An intense recent research effort into adenovirus (Ad) biology has revealed crucial steps involved in gene transfer. The tro-pism of Ads is determined by two distinct virus-cell interactions. Initially, there is high-affinity binding of the fiber knob to the primary receptor, which is the coxsackie-adenovirus receptor (CAR) for most serotypes, including 2 and 5, which are most commonly used for gene therapy approaches (1,2). Binding is followed by interaction between cellular integrins and an arginine-glycine-aspartic acid (RGD) motif located at the penton base (3). The latter event leads to formation of endosomes and viral internalization. Subsequently, the adenoviral DNA is transported to the nucleus and adenoviral protein synthesis, or in the case of replication-deficient Ads, transgene expression, begins. Ad DNA is not frequently integrated into the host genome, thereby resulting in a low risk of mutagene-sis. Nevertheless, the limited duration of gene expression may render Ads less desirable for the therapy of metabolic diseases, where long-term expression is needed, but is adequate for cancer gene therapy approaches, where the purpose typically is to kill the target cells. Infection is not dependent on cell-cycle phase. Therefore, both cycling and nondividing cells are infected. Importantly, appealing features of Ad for cancer therapy include stability and unparalleled capacity for gene transfer and expression in vivo. Further, production of high titers of current good manufacturing practices quality Ad, necessary for clinical trials, is well established. Nearly 80% of patients enrolling into gene therapy clinical trials are cancer patients (4). Therefore, Ad is currently the most commonly used clinical agent. Many examples given here will be from the field of cancer gene therapy.

* Current affiliation: Helsinki University Central Hospital, Helsinki, Finland.

Considering the biology of viral entry, it is logical the degree of gene transfer is determined chiefly by the degree of CAR expression (5-17), reviewed in (18,19). Nevertheless, integrin levels may also play a role. This aspect is not as well understood as various avp-class integrins can mediate the second step of virus entry. avp3 and avp5 integrins were the earliest receptors implicated (3,20), but others, such as avp1 may also be involved, and further molecules may be currently unidentified (21). Also, alternative receptors, such as major histocompatibility complex and heparan sulfate pro-teoglycan have been implicated although their role is not yet clear (22,23). Finally, some recent data suggests that a heparin sulphate proteoglycan-binding moiety present in the midsec-tion of the fiber may have a role in mediating transduction of hepatocytes (24).

The biodistribution of Ad is not determined only by receptor tropism. In fact, in mice, intravascular Ad results in accumulation mainly in the liver, spleen, heart, lung, and kidneys (25-28), although these tissues may not necessarily be the highest in CAR expression. Instead, the degree of blood flow and the structure of the vasculature in each organ probably contribute to the biodistribution. Importantly, tissue macrophages such as Kupffer cells of the liver have a major role in clearing Ad from blood (29,30). This is an active and non-CAR-mediated process, and uptake via this mechanism leads to rapid degradation of Ad DNA and ineffective transgene expression. Uptake by Kupffer cells saturates when the dose of Ad is increased, resulting in more effective circulation of Ad. This threshold is approximately 1-2 X 1010 viral particles in mice, but is not known for humans (28). The threshold effect is probably a major factor contributing to discrepancy between different studies reporting discordant biodistribution and gene transfer rates, as variable dosing has been used. These phenomena also complicate preclinical specificity stud

4104-8_Ch3_Templeton ies, and it is unclear how well murine results correlate with human data.

Although CAR is expressed ubiquitously on most normal epithelial tissues, lack or down-regulation of CAR has been reported for various tumor types, such as ovarian, prostate, lung, breast, and colorectal cancer, as well as melanoma, gli-oma, and rhabdomyosarcoma (10-13,14,15,17,31-36), reviewed in (18,19). Further, this could be a general phenomenon associated with the carcinogenesis of various tumor types, as inverse correlation with tumor grade has been suggested (14,15). The function of CAR is not well understood but it may be associated with cell adhesion and perhaps cell-cycle regulation. Localization to tight junctions has been suggested (16) as has a role in suppression of tumor growth (15). Interestingly, a preliminary report has suggested inverse correlation between activity of the RAS-MAPK pathway and CAR expression (37). For treatment of cancer, these associations are concerning, if confirmed. In particular, the population most desperately needing novel treatments is patients with advanced disease, and if CAR is variably expressed in these tumors, CAR-dependant approaches may not be useful. Thus, targeting of Ad to tumor cells may useful for increasing the clinical efficacy and safety of approaches. Further, considering the widespread expression of CAR, targeting approaches may be advantageous for increasing the specificity of any clinical Ad gene therapy application.

Two main approaches have been used for modification of Ad for the purposes of increased safety and efficacy. Tran-scriptional targeting restricts expression of transgenes to target tissues, whereas transductional targeting approaches limit the entry of agents to target cells. The former can be achieved by using tissue-specific promoters (TSPs). These promoters are typically associated with genes highly expressed in tumor tissue with lower expression in normal tissues, of which the liver is the most important. Transductional targeting can be achieved by using adapter molecules, genetic modification of the fiber, serotype chimerism, or completely replacing the fiber with heterologous targeting moieties. Also, attempts have been made to use other regions of the capsid for targeting, but little data exists on the feasibility of the approach in vivo. These approaches are discussed in the following section. In addition to introducing new tropism, many applications would benefit from ablation of binding to CAR (38). For systemic treatment, blocking of Kupffer cell uptake may be even more important (30).

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