Gene Therapy Vectors
Non-Viral Vectors
Naked Plasmid DNA
While primarily used for in vitro gene transfer, plasmid DNA
remains the most accessible tool for gene transfer in vivo .
Plasmids are circular DNA constructs that can be customized with a
versatile combination of transgenes and regulatory elements. Compared to
other vectors, naked plasmids can hold significantly larger quantities
of genetic information.6,7 Plasmids are also easy to
produce, with adequate infrastructure for clinical-grade plasmids
already in place.8 Naked plasmid DNA is
non-immunogenic; while an immune response can be mounted against the
foreign transgene product, there is no immune response generated against
the plasmid itself.9 This lack of vector-directed
immune response enhances safety and allows for potential repeated
administration of plasmid-based gene therapies.
However, naked plasmid alone transduces cells at therapeutically
irrelevant levels,10 and enhancement with transfection
reagents is only marginally effective for gene
uptake.11,12 Overcoming this limitation requires
select methods of administration, which will be discussed later in this
review. In addition, plasmid DNA is not integrated in the genome,
leading to a limited duration of expression. Prolongation of this
expression is under investigation through numerous studies on select
promotors. Our group has previously demonstrated expression of a
dominant-negative TGFβ II receptor under the control of a long-acting
polyubiquitin C (UBc) promoter for at least 3-4 weeks in a canine heart
failure model of atrial fibrillation.13 Similarly,
intermediate term gene expression has been demonstrated by others in
murine myocardium, bone, skeletal muscle, and
lung.14-18 While long term data has yet to be
reported, the option of repeated rounds of plasmid gene therapy could
compensate for loss of transgene expression over time.
Nanoparticles
Another choice of non-viral vector for myocardial gene transfer are
nanoscale liposomes. Lipid-based nanoparticles offer biocompatibility,
good cellular uptake, and can be deployed with targeting ligands to
enhance tissue specificity. The liposomal delivery mechanism for small
molecule drugs is already in clinical use as a chemotherapeutic vehicle,
and lipid-based nanoparticles containing a genetic construct have a
demonstrated ability for transducing cardiac
cells.19,20 However, off-target tissue effects may
still be encountered when nanoparticles are administered via systemic
circulation, and charged lipid particles are subject to rapid clearance
by the reticuloendothelial system. Future advances in liposomal
stability, distribution, and release offer potentially exciting avenues
for cardiac gene delivery.20,21
Another non-viral vector, modified-mRNA
(modRNA)
In the early 1990s, mRNA was successfully delivered to brain and
skeletal muscle.22,23 However, the use of mRNA as gene
delivery vector to mammalian tissue did not evolve since then. This is
mostly due to mRNA induced innate immune response via stimulation of
Toll-like receptors.24 Furthermore, mRNA is likely
cleaved by RNase in vivo.24 In 2005, Dr. Katalin
Karikó, who contributed to recent development of COVID-19 mRNA vaccine,
demonstrated that modifying mRNA’s secondary structure by replacement of
uridine with pseudouridine prevented innate immune system recognition
and RNase degradation.25
Compared to DNA vectors, modRNA has advantages and disadvantages as a
gene delivery tool. One advantage is that mRNA does not require
localization of nucleus or transcription process. modRNA gene delivery
has minimal risk of integration into the host
genome.26 modRNA has been shown to be highly efficient
with robust transient expression with no sign of innate immune
response.27 ModRNA is translated within minutes and
lasts up to 10 days in vivo.28 The use of modRNA in
heart is mainly for myocardial ischemia/reperfusion injury in ventricle
because of its transient pharmacokinetic profile.29,30Disadvantages of modRNA are unstable modRNA generation and the need for
repeated delivery due to its short expression pattern. To date, modRNA
has not been tested in AF treatment yet. If translation efficiency of
modRNA is improved, modRNA can be another non-viral vector for AF.
Viral Vectors
Viral vectors are live, replication deficient viruses which have been
genetically modified to replace the native viral genes with therapeutic
transgenes. Any cell that the vector infects integrates the transgene
payload to produce or inhibit a genetic product. Compared to non-viral
plasmids which must be delivered directly to the tissue of interest,
viral vectors have the theoretical advantage of minimally invasive
delivery via the bloodstream. There are three main types of viral
vectors used today in gene therapy, though the Adeno-associated virus
(AAV) is currently best suited for cardiac gene therapy.
Adeno-associated virus
(AAV)
First isolated as an unrelated contaminant in adenovirus samples, AAV is
a non-enveloped, non-integrating single-stranded DNA parvovirus. AAV
emerged as a focus of gene therapy vector development due to low
immunogenicity, potential for long duration of expression, and a robust
safety profile.31-33 AAV is capable of durable,
possibly life-long transgene expression in vivo : no upper limit
to duration of expression has been determined, with numerous studies
showing transgene expression years after a single administration.
Notably, AAV alone is incapable of productive replication and requires
coinfection with a helper virus, usually adenovirus or herpesvirus. The
lack of self-replication machinery increases the safety of AAV, but also
limits the size of its genome to about 4.7kb.
The primary AAV serotypes are AAV 1-9. While more serotypes and variants
have been characterized and silent infection is highly prevalent in
humans, no associated pathogenicity has been
identified.34 Each serotype has a distinct capsid
protein sequence correlating to variable tissue tropism, with AAV
serotypes 1, 6, 8, and 9 exhibiting the highest cardiac
tropism.35,36 By engineering the makeup of the viral
capsid proteins, it is possible to generate novel, chimeric AAVs with
improved transduction efficiency and tropism in rodent
models.37-39 Tissue specificity can also be achieved
with the use of site specific promoters to drive transgene expression
only in the atria.40 While transduction efficiency is
often more limited in scale-up from rodent to large animal models, these
emerging strategies are accompanied with recent FDA-approval for
non-cardiac gene therapies and a number of clinical trials utilizing an
AAV vector.41-43
The primary disadvantage of AAV vectors is limited transgene size. When
including a cardiac-specific promoter, many transgenes exceed the
maximal size for an AAV construct. Additionally, a clinical effect may
be delayed as gene expression requires conversion of the single-stranded
viral genome to the double stranded host genome.44AAV-mediated gene therapy is further hindered by the potential
pre-existing neutralizing immune response generated against AAV capsid
proteins, described in further detail in a following section.
Adenovirus (Ad)
The wild-type Ad is a non-enveloped, non-integrating double stranded DNA
virus, ubiquitous in the environment and one of the causative agents of
the common cold. Ad vectors are simple to produce, transduce both
dividing and non-dividing cells with high efficiency, and have a
packaging capacity for moderate sized genes.45However, in the heart, gene expression after Ad vector transduction is
robust but transient and Ads can trigger a strong innate immune response
and toxicity due to viral gene products.46 The use of
Ads came into serious question in 1999 after the death of a patient with
ornithine transcarbamylase deficiency due to a massive immune response
following injection of Ad vector.47
Recombinant modifications have given rise to first, second, and
third-generation Ad vectors, with key immunogenic components deleted.
These vectors show promise for evading the host immune response and
producing a prolonged gene expression, but are more difficult to
produce.48
Lentivirus (LV)
Lentiviral vectors are enveloped, integrating, single-stranded RNA
retroviruses42. In gene therapy, LV vectors are
usually derived from the HIV-1 virion, modified to be
replication-defective to safeguard against off-target continued
infection.49,50 Retroviral vectors typically require
active cellular division to integrate and express a transgene, but the
machinery of HIV conveys an ability to transduce intact nuclear
membranes in post-mitotic cells (such as cardiomyocytes), and
accomplishes long-term gene expression with moderate packaging
capacities.51-53 Despite this attractive profile for
efficacy, the LV apparatus of random genome integration with a
preference for coding regions poses a clinical safety precedent for
oncogenic transformation.34,48 While terminally
differentiated cardiomyocytes pose a lower mutagenic risk than
mitotically active tissues, the safety and efficacy of lentiviral
vectors for cardiac use have yet to be demonstrated in clinical trials.
Immunogenicity of Viral
Vectors
The promise of viral vectors is inseparable from the perennial obstacle
of inherent immunogenicity. Viral capsids are targets for the innate and
humoral immune responses, and foreign transgene products can trigger the
adaptive immune response. Adenoviruses are most notoriously associated
with immune provocation, resulting in declining use following adverse
events in previous clinical trials.54 Though the
advent of AAV-mediated gene therapy has alleviated many of the safety
concerns associated with the use of viral vectors, AAV infections are
silently endemic to many human populations. A geographically variable
but significant percentage (20 – 60%) of humans are predicted to have
pre-existing neutralizing antibodies (NAbs) against one or more AAV
capsids, rendering AAV-mediated treatments
ineffective.55,56 Furthermore, in naïve patients,
initial exposure to an AAV therapy results in generation of NAbs against
the AAV capsid, eliminating the potential for readministration of AAV
vector-mediated gene therapy.57 A complete
understanding of the significance of AAV NAb titers and cross-reactivity
between serotypes has yet to be established, posing a challenge for
clinical study enrollments. Lentiviral vectors possess an advantageous
ability to mostly evade the host immune system, however, the foreign
transgene product itself can still incite an immune response and
subsequent suppression. These altered proteins, while therapeutic, can
present to the adaptive immune system as a potent neo-antigen. In an
effort to overcome these sophisticated, protective host defenses,
immunomodulation at the time of vector administration is an area of
active research.58