Heart and Metabolism

Methods of gene transfer to the myocardium

Jean-Jacques Mercadier1,2, Damien Logeart2,3
1Departments of Physiology and Cardiology, Groupe Hospitalier Bichat–Claude Bernard,
Paris, France 2INSERM U 460, Faculté de Médecine Xavier Bichat, Paris, France
3Department of Cardiology, Hôpital Beaujon, Clichy, France
Correspondence: Dr Jean-Jacques Mercadier, Service de Physiologie–Explorations Fonctionnelles, Groupe Hospitalier Bichat–Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France. Tel: +33 1 40258404, fax: +33 1 40258800, e-mail: jjmercadier@wanadoo.fr

Abstract

Myocardial gene therapy was born at the beginning of the 1990s from the marriage of recombinant adenovirus technology with the characterization of well-defined pathophysiological mechanisms in the heart. The recent development of relatively simple vector delivery procedures has raised the possibility of treating diseases such as ischemic cardiomyopathies with angiogenic factors. Studies of experimental heart failure in the rat have shown that myocardial gene therapy can improve cardiac performance and prolong life. It is conceivable that within the near future this approach may be applied to human heart failure in specific settings such as open chest cardiac surgery. However, there are a number of obstacles to be overcome before myocardial gene therapy can spread to the field of routine clinical cardiology, among them the identification of a safe vector enabling good transduction efficiency rates to cardiac myocytes through the coronary arteries. The present article summarizes the various methods that have been developed during the past decade to transfer genes to cardiac myocytes, from the isolated perfused rat heart setting to vector delivery directly into specific branches of the coronary arteries in vivo. n Heart Metab. 2002;18:42–47.

Keywords: Gene therapy, biotherapies, myocardium, heart failure

Introduction
It is acknowledged that, in the future, gene transfer to somatic cells will provide a new therapeutic approach to treat various pathogenic processes, including those responsible for common acquired or degenerative diseases such as heart failure. However, there remain a number of methodological obstacles regarding, for instance, vector safety and infection efficiency, cardiac restriction, regulation of transgene expression, and, in particular, myocardial gene therapy and vector delivery procedures. Indeed, gene transfer to the myocardium should be as diffuse and homogeneous as possible, especially in heart failure therapy. Various vector delivery methods have been tried and have achieved variable results.
This paper summarizes the specificities of the adult mammalian myocardium with respect to gene transfer and the various methodologies that have been developed during the past decade, together with their relative efficacy.

Specificities of adult myocardium and consequences for gene therapy
The adult myocardium has a number of properties that represent either an advantage or a disadvantage for gene therapy. The adult myocardium comprises an almost equal amount of cardiac myocytes and nonmuscle cells, the former constituting the main part of its volume. Although some recent papers have suggested a low level of cardiomyocyte division within the adult myocardium [1], adult cardiac myocytes are terminally differentiated cells which have lost the capacity to divide. This has posed a major problem for a generation of researchers in the field of cellular cardiology, since it was almost impossible to transfer any cDNA into isolated adult cardiac myocytes. At the beginning of the 1990s, a major change came in the form of recombinant adenoviruses, allowing a very high rate of transduction of adult cardiac myocytes. This discrepancy is due to the fact that, at least schematically, naked cDNA can enter the cell only when cells divide, whereas viruses have specific, well-characterized receptors on the membrane of the target cell, the role of which is to facilitate virus penetration within the cell. Similarly, retroviruses cannot be considered for targeting cardiac myocytes, since they also require cell division. Therefore, only three types of vectors are currently able to transfer genes to adult cardiac myocytes with any significant efficiency: recombinant adenoviruses [2], adeno-associated viruses [3, 4], and lentiviruses (HIV family) [5]. However, despite their efficiency, the use of these vectors remains limited by a number of problems, notably the difficulty of obtaining high viral titers, their immunogenicity, some uncertainties about their safety, and their low capacity to allow long-term expression of the transgene. The experimentally widely used adenoviruses very easily infect adult cardiac myocytes, do not incorporate into the host cell genome (thus limiting the risk of insertional mutagenesis), and are not diluted with time, since myocytes do not divide. But adenoviruses are still responsible for a significant immune response due to the expression of residual viral proteins; and, despite the use of strong and/or tissue-specific promoters, the expression of the transgene decreases rapidly with time. The so-called “new generation” of adenoviruses should be less immunogenic, but the possibility of producing high viral titers in amounts sufficient to enable wide clinical use seems unlikely at present. Adeno-associated viruses and lentiviruses should enable a longer duration of gene expression, but it is also difficult to obtain high titers of these viruses. This is the reason why many research groups are currently working very actively at developing nonviral vectors which could allow significant DNA transfer to cardiac myocytes [6].

Methods to deliver genes to the myocardium
The possibility to express a foreign gene in the heart in vivo was first demonstrated during the early 1990s after intravenous or intramyocardial injection of naked cDNA [7] or cDNA-carrying adenoviruses [8, 9]. Once the superiority of adenoviruses had been demonstrated in terms of transfer efficiency, the limitations of these routes of delivery soon became apparent: less than 1% of cardiac myocytes stain positive for the reporter gene following intravenous delivery. However, these pioneering experiments heralded a period of active research to optimize the methods of vector delivery. Figure 1 illustrates the main methods that have been developed to date. Most, if not all, have used recombinant adenoviruses as the vector for gene transfer.

Figure 1. Main techniques used to transfer genes to the myocardium in vivo [10]. (A) Intracoronary injection; (intramyocardial injection; (C) intrapericardial injection; (D) injection into the left ventricular cavity with aortic clamping; (E) same procedure as in D, plus clamping of the pulmonary artery. SVC, Superior vena cava; IVC, inferior vena cava.
Reproduced from: Hajjar RJ, del Monte F,
Matsui T, Rosenzwieg A. Prospects from gene
therapy for heart failure Circ. Res. 2000;86:616–621.

Intramyocardial delivery
Even if transgene expression following direct intramyocardial injection is limited to the area of the needle track, this technique has a reasonable chance of development in the future in two areas. First, it is an ideal technique for myocardial gene transfer through the epicardium once the chest is opened, and it is likely that surgeons will take advantage of it to deliver genes encoding angiogenic factors or a combination of genes and “wild” or transfected cells in the context of the future “biotherapy” of myocardial diseases. Second, the approach has been used successfully to deliver genes through the endocardium using electromagnetic guidance of the catheter in the left ventricle [11]. However, it is important to point out that among the specifications of myocardial therapy, an important one is the need for homogeneous expression throughout the myocardium and therefore delivery of the transgene throughout the myocardial wall to avoid heterogeneous expression. It is unlikely that direct myocardial injection will ever fulfill this requirement. Alternatively, if the above requirement is essential for the transfer of a gene that modifies the electrical activity of cardiac myocytes, its importance diminishes if myocyte contraction and/or relaxation is the targeted function, and still further if the goal is to stimulate myocardial angiogenesis.

Intrapericardial delivery
Injection of recombinant adenoviruses into the pericardium leads to significant expression of the transgene in the myocardium [12]. However, because of the barrier formed by the epicardium, this expression is limited to the subepicardial area except in newborn animals in which the epicardium is loose and the myocardium rather thin. Nevertheless, a recent study has shown that the injection of various enzymes — collagenase, hyaluronidase — into the pericardium before adenovirus delivery facilitates the diffusion of the vector throughout the ventricular wall [13]. Several studies have used this route to deliver angiogenic factors, with contrasting results [14]. The clinical relevance of this route of vector delivery must, however, be strengthened by a demonstration of its efficacy and tolerance in larger mammalian species.

Endovascular delivery
Intravenous injection of high doses of adenoviruses cannot yield significant amounts of transduced cardiac myocytes. Therefore, it became clear that direct intracoronary vector delivery would be necessary if a significant degree of gene transduction to the myocardium were to be achieved. This was reinforced by the results of the pioneering study of Barr et al. [15], who showed that the simple direct injection of 1010 plaque forming units of an adenovirus carrying the lacZ gene (gene coding for b-galactosidase, enabling identification of transduced myocytes by a blue colorimetric reaction) through the ostium of the left coronary artery yielded 32% of transduced myocytes. Clearly, infection was not specific to the cardiac myocytes and most of the endothelial cells of the great coronary arteries and capillaries were also infected. Such spectacular efficiency of such a simple method of delivery has proved difficult to reproduce: Mühlhauser et al [16], for instance, showed that intracoronary adenovirus delivery was inefficient in comparison with direct intramyocardial injection. A number of research groups, including ours [17], have sought pharmacological means to overcome this barrier. Using the Langendorff isolated perfused rat heart model, we and others showed that the permeability of the endothelium for adenoviruses can be markedly increased by pretreatment of the coronary vasculature with mediators of inflammation, such as histamine, serotonin, or bradykinin [18–20]. Moreover, we showed that the facilitating effect of these agents was almost nil if it was not accompanied by a 1-minute interruption of the coronary flow once adenoviruses were present in the coronary microcirculation [20].
In order to get closer to the delivery conditions applicable in the clinical setting, several methods have been developed in various mammalian species. The method shown in Figure 1D and 1E, first described by Hajjar et al. [21] in rats, consists in injecting the vector under high pressure through a catheter inserted into the left ventricle or aortic root, just above the aortic cusps, while the aorta, or aorta and pulmonary artery, is clamped for 10 to 40 seconds. This method allows sufficient gene transduction to achieve a functional effect on cardiac contractility [21, 22]. This initial work by Hajjar et al should not be underestimated, as it was the first to demonstrate that gene transfer to the myocardium is able to improve cardiac function in vivo. Although the pressure in the left ventricle and coronary arteries was not measured in this study, it is likely that left ventricular contraction against a clamped aorta forced the adenovirus-containing blood to pass through the coronary vessels under such a high pressure that it opened spaces between the endothelial cells, thus allowing adenoviruses to reach the interstitial space and cardiac myocytes. However, because of the high systolic pressure in the left ventricle, this technique favors the transduction of the subepicardial layers of the myocardial wall. The advantage of clamping the pulmonary artery is that, by decreasing the venous return to the left ventricle during clamping, it lowers left ventricular filling pressures, thus enabling better perfusion of the subendocardial myocardial layers and better viral transduction at this level. To date, no transfer of this method to a larger mammalian species appears to have been attempted. One may speculate whether such a method will eventually be applied in humans.

Figure 2. Comparison of the various methods used, to date, to deliver adenoviruses (containing a cDNA encoding luciferase) to rabbit myocardium in vivo [23]. RLU, Relative light units. After [24].

In a recent study we compared the transfer efficiency of the various methods of adenovirus delivery in the rabbit (Figure 2) [23]. We found in vivo what we first observed ex vivo using the isolated rat heart model, namely that a coronary flow interruption of 30 to 40 seconds at the moment of adenovirus delivery is necessary to achieve significant myocardial gene transduction. Transduction efficiency doubled when the injection pressure was increased up to 400 mm Hg for a few seconds, a procedure which showed no obvious deleterious consequences for the rabbits during up to 3 days of follow-up. The rate of transduction was further increased fivefold, without the need to increase intracoronary pressure, by closing the coronary sinus on the venous side during virus injection. A significant transduction efficiency was also observed after adenovirus delivery through the sole retroperfusion of the pig coronary sinus [25]. It seems, therefore, that the combination of a few relatively simple techniques that can easily be performed in the setting of interventional cardiology enables a very significant rate of gene transfer to the myocardium, at least in the perfusion area of a given coronary artery branch. Last, a new method of intravenous gene delivery has recently been developed, based on the systemic delivery of vectors mixed with particles which burst within cardiac capillaries under the effect of an ultrasound beam delivered to the myocardium through the chest wall, thus favoring vector transfer to cardiac myocytes [26].

Myocardial gene therapy: for which cardiac diseases?
Because we are constantly learning more about the molecular mechanisms underlying the alterations in cardiac myocyte function in cardiac diseases, including heart failure, the field of potential application of myocardial gene therapy is continually increasing. For a number of reasons, which cannot be detailed here, it seems unlikely that myocardial gene therapy will be suitable for inheritable ion channel disorders for some time. The same caution may be applied to familial hypertrophic cardiomyopathies, the pathophysiology of which is very complex and leads to a number of phenotypes with varying degrees of mortality risk. By contrast, familial dilated cardiomyopathies at the stage of overt heart failure, which share a number of pathophysiological processes with common heart failure at the late stages of the disease, could be considered as candidates for myocardial gene therapy. Finally, and somewhat unexpectedly, it seems that it will be the most common form of heart disease, congestive heart failure, with its large number of functional alterations and changes in gene expression, that will probably be the first to benefit from myocardial gene therapy. Indeed, because of the poor short-term prognosis of this disease state, despite currently available pharmacological therapy, and the relatively low risk of myocardial gene therapy together with the shortage of donor hearts, the first clinical trials could soon be undertaken in patients in NYHA stage IV awaiting cardiac transplantation, once the problems of vector delivery have been overcome.

REFERENCES

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