Myocardial gene therapy is an emerging therapeutic option for the treatment of patients with advanced ischemic heart disease. Early clinical experiences show feasibility and potential efficacy of gene-mediated therapeutic angiogenesis. As percutaneous methods of gene delivery mature, the interventional cardiologist will undoubtedly play a major role in the delivery of these agents and will require a working knowledge of this field. Future placebo-controlled, large, randomized trials with both current and new angiogenic factors will define the ultimate role of this therapy.
Vipul R. Panchal, MD
is a Fellow in Interventional Cardiology at the Krannert Institute
of Cardiology, Indiana University School of Medicine, Indianapolis,
IN. He completed his General Cardiology Fellowship and his Internal
Medicine Residency at the same institution. He received his MD from
Northeastern Ohio Universities College of Medicine in 1996.
Vijay Kalaria, MD
is an Assistant Professor of Clinical Medicine and an Associate
Director of Cardiac Catheterization Laboratories and Interventions,
Krannert Institute of Cardiology, Indiana University School of
Medicine, Indianapolis, IN.
Jeffrey A. Breall, MD, PhD
is a Clinical Professor of Medicine and the Director of Cardiac
Catheterization Laboratories and Interventions, Krannert Institute
of Cardiology, Department of Medicine, Indiana University School of
Medicine, Indianapolis, IN.
Keith L. March, MD, PhD
is an Associate Professor of Medicine at the Krannert Institute of
Cardiology, Department of Medicine, Indiana University School of
Medicine, Indianapolis, IN.
Myocardial gene therapy is an emerging therapeutic option
for the treatment of patients with advanced ischemic heart
disease. Early clinical experiences show feasibility and
potential efficacy of gene-mediated therapeutic angiogenesis. As
percutaneous methods of gene delivery mature, the interventional
cardiologist will undoubtedly play a major role in the delivery
of these agents and will require a working knowledge of this
field. Future placebo-controlled, large, randomized trials with
both current and new angiogenic factors will define the ultimate
role of this therapy.
Patients with coronary artery disease refractory to medical
therapy and without revascularization options continue to challenge
the cardiologist. In recent years, myocardial gene therapy for the
treatment of heart disease has evolved from bench to bedside. Early
clinical trials of myocardial angiogenesis have shown promise
and emphasize the need for further studies to determine the optimal
delivery method, agent, and dose for safe and effective
This review will focus on current aspects of catheter-based gene
therapy for myocardial angiogenesis that are relevant to the
interventional cardiologist, including possible delivery methods,
agents, vectors, and potential problems with each strategy.
The prevalence of symptomatic patients with unrevascularizable
end-stage coronary artery disease is increasing. Many of these
patients remain symptomatic despite maximal anti-anginal therapy
and may not be amenable to or may have failed surgical or
percutaneous forms of myocardial revascularization.
It is estimated that approximately 12% of symptomatic patients with
coronary disease undergoing cardiac catheterization are not
candidates for percutaneous coronary intervention or coronary
artery bypass grafting (CABG). These patients suffer high rates of
cardiac events and have limited therapeutic options.
A potential therapeutic option for these patients is therapeutic
Therapeutic angiogenesis is an intervention aimed at inducing
neovascularization and promoting collateral formation in ischemic
myocardium through the administration of angiogenic growth factors
or their genes.
It is well known that coronary collateralization improves the
prognosis of patients with obstructive coronary artery disease by
limiting infarction size, preserving ventricular function, and
enhancing overall perfusion in ischemic myocardium.
Thus, augmentation of collateral vessel growth may be a valuable
approach in these patients.
Angiogenesis is a process in which new capillaries sprout from
the existing vasculature by way of proliferation and migration of
endothelial cells. This is mediated, in part, by angiogenic growth
Wound healing and reproductive growth are examples of
physiologically normal forms of angiogenesis. Pathologic forms
include tumors and diabetic retinopathy.
Angiogenesis can also be a response to ischemia. It is suggested
that patients with extensive coronary disease may have an impaired
endogenous angiogenic response, and enhancement of this process is
the basis of therapeutic angiogenesis.
The feasibility of therapeutic myocardial angiogenesis was shown
in initial animal studies, primarily using the angiogenic proteins,
such as vascular endothelial growth factor (VEGF) and fibroblast
growth factor (FGF). Early attempts to enhance collateral perfusion
in ischemic myocardium used local administration of recombinant
forms of these proteins.
These studies showed the efficacy of exogenous supplementation of
growth factors by demonstrating enhancement of collateral flow as
well as improvement in myocardial perfusion and in wall motion.
Rationale for gene therapy
Administration of recombinant proteins for angiogenesis has
shown benefits in several phase I trials.
However, there are several limitations to protein delivery methods.
A critical problem is inadequate myocardial exposure time due to
brief serum and tissue half-life of the protein. Higher initial
concentrations and multiple administrations of the angiogen would
provide efficacy, but at the expense of potential side effects.
In contrast, gene therapy modalities offer a sustained, but
self-limited, expression of therapeutic factors that can achieve
desired biological effects in a single dose.
The goal of cardiac gene therapy is to deliver genetic material
(frequently by plasmid or viral vectors) to the myocardium in order
to induce sustained and local overexpression of the transgene. This
process is called transfection.
The degree of physiologic effect derived from gene transfer is
dependent on the efficiency of the transfection process. Because of
variable transfection efficiencies, gene therapy has a more
unpredictable dose-response relationship in comparison with protein
Successful gene therapy is contingent on several consecutive
steps (Figure 1): (1) the method of gene delivery to the target
tissue, (2) a capable vector system allowing intracellular passage
of genetic material (plasmid or virus), and (3) robust local
expression of an appropriate transfected gene (angiogenic agent)
within the target tissue.
Various routes of administration have been used in delivering
angiogenic proteins and genes.
The optimal technique maximizing safety and efficacy for myocardial
gene delivery is not well established.
An ideal technique would deliver angiogenic factors in a
site-specific manner to the target tissue, minimizing systemic
exposure. It should achieve high local concentrations as well as
provide sufficient tissue retention time to allow cell interaction
and subsequent gene transfer.
Finally, the technique should be simple to perform, relatively
inexpensive, and use existing technologies.
Catheter-based myocardial delivery can be divided into three
methods: intravascular, intramyocardial, and intrapericardial.
Intravenous administration is the simplest form of delivery. Its
major drawback, however, is very low myocardial tissue uptake. In
radiolabeled distribution studies, only 0.26% of bFGF was isolated
in ischemic myocardium 1 hour after IV injection and 0.04% after 24
This results in significant systemic exposure of the agent,
consequently increasing the risk for side effects. Accordingly, IV
administration of agents will not be effective in inducing
Intravenous administration of an agent that is designed with a
"homing" mechanism can improve myocardial delivery. This has been
performed by complexing therapeutic agents with ultrasound contrast
bubbles that can be injected intravenously and subsequently lysed
at the myocardium with transthoracic high-frequency ultrasound.
The intracoronary route has been used in many clinical trials of
It is favored because it is relatively simple to administer in the
catheterization laboratory, requires no special equipment, is
widely available, and is suited to most patients with coronary
The intracoronary route is limited by poor myocardial tissue
uptake, although it is slightly better than the IV route.
Approximately 3% to 5% of bFGF was recovered from the myocardium
several hours after intracoronary injection, and with a low 0.04%
retention after 24 hours.
In a phase I study, patients receiving selective intracoronary
injections of FGF-2 incurred similar systemic exposures to those
receiving IV infusions.
Despite relatively poor first-pass uptake, animal studies and phase
I studies seemed to indicate potential efficacy of single bolus
intracoronary protein injections.
Coronary venous retroinfusion--
Coronary venous retroinfusion is gaining interest as an efficient
myocardial gene-delivery modality. Retroinfusion into the coronary
vein is very effective in delivering drugs to the myocardium and
Deposition is enhanced significantly in ischemic myocardium during
balloon occlusion of a corresponding coronary artery. Significantly
longer tissue retention has also been reported by this route.
This method is performed by cannulation of a selected cardiac
vein via the coronary sinus using a modified balloon-tipped
perfusion catheter. The coronary vein is then occluded to prevent
drainage. Pressure-regulated infusion of the gene agent is
performed at an internal venous pressure of 20 mm Hg for several
modification of this technique is selective high pressure (100 mm
single-bolus injection (10 mL). The technique appears to be safe
because no echocardiographic evidence of myocardial dysfunction has
been noted, and myocardial marker release is minimal.
Gene delivery with this method has been shown to be feasible.
Pressurized retrograde coronary venous delivery of an adenovirus
has shown considerably more gene expression than that of antegrade
intracoronary administration in pigs.
The technique is also effective for plasmid-driven angiogenesis as
well. Administration of plasmid developmental endothelial locus-1
(del-1, an angiogenic factor) improved myocardial perfusion in
In addition to gene delivery, coronary venous retroinfusion
provides widespread distribution of cells and microspheres to the
Until recently, all myocardial gene delivery methods were
surgical in nature. Early phase I clinical trials of gene-based
myocardial angiogenesis administered growth factors by direct
transepicardial injection at the time of CABG or using
This approach was favored over intravascular methods due to the
lack of significant myocardial washout and dissemination to distant
tissues after initial injection.
Subsequent animal studies have shown, however, that incomplete
retention of injectate also occurs with intramyocardial delivery,
although to a lesser degree.
Currently, the only clinically feasible catheter-based method of
intramyocardial administration is transendocardial intramyocardial
delivery via an intracavitary left ventricular approach. This
technique has been validated in preclinical and clinical studies.
In a biodistribution study comparing epicardial open chest
injection of microspheres with percutaneous endomyocardial delivery
in pigs, the endomyocardial tissue retention was better (43% versus
15%) than thoracotomy-based epicardial injection.
Transendocardial injections are performed with various
specialized catheters that have retractable sheathed needles
or helical infusion needles.
The catheter is advanced intra-arterially across the aortic valve
into the left ventricle and endocardial injections are performed.
There are several adaptations to this technique, using different
guidance methods such as intracardiac echocardiography (ICE),
and electromechanical mapping.
The only published clinical experience to date is with
Fluoroscopic-guided endocardial gene transfer alone is not
likely to be useful clinically because it does not provide adequate
anatomic information about injection sites or the nature of the
myocardium. However, ICE guidance in conjunction with fluoroscopy
provides useful information, such as directing needle position and
monitoring systemic leakage of gene injectate, and it provides the
ability to see injected sites to avoid repeat administration.
Transendocardial injection guided by a left ventricular
electromechanical mapping system (NOGA, Biosense Webster, Inc.;
Diamond Bar, CA) has gained considerable interest. Using a magnetic
field, the system is able to precisely locate the catheter in the
ventricle in three dimensions. In addition, electromechanical
mapping can delineate normal, ischemic, or hibernating myocardium
based on the amplitude of unipolar endocardial potentials and
assessment of local myocardial cell shortenening.
Disadvantages of this technique include the requirement of
multiple injection sites, relatively small injection volumes (0.1
to 1 mL), and a relatively complex mapping method.
Another catheter-based method that provides efficient gene
transfer is intrapericardial administration. Catheters have been
developed to access the pericardial space percutaneously in a safe
manner in animals.
A helical-tipped transventricular penetrating catheter has been
used to instill various genes into the pericardial sac of dogs.
Under fluoroscopic guidance, the catheter is advanced into the
right ventricle, and the tip is passed through the apex.
Alternatively, a percutaneous transthoracic catheter incorporates a
sheathed needle with a suction device to grasp the pericardium and
access the pericardial space.
Robust gene expression has been seen in the pericardium and
epicardium with these techniques, while serum and distant tissue
levels were undetectable or at background levels.
Minimal systemic exposure, high transgene expression from
adenoviral vectors, and potentially widespread myocardial delivery
are valuable characteristics. However, the clinical use of this
technique may be limited because of prior pericardial manipulation
in many patients with cardiac surgery.
Furthermore, assessment of theoretical risks such as
intrapericardial bleeding with angiogenesis therapy needs further
Gene transfer vectors
Cardiovascular gene transfer requires effective and durable
vectors that carry and facilitate entry of genetic material into
the target cell and to the nucleus.
Once this is accomplished, sustained expression of the transgene
can occur. The vector usually contains a promoter region that
allows for expression and translation of the gene, a copy DNA
segment encoding the angiogenic factor, and various signals for
ending translation and for posttranslational processing.
The traditional types of vectors used for gene therapy are nonviral
Nonviral gene transfer
Nonviral plasmid-mediated DNA transfer is used because of its
safety profile and transient expression. Many viral vectors produce
an immune response to some degree, which is avoided with nonviral
methods. In addition, transient transgene expression occurs in the
nucleus without incorporation into chromosomal DNA.
Plasmids can also incorporate larger cassettes to code multiple
genes or larger proteins. A major disadvantage, however, is that
naked DNA has a short half-life due to degradation by circulating
nucleases and scavenger receptors.
Plasmids are also very inefficient in transfection.
The formulation of plasmid DNA complexes with macromolecules has
enhanced stability and transfection.
The DNA is combined with polymers, such as cationic liposomes or
poloxamers. Negatively charged DNA combines with positively charged
cationic lipids that form stable structures, which isolate plasmids
from nucleases and facilitate entry into the cell by endocytosis.
Another novel way to enhance and target nonviral DNA delivery is
with the use of microbubbles. Plasmid DNA is complexed with
microbubbles, delivered to the myocardial circulation, and
subsequently disrupted with high-power ultrasound releasing the
genetic material to the target area. It is based on contrast
microbubbles similar to those used in contrast echocardiography and
can be formulated of albumin, polymeric shells, or cationic lipid
In a porcine study of systemically delivered plasmid DNA,
transfection occurred only in myocardium that was exposed to the
Similarly, a 10-fold increase in transfection efficiency was noted
with an adenovirus formulated with microbubbles. There was,
however, extensive transgene expression in the livers of all
animals in which this method was used,
suggesting that site-specific delivery may still be necessary.
Viral gene transfer
Gene transfer with the use of engineered viruses is very
efficient in transfecting cells. This is primarily due to the
innate ability of a virus to infect a cell and transfer its genetic
material. Viruses are not subject to intravascular degradation in
contrast to plasmids. Several different types of viruses can be
used for gene therapy. The concerns of viral gene therapy are of
host immune responses and prolonged gene expression leading to
overexposure to the angiogenic factor.
Adenoviruses have been studied extensively and provide
short-term, 1- to 2-week, transgene expression without integrating
into host DNA. They are capable of transfecting both the
proliferating and nonproliferating cells. These viruses are
rendered replication-deficient, and the genome is replaced with the
desired angiogenic expression cassette.
Newer generation adenoviruses have a longer duration of expression
and less immunogenicity. Adeno-associated viruses provide longer
duration of expression as well.
Retroviral vectors transfer RNA, which is transcribed to DNA and
integrated into the host genome resulting in long-term expression.
Retroviruses have limited use in the myocardium, as they can only
transfect proliferating cells.
Many angiogenic growth factors have been identified (Table 1).
However, all clinical trials of angiogenesis to date have studied
the VEGF and FGF classes.
Human cardiovascular gene therapy trials in progress or in early
development are now expanding to trials using platelet-derived
growth factor, hypoxia inducible growth factor-1*, del-1, and
nitric oxide synthase.
Vascular endothelial growth factor is an endothelial
cell-specific mitogen and exerts its influence by means of specific
There are several isoforms of the VEGF protein and all isoforms
impart angiogenic effects.
However, of the several receptors for VEGF, only the VEGFR-2 (KDR)
receptor mediates angiogenesis.
Fibroblast growth factors are a family of several angiogens that
share a similar protein structure. In contrast to VEGF, their
effects include proliferation of multiple cell types including
endothelial cells, smooth muscle cells, and fibroblasts.
Both VEGF and FGF can enhance production of various proteases
essential to matrix degradation for angiogenesis.
Safety concerns of gene therapy
Despite the promise seen in both preclinical and early clinical
gene transfer studies, concerns regarding safety of gene delivery
and toxicity of therapy have arisen. The death of a patient in a
phase I gene therapy trial for treatment of ornithine
transcarbamylase deficiency due to an adenovirus-mediated reaction
has triggered concern.
Adenoviral vectors are known to induce inflammatory and immunologic
reactions that are sometimes severe. Furthermore, VEGF trials have
reported hypotension, edema, and formation of spider angiomas.
In some cases, overexpression of VEGF has produced vascular tumors,
vessel clusters, and severe localized edema.
Similarly, FGF has shown potential toxicities including
hypotension, proteinuria, and thrombocytopenia.
The risk of pathologic angiogenesis is also of concern due to
the possibility of enhanced growth of clinically silent
malignancies. Retinal neovascularization (diabetic retinopathy and
retinal vasculopathies) is associated with elevated levels of VEGF.
Trials of catheter-based angiogenesis
Initial catheter-based phase I clinical studies administered
recombinant protein by the intracoronary route. These studies were
small and without placebo controls, but did show potential benefit
including improvements in exercise duration, perfusion, and fewer
Larger phase II placebo-controlled clinical studies with the same
approach, however, did not yield the same results. Patients treated
with intracoronary and IV VEGF in the VEGF in Ischemia for Vascular
Angiogenesis Trial failed to meet endpoints of improved exercise
time and angina class.
In the FGF-2 Initiating Revascularization Support Trial,
intracoronary injection of FGF-2 did not improve exercise time or
These findings are not surprising given the unfavorable
pharmacokinetics of intracoronary delivery described
To date, there are three published clinical trials of
catheter-based gene transfer for angiogenesis (Table 2).
These trials evolved from the need for a placebo control arm. Early
phase I gene trials used intraoperative direct myocardial injection
for gene transfer that precluded randomization to placebo due to
the inherent risks of surgery and anesthesia.
Percutaneous methods allowed a control arm to the study design.
The first percutaneous trial of catheter-based gene therapy was
a pilot study to show the safety and feasibility of
transendocardial gene transfer using left ventricular
electromechanical mapping. Vale and colleagues
randomized 6 patients to plasmid VEGF-2 gene transfer or a placebo
procedure. Improvements in anginal episodes and nitroglycerin use
were noted in both groups, though the placebo group later
experienced worsening angina, whereas the patients in the gene
transfer group continued to improve. Perfusion scores and
electromechanical function in ischemic sites were significantly
better in the VEGF-treatment group than in the placebo group.
Based on these initial findings, a phase II dose-escalation
study of 19 patients using the same method was performed and
significant improvements were found in the Canadian Cardiovascular
Society anginal class (3.5 versus 2.2,
= 0.012) and a significant mean change in angina class for patients
treated with plasmid VEGF-2 (1.2 versus 0.1,
= 0.04). There were trends toward improvements in exercise
duration, angina questionnaire data, and perfusion on single-photon
Finally, in the Angiogenic GENe Therapy Trial, a phase II,
double-blinded, randomized, placebo-controlled trial of 79 patients
showed safety of intracoronary adenoviral gene transfer of FGF-4.
Treated patients showed a nonsignificant increase in exercise
duration at 4 weeks (1.3 versus 0.7 min,
= not significant). However, in a subgroup analysis of patients
excluded with exercise times >10 minutes, this difference became
significant (1.6 versus 0.6 min,
It is important to note the limitations of these and other
trials of gene therapy. The studies are relatively small, and many
are nonrandomized or uncontrolled. Furthermore, endpoints such as
exercise duration and health status measures are "soft" endpoints
and can be subjective.
These issues underscore the need for larger phase II, randomized,
placebo-controlled trials to definitively prove efficacy.
The application of percutaneous catheter-based gene therapy
techniques will allow larger scale studies to be performed. These
same techniques may apply to other emerging technologies (such as
cell delivery/transplantation for myocardial regeneration), thus
continuing to expand the role of interventional cardiologists.