Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors


1997;94:3352C3356. We then generated a recombinant adenovirus made up of this modified fiber and showed PD0166285 that this short peptide sequence designed in the knob is compatible with the biological functions of the fiber. In addition, by using a ligand-specific antibody, we have shown that this peptide incorporated into the knob remains available for binding in the context of P1-Cdc21 mature virions made up of modified fibers. These findings suggest that heterologous ligands can be incorporated into the HI loop of the fiber knob and that this locale possesses properties consistent with its employment in adenovirus retargeting strategies. Recombinant adenovirus vectors have found wide employment for a number of gene therapy applications (22, 36, 40). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo. Indeed, recombinant PD0166285 adenovirus vectors are distinguished from other available systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 21, 26, 28, 30, 32). Despite this property, specific aspects of the adenovirus biology have prevented the full realization of the potential of such vectors. In this regard, the broad tropism profile of the parent computer virus for cells of diverse tissues potentially allows unrestricted gene delivery. Thus, for the many gene therapy applications requiring targeted, cell-specific gene delivery, the promiscuous tropism of the adenovirus vector represents a confounding factor. Based on this concept, strategies to change the native tropism of adenovirus have been developed to allow the derivation of vectors capable of targeted gene delivery. Strategies to achieve this end are directed at modifying specific actions in the adenovirus contamination pathway. Adenoviruses of serotypes 2 and 5 normally achieve initial recognition and binding to target cells by means of interactions between the carboxy-terminal knob domain name of the fiber protein and the primary receptor (4, 19, 39). After binding, RGD motifs in the penton base interact with cellular integrins of the V3 and V5 types (1C3, 43, 44). This conversation triggers cellular internalization whereby the virions achieve localization within the endosome. Acidification of the endosome elicits conformational changes in capsid proteins, allowing their conversation with the endosome membrane in a manner that achieves vesicle disruption and particle escape (41). Following endosomolysis, the virion translocates to the nucleus, where the subsequent steps of the viral life cycle occur. This understanding of the key role played by capsid proteins in the viral infectious pathway has suggested strategies to alter this process via modifications of these proteins. In this regard, genetic retargeting of adenovirus vectors via modification of viral genes encoding coat proteins, if successful, offers a simple way to achieve a significant improvement in the present generation of these gene-delivery vehicles. To this end, PD0166285 several groups have reported genetic modifications to the knob domain name of adenovirus fiber protein and incorporation of such chimeric fibers into virions. For instance, Stevenson et al. (37) and Krasnykh et al. (25) reported successful generation of adenovirus type 5 (Ad5) virions made up of fibers consisting of the tail and shaft domains of Ad5 fiber and the knob domain name of Ad3, respectively. In addition, Michael et al. (31) exhibited the incorporation of the gastrin-releasing peptide into the carboxy terminus of recombinant Ad5 fiber. This obtaining was extended by Legrand et al. (30a), who achieved rescue of recombinant adenovirus vectors made up of such fibers. Another report published by Wickham et al. (45) described the generation of recombinant computer virus containing fibers with carboxy-terminal polylysine sequences. These studies have established key feasibility issues with respect to this genetic approach but have also demonstrated a number of potentially limiting factors. Of note, all the modifications of adenovirus fiber reported so far were directed towards carboxy terminus of the protein. In addition, these efforts were initiated without prior knowledge of the three-dimensional (3D) structure of the fiber knob. Thus, the employment of the carboxy terminus of the fiber represented a choice of convenience without consideration of the knob tertiary structure. Clearly, 3D structural information has important bearing upon the placement of heterologous protein sequences within the knob for targeting purposes. Such localization of targeting ligands would ideally be achieved in such a manner as to allow their surface presentation and to minimally perturb the fiber quaternary PD0166285 structure. Thus, the recent crystallization of the fiber knob by Xia et al. (47, 48) has provided a level of structural resolution potentially allowing such a rational modification of the fiber protein. According to the proposed 3D model of the knob (Fig. ?(Fig.1),1),.