Copyright © 2006, European Society of Cardiology
AAV vector re-targeting: A small step on the way to cardiac-specific gene transfer
Institut für Herz-und Kreislaufphysiologie, Heinrich-Heine-Universität, Universitätsstr.1, 40225 Düsseldorf, Germany
* Corresponding author. Tel.: +49 211 8112675; fax: +49 211 8112675. Email address: Axel.Goedecke{at}uni-duesseldorf.de
Received 6 February 2006; accepted 9 February 2006
See article by Müller et al. [8] (pages 70–78) in this issue.
Gene and stem cell therapy are two visions of modern medicine that could have the potential to redirect patient treatment from a pharmacological/interventional to a molecular approach. The idea behind both strategies is the perspective to causally treat disease. Both visions are attractive, both promise causal therapies. Therefore, it is not too astonishing that both approaches have caused hopes and hypes. However, as sometimes observed when a new field moves from the first spectacular results into broader investigation, problems and pitfalls become apparent that dampen the expectations of fast solutions.
In 1999, the field of gene therapy was shaken by the case of Jesse Gelsinger, who died in a clinical gene therapy trial for liver disease. Three years later, in French clinical trials to cure children with severe combined immunodeficiency, three children treated with retrovirally transduced cells developed cancer, most likely as a result of insertional mutagenesis by the retrovirus. Consequently, gene therapy trials were put on hold and doubts came up whether gene therapy represents a realistic future option in medicine [1]. These cases also showed in a tragic manner the need to send researchers from the bedside back to the bench to solve basic problems of somatic gene therapy.
In general, somatic gene therapy requires the solution of a complex set of problems that are all connected to the following three questions briefly addressed below:
- 1 What is my therapeutic gene?
The identification of the therapeutic gene is, of course, closely related to the biological dysfunction that is to be cured. In the cardiovascular field, local expression of cell cycle inhibitors, suicide genes, or NO synthases were able to attenuate the development of restenosis after balloon angioplasty. In addition, therapeutic angiogenesis still represents an interesting approach for the treatment of cardiac and peripheral ischaemia [2]. Experimental studies demonstrated that cardiac function is also amenable to modulation by somatic gene transfer. Overexpression of a mutant phospholamban to improve cardiac function in cardiomyopathic hamsters [3] or the generation of new pacemakers by local expression of dominant negative Kir2 potassium channels are encouraging examples [4].
- 2 Which is the appropriate delivery system to introduce the vector into an adult organism?
A beneficial therapeutic effect of a gene when expressed in the target cell type may turn into the opposite when it is expressed at another site. Thus, a spatially restricted expression of the target gene is in most cases an absolute requirement for successful gene therapy. Spatial specificity maybe achieved, at least in part, by a specific route to deliver the vectors. For vascular gene therapy, many specific devices for local gene transfer have been developed on the basis of the well-established catheter technology that is routinely used in interventional cardiology [5]. A gene transfer to the heart, however, is much more demanding. Direct injection into the ventricular wall is possible but does not allow a global transfection of myocardial cells. The problem of an inhomogeneous transfection was solved by injection of vectors via a catheter into the aortic root [6]. After cross clamping of the aorta and pulmonary artery, the beating heart then pumps the vector particles with high pressure into the coronary circulation and across the endothelial barrier into the myocardium. Whereas this technique has widely been used in experimental animal models, a similar application in humans with the intention to cure a diseased heart is not possible. Rather, more gentle methods such as retroinfusion of vectors or a catheter-based strategy [7] may be preferable methods.
- 3 What is the best vector and promoter to achieve sufficient levels of expression?
To date efficient gene transfer into the heart requires high titres of viral vectors to achieve homogeneous transfection of the heart because transfection times in the ischaemia-sensitive heart must be kept short. A high titre of viral vectors compensates for the time restrictions. The most successful vector system for cardiac gene transfer is made up of adenoviral vectors, which can easily be produced with titres of >1011 per ml. However, transfection for a short time using high viral titres comes at the cost of specificity. Once the heart is reperfused, the majority of the viral particles instilled in the coronary circulation are transferred to the systemic circulation. Adenoviral vectors are then trapped in the liver, which in most experiments is massively transfected.
Thus, it appears that a bottleneck in cardiac gene therapy approaches resides in a low affinity of viral capsids for cardiac myocytes as compared to liver cells. Theoretically, an improved level of vector specificity for the heart can be achieved by alteration of the viral tropism towards a cardiotropic vector, and on the transcriptional level by specific cardiac promoters. Whereas the first strategy would allow minimization of transfection outside of cardiac myocytes, the transcriptional strategy would restrict expression to the target cells.
In this issue of Cardiovascular Research, Müller et al. [8] used a combination of both strategies to develop vectors based on adeno-associated virus 2 (AAV-2) to improve myocardial gene transfer. Although not all of the critical problems are solved, the combination of an altered tropism with a promotor which predominantly directs gene expression to cardiac myocytes enabled efficient transfection of the heart by simple tail vein injection in a mouse model.
AAV belongs to the group of parvoviruses that are unable to pass through a full infectious cycle. Rather, AAV depends on gene products of adenoviruses to produce offspring particles [9]. One advantage of this vector system is that high-titre virus stocks can be prepared. In addition, no obvious pathology is connected to an AAV virus infection and therefore it is assumed that future human applications would be associated with minor risks. However, AAV exhibits a strong liver tropism that makes this virus prima vista not an ideal tool to transfect the heart. Investigation of the infection process showed that the primary cell-surface receptor for AAV2 appears to be heparan sulfate proteoglycan (HSPG). In addition,
vβ5-integrins and the FGF-1 receptor act as co-receptors that support the infection by AAV2. In an earlier work, the HSPG binding motif on AAV2 capsids was identified [10], and exchange of two basic arginine residues for glutamic acid resulted in detargeted particles that greatly lost their ability to bind to HSPG and to transfect the liver. Müller et al. now combined these detargeted vectors with cardiac-specific promoters. This combination resulted in an undetectable expression and transfection of the liver, demonstrating the power of the chosen strategy. Thus, the work by Müller represents an important progress on the way to cardiac-specific gene transfer.
However, there is still a long way to go until a safe cardiac myocyte-restricted gene transfer can be achieved. Despite all the improvements, the work by Müller unmasked the promoter specificity as the weak point of the strategy. When packaged in AAV wild-type capsids, luciferase activity was detected not only in heart but also to a substantial amount in skeletal muscle, liver, and lung. Whereas basal activity in the liver was eliminated by the use of the detargeted capsids, the expression in skeletal muscle and lung remained. Thus, better promoters with higher specificity are required to achieve a cardiac myocyte-specific expression. In transgenic mice, the-MHC promoter displays the highest level of cardiac myocyte-specific expression [11]. Unfortunately, the length of the promoter is 5.7kb, much larger than the maximal packaging capacity of an AAV capsid, and therefore this promoter cannot be used with the AAV vector system.
Although Müller et al. do not use the term "gene therapy" anywhere in their paper but rather refer to cardiac-specific gene expression as an alternative to transgenic animals, the link to cardiac gene therapy is obvious. This work clearly underscores the problems that still have to be solved to achieve cardiac myocyte-specific gene therapy in humans. The strategy to combine an altered viral tropism with a cell-type specific promoter is a step in the right direction. However, we have to wait and see whether the small AAV genome size will offer enough space to solve all of these problems.
- 2 Which is the appropriate delivery system to introduce the vector into an adult organism?
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