Adenovirus Vectors

The wild type adenovirus genome is approximately 35 kb of which up to 30 kb can be replaced with foreign DNA (Smith, 1995, Verma & Somia, 1997). There are four early transcriptional units (E1, E2, E3 & E4), which have regulatory functions, & a late transcript, which codes for structural proteins. Progenitor vectors have either the E1 or E3 gene inactivated, with the missing gene being supplied in trans either by a helper virus, plasmid or integrated into a helper cell genome (human fetal kidney cells, line 293, Graham et al, 1977). Second generation vectors additionally use an E2a temperature sensitive mutant (Engelhardt et al, 1994) or an E4 deletion (Armentano et al, 1997). The most recent "gutless" vectors contain only the inverted terminal repeats (ITRs) & a packaging sequence around the transgene, all the necessary viral genes being provided in trans by a helper virus (Chen et al, 1997).

Adenoviral vestors are very efficient at transducing target cells in vitro & vivo, & can be produced at high titres (>10¹¹/ml). With the exception of Geddes et al (1997), who showed prolonged transgene expression in rat brains using an E1 deletion vector, transgene expression in vivo from progenitor vectors tends to be transient (Verma & Somia, 1997). Following intravenous injection, 90% of the administered vector is degraded in the liver by a non-immune mediated mechanism (Worgall et al, 1997). Thereafter, an MHC class I restricted immune response occurs, using CD8+ CTLs to eliminate virus infected cells & CD4+ cells to secrete IFN-alpha which results in anti-adenoviral antibody (Yang & Wilson, 1995). Alteration of the adenoviral vector can remove some CTL epitopes, however the epitopes recognised differ with the host MHC haplotype (Sparer et al, 1997, Jooss et al, 1998). The remaining vectors, in those cells that are not destroyed, have their promoter inactivated (Armentano et al, 1997) & persisting antibody prevents subsequent administration of the vector.

Approaches to avoid the immune response involving transient immunosupressive therapies have been successful in prolonging transgene expression & achieving secondary gene transfer (Jooss et al, 1996; Kay et al, 1997). A less interventionist method has been to induce oral tolerance by feeding the host UV inactivated vector (Kagami et al, 1998). However, it is desirable to manipulate the vector rather than the host. Although only replication deficient vectors are used, viral proteins are expressed at a very low level which are presented to the immune system. The development of vectors containing fewer genes, culminating in the "gutless" vectors which contain no viral coding sequences, has resulted in prolonged in vivo transgene expression in liver tissue (Schieder et al, 1998). The initial delivery of large amounts of DNA packaged within adenovirus proteins, the majority of which will be degraded & presented to the immune system may still cause problems for clinical trials. Moreover the human population is heterogeneous with respect to MHC haplotype & a proportion of the population will have been already exposed to the adenoviral strain (Gahry-Sgard et al, 1997)

Until recently, the mechanism by which the adenovirus targeted the host cell was poorly understood. Tissue specific expression was therefore only possible by using cellular promoter/enhancers e.g. the myosin light chain 1 promoter (Shi et al, 1997) & the smooth muscle cell SM22a promoter (Kim et al, 1997), or by direct delivery to a local area (Rome et al, 1994). Uptake of the adenovirus particle has been shown to be a two stage process involving an initial interaction of a fibre coat protein in the adenovirus with a cellular receptor or receptors, which include the MHC class I molecule (Hong et al, 1997) & the coxsackievirus-adenovirus receptor (Bergelson et al, 1997). The penton base protein of the adenovirus particle then binds to the integrin family of cell surface heterodimers (Wickham et al, 1993) allowing internalisation via receptor mediated endocytosis. Most cells express primary receptors for the adenovirus fibre coat protein, however internalisation is more selective (Harris & Lemoine, 1996). Methods of increasing viral uptake include stimulating the target cells to express an appropriate integrin (Davison et al, 1997) & conjugating an antibody with specificity for the target cell type to the adenovirus (Wickham et al, 1997b, Goldman et al, 1997). The use of antibodies though increases the production difficulties of the vector & the potential risk of activating the complement system. By incorporating receptor binding motifs into the fibre coat protein, Wickham et al (1997a) were able to redirect the virus to bind the integrin expressed by damaged endothelial or smooth muscle cells, or heparin sulphate receptors which is expressed by numerous cells types.

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