Human adenoviruses (Ad) of serotypes 2 and 5 (Ad2 and Ad5) have been extensively used for a variety of gene therapy applications. This is largely due to the ability of these vectors to efficiently deliver therapeutic genes to a wide range of different cell types. However, the promiscuous tropism of Ad resulting from the widespread distribution of its primary cellular receptor, the coxsackievirus and Ad receptor (CAR) (1
), limits the utility of Ad vectors in those clinical contexts where selective delivery of a therapeutic transgene to diseased tissue is required. Uncontrolled transduction of normal tissues with Ad vectors expressing potentially toxic gene products may lead to a series of side effects, thereby undermining the efficacy of the therapy. Furthermore, cellular targets expressing CAR below certain threshold levels are not susceptible to Ad-based therapies due to their inability to support Ad infection. Therefore, the dependence of the efficiency of the Ad-mediated cell transduction on the levels of CAR expression by the target cell presents a serious challenge for the further development of Ad-based gene therapeutics.
In order to overcome this limitation, the concept of genetic targeting of Ad vectors to specific cell surface receptors has been proposed. Strategies to retarget Ad vectors are based on the currently accepted model of Ad infection (for a review, see reference 16
), which postulates that the initial binding of the Ad virion to the cell is mediated by the attachment of the globular knob domain of the Ad fiber protein to CAR. This is then followed by an internalization step triggered by the interaction of the RGD-containing loop of a second Ad capsid protein, the penton base, with cellular integrins. Although recent studies have shown that representatives of different Ad serotypes may utilize cell receptors other than CAR, the two-step mechanism of cell entry established for Ad2 and Ad5 appears to be common to the majority of human Ad serotypes. As the fiber protein is the key mediator of the cell attachment pathway employed by Ad, genetic incorporation of targeting ligands within this viral protein was originally proposed as the strategy to derive targeted, cell type-specific Ad vectors (21
Each of the rodlike fiber proteins localized at the vertices of icosahedral Ad5 capsid is a homotrimer which consists of three identical copies of a 62-kDa polypeptide (for a review see reference 4
). This trimeric molecule has a domain organization, with each domain playing important roles in the structure and function of the virion. Whereas the amino-terminal tail domain is responsible for association of the fiber with the penton base, the carboxy-terminal knob domain, which has a propellerlike structure formed by two β-sheets (34
), performs two distinct functions, both vital for virus assembly and propagation. In addition to forming the CAR-binding site, which is formed by residues from the AB loop, β-strand B, and the DE loop (for details, see reference 25
), the knob initiates and maintains the trimerization of the entire fiber molecule. This trimerization is critical for the successful formation of the virion, as monomeric fibers cannot associate with the penton base (24
). The knob and the tail of the fiber are connected by the β-spiral shaft domain (30
), which extends the knob away from the surface of the virion, thereby facilitating interaction with CAR.
Early attempts to generate Ad vectors possessing expanded tropism involved incorporation of short peptide ligands into either the carboxy terminus (32
) or the so-called HI loop (7
) of the knob of the Ad fiber protein. Although these studies demonstrated the feasibility of genetic targeting of Ad and showed the potential utility of such vectors in the context of several disease models (14
), further progress in this direction has been hampered by the structural conflicts often observed as a result of modification of the fiber structure (33
). Due to the rather complex structure of the fiber knob domain, even minor modifications to this portion of the molecule may destabilize the fiber, thereby rendering it incapable of trimerization and hence nonfunctional. According to the published data, the upper size limit for a targeting ligand to be incorporated into Ad5 fiber is about 30 amino acid residues (13
), which dramatically narrows the repertoire of targeting moieties, thereby restricting the choice of potential receptors and, therefore, cell targets. As a result of this limitation, only a handful of heterologous peptide ligands [oligolysine, FLAG, RGD-4C, RGS(His)6
, and HA epitope] have been successfully used in the context of Ad5 fiber modification. The task of Ad targeting is further complicated by the need to ablate the native receptor-binding sites within the fiber of an Ad vector to make it truly targeted.
This study presents an alternative approach to Ad targeting based on replacement of the native fiber in an Ad capsid with a chimeric protein, which results in permanent ablation of native Ad receptor tropism and simultaneously offers flexibility in the generation of novel vector tropism.