Adeno-associated viruses (AAV) are members of the family Parvoviridae, and constitute non-pathogenic, non-enveloped viruses containing a 4.7 kb single-stranded DNA genome that encodes the structural proteins of the viral capsid, encoded by the cap gene and the non-structural proteins necessary for viral replication and assembly, encoded by the rep gene, flanked by short inverted terminal repeats. Their life cycle involves two phases, the replicative and the latent phase. In the productive phase, it co-infects the host cell only when a helper virus is present [29]. In the absence of a helper virus, AAV usually enters the latent phase, integrating into the human genome, commonly within a specific region of chromosome 19, although this has been observed only in cell lines. Its principal advantage for gene therapy is the poor inflammatory response to infected cells. As a therapeutic vector, AAV consists only of the inverted terminal repeats which are necessary for replication, packaging, and integration, while the viral coding sequences are entirely removed, rendering AAV vectors replication-deficient. The resulting recombinant vectors can efficiently deliver a transgene and safely mediate long-term gene expression in dividing and non-dividing cells of numerous tissues. AAV has the potential to facilitate long-term transgene expression in the absence of destructive T-cell responses, and such vectors have been generally proven safe. However, naturally occurring AAV variants are typically inefficient in infecting a number of stem cell types, particularly human embryonic stem cells [29].

There are 13 reported serotypes of AAV that display different tissue tropism, depending on the structure of the capsid protein. Serotypes AAV-1, 6, 8 and 9 have been shown to be the most cardiotropic. However, significant transduction in non-target tissues such as liver, skeletal muscle and lung has been documented [4]. In order to improve AAV cardiotropism, several methods have been introduced and novel viral capsid AAV libraries were constructed through DNA shuffling and chimeric strain production. This strategy simultaneously enhances tissue tropism and also helps AAV-based vectors to evade naturally occurring neutralizing antibodies [30–34]. Neutralizing antibodies to various AAV serotypes are present in approximately 20–80 % of the population, severely limiting the potential therapeutic use of AAV. Thus, the presence of such antibodies constitutes a major exclusion criterion in many AAV-based clinical trials [35]. There are numerous publications describing the efficient transduction of cardiac cells by AAV vectors [36, 37], and it appears that the AAVs constitute the most effective way for gene transfer in cardiac cells to date. Moreover, there are ongoing gene therapy clinical trials for HF in which gene transfer is mediated by AAV vectors.

Specifically, the first clinical trial of gene therapy was launched in the United States in 2007 [35, 38] and was based on gene delivery of the SERCA2 cDNA via a recombinant AAV1 (AAV1.SERCA2a) for calcium up-regulation in patients with advanced heart failure (CUPID). The study was a two-part, Phase 1/2 multicenter trial designed to evaluate the safety and the biological effects of gene transfer of the SERCA2a cDNA via the recombinant AAV1. In part 1, participants were administered a single intracoronary infusion of AAV1.SERCA2a in an open-label approach. A 12-month follow-up of these patients documented an acceptable safety pattern; moreover, improvement of cardiac function was detected in several patients. Overall, the results of the specific study suggested that gene transfer of AAV1.SERCA2a confers quantitative biological benefit. During the second part of the trial, 39 patients with advanced HF, received the AAV1-mediated SERCA2a transgene in one or three doses of DNase resistant particles (DRPs), i.e. low dose (6 × 10¹¹ DRPs), middle dose (3 × 10¹² DRPs), and high dose (1 × 10¹³ DRPs), or placebo [39]. Over a 1-year period, the cumulative recurrent cardiovascular events such as death, heart failure admission, left ventricular assist device (LVAD) insertion, cardiac transplantation, and myocardial infarction, increased in the placebo group but not in the low and middle-dose AAV1.SERCA2a groups which exhibited diminished recurrent cardiovascular events for the first 6 months. However, from months 6 to 12, these groups demonstrated events that were similar to placebo. The high-dose AAV1.SERCA2a group, continued to do significantly better than all groups at 12 months without any increase in adverse events, laboratory abnormalities, or arrhythmias [39]. Furthermore, the CUPID 2 Phase 2b trial (NCT01643330) is underway, designed to evaluate whether increasing SERCA2a activity via gene therapy improves clinical outcome [40]. Available data so far, indicate that calcium up-regulation by AAV1/SERCA2a gene therapy is safe and of potential benefit in advanced HF [40]. The promising results of the CUPID clinical trial, have prompted researchers to conduct two other clinical trials targeting SERCA2a, that will be enrolling patients soon. The first trial, which is conducted in the United Kingdom, includes patients with advanced heart failure who received LVAD at least 1 month prior to treatment. These patients will be treated either with AAV1.SERCA2a or saline. Simultaneously, a Phase 2, single-center, double-blind, randomized, placebo-controlled, parallel-group study will be conducted at the Institute de Cardiologie Pitié-Salpêtrière in Paris, France, with the primary objective of investigating the impact of AAV1.SERCA2a on cardiac remodelling parameters in patients with severe HF [4].

Human adenoviruses constitute another viral family used in gene therapy approaches, which are classified into 51 immunologically distinct serotypes divided into six subgroups, namely subgroups A–F. Adenoviruses are non-enveloped, double-stranded (ds) DNA viruses and contain large protein complexes that bind to CD46 or to coxsackie-adenovirus receptor (CAR), depending on the serotype for viral binding and cellular entry. Upon cellular entry via clathrin-mediated endocytosis, the dsDNA is transported to the nucleus across nuclear pores, allowing efficient transduction of both mitotic and non-mitotic cells. Their genome size ranges from approximately 30 to 35 kb and contains five segments that encode early gene products (E1a, E1b, E2a, E2b, E3, and E4), and five segments that encode late gene products (L1–L5). The E1, E2, and E4 gene products regulate transcription and translation of the late genes and therefore, are indispensable for viral replication [41]. Adenoviral vectors are among the most promising gene transfer vehicles for the in vivo treatment of a range of human diseases, e.g. cystic fibrosis and haemophilia, because of their ability to infect a wide spectrum of cell types, including quiescent cells. Adenoviral vectors present the following advantages: (a) they provide high efficiency of gene transfer because they are able to deliver many genome copies per target cell that typically translates into very high expression, (b) they can deliver relatively large therapeutic genes of approximately 25–30 kb in size especially with the latest generation vectors, and (c) they confer high fidelity of gene transfer because the vector genomes are genetically stable. However, although replication-deficient vectors based on adenoviruses can be produced easily and at high titres, two main disadvantages occur, i.e. (a) the transgene’s expression is transient and typically lasts 1–2 months in non-dividing cells, while is much shorter in dividing cells and (b) there is a significant induction of both cellular and humoral host immune response. First-generation adenoviral vectors may be applied where short-term expression and single dosing is desirable, such as cancer vaccine therapies. Initial enthusiasm toward the use of first-generation adenoviral vectors in gene replacement therapy has diminished, because not only they did fail to achieve sustained gene transfer, but also resulted in significant toxicity and death of an individual [41]. Furthermore, the pre-existing immunity against adenoviruses in patients, may result in low levels of transgene delivery and expression. Third-generation gutless or helper-dependent adenoviral vectors have attenuated immunogenic potential, but still a significant risk remains. Finally, CAR receptors are particularly prevalent in liver tissue, thus limiting adenoviral tissue specificity. Due to the aforementioned limiting factors, adenoviral mediated strategies are limited in cardiovascular gene therapy trials [42].