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