Gene Therapy for Atrial Fibrillation

Introduction

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a frequent cause of stroke. ^, Despite its clinical importance, AF is a difficult condition to treat, with limited efficacy of current therapeutic strategies including antiarrhythmic drugs and catheter ablation. A major reason for the low efficacy of the current therapies is that they do not target the molecular mechanisms underlying AF. Antiarrhythmic drugs, which predominantly function as ion channel blockers, are limited by the lack of tissue specificity, with a significant risk for ventricular proarrhythmogenesis. In recent years, some investigators have used gene-based approaches to directly and specifically target the signaling pathways in the atrial myocardium that underlie the creation of electrical and structural remodeling in AF. All studies of gene therapy for AF are in the preclinical stage, with the most promising results having been obtained in large animal models that parallel the electrical and structural remodeling seen in humans (e.g., canine and porcine). There are two main problems that must be solved for a gene therapy to be successful. The first is a question of genetics and molecular biology: Which genetic program will, if run in target cells, reverse the disease phenotype? This answer is different for every disease and can range from a simple genetic knockout to the expression of an engineered protein with novel functionality. The second question is how to deliver and express that genetic solution (herein referred to as a transgene) in vivo in a tissue- and cell type–specific manner.

This chapter thus focuses on both aspects of gene therapy for AF, first summarizing current technology for myocardial transduction and then detailing contemporary gene-based strategies for the treatment of AF.

Myocardial Transduction

Myocardial transduction, or the delivery and expression of a therapeutic transgene in the cells of the myocardium, is the primary metric that governs efficacy for cardiac gene therapy. Myocardial transduction is governed by three interrelated variables. The first of these variables is the vector itself. Vectors can take many forms, from naked plasmid DNA to genetically modified viruses to highly engineered nanoparticles ( Table 55.1 ). The second is the route of administration. Although the most common route of administration is intravenous (IV), a wide variety of techniques have been used to bring the vector into contact with the myocardium. Finally, the transgene itself and its associated regulatory elements represent the third factor governing myocardial transduction. All three can have profound consequences on the efficacy of a cardiac gene therapy and will be discussed in detail.

TABLE 55.1

Cardiac Gene Delivery Strategies

Vector	Transfer Material	Diameter	Duration of Expression	Packaging Capacity	Cardiac Specificity	Immunogenicity	Comments
Nonviral Vectors
Nanoparticles (liposomes, polymerosomes)	Any nucleic acid	20 nm–200 μm	Dependent on cargo	Dependent on cargo	Possible with targeting ligand, liver off targets	Low	Customizable tropism when conjugated to targeting ligand
Naked plasmid	Plasmid DNA	N/A	Intermediate	Large	Highly specific via direct injection, higher with cardiac electroporation	Low	Low transduction without secondary transfection technique
Viral Vectors
Adenovirus	dsDNA	70–100 nm	1 month	36 KB	Nonspecific	High	Not suitable for long-term therapy
AAV 6	ssDNA	20 nm	Long term, episomal	4 KB	High	Low	Superior via direct administration
AAV 9	ssDNA	20 nm	Long term, episomal	4 KB	High cardiac, high liver	Low	Superior via systemic administration
Lentivirus	ssRNA	80–120 nm	Long term, integrating	10 KB	Nonspecific	Moderate	Concerns of insertional mutagenesis

AAV, Adeno-associated virus; dsDNA , double-stranded DNA; ssDNA , single-stranded DNA; ssRNA , single-stranded RNA.

Vectors

Nonviral Vectors: Naked Plasmid DNA

Although primarily used for in vitro gene transfer, plasmid DNA remains the most easily accessible tool for gene transfer in any form. These circular DNA constructs can be customized with any number of transgenes and regulatory elements, allowing for maximum freedom in transgene engineering. Compared with other types of gene therapy vectors, which are limited in their packaging capacities, naked plasmids can hold massive amounts of genetic information. ^, Plasmids are also much easier to produce; significant infrastructure already exists for clinical scale manufacturing of guanosine monophosphate (GMP) plasmid. Furthermore, naked plasmid DNA is nonimmunogenic; there is no immune response generated against the plasmid itself, although an immune response against the foreign transgene product can still occur. This lack of vector-directed immune response allows for readministration of plasmid-based gene therapies, in addition to being an added safety feature of plasmid-mediated gene therapy.

Although potential for readministration is a desirable trait for all gene therapies, it is especially important for plasmid-based therapies. Indeed, recurrent dosing of plasmid-mediated gene therapy can compensate for many of the weaknesses associated with this vector platform. An open question that overshadows plasmid-mediated gene therapy is the duration of expression. Plasmid DNA does not integrate into the genome, so it is unfit for gene therapy in cell populations that are dividing. Cardiomyocytes, however, are postmitotic, so there is no concern that cell division will dilute the transgene and render a gene therapy ineffective over time. The duration of plasmid DNA expression in vivo, however, remains limited in time, even for postmitotic cells. Transgene silencing can occur through a wide array of silencing techniques. Interferon-γ and tumor necrosis factor-α have both been implicated in the mRNA-mediated silencing of transgenes expressed from viral promoters. Other studies have shown that unmethylated CpG repeats contained in the bacterial plasmid backbone are similarly immunogenic and lead to silencing. Although transgene silencing is thus problematic for some gene therapy constructs, numerous studies have shown longer expression of plasmid-mediated gene expression in vivo, particularly for polymerase III–dependent promoters. Our group has previously demonstrated expression of a dominant-negative transforming growth factor (TGF)-β II receptor under the control of a long-acting polyubiquitin C (UBc) promoter for at least 3 to 4 weeks in a canine heart failure (HF) model of AF. Studies have also demonstrated plasmid-based gene expression for 4 to 5 weeks in murine myocardium and for 6 weeks in murine bone. Escoffre et al. studied plasmid-based gene expression after electroporation of plasmids in murine skeletal muscle. They detected enhanced green fluorescent protein (EGFP) expression until the end of the study at 78 days. In a related study, Eefting et al. showed evidence of plasmid-mediated small interfering RNA (siRNA) gene knockdown for longer than 100 days in the skeletal muscle. Yew et al. further showed robust transgene expression in mouse lung 35 days after administration using the human ubiquitin B promoter, which was further enhanced over the viral cytomegalovirus (CMV) promoter by the depletion of CpG motifs in the plasmid itself. Although intermediate-term gene expression has proven possible, truly long-term data have not been reported. Even if longer term (>1 year) expression is possible, multiple rounds of gene therapy could compensate for loss of transgene expression over time were it to occur.

Naked plasmid alone transduces cells at therapeutically irrelevant levels, so the attractiveness of naked plasmid DNA as a gene therapy vector is limited. Combining naked plasmid with transfection reagents is similarly ineffective; lipid-mediated transfection technology only achieves a negligible amount of gene transfer into the myocardium, and complexing agents, such as calcium phosphate, only marginally improve the efficiency of gene uptake. These limitations can be overcome, however, via creative routes of administration, discussed later in this chapter.

Nonviral Vectors: Nanoparticles

Another type of vector for myocardial gene transfer is nanoparticles. These nanoscale molecular entities can be created using a variety of combinatorial chemistry techniques and range from 20 nm to 200 μm in diameter. Nanoparticles are generally nonimmunogenic and can be functionalized with targeting ligands to enhance tissue and cell type specificity. Lipid-based nanoparticles have high capacities for cellular uptake and endosomal escape, in addition to enhanced protection of the encapsulated genetic transfer material during circulation. Lipid nanoparticles have been used in a wide variety of studies for transducing cardiac tissue ^, and show great promise for further exploration. One issue plaguing nanoparticle-mediated delivery is the on-target delivery rate. Nanoparticles have been shown to effectively increase drug concentrations in target tissues; however, focusing on this increase in on-target concentration ignores the extensive (around 95%) off-target delivery of these vectors. Specific targeting of nanoparticles using conjugated antibodies or protein motifs against target cell receptors can enhance this specificity, but current methods of nanoparticle targeting are limited by the body’s natural clearance of these particles.

Viral Vectors

The field of gene therapy would not be possible without viral vectors. At a basic level, viruses are molecular entities that transport genetic material from one cell to another. In the wild, this genetic material is the viral genome, which contains instructions for taking over a host cell and creating more viral particles. In gene therapy, however, the viral genes are replaced with a transgene, creating a viral vector. Any cell that the vector “infects” receives a copy of the transgene instead of pathogenic viral genes. In early studies to test transduction, this transgene has usually been a reporter gene, such as green fluorescent protein (GFP) or β-galactosidase (LacZ). Once a gene therapy is more advanced, however, that reporter will be replaced with a therapeutic transgene. There are three main types of viral vectors used today in gene therapy, but one is currently best suited for cardiac gene therapy.

Adenovirus

The first vector to be widely used for cardiac gene therapy was the adenovirus. ^, The wild-type adenovirus is a double-stranded DNA virus that is one cause of the common cold. Adenoviral vectors have a 35 KB packaging capacity, which allows for the delivery of moderate-sized genes. Adenoviral vectors are simple to produce and transduce target cells with high efficiency. Nevertheless, adenoviral vectors are highly immunogenic, which makes them unappealing for long-term gene therapy applications. This immunogenicity leads to limited duration of expression (2–4 weeks) because of immune cell clearance of transduced cells. Indeed, a 2016 clinical trial using adenovirus to express adenylyl cyclase 6 in patients with heart failure (HF) showed improved ejection fraction at 4 weeks but not at 12 weeks. Immunogenicity can also cause intense immunological host responses, causing short-term morbidity, organ damage, and even death. For this reason, adenovirus has largely fallen out of use for clinical gene therapy. ^, The high short-term expressivity of adenovirus, however, makes it appealing for the prevention of short-term diseases, such as postoperative AF. In a porcine model, Donahue et al. demonstrated that KCNH2 gene transfer could prevent postoperative AF for up to 10 days, after which the percentage of animals in sinus rhythm decreased commensurate with transgene expression (see later).

Lentivirus

Lentiviral vectors, a genus of retroviruses, are common tools for in vitro molecular biology but are also the primary vectors used for ex vivo gene therapy. Lentiviral vectors are based on the human immunodeficiency virus (HIV). These vectors can transfect intact nuclear membranes, allowing for transgene expression in terminally differentiated cells such as cardiomyocytes. A major advantage of lentiviral technology is long-term gene expression and multiple safeguards to protect against wild-type reversion. Compared with other viral vectors, lentivirus appears to have similar transfection efficiency when injected into the myocardium. Additionally, lentiviral vectors have moderate packaging capacities, with transgenes cassettes of up to 10 KB having sufficiently high expression levels in vitro. The safety and efficacy of lentiviral vectors for cardiac use have yet to be demonstrated in any clinical trials. One major issue with in vivo use of lentivirus is the fear of insertional mutagenesis. In ex vivo applications, insertional mutagenesis is not a concern because all cells that will be reintroduced into the body can be screened for transgene insertions in an oncogene or tumor suppressor. Because cardiac gene therapy requires in vivo transduction, lentiviral gene therapy would have a formal risk for causing cancer. Although a case of lentivirus-mediated oncogenesis has yet to be observed, the risk has made researchers hesitant to pursue lentiviral gene therapy in vivo .

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here