Praveen S.V., MBBS, MD, DNB, Johnson Francis, MBBS, MD, DM,
FCSI, Venugopal K., MBBS, MD, DM, FCSI.
Department of Cardiology, Medical College, Calicut, Kerala, India
Address for
correspondence: Dr. Praveen S.V., MBBS, MD, DNB, Department of
Cardiology, Medical College, Calicut-673008. Kerala, India. Email:
praveensv2001@yahoo.com
Abstract
Gene therapy has
progressed from a dream to a bedside reality in quite a few human
diseases. From its first application in adenosine deaminase
deficiency, through the years, its application has evolved to
vascular angiogenesis and cardiac arrhythmias. Gene based biological
pacemakers using viral vectors or mesenchymal cells tested in
animal models hold much promise. Induction of pacemaker activity within
the left bundle branch can provide stable heart rates. Genetic
modification of the AV node mimicking beta blockade can be therapeutic
in the management of atrial fibrillation. G protein overexpression to
modify the AV node also is experimental. Modification and expression of
potassium channel genes altering the delayed rectifier potassium
currents may permit better management of congenital long QT
syndromes. Arrhythmias in a failing heart are due to abnormal calcium
cycling. Potential targets for genetic modulation include the
sarcoplasmic reticulum calcium pump, calsequestrin and sodium calcium
exchanger.Lastly the ethical concerns need to be addressed.
Key Words: Gene therapy;
biological pacemakers; AV node modification; long QT syndromes;
sarcoplasmic reticulum calcium pump .
Introduction
Gene therapy is defined as the transfer of nucleic acids to
somatic cells as therapeutically useful molecules. Genetic defects can
be corrected or gene products be expressed by gene therapy.
This approach has many potential applications, the most obvious being
the treatment of inherited monogenic disorders like cystic fibrosis.
Human genome has approximately 30,000 genes. The genetic diversity is
amplified by alternate splicing of mRNA and post translational
modification of proteins. The possible gene targets for
arrhythmias is very large. Anti arrhythmic agents act by blocking the
ion channels. These antiarrhythmics have poor channel specificity and
may cross react with other ion channels. In addition they have poor
affinity for the channels and have a narrow therapeutic window. The
molecular targets of arrhythmia management are the ion channels and the
modulators of ion channels like G proteins
1.
Vectors for gene therapy
A
vector is the vehicle commonly used to introduce the gene to the target
cell. Vectors may be RNA or DNA viruses or non viral in nature. Viruses
which have the capacity to incorporate themselves in the host genome
are used as vectors for gene therapy. The commonly used viral vectors
are genetically modified retroviruses, adenoviruses, adeno associated
viruses and lentiviruses. These viral vectors are made replication
deficient to ensure safety, but requires large amounts of vector
particles for efficacy. Non viral vectors based on plasmids, DNA- lipid
complexes and naked DNA are also used since they lack foreign proteins
and avoid immunological problems. None of the currently available
vectors satisfy the criteria of an ideal gene therapeutic system. The
feasibility of gene transfer has been demonstrated in both animals and
humans.
In
contradistinction to the experimental laboratory, the extent of gene
transfer and expression is low in clinical settings. The period
during which a newly introduced gene is expressed is variable and
differs with the tissue, but is often short. For example,
early-generation non-viral vectors express the gene at maximum levels
only for a few days
2. Many
adenoviral vectors express the gene for 2-3 weeks
3. Non viral vectors also have short
duration of gene expression. Short duration of gene expression
necessitates repeat dosing, although less efficacious. By contrast,
expression from adeno-associated viral vectors may not peak for several
weeks, but then remain constant in some tissues for several months
4. Retroviruses produce a long lasting
effect by integration of the transfected gene into the host genome
5
Methods of gene delivery
The
classical methods of vector delivery are direct injection into the
myocardium, infusion through the coronary arteries or administration to
the epicardium.Various novel methods of transfection have been tried in
animal models, including DNA polymer coating on inert materials and
subsequent transfer to the atrial myocardium, with sustained gene
activity
6. Intracoronary
perfusion is another modality of gene transduction with near
complete expression under optimal conditions
7.
The gene transfer efficiency depends on the coronary flow rate, virus
concentration, virus exposure time and microvascular permeability.
Agents which increase the microvascular permeability have been used to
enhance the delivery. Each disease has its own target tissue and the
amount of gene product required for treatment. So only few
generalizations can be made about the vector selection and the method
of gene delivery.
The need for gene therapy in
cardiac arrhythmias
Antiarrhythmic
medications suppress arrhythmias, but their systemic effects are often
poorly tolerated and their proarrhythmic effects increase mortality.
Radiofrequency ablation can cure only a limited number of arrhythmias.
Implantable devices can be curative for bradyarrhythmias and lifesaving
for tachyarrhythmias, but require a lifetime commitment to repeated
procedures, have a significant expense, and may lead to severe
complications. The need for new treatment strategies for cardiac
arrhythmias has motivated the continuing development of gene
therapeutic options Gene therapy may prove to be a less
intrusive, long-term solution to arrythmias than pacemakers or
antiarrhythmic agents. Gene therapy for arrhythmias is a field still in
its infancy.
The ongoing
research into gene therapy for cardiac arrhythmias can be briefly
discussed under:
1) Biological pacemakers
2) AV nodal modification
3) Long QT syndromes
4) Arrhythmias of cardiac failure
5) Ventricular arrhythmias
Biological pacemakers
Conventional
electronic pacemakers although highly efficacious, have a finite
battery life and problems related to lead and circuitry. Biological
pacemakers aim a near physiological pacing by molecular or cellular
targeting.Biological pacemakers can be cell therapy based or gene
therapy based. Strategies have included gene therapy using naked
plasmids or viral vectors and cell therapy with both adult human
mesenchymal stem cells (hMSCs) and human embryonic stem cells
8,9. Over the past
few years, gene therapy has been used to explore the
overexpression of β2-adrenergic receptors, the down-regulation of
inward rectifier current, and the overexpression of pacemaker current
as potential sources of biological pacemakers
10.
The protein product of HCN2 gene is a candidate for the pacemaker
current. Rosen et al from Columbia university have recreated the
biological pacemaker by administering the pacemaker gene
HCN2 via viral vector or in an hMSC platform to produce
pacemaker function in the intact canine heart. Adenovirus containing
the HCN2 gene [ad HCN2] on introduction in canine models
produced spontaneous rhythms.
Another
potential approach to a biological pacemaker is the induction of
pacemaker activity within the left bundle branch, thereby providing a
ventricular escape rhythm with physiologically acceptable rates. The
long-term stability and feasibility of this approach remain to be tested
11. Adeno viral vectors have been tried
in animal models to create pacemaker clones from ventricular myocytes.
The inward rectifier current [I
K1] is responsible for the
maintenance of the resting membrane potential. The main subunit of this
is Kir 2.1. The Kir 2.1 gene was mutated to make it a
dysfunctional channel (a dominant-negative), inserted into an
adenoviral vector and delivered to the hearts of guinea pigs. The
enhanced automaticity of the ventricular myocytes subsequent to the
gene transfer conferred pacemaker like properties
12.
AV nodal modification
As
in case of biological pacemakers, the field is limited to animal
studies. The major area of research in gene therapy for
tachyarrhythmias now is on AV nodal modification to produce AV nodal
block mimicking beta blockade.
Genetic
modification of AV node in an intracoronary perfusion model of porcine
heart was reported by Donahue et al from John Hopkins University
13. They infected porcine hearts with
Adbetagal (recombinant adenovirus expressing Escherichia coli
beta-galactosidase) or with AdGi [adenovirus encoding the Galphai2
subunit]. Galphai2 overexpression suppressed baseline atrioventricular
conduction and slowed the heart rate during atrial fibrillation without
producing complete heart block. In contrast, expression of the reporter
gene beta-galactosidase had no electrophysiological effects. Reporter
genes are nucleic acid sequences encoding easily assayed proteins.
AV nodal gene
transfer can decrease heart rates in animal models of atrial
fibrillation. Inhibitory G protein overexpression can prolong the
AV nodal refractory period with slowing of conduction resulting
in reduced ventricular rates in atrial fibrillation
14. In mouse models, cardiac
overexpression of adenosine receptor [A(3)AR] resulted in gene
dose-dependent AV block and pronounced sinus nodal dysfunction in vivo.
These may have future therapeutic implications for SA and AV nodal
modification
15. Focal gene
transfer to the AV node to produce a genetic calcium channel
blocker has been successful in experimental settings. Over
expression of the ras related small G- protein [Gem] in AV node
slowed AV nodal conduction controlling the heart rate in
atrial fibrillation
16.
Long QT syndromes ( LQTS)
Sympathetic imbalance was previously thought to be responsible
for this disease. Since 1991, 7 LQTS genes have been discovered and
more than 300 mutations have been identified to account for the
disease. Acquired LQTS, is presumed to be due to the blockade of the
rapid component of the delayed rectifier K+ current (I
Kr).
Blockage of the I
Kr prolongs the QT interval and increases
the dispersion of repolarization, predisposing to torsades de pointes.
Molecular genetic analysis could be useful to unravel subclinical
mutations or polymorphisms.
Individuals
with cardiac potassium channel missense mutation, Q9E-hMiRP1 are
predisposed to develop QT prolongation after clarithromycin
administration. Experimental studies have demonstrated that cells
transfected with plasmid DNA containing Q9E-hMiRP1 have reduced
potassium currents on exposure to clarithromycin.
Site specific
gene therapy for arrhythmias by transfecting cell clones with the K+
channel genes is a feasible approach to the management of LQTS
17. Mutated K+ channels resulting in loss
of function have been implicated in LQT 1 and 2. The potassium channel
alpha subunit genes KCNH2 [HERG] and KCNQ1 [KvLQT1] responsible
for Ikr and Iks respectively are mutated in LQTS. In normal epithelia,
KCNE3 [E3] interacts with the KVQT1 [Q1] thereby augmenting the
potassium currents. E3 subunit can be genetically expressed in cardiac
tissues [where it is normally scarce] to abbreviate the action
potential duration and enhance the potassium current.This potentially
prevents arrhythmias in LQTS. Adenovirus encoded E3 introduced into
guinea pig ventricles shortened QT interval on homogenous transduction,
but could be potentially arrhythmogenic if transduction is heterogenous
18.
Arrhythmias of cardiac failure
Abnormal
calcium cycling plays an important role in the genesis of contractile
dysfunction and arrhythmias in the setting of heart failure. Genetic
modulation of the sarcoplasmic reticulum calcium ATP ase pump [SERCA]
can have an ameliorating effect on the arrhythmias of cardiac
failure.
Delayed
repolarization predisposes the failing heart to ventricular
arrhythmias and this represents a logical target for gene therapy. The
calcium ATPase SERCA1 was coexpressed with the potassium channel Kir2.1
in guinea pig hearts. Such myocytes had bigger calcium transients
and shorter action potentials. In vivo, repolarization was abbreviated,
but contractile function remained unimpaired
19.
This shortening of repolarization prevents arrhythmias. Coexpression of
SERCA1 prevented the decrease in contractility due to shortening of
action potential. This interesting observation is useful in prevention
of arrhythmias in the setting of heart failure.
Calsequestrin,
the high capacity calcium binding protein expressed in sarcoplasmic
reticulum, also positively controls the rate of calcium release during
excitation-contraction coupling. Mutations in the calsequestrin gene
have been linked to arrhythmias and sudden death. For example,
the recessive form of catecholaminergic polymorphic ventricular
tachycardia is associated with calsequestrin mutations
20. Modulation of the gene for
calsequestrin could be one of the numerous potential targets for
therapy.
The main
pathway for calcium efflux from the cell is the Na - Ca exchanger
(NCX), a membrane antiporter and a determinant of both the
electrical and contractile state of the heart. NCX causes efflux of one
Ca(2+) for three Na+ transported into the cell. Enhanced expression of
NCX has recently been recognised as one of the molecular
mechanisms that contributes to reduced Ca(2+) release, impaired
contractility and an increased risk of arrhythmias during the
development of cardiac hypertrophy and failure. The NCX also plays a
crucial role in the pathogenesis of arrhythmias and cellular injury
associated with ischaemia and reperfusion. Hence, NCX blockade
represents a potential therapeutic strategy for treating cardiac
disease.However, its reversibility and electrogenic properties must be
taken into consideration when predicting the outcome. NCX inhibition
has been demonstrated to be protective against ischaemic injury and to
have a positive inotropic and antiarrhythmic effect in failing heart
cells
21.
Failing
hearts show a delay of repolarization and prolongation of
action potential due to diminished potassium currents, which is
proarrhythmic. Adenovirus over- expressing the potassium channels
[AdShK] can reverse the prolongation of action potential duration,
thereby potentially preventing arrhythmias
22.
Ventricular arrhythmias
Electrical
alternans has been linked to the development of ventricular
arrhythmias.Increasing the rapid component of the delayed rectifier
current (I
Kr) may suppress electrical alternans and may be
antiarrhythmic. I
Kr in isolated canine ventricular myocytes
was increased by infection with an adenovirus containing the gene for
the pore-forming domain of I
Kr [human ether-a-go-go gene
(HERG)]. The voltage at which peak I
Kr occurred was
significantly less negative in HERG-infected myocytes, thereby shifting
the steady-state voltage-dependent activation and inactivation curves
to less negative potentials
23.
This observation lend support to the idea that increasing I
Kr
may be a viable approach to suppressing electrical alternans thereby
suppressing arrhythmias.
Current problems with gene therapy
As alluded to
earlier, gene medicine is still in its infancy. Except for a
few human trials, majority of trials to date have been in
experimental animals.The expected result from gene therapy is a
permanent cure of arrhythmias with a single stage treatment with
minimal or no adverse effects. Obviously we are far from the
ideal. In the field of arrhythmias, an
expectant waiting for the scenario to unfold in full is needed.
The available vectors to date are far from ideal.Problems with vectors
include variability in transfection capabilities,inefficient delivery
at site,limited period of gene expression, and immunogenicity.
The tissue expression of many genes are transient. The level and
efficiency of expression of many trans genes are suboptimal. Many viral
vectors are potentially immunogenic and carcinogenic. The
interaction between vector and host genome can result in the vector
being rendered replicant and lose the therapeutic gene. Another
area of concern is that the currently available vectors have less
ability to transduce vascular cells than nonvascular cells. This could
hamper efforts at cardiovascular gene transfer. Traditional vectors
need to be engineered to increase their affinity for the target tissue
or cell and prevent transduction to other cells
24.
Succesful
transfer of the therapeutic gene to all the myocytes at the target site
is not fully achieved in the experimental settings. The receptors for
many viral vectors are present in many tissues therby limiting the
specificty of gene delivery.
Many an
arrhythmia with a diffuse substrate like atrial fibrillation needs the
gene to be delivered to a wide area [the whole of the atrium]. The
transfer methods like direct injection into myocardium fails to deliver
the gene a short distance from the injecton site. A
special area of concern in arrhythmia gene therapy is the potential for
the remedy itself being arrhythmogenic. Incomplete restoration may in
also be arrhythmogenic. In a non linear system like biological
organisms, making an isolated change in a specific aberration will
result in restoration of normal function only if the defect is truly
isolated and is the direct cause of the phenotypic response.The long
term response of a genetic modification in the myocardium is unknown at
present. Only continued research and time can answer these problems
with certainty.
The Future
Newer
refinements in vector development and design are needed to have better
transduction in cardiovascular tissue. Cell specific regulatory
elements and promoters to selectively target the cardiac tissue is a
potential area of interest
25.
Application at bed side awaits further refinement in gene delivery.
Bactofection (bacterial gene delivery) as an alternative to viral
vectors has been proposed
26.
Hybrid vectors, gutted vectors and new generation non viral vectors may
hold the key to future research.
Conclusion
Given a wide
plethora of potential targets for gene therapeutic strategies, the
possible applications are unlimited. We have still a long way to go
from animal models to the level of safe and efficacious application at
the bedside. This awaits more refinements in gene delivery methods and
vector designs. Not to be forgotten are the increasing concerns about
safety. A regulated and sustained target tissue expression of the
transduced gene with a wide index of safety should be the
ultimate goal of any genetic intervention.
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