text

Research Article Open Access
Fishbein et al., Angiol 2013, 1:2
http://dx.doi.org/10.4172/2329-9495.1000109
Review Article Open Access
Angiology: Open Access
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
Keywords: Endovascular stents; Restenosis; Vascular gene delivery
Abbreviations: Ad: Adenovirus; AAV: Adeno-Associated
Virus; BMS: Bare Metal Stents; DES: Drug-Eluting Stents; ECM:
Extracellular Matrix; E-NTPDase: Ectonucleoside Triphosphate
Diphosphohydrolase; GFP: Green Fluorescent Protein; FGF-2:
Fibroblasts Growth Factor-2; HC: Hydrolyzable Cross-Linker;
IGF: Insulin-Like Growth Factor; IL-1β: Interleukin-1β; IL-6:
Interleukin-6; IL-8: Interleukin-8; ISR: In-stent Restenosis; LMW: Low
Molecular Weight; LST: Late Stent rombosis; MCP-1: Monocyte
Chemoattractant Protein-1; MNP: Magnetic Nanoparticles; NFκB:
Nuclear factor κB; NO: Nitric Oxide; NOS: Nitric Oxide Synthase;
ODN: Oligodeoxynucleotides; PAB: Polyallylamine Bisphosphonate;
PABT: Polyallylamine Bisphosphonate with Latent iol Groups; PC:
Poly-Phosphorylcholine; PCI: Percutaneous Coronary Interventions;
PDGF: Platelet-Derived Growth Factor; PEI (PDT): Polyethyleneimine
Modied with Pyridyldithio Groups; PFU: Particle Forming Units;
PLGA: Poly [lactic-co-glycolic acid]); PVA: Poly Vinyl Alcohol; ROS:
Reactive Oxygen Species; SMC: Smooth Muscle Cells; TIMP-3: Tissue
Inhibitor of Metalloproteinases-3; TNF: Tumor Necrosis Factor;
VEGF: Vascular Endothelial Growth Factor
DES for the Prevention of In-Stent Restenosis
A percutaneous vascular intervention (PCI) is a standard
therapeutic approach for treating patients with symptomatic coronary
disease. PCI are carried out annually in more than one million patients
in USA alone [1]. An overwhelming majority of PCI cases involve stent
angioplasty performed with either bare metal stents (BMS) or drug-
eluting stents (DES). Compared to BMS, DES achieve better outcomes
related to early and mid-term arterial patency and are associated with a
reduced need for target vessel revascularization [2]. However, in some
studies DES have been linked to a higher rate of late stent thrombosis
(LST) [3]. Aborted healing response, lack of re-endothelialization and
unresolved inammation have been suggested as the major factors
contributing to this complication [3]. LST is a grave medical condition
carrying a high risk of sudden cardiac death. To minimize the risk
of thrombosis, patients implanted with DES are routinely prescribed
prolonged aggressive dual anti-platelet therapy that causes bleeding
complications in a signicant fraction of post-PCI patients [4]. e
incidence of ISR and LST varies widely among dierent groups of
patients and lesion characteristics. Most registries based on unselected
patient populations report 20-30% and 8-10% ISR, and 0.8-1% and 1.5-
2% LST in the rst year following stent deployment, for the patients
receiving BMS and DES, respectively [5,6].
Moreover, the anti-restenotic eect of DES is less pronounced in
patients with peripheral artery disease [7], as well as coronary disease
patients aicted by diabetes or renal failure [8]. Furthermore, the
presence of ostial lesions, pre-existing ISR, smaller arterial diameter
and longer lesion length are also associated with poor outcomes [8].
e binary rate of restenosis in patients with these and some other
anatomical and clinical circumstances is currently in double digits
even with the use of the 2nd and 3rd generation DES devices [9]. It is
also noteworthy that ISR developed within DES is especially resistant
to further interventional therapeutic approaches and, thus, is oen an
indication for bypass surgery.
Vascular Gene erapy and the Concept of Gene-
Eluting Stents (GES)
e use of specialized catheters to deliver and lodge gene vectors
in the vessel wall preceded stent-based technologies for vascular gene
therapy by 15 years [10]. ese initial studies provided important
insights in transfectability/transducibility of adventitia, media and
intima (neointima) in various experimental species and established
multiple potential molecular targets for restenosis prevention [11].
However, the extent of vascular tissue transduction even with
sophisticated catheter devices (Dispatch®, Inltrator®) remained
inadequate for clinical translation of vascular gene therapy mostly
due to the short intraluminal retention time of the delivered gene
therapeutics [10].
Immobilization of gene vectors on stents embraces the best of both
DES and catheter-based methods of vascular gene therapy, by providing
the means to overcome the limitations of DES. First, gene therapy can
*Corresponding author: Ilia Fishbein, Dept of Pediatrics, Division of Cardiology,
The Children’s Hospital of Philadelphia, USA, Tel: (215) 590-8740; Fax: (215) 590-
5454; E-mail: FISHBEIN@email.chop.edu
Received June 27, 2013; Accepted July 25, 2013; Published July 27, 2013
Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013)
Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents. Angiol 1:
109. doi: 10.4172/2329-9495.1000109
Copyright: © 2013 Fishbein I, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Abstract
A synergistic impact of research in the elds of post-angioplasty restenosis, drug-eluting stents and vascular
gene therapy over the past 15 years has shaped the concept of gene-eluting stents. Gene-eluting stents hold
promise of overcoming some biological and technical problems inherent to drug-eluting stent technology. As the
eld of gene-eluting stents matures it becomes evident that all three main design modules of a gene-eluting stent: a
therapeutic transgene, a vector and a delivery system are equally important for accomplishing sustained inhibition of
neointimal formation in arteries treated with gene delivery stents. This review summarizes prior work on stent-based
gene delivery and discusses the main optimization strategies required to move the eld of gene-eluting stents to
clinical translation.
Endovascular Gene Delivery from a Stent Platform: Gene- Eluting Stents
Ilia Fishbein1,2*, Michael Chorny1,2, Richard F Adamo1, Scott P Forbes1, Ricardo A Corrales1, Ivan S Alferiev1,2 and Robert J Levy1,2
1Dept of Pediatrics, Division of Cardiology, The Children’s Hospital of Philadelphia, USA
2The University of Pennsylvania, USA

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 2 of 9
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
attain a longer lasting therapeutic modication of vascular substrate;
second, gene interventions allow for selective inhibition of SMC
proliferation and migration while maintaining and even enhancing
endothelial re-growth; third, the underlying atherosclerotic process
can be addressed; and fourth, a modulation of the biological activity of
gene-eluting stents can be achieved with systemically administered low
molecular weight compounds.
Furthermore, compared with the scaold-less vascular gene
delivery as accomplished with the use of delivery catheters, stents
present an advantageous physical platform for local arterial gene
transfer. Indeed, higher arterial concentrations of gene vectors can be
achieved with the immobilization on stent surface when compared to
non-immobilized vectors following administration of a smaller vector
input dose. Superior vascular wall retention is a combined result of
physical vector association with a permanently implanted scaolding
device, and a shielding eect of the vector deposited on the adluminal
surface of the stent (i.e. at a stent/tissue interface) from the shearing
eect of blood ow. As a consequence of protracted vascular residence,
immobilization on the stent minimizes the distal spread of gene vector,
reducing inadvertent inoculation of non-target tissues. Moreover,
stent-based delivery of gene therapeutics is feasible in diseased
arterial segments with side branches. Human arteries susceptible to
atherosclerosis, with the exception of the common carotid artery, are
extensively branched and therefore scaold-less delivery of uid phase
vector formulations leads to immediate run-o of the vector to the
circulation through the side branches. Furthermore, since stent-based
gene transfer does not require isolation of the treated vascular segment,
procedure-related ischemic complications can be minimized. Finally,
stent-based gene delivery provides an optimal spatial conguration
to prevent cellular proliferation and migration in the setting of ISR,
since cell activation aer stent deployment is typically observed in the
vicinity of stent struts [12]. Physical association of gene vector with a
stent establishes a transgene expression gradient that overlaps with the
cell activation gradient, thus approximating vectors to the projected
site of the action.
us, the use of gene delivery stents for atherosclerotic vascular
disease oers the appealing possibility of site specic delivery of
gene therapy with long lasting and controllable transgene expression
that can prevent in-stent restenosis, and also provide therapy for the
underlying vascular disease.
Minimal requirements for GES function and performance
e design requirements for GES share several characteristics with
DES platforms. First, deliverability of both types of devices should not
be compromised by the accommodation of a therapeutic agent and its
matrix on the stent struts. Second, the matrix associated with DES and
GES struts has to withstand the mechanical stretch associated with a
change of the stent diameter during deployment. Cracking, peeling
and delamination of the matrix during stent implantation should be
prevented since these post-deployment defects lead to irregularity in
the release rate of a therapeutic agent and can cause distal embolization.
ird, the matrix has to be highly biocompatible, causing no more than
a minimal thrombogenic and inammatory response.
ere are several important distinctions, however, in the design
characteristics of DES and GES. While DES have been devised to be able
to provide sustained release and vessel wall delivery of low molecular
weight therapeutic agents for as long as 3 months, this may not be
necessary for the GES, since transfected/transduced cell populations in
the stented artery are able to produce and secrete a transgene product
for prolonged period of time. A minimal required duration of stent-
associated vector perseverance in its active transduction competent
form is not entirely clear. Since the medial and neointimal SMC
proliferation starts no earlier than 1 week aer stent deployment in
human and higher animal models [13], the target cell population is not
yet present at the time of vascular injury. is fact argues for at least
2-3 week-long release period of a transduction competent gene vector
from the stent, posing additional requirements to GES technology
related to extended vector stability in vivo. Unlike LMW drugs, which
are oen stabilized by matrix immobilization, gene vectors (both non-
viral and especially virus-based) are vulnerable and lose their activity if
not properly protected. Elicitation of immune response to a therapeutic
moiety is another specic problem of GES since LMW drugs used
in DES are rarely strong immunogenes unless bound to proteins.
erefore, preventing distal vector spread to regional lymph nodes and
spleen, as well as physical shielding of the vector from direct contact
with pre-formed antibodies and eector T-cells are of paramount
importance for sustaining therapeutic levels of transgene expression
following GES use. Finally, the 1-2 orders of magnitude size dierence
between LMW drugs and gene vectors dictates use of dierent matrices
to enable timely release of a therapeutic agent from a stent platform,
aecting the predominant mechanism of the agent release (diusion vs
matrix degradation).
Conceptual Technologies in GES Design
Research concerning gene delivery stents over the past 15 years
illustrates a number of principles and mechanisms involved in stent-
facilitated delivery of gene therapy vectors to the arterial wall. Several
dierent approaches have been exploited to achieve labile association
of gene vectors with stent struts (Table 1).
Bulk immobilization (polymer coatings)
Initial investigations concerning gene delivery stents utilized
polymer coatings on the surface of metallic stents in which gene vectors
were dispersed or pseudo-dissolved. ese coatings (typically 50-250
µm thick) were deposited on a primed stent surface either by a multiple
dip technique or by aerosol deposition of a polymer solution containing
a suspension of the vector. Aer solvent evaporation and polymer
precipitation on the stent surface, the gene vector stays contained
between the polymer bers. A subsequent release of vector particles is
then controlled by diusion though the polymer matrix (determined by
the relative hydrophilicity/hydrophobicity of the matrix and the pore
size), matrix degradation and/or dissolution of matrix in blood and
tissue uid. Depending on the polymer type, these three mechanisms
have varying contributions to the overall release kinetics of the vector
from the stent coating. A variety of synthetic polymers (PLGA [14],
poly(beta-aminoester) [15-18], polyurethane [19,20], PVA [21], poly
[phosphorylcholine-lauryl methacrylate] [22-28]), semi-synthetic
polymers (dopamine-modied hyaluronic acid [29,30], cationized
gelatin [31], styrene-modied gelatin [32], cationized pullulan [33] or
Immobilization method Advantages Disadvantages
Bulk coating High loading of gene
vectors, ease of scale-up
Inadequate release
kinetics, polymer-induced
inammation
Surface tethering Controllable release rate,
no inammation
Low loading of gene
vectors
In-situ stent loading using
magnetic targeting
No vector loss on route to
delivery site, possibility of
repeated loading
A limited choice of stent
materials, MNP safety
issues
Table 1: Comparison of gene vector immobilization methods on stent surfaces.

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 3 of 9
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
naturally occurring macromolecules (collagen [34], denatured collagen
[35], gelatin [17,18]) have been employed for the bulk incorporation of
genetic material on stent wires.
An obvious advantage of the bulk immobilization is the ability to
integrate substantial amounts of the vector in a thick coating. Plasmid
DNA quantities as high as 4 mg [20] or Ad doses up to 5×1010 particles
[34] have been reported. ere are several important disadvantages of
the bulk immobilization as well. Typically, 80-90% of the vector load
is released within the rst 24 hours [17,19,21,23,24,33,36], especially
in the case of non-viral systems. Part of this eect is due to the “burst
release” of a fraction of the vector payload, which is either deposited
in the outermost layer of the polymer coating or has been adsorbed
onto the polymer layer aer going out of solution during the solvent
evaporation step. A partial solution for preventing this undesired
rapid release of the vector is provided by a super-coating of a vector/
polymer composite with additional layers of vector-free polymer
[14,17,18]. Another potential method to modulate the release rate of
gene therapeutics from bulk polymer coatings uses controlled cross-
linking of a polymer as exemplied by Nakayama [32] who developed a
photoactivatable coating system consisting of styrene-modied gelatin,
a polymerization initiator, carboxylated camphoroquinone and a
reporter Ad-LacZ adenovirus vector. Upon irradiation of dipped stents
with a broad spectrum halogen lamp, gelatin chains become cross-
linked forming a hydrogel that entraps Ad.
Another important disadvantage of the bulk polymer coatings is
an inammatory reaction triggered by native and especially synthetic
polymers in the arterial segments placed in contact with the polymers
[37]. us, unless potent agents, such as the anti-cancer drugs commonly
used in drug-eluting stents are utilized, the inammatory response to
the polymer coating negates the therapeutic eect of a transgene. One
important exception to the uniformly pro-inammatory character of
polymer coatings is an almost complete lack of inammatory inltrates
and generally high biocompatibility of poly [phosphorylcholine-
lauryl methacrylate] co-polymer utilized in the BiodivYsio stent [23-
28] approved by FDA for clinical use in 2000. A hydrophilic poly-
phosphorylcholine (PC) moiety of this macromolecule mimics an
outer phospholipid layer of cell membranes, thereby reducing platelet
activation on the metal surface. Platelet passivation makes this stent
a device of choice in patients with absolute contraindications to dual
anti-platelet therapy aer stenting. A positive surface charge on the
BiodivYsio stent imparted by choline residues renders the PC coating
useful for electrostatic binding of negatively charged DNA plasmids,
as well as Ad and AAV vectors. e latest generation of BiodivYsio
stents feature extremely thin coatings (50 nm) precluding signicant
incorporation of the vector in the bulk of the polymer. erefore, this
system constitutes a paradigm that includes both elements of bulk
immobilization and the coatless surface immobilization systems as
described below.
Surface immobilization of gene vectors on coatless metal
substrate
Surface immobilization of gene vectors on the surface of
endovascular stents represents a particular case of substrate mediated
gene delivery, a concept put forward by the work of Shea [38,39]
primarily to increase biocompatibility of bioprosthetic scaolds.
e main premise of substrate-mediated gene transfer is to use the
tissue-facing surface of an implanted device to append (rather than
bulk-incorporate) therapeutic gene vectors in an arranged pattern
to facilitate transduction of tissue elements on the interface with the
bioprosthesis, thereby improving functional integration of the device.
Protein anity adaptors for vector immobilization: Studies by
our group further developed the idea of substrate mediated delivery for
use in conjunction with endovascular stents. Driven by the established
data on detrimental pro-inammatory and pro-restenotic eects of
stent coatings we designed and implemented a chemical strategy for
reversible attachment of both viral and non-viral gene vectors that
does not rely on surface coating. Our method of vector immobilization
on non-coated metal surface is based on surface modication with
poly(allylamine)-bisphoshonate (PAB) compounds bridging the metal
surface and gene vectors [40]. Bare metal gene delivery using PAB
involves rst forming a polymer monolayer on the metal surface of the
stent that is established due to the formation of strong coordination
bonds between pendant bisphosphonic groups of the polymer and
Fe, Ni and Cr atoms and their oxides on the steel surface. is ultra-
thin (less than 5 nm) permanently attached polymer lm is rapidly
formed upon exposure of metal samples to an aqueous PAB solution.
e molecular monolayer of PAB can be further covalently modied
in order to permit a covalent attachment of vector binding agents
using well established amine conjugation chemical strategies targeting
multiple primary amines of the allylamine backbone [40]. High anity
vector binding proteins such as anti-Ad knob [40], anti–DNA [41]
antibodies or a recombinant D1 domain of Coxsackie-Adenovirus
Receptor [40] can be used as binding adaptors enabling high-anity
binding of gene vectors to stents. Stent loading with gene vectors is
then achieved by simple immersion of a stent in a vector suspension.
We further demonstrated that agents with dierent binding anities
can be exploited to adjust a vector capacity of the gene-eluting stent
and to modulate the release rate of gene therapeutics both in vitro and
in vivo [40]. However, limited control over the vector release kinetics
remains a disadvantage of the protein anity binding immobilization
technology. Although we have demonstrated the vector presence at
the interface between stent struts and the arterial wall 24 hours post-
deployment and associated reporter (GFP) activity in all 3 arterial
layers 7 days post-stenting [40], the duration of the vector binding to
a stent in vivo may be far from optimal. It is challenging to prolong
vector association with the stent surface using this technique, since
the vector association with stents is determined by the antigen/
antibody or receptor/ligand anity, local pH, and the protein
content of blood and tissue uid; the factors that cannot be changed
deliberately. Furthermore, the protein anity binding technology
cannot be immediately adapted to any given vector, since it implies
availability of a vector-binding molecule with Kd in the range of 10-8-
10-9 M. Moreover, vector-binding strategies based on protein anity
interactions are dicult to adapt to manufacturing scale because of
protein stability and species specicity issues.
Viral vector tethering to bare metal stents via hydrolyzable
cross-linkers: Motivated by the limitations of anity immobilization
we developed an alternative methodology for the reversible binding
of recombinant replication-defective adenoviruses to metal surfaces
that completely avoids using protein adaptors. is method is based
on hydrolyzable cross-linker (HC) molecules that directly append
vectors to PAB-activated steel (Figure 1). e subsequent release
of the vectors is governed by the kinetics of cross-linker hydrolysis
(Figure 1) and can be modulated by the usage of HC with variable
hydrolysis rates. A new conjugation strategy necessitated a change of
PAB chemical design with the introduction of latent thiol groups in
the side chains of the polymer molecule. is novel compound, PABT,
was successfully synthesized and characterized by our group [42]. e

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 4 of 9
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
devised linking strategy by itself was sucient to achieve signicant
binding of thiol-reactive Ad particles to the surface of model steel
samples. However, we choose to expand the amount of available thiol
group on the metal surface using additional exposure of thiol-activated
metal samples to an aqueous solution of pyridyldithio (PDT)-engraed
polyethyleneimine, PEI (PDT) followed by reduction of PDT to thiols
with dithiothreitol. We demonstrated that the “amplication” protocol
resulted in a more eective Ad tethering when compared with the basic
“no-amplication” protocol [42].
Synthetic, biodegradable HC are particularly promising tools for
achieving a site-specic tunable release of gene therapy vectors since
they can be synthesized to have a broad range of hydrolysis rates and
thus the vector release rate from the surface of a stent can programmed
per the formulation parameters [43].
Magnetic stent targeting
Both bulk immobilization and surface tethering approaches make
use of stent-based gene delivery systems that are assembled prior
to stent implantation in the artery. Introduction of a stent into the
vasculature through the hemostatic valve of a vascular sheath and
routing it to a deployment site through the atherosclerotic and oen
calcied arterial conduit always involves some physical damage to the
stent coating and vector depot. As an alternative approach, stent loading
can be accomplished in situ aer the stent has already been implanted.
Since the deployed stent is freely accessible to blood ow, its surface
can be actively targeted with therapeutic agents delivered to systemic
or regional circulation, provided the targeting forces are strong enough
to capture and retain a therapeutic agent on the stent surface.
is concept of in situ stent loading with gene vectors was recently
implemented by our group using a magnetic targeting paradigm
[44]. Stents made of magnetic alloys, when placed in a uniform
magnetic eld, such as that created within an MRI coil or induced
by paired electromagnets generate strong highly localized magnetic
A B
Catheter
Magnet
Magnetic
nanoparticles
Figure 2: A targeted delivery of MNP co-formulated with Ad-Luc vectors to a deployed stent mediated by the uniform eld induced magnetization
effect. The uniform eld generated by paired electromagnets (A) both induces high gradients on the stent and magnetizes Ad-loaded MNP, thus
creating a magnetic force driving MNP to the stent struts and adjacent arterial tissue (B). (Adapted from [45] with permission).
A
5 nm
H2O
O
O
O
O
O
O
O
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P
S
S
P
P
P
NH
NH NH
NH
NS
S
S
S
S
S
N
N
NH
n
H
N
NH NH
HL
HL
HN
OH
Z2
Z1
Z1Z2
Z1
O
O
HO
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2
-HN
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OO
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SO2Na
CH CH2CH CH2CH CH2
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NH2NH NH
n-k-m k m
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Fe Ni Cr Fe Ni Cr Fe Ni Fe Ni Cr Fe Ni Cr Fe Ni
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H
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coordination
layer
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=
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Figure 1: A scheme illustrating specic chemical interactions to enable adenovirus binding to a stent surface. Ad vectors were modied by
reacting lysine residues of capsid proteins with a bifunctional amine/thiol-reactive Hydrolyzable Cross-Linker (HL) possessing a hydrolyzable
ester bond separating fragment Z1 and Z2. Stainless steel stents were consecutively exposed to a solution of Polyallylamine Bisphosphonate
Comprising Latent Thiol Groups (PABT) and a reducing agent, TCEP, to activate thiol groups on the surface. To expand the amount of available
thiol functions, a subsequent treatment with polyethyleneimine modied with pyridyldithio groups, PEI (PDT), and DTT was used. Finally,
HL-modied Ad vectors were reacted with thiolated stent surfaces to achieve covalent tethering of Ad. The subsequent release of covalently
immobilized Ad is driven by hydrolysis of the ester bond in the cross-linker’s backbone. (Adapted from [42] with permission).

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 5 of 9
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
forces due to steep eld gradients between stent struts [44-47]. ese
magnetic forces enable the targeted capture of systemically or locally
administered gene vectors formulated in MNP [44-47] (Figure 2).
e same external magnetic source can be used to magnetize both
stents and MNP. In our study [44], aer implanting stents made of
magnetically responsive 304-grade stainless steel in the common
carotid arteries of rats, suspension of MNP incorporating 2.5×109
Ad-Luc was slowly injected through the catheter into the aortic arch.
e delivered vector was then transported en masse with antegrade
blood ow to carotid circulation across the stented segment. During
and immediately aer MNP/Ad-Luc delivery a uniform magnetic eld
of 0.1 Tesla was established across the neck area of the rats. While
no direct quantication of the amount of Ad vector attached to stent
struts following magnetic targeting was pursued, the vector uptake was
signicant enough to cause a localized transduction of stented carotid
segment, greatly exceeding that achievable with free Ad, as determined
by bioluminescence imaging [44].
Magnetic reloading of depleted GES: Prolonged arterial expression
of a transgene with an established anti-restenotic activity following a
single intervention is the ultimate goal of stent-based vascular gene
therapy. Despite ongoing research in the eld for the past decade,
it is still too early to conclude whether this goal is realistic. Indeed,
even with protracted release kinetics of gene vectors from stents,
such as one achieved with hydrolyzable cross-linker immobilization,
the duration of the transgene expression and hence the durability of
the therapeutic eect is a concern. In most mammalian tissues, aer
reaching peak level, transgene expression tends to decrease over time
due to both promoter attenuation and mounting immune response
to the vector and the transgene product. Previous reports [48,49]
have documented that suppressed neointimal growth can recur if the
inhibiting modality is withdrawn early. e vulnerability period for
restenosis recurrence following premature treatment withdrawal in
human arteries is unknown, but appears to be longer for peripheral
compared to coronary vasculature. erefore, the potential to
rejuvenate therapeutic transgene expression by reloading stents with
additional payloads of the same or other gene vectors is of paramount
importance in designing translatable vascular gene therapy approaches
to prevent restenosis, especially in peripheral vasculature. Uniform
eld high-gradient magnetic targeting provides a potential way of
addressing reloading of depleted GES at delayed time points. In order
to reload a stent in situ, a gene vector needs to be physically partitioned
at or near the depot for sucient time to saturate binding sites in the
depot matrix with the re-delivered vectors. Magnetic eld mediated
targeting provides a unique physical opportunity to achieve this task.
Reporter Studies and Pharmacokinetics of Transgene
Expression
Reporter studies are instrumental for optimizing stent-based
gene delivery systems in regards to transgene expression strength
and durability. GFP [14,16,20,34-36,40,42,50,51], β-galactosidase
[20,21,23,26,27,32] and luciferase [20,36,40,42,43] have been
extensively employed to map temporal and spatial expression patterns
of stent-delivered transgenes. It is noteworthy that the information
provided by dierent reporter systems is not redundant. For example,
while luciferase is typically more sensitive than the other two reporter
systems, it does not readily provide a tissue level resolution. Use of
GFP and β-galactosidase, on the other hand, is compatible with direct
histological examination of transduced tissue, providing valuable
information about transduced cell populations and their distribution
in distinct compartments of the vessel wall. Native GFP uorescence is
labile and extremely sensitive to tissue xation methodology. Moreover,
GFP emits light in the same part of spectrum as elastin, making
unambiguous detection of GFP positive cells dicult, especially in the
injured tissue, where elastin is partially disintegrated. A recent advent
of high quality commercial anti-GFP antibodies makes this task easier
by streamlining immunouorescence and immunohistochemical
approaches for the identication of transduced cells.
A direct comparison of the extent and duration of reporter arterial
expression among the published studies is challenging because of
dissimilarities between animal models, reporter systems and reporter
detection methods employed by dierent groups. Nevertheless, several
common traits of arterial tissue transduction from a stent platform
are preserved throughout most of these studies regardless of the
specic experimental setup. First, gene transfer is highly site-specic,
leading to more eective transduction of cells underlying stent struts
compared to cells located between the stent wires [20,21,26,27,40,42].
Second, despite neointimal growth not occurring until 4-14 days aer
a stent deployment, a nascent neointima is more eectively transduced
than media and adventitia [20,26,36,40,42]. ird, duration of gene
expression may exceed one month following implantation of GES
[26,27]. Fourth, no massive distal spread of a transgene to liver, spleen
and lungs is apparent [14,20,34,40,42]. Fih, dierent vector systems
may have predilection for transducing specic cell populations in the
arterial wall [20,26].
Disease-related Targets and erapeutic Transgenes
In-stent restenosis is a multifactorial disease [52,53]. e stenting
related factors with proven pathogenic bearing for ISR include
endothelial denudation, direct trauma to medial SMC resulting in
altered SMC apoptosis, proliferation and ECM synthesis programs,
parietal thrombus formation, unresolved inammation and foreign
body reaction to the stent and its coating. Accordingly, the number
of molecular targets relevant to inhibition or stimulation of signaling
pathways altered during ISR development is extremely high [52,53].
Comprehensive reviews [11,54,55] focused on gene therapy of
restenosis and related vasculoproliferative conditions list more than
150 dierent genes implicated in regulation of cell proliferation,
migration, ECM turnover, thrombosis and inammation that were
targeted with gene therapy interventions to mitigate restenosis. Only a
small fraction of these therapeutic gene constructs was investigated in
conjunction with GES.
GES targeting re-endothelialization
Since the seminal study by Asahara et al. [56] describing inhibition
of restenosis in a rat model with locally administered vascular
endothelial growth factor (VEGF) was published in 1995, strategies
aimed at attenuating restenosis via enhanced re-endothelialization
have come into focus. In line with this concept, Walter et al. [28] have
used poly[phosphorylcholine-lauryl methacrylate]-coated stents to
electrostatically adsorb 200 µg plasmid DNA encoding human VEGF-
2. When deployed in Fogarty balloon-denuded external iliac arteries
of hypercholesterolemic rabbits, VEGF-2 plasmid-eluting stents
enhanced endothelial recovery in the stented arteries in comparison
with the animals receiving control stents and resulted in a 60%
reduction of cross-sectional arterial narrowing of the stented region 3
months aer the procedure [28].
GES targeting SMC proliferation, migration and ECM
remodeling
Serine/threonine kinase, Akt1, also known as protein kinase B

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 6 of 9
Volume 1 • Issue 2 • 1000109
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ISSN: 2329-9495 AOA, an open access journal
(PKB) is an important signaling hub integrating input from several pro-
proliferative pathways, in particular from PI3K [57]. Akt1 activation
during neointimal tissue maturation [58] as well as the anti-restenotic
eectiveness of Akt1 inhibition [59] are well established. Recently
Che et al. [29] applied coordination chemistry to achieve stable
attachment of dopamine-conjugated hyaluronic acid (HA) to stents via
bonds formed between dopamine residues and metal ions on the stent
surface. e HA-coated stents were further modied with polyplexes
made of siRNA to Akt1 and disulde cross-linked PEI. A sustained
release of siRNA from the stent surface was observed in vitro for at
least 72 hours. In in vivo study, an undisclosed amount of Akt1 siRNA
polyplexes was deposited on HA-coated stents. Akt1 siRNA stents
implanted in rabbit iliac arteries decreased the extent of ISR by 50% at
4 weeks aer deployment compared to BMS [29].
Platelet-derived growth factor (PDGF) existing in two
homodimeric forms (PDGF-AA, PDGF-BB) and as a heterdimer
(PDGF-AB) is a well established SMC mitogen [60] and pro-migratory
chemoattractant [61]. All three isoforms were shown to promote SMC
accumulation in vitro and in vivo, while demonstrating relatively low
activity toward endothelial cells [62]. erefore, LMW inhibitors of
PDGF receptor tyrosine kinase activity [63], as well as gene therapy
modalities of inhibiting the PDGF-triggered signaling cascades [64,65]
are appealing directions to selectively inhibit SMC growth without
aecting endothelial cell regeneration. To this end, Li et al. [66]
synthesized phosphorothioate antisense oligodeoxynucleotides (ODN)
to the 15 base-long conserved coding sequence of PDGF-A gene and
complexed them with PEI to make a polyplex formulation that was
absorbed on hydrogel coated stents. When implanted in the coronary
arteries of healthy young pigs, the anti-sense-eluting stents resulted in
a 50% reduction of neointimal thickening in comparison with non-
sense ODN-eluting and plain hydrogel stents [66]. Importantly, the
endothelial lining was complete 28 days aer stenting in the arterial
sections from the animals treated with the antisense PDGF-A –eluting
stents, while the endothelialization was patchy at the 28 days time point
in the arteries of animals receiving commercial sirolimus-eluting stents
[66]. Likewise, complete endothelialization of stented pig coronaries
and a 40% reduction of restenosis were observed with a six-ring
phosphorothioate morpholino backbone anti-sense construct eluted
from a PC-coated stent and targeting expression of a cell cycle check
point transcription factor c-myc [67].
In-stent neointima is primarily formed by the progeny of medial
SMC and adventitial myobroblasts that populate the intimal space
of an injured artery. Matrix remodeling triggered by vascular trauma
makes possible unhindered migration of these cell populations along
the gradients of specic chemoattractants [68]. us, stabilization
of ECM was proposed for limiting SMC migration toward (neo)
intima. In accordance with this idea, Johnson and colleagues [23]
bulk-immobilized a suspension of Ad vector (4×108 pfu) encoding
synthesis of the tissue inhibitor of metalloprotease-3 (TIMP3) on PC-
coated stainless steel stents. At 28 days aer stent implantation in pig
carotid arteries neointimal volume was reduced by 40% and 50% in the
animals treated with Ad-TIMP3-decorated stents in comparison with
pigs receiving BMS or Ad-LacZ immobilized stents, respectively [23].
GES targeting inammatory cell recruitment
Acute inammatory response in the form of platelet and
brin deposition and massive neutrophil recruitment to the site
of vascular trauma via P-selectin and CD11b/CD18 engagement is
triggered immediately upon stent deployment [52,69]. Days later, an
escalating production of IL-6, IL-8 and MCP-1 by the injured SMC
and ingressing neutrophils attracts monocytes that transmigrate into
the tissue becoming resident macrophages thus denoting a stage of
chronic inammation. Macrophages are the key cell type bridging
inammation to neointimal formation, since macrophage-produced
and secreted chemokines (TNF, IL-1β) and growth factors (PDGF,
FGF-2, IGF) stimulate luminal migration and proliferation of medial
SMC and adventitial myobroblasts that collectively form neointima
[52,69,70]. erefore, eectively curbing the inammatory response
represents a well-recognized anti-restenotic strategy [71,72].
In keeping with this therapeutic approach, Egashira and co-
workers [21] evaluated stents formulated with plasmid DNA encoding
dominant negative variant of MCP1. e stents deployed in the iliac
arteries of hypercholesterolemic rabbits and cynomolgus monkeys
resulted in approximately a 20% reduction of macrophage inltration
at the stent implantation site and a 20-30% restenosis attenuation in
comparison with BMS [21].
NFkB is a master transcription factor involved in the regulation
of multiple inammatory mediators. Ohtani et al. [19] used NFkB
decoy ODN (500 µg/stent) incorporated in a polyurethane coating.
Stents deployed in femoral arteries of hypercholesterolemic rabbits
demonstrated approximately a 30% decrease of neointimal thickening
in comparison with BMS and polyurethane coated stents at 4 weeks
post-stenting and a signicant decrease of inammatory marker
expression at 3 and 10 days post-implantation [19].
GES targeting thrombus formation in stented arteries
Platelets are the rst cellular elements to contact the surface of
newly deployed stents. Since both human patients and experimental
animals undergoing stent placement are pretreated with anti-platelet
and anti-coagulant drugs, acute blockage of a treated artery is seldom
an issue. However, a non-occluding parietal thrombus is routinely
formed at the angioplasty/stenting site. If the parietal thrombus
persists, it gets populated with the migrating SMC, thus facilitating fast
neointimal growth.
In an attempt to minimize platelet activation at the site of stent
implantation, Takemoto et al. [31] formulated gene-eluting stents
incorporating plasmid DNA encoding E-NTPDase, a crucial regulator
of platelet thrombus formation. is molecule is a membrane bound
enzyme that locally hydrolyses ATP, thus opposing platelet aggregation.
e plasmid DNA (78 ± 5 µg) was absorbed into cationized gelatin
hydrogel coating on the stent struts. Implantation of E-NTPDase-
eluting stents in a rabbit model of repeated femoral injury, which
is characterized by a high rate of thrombus formation completely,
prevented the occurrence of intravascular thrombi, while 25% and 50%
of femoral arteries treated with control βGal plasmid-eluting stents
had compromised patency at 3 and 7 days post-stenting. Moreover,
since thrombus serves as a nidus for SMC colonization, thus enhancing
neointimal formation, the extent of early restenosis (at day 7) was
signicantly reduced in the vessels treated with E-NTPDase stents [31].
Stents targeting nitric oxide production
Nitric oxide (NO), a by-product of enzymatic conversion
of arginine to citrulline has recently emerged as a key signaling
molecule in vascular homeostasis [73]. Increased NO production and
availability were shown to prevent platelet aggregation [74], inhibit
SMC proliferation [75,76], and migration [77], promote endothelial
growth [78], and decrease expression of cell adhesion molecules
by endothelium, inhibiting ingress of activated leukocytes into the

Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
Page 7 of 9
Volume 1 • Issue 2 • 1000109
Angiol
ISSN: 2329-9495 AOA, an open access journal
arterial intima [79]. Additionally NO promotes viability of endothelial
progenitor cells and enhances their capacity to reconstitute damaged
endothelium [80]. e pleiotropic nature of its benecial actions makes
NO-related interventions a plausible strategy for the prevention of ISR.
Both endothelial [81,82] and inducible [83,84] forms of NO
synthase (eNOS and iNOS, respectively) were investigated as genes
with potential anti-restenotic activity in animal models of arterial
injury/local delivery generally showing strong inhibiting eects on
neointimal formation. Driven by the wide scope of anti-restenotic
mechanisms bestowed by NO, we [40,42] and others [17,25,26,51] have
pursued the idea of the stent-based delivery of NOS-encoding vectors.
e studies by Sharif and others [25] employed PC coated stents
congured with 5x109 pfu of Ad-eNOS to address the hypothesis
that enhanced reendothelialization and reduced restenosis can be
achieved in a rabbit model with GES-induced eNOS overexpression.
e authors demonstrated signicantly enhanced restoration of the
endothelial layer and reduced neointimal thickness in both normo-
and hypercholesterolemic rabbits treated with Ad-eNOS stents in
comparison with animals that received Ad-βGal-eluting stents or
blank PC-coated stents. In an attempt to formulate a gene-eluting
stent devoid of immunogenic and pro-inammatory adenoviral
proteins, thus facilitating clinical translation of stent-based eNOS
overexpression, the same group recently reported [26] on a Lipofectin-
based lipoplex formulation containing human eNOS plasmid. Ten µg
of the lipoplex formulation were uniformly deposited on PC-coated
stents. e non-viral gene-eluting stents deployed in iliac arteries of
hypercholesterolemic rabbits achieved levels of transgene expression
comparable to Ad-containing GES. Moreover re-endothelialization
at 28 days was similarly enhanced by eNOS lipoplex-eluting stents.
However, no signicant reduction in restenosis indices was observed
in this study. e authors explain this discrepancy by the fact that
liposomal formulation primarily targets the macrophage population
in the diseased artery and not SMC, thus missing the vital cell type
aecting restenosis.
In contrast, using a similar rabbit model for therapeutic assessment
of eNOS-lipoplex-eluting stents Brito [17] demonstrated a 30%
reduction of restenosis accompanied by a 50% decrease of proliferation
in the eNOS lipoplex-treated rabbit arteries in comparison with an
empty vector control. e dierence in the vector dose (10 µg vs 25 µg)
and the delivery system used (adsorption of lipoplexes on a PC-coated
stent vs stent coating with lipoplexes dispersed in a gelatin/mannitol
formulation with additional lipoplex-free gelatin/PLGA supercoating)
between the former and the latter studies may account for the dierent
outcomes, highlighting the importance of proper pharmaceutical
adjustments for revealing full therapeutic potential of GES.
In our studies, Ad-iNOS delivered in a rat model from PAB- or
PABT-modied bare metal stents using anity adaptors [40] or
hydrolyzable cross-linkers [42] for vector tethering, respectively,
resulted in a 50%-60% reduction of neointimal thickening in
comparison with the BMS-implanted arteries. ese eects were
achieved using 109-1010 Ad-iNOS particles (107-108 pfu) appended to
a stent. Our recent studies assessing NO synthesis and ROS production
in cultured SMC and endothelium transduced with Ad-iNOS tethered
to stainless steel disks have determined that the super-physiological
levels of iNOS expression driven by the immobilized vector can lead to
NOS decoupling due to deciency of arginine and tetrahydrobiopterin
[85]. Decoupled NOS is unable to generate NO, producing superoxide
instead [86]. If not eectively removed by superoxide dismutases,
superoxide triggers a sequence of redox reactions that result in the
formation of multiple additional ROS, which can cause extensive
tissue damage [86]. erefore, pharmacological supplementation with
arginine and tetrahydrobiopterin may be required to modulate the
function of NOS overexpressed in the arterial wall using stent based
delivery of a respective vector [85].
Which of three known isoforms of NOS is the best choice for the
use in conjunction with GES is not immediately obvious. A study
by Cooney [82] compared the antirestenotic eectiveness of Ad-
iNOS and Ad-eNOS in a model of intraluminal dwell delivery of the
vectors to temporarily isolated segment of balloon-injured rabbit
carotid arteries. While a 20-min incubation of both vectors (109 pfu)
resulted in an identical extent of neointimal inhibition in comparison
with Ad-βGal-treated counterparts, this was allegedly achieved by
dissimilar mechanisms, since eNOS overexpression promoted and
iNOS overexpression inhibited endothelial regeneration in the treated
arterial segments [82]. Additional in-depth studies are required to
elucidate the actual mechanisms involved in the antirestenotic eects of
dierent NOS isoforms and their utility in the setting of GES-induced
overexpression.
Conclusions
Gene-eluting stents represent an interesting alternative to drug-
eluting stents in clinical circumstances where current DES formulations
oen fail to provide satisfactory results. Research related to GES has
now come to a phase when the perspectives of clinical translation
should always be taken into consideration. However, further work
related to optimization of all three main design components of GES:
a therapeutic transgene, a vector and a delivery system has to be done
before stent based vascular gene therapy comes to clinical fruition.
Acknowledgement
Studies carried out as part of the experimental work discussed in this review
were supported by American Heart Association Scientist Development grants
(I.F. 0735110N and M.C. 10SDG4020003), U.S. National Heart, Lung, and Blood
Institute grant HL 72108 (R.J.L.), U.S. National Heart, Lung, and Blood Institute
grant HL 111118 (M.C.) and U.S. National Heart, Lung, and Blood Institute
T32 007915 (S.P.F. and R.A.C.). The authors wish to acknowledge secretarial
assistance of Ms. Susan Kerns.
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Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
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Citation: Fishbein I, Chorny M, Adamo RF, Forbes SP, Corrales RA, et al. (2013) Endovascular Gene Delivery from a Stent Platform: Gene- Eluting
Stents. Angiol 1: 109. doi: 10.4172/2329-9495.1000109
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Angiol
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