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Symposium Report |
1 British Heart Foundation Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, Church Street, Glasgow G11 6NT, UK
Abstract
Clinical gene therapy for cardiovascular disease remains achievable. To date, however, preclinical studies and clinical trials have highlighted shortfalls in viral gene delivery to vascular cells. These include poor efficiency, poor target tissue selectivity, the presence of pre-existing neutralizing antibodies and immunogenicity generated by the host to vectors such as adenovirus. These important issues require careful consideration when applying viral vectors for gene therapy. Each delivery vector requires precise optimization and tailoring for each disease application since parameters relating to vector : tissue exposure time, route of delivery and target cell type vary considerably. Optimization can be achieved through modification of the structure of the virus capsid proteins and expression cassette to generate vectors that are highly selective and efficient for target cell binding and entry as well as instilling transcriptional control and/or longevity on transgene expression. This ultimately will improve the efficacy and toxicity profiles of gene delivery vectors and has become a very important area in gene therapy. Here, we review recent advances in the targeting of viral gene delivery vectors to the vasculature.
(Received 19 August 2004;
accepted after revision 15 October 2004; first published online 12 November 2004)
Corresponding author Andrew H. Baker, British Heart Foundation Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, Church Street, Glasgow G11 6NT, UK. Email: ab11f{at}clinmed.gla.ac.uk
Many setbacks have hit the gene therapy community in recent years, not least the death of Jesse Gelsinger (Lehrman, 1999) and the development of leukaemia in two patients on the severe combined immune deficiency syndrome (SCID) trials in France (Hacein-bey-abina et al. 2003; Kohn et al. 2003). Concerns relating to viral vectors for gene delivery are further compounded by immunogenicity, pre-existing antibodies throughout the general population and the potential for insertional mutagenesis by certain viral systems. These problems, however, are counterbalanced by some extremely positive results in clinical gene therapy to date. Many patients on the retroviral severe combined immunodeficiency (SCID) trials, having a disease fatal for those patients without matched bone marrow transplantation, are in disease-free remission many years after gene delivery. In the vasculature, delivery of adenoviruses to ischaemic myocardium has been well tolerated with some limited signs of clinical benefit (Grines et al. 2002; Hedman et al. 2003). Many other trials are on-going for diverse clinical problems and the outcomes (both in terms of therapeutic gain and vector-associated toxicity issues) are keenly awaited.
It is over a decade since the first studies defined the capacity to deliver genes to vascular cells in vivo (Nabel et al. 1991). Historical examples of therapeutic avenues of research pursued include gene therapy for postangioplasty restenosis (Baek & March, 1998), prevention of vein graft failure (Baker et al. 1997) and therapeutic angiogenesis to alleviate myocardial or peripheral ischaemia (Yla-Herttuala & Alitalo, 2003). During the course of early experiments it became obvious that direct access to the target tissue combined with the use of high titres of viral vectors were required to achieve sufficient levels of transgene at the target site to provide therapeutic benefit. This was achieved either by local vessel dwell procedures, ex vivo vein transduction prior to grafting or direct injection via invasive surgery or through catheters. Intravenous routes of gene delivery to the vasculature were not appropriate unless liver transduction was the target (e.g. for modification of lipid metabolism or use of the liver as a factory for soluble transgene production). While these studies have produced valuable preclinical data and some encouraging clinical data in early trials (Yla-Herttuala & Martin, 2000; Grines et al. 2002; Hedman et al. 2003), it would be highly advantageous if gene delivery to vascular cells could be improved in terms of efficiency (thus allowing reduced doses to be used and potentially reduced immunogenicity) and improved selectivity (thus reducing potential deleterious side-effects of transgene expression at non-target sites). This would improve rates of local gene delivery with modified vectors as well as (potentially) allowing the construction of vectors that can home to defined sites in vivo following systemic application (Fig. 1). The latter would open new avenues for preclinical gene therapy providing such vectors can be constructed. Proof-of-concept for this has already been established in vivo. For example, antibodies against angiotensin converting enzyme (ACE) can target adenoviral vectors to the pulmonary endothelium following I.V. injection (Reynolds et al. 2001). These so-called ‘vector targeting’ strategies are varied and have advanced substantially in the recent past owing to our increasing knowledge of viral biology and pathology as well as the detailing of mechanisms through which to alter virus–cell binding and/or internalization. Since adenovirus (Ad) and adeno-associated viruses (AAV) are commonly used for cardiovascular gene therapy, this short review will focus on recent accomplishments in the development of targeted Ad and AAV vectors. A more comprehensive review including non-viral gene delivery has recently been published (Baker, 2004).
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The most commonly used Ad vectors for gene therapy are based on serotypes 2 and 5. In cardiovascular disease, adenoviruses can transduce the endothelium and smooth muscle cells within the vessel wall, albeit using very high titres (Lemarchand et al. 1993; French et al. 1994). This has not hindered preclinical progression of Ad serotype 5 vectors to clinical trials for ischaemia (Grines et al. 2002; Hedman et al. 2003) although the results thus far have been somewhat disappointing. It would therefore be of substantial benefit for cardiovascular gene therapy to develop adenoviruses with a more favourable infectivity profile. This can be achieved in a number of ways, including the use of antibodies to retarget the virus to alternative receptors such as ACE or E-selectin (Harari et al. 1999; Reynolds et al. 2000), targeting peptides inserted into the HI loop of the fibre structure to modulate receptor binding (Nicklin et al. 2000, 2004; Xia et al. 2000), molecular adaptors (Trepel et al. 2000), or by pseudotyping (involving the exchange of the Ad fibre for a fibre from an alternative serotype that possesses a more favourable cell binding profile; Chillon et al. 1999; Havenga et al. 2001). All these strategies have provided proof-of-concept for improvement in the transduction of vascular cells (reviewed by Nicklin & Baker, 2002). One advantage of using phage display-derived peptides is that knowledge of the target receptor is not required in identification of cell-selective peptides (Arap et al. 2002a, 2002b; Rajotte, 1998). Furthermore, many studies have proven, notably for the RGD-4C peptide (Pasqualini et al. 1995), that incorporation of targeting peptides into complex virion structures does not compromise either virus assembly or the targeting capacity of the inserted peptide (Dmitriev et al. 1998; Shi & Bartlett, 2003). Such strategies must be combined with mutations to modulate native Ad–receptor interactions to achieve efficient retargeting, an area that requires further experimentation, particularly in the in vivo setting. An excellent example of pseudotyping is replacing the Ad 5 fibre with that of Ad 16, a subgroup B virus whose receptor is CD46 (Gaggar et al. 2003), leading to enhanced transduction of vascular cells in culture and in intact human saphenous vein segments (Havenga et al. 2001). This has allowed efficient transduction at titres far lower than that required if using Ad serotype 5 and clearly has advantages relating to acute viral toxicity and post-transduction immune responses. This study has also highlighted the species specificity of certain Ad serotypes (in this case Ad 16), an important consideration in the design of preclinical experiments. The issue of species variability in Ad–host interaction (at the level of in vivo liver transduction and red blood cell haemagglutination) has been further highlighted recently (Nicol et al. 2004).
Adeno-associated viruses
Adeno-associated viral vectors, particularly serotype 2 (AAV-2), afford long-term transgene expression through either episomal or integrative maintenance in the nucleus, are minimally immunogenic and are not associated with any known human pathology. Unfortunately, AAV-2 has a relatively poor tropism for vascular cells, although reasonable levels of transduction have been achieved in smooth muscle cells (Richter et al. 2000) and cardiac myocytes (Melo et al. 2002) in vivo. Endothelial cells, however, are very poorly transduced by AAV-2 (Richter et al. 2000; Nicklin et al. 2001). The deficiency of AAV-2 for endothelial transduction was realized a number of years ago by Richter et al. (2000), who showed that local delivery of AAV-2 to blood vessels led to transduction of underlying vascular smooth muscle cells, even in the presence of an intact endothelium. Subsequent studies have reported sequestration of AAV-2 with the extracellular matrix around endothelial cells (thus preventing cell binding and entry) and degradation of internalized AAV-2 particles in the proteasome as factors that restrict efficient endothelial cell transduction (Nicklin et al. 2001; Pajusola et al. 2002). Hence, targeting AAV-2 to alternative receptors is an attractive route to improve vector binding profiles. This re-routing would probably reduce or eliminate matrix sequestration and (potentially) direct the virus through alternative intracellular pathways, thereby bypassing proteasome degradation. As for Ad, strategies include non-genetic modification of AAV-2, genetic insertion of targeting peptides and pseudotyping with alternativee serotype capsids (reviewed by Nicklin & Baker, 2002). In the case of targeting peptides, two main methods have been assessed thus far for vascular cells. First, phage display-derived peptides have been incorporated into AAV-2 capsids at position 587 (predetermined for optimal peptide insertion within the capsid to display the inserted peptide on the surface of the virion; Girod et al. 1999). These studies showed increased efficiency and selectivity of endothelial cell transduction in vitro and in vivo following I.V. injection using either 7-mer or 12-mer targeting peptides (Nicklin et al. 2001; White et al. 2004). In an alternative strategy, Muller et al. (2003) developed an AAV-2 peptide library displaying random peptides within the AAV-2 capsid (thus bypassing the need to use phage display to isolate cell-selective targeting peptides) and isolated viruses selective for endothelial cells in vitro (using human primary coronary endothelial cells). However, only limited evidence of in vivo efficacy for heart-homing was provided (Muller et al. 2003). The potential of such strategies remains to be tested in preclinical disease models. Considering the heterogeneity of the vascular endothelium, it can be envisaged that organ- or disease-selective vectors could be engineered (see Fig. 1).
Since the AAV vector family available to the gene therapy community has increased significantly in recent years with the isolation of at least eight serotypes (Gao et al. 2002, 2004), many studies have addressed the potential to increase transduction of target cells and tissues using vectors with alternative capsids. In the case of liver transduction, AAV-8 is remarkably more efficient than AAV-2 (Gao et al. 2002; Sarkar et al. 2004). Disappointingly, however, no AAV serotypes appear any more effective than AAV-2 for transduction of vascular endothelial cells (Dishart et al. 2003), although detailed analysis of all available serotypes in vitro and in vivo has yet to be reported. Recently, the potential of AAV-6-based vectors for cardiac gene therapy has been shown (Gregorevic et al. 2004). In this remarkable study, highly efficient cardiac gene delivery was achieved following systemic administration of low-dose pseudotyped AAV-6 when combined with acute vascular permeabilization using vascular endothelial growth factor.
Conclusions
The rational design, construction and testing of vascular-targeted gene delivery vectors is a promising route to improve the safety and efficacy of vascular gene therapy. This area of research remains in its infancy but has broad applications to improve existing strategies as well as to open new avenues of research for pertinent disease applications within the cardiovascular system.
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