Strategies and Considerations

The capacity of an Ad vector to infect a given cell is therefore dictated by the CAR- and integrin-expression levels of the cell. It has been shown that cells expressing both receptors below a certain threshold level are refractory to Ad infection [16]. Recent studies have also demonstrated that a number of cell types such as endothelial, smooth muscle cells, differentiated airway epithelium cells, lymphocytes, fibroblasts, hematopoietic cells, and some others demonstrate either complete or partial resistance to Ad infection [10, 17-23]. Importantly, the employment of Ad vectors for cancer gene therapy has revealed that many types of tumor cells express CAR at marginal or even undetectable levels and are thus Ad-refractory [24-26]. An interesting finding in this regard was recently published by Okegawa et al., who demonstrated a striking inverse correlation between the level of CAR expression by prostate cancer cell lines and their tumorigenicity, thereby suggesting that in general the most aggressive tumors may be CAR-deficient and therefore refractory to therapeutic intervention utilizing unmodified Ad2- or Ad5-derived vectors [27]. The authors have also observed the same phenomenon on human breast cancer cells. If the results of this work are further corroborated by data from other laboratories, CAR deficiency in tumors may become a major obstacle in employing Ad vectors for cancer gene therapy, therefore necessitating the derivation of Ad vectors capable of infecting these tumors in a CAR-independent fashion. Another reason to develop tropism-modified Ad vectors is the fact that many normal human tissues express high levels of CAR [7] and may thus become random targets for therapeutic Ad agents. As the products of some therapeutic genes may be toxic or otherwise deleterious to normal cells, such uncontrolled transduction may result in destructive side-effects which can compromise the efficiency of the therapy.

An overview of the native cell-entry pathway utilized by Ad suggests that it may be modified by altering the mechanism of the virus-cell interaction. Theoretically, this goal may be achieved by modifying the structure of the receptor-binding components of the Ad virion, the fiber and the penton base, in a way which promotes interactions of the modified capsids with cell surface-localized molecules distinct from the native Ad receptors. The accomplishment of this goal would result in Ad vectors possessing expanded tropism, which would be able to achieve cell entry by either of two routes, the natural or newly created pathway. Obviously, although such infectivity-enhanced vectors would be of utility in those clinical applications where tropism to CAR is not a confounding issue, they would still be a suboptimal means of cell-specific gene delivery in most therapeutic strategies requiring stringent control over vector dissemination in patients. Therefore, in order to achieve the maximum targeting gain, the development of truly targeted Ad vectors will necessitate the ablation of the native CAR tropism of the vector.

These two goals may be realized by a variety of strategies, which differ from each other in the means utilized for engineering the novel viral tropism and the ablation of CAR tropism. In essence, there are two conceptually different approaches, which may be referred to as conjugate-based targeting and genetic targeting. These strategies are similar in that they are both based on establishing a physical link between the Ad virion and a targeting molecule, or ligand, such that binding of the ligand to a target receptor attaches the virion to the cell expressing that receptor. The basic difference between these strategies is that whereas the conjugate-based approach employs methods of complexing the Ad vector with the targeting moieties which do not usually require any modifications of the Ad virion, and results in a multicomponent vector, in genetic targeting no extraneous complexes or conjugates are involved as the targeting is achieved by genetic modification of the Ad virion itself, thereby resulting in a single-component vector.

A variety of different types of molecules may be employed as targeting ligands in these two approaches. Perhaps with the exception of small inorganic molecules which possess specificity to selected cell surface receptors, any natural receptor-binding ligand can be linked to an Ad capsid. This covers a wide spectrum of ligands ranging from relatively simple organic substances such as folate to complex chemical conjugates or genetic fusions of antibodies (see "Conjugate-Based Targeting" below). However, this spectrum of naturally available targeting moieties, although quite broad, cannot meet the needs of Ad targeting in the most general sense, as it is not applicable to cell surface molecules which do not perform any receptor functions and thus do not have any natural ligands. To direct Ad vectors to this type of molecule, relevant targeting ligands should be engineered de novo. This task may be achieved by developing mono- or polyclonal antibodies against the target molecule and using these antibodies for Ad targeting. However, this approach can be used only in a conjugate-based strategy, since the incorporation of an entire antibody molecule into the Ad capsid is not yet possible (see discussion below). Alternatively, a more versatile approach which is compatible with both targeting strategies may be employed for the identification of ligands. Specifically, phage libraries which are designed to display an enormous diversity of random peptides or single-chain antibodies may be utilized for the identification of the ligands of interest in a so-called "biopanning" procedure. Such biopanning usually involves several rounds of interactions between the phages constituting the library with the target, which may be represented either by purified target molecules or cells expressing these molecules or, in the extreme, the entire organism [28-34]. Each round of selection leads to the isolation of an enriched subpopulation of phage particles demonstrating some degree of binding to the target, which is then used in a subsequent round of selection. After being repeated several times, this sequential procedure normally results in the identification of ligands possessing specificity to selected targets, which may be used for the Ad rerouting strategies.

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