7.1.2.4 Replication of Adenoviral vectors

Adenoviral replication depends on the presence of the E1A, ELB region of the viral genome, therefore these regions must be deleted from genome. Such vectors are capable of infecting a cell only once and no viral propagation occurs.  This deletion can make room for inserting a foreign DNA, up to size of about 6kb. The process of substitution can be carried out by either recombination or by molecular biological techniques (Rosenfeld, et al 1990)

An alternative method, the foreign genes are inserted into the E3 region of an E1 deleted vector (Mittal et al 1993).  It is also possible to make adenoviral vectors by recombination employing cells infected with adenovirus or adenoviral vectors and co-transfected with the foreign gene sequence in a plasmid carrying appropriate adenovirus DNA sequence. This forms recombinant adenoviral molecules with the foreign gene inserted in the vector.

The strategy for recombinant adenoviral construction involves that particular region of the genome EIA and EIB that are essential for replication are replaced by the desired gene of interest. The adenoviral vector is composed of two parts, viral DNA vector and a packaging cell line. The adenoviral DNA vector is a plasmid DNA that contains a portion of the viral genome after deleting the E1A region, and replaced the gene of interest.

The adenoviral vector is produced using either in vitro ligation or homologous recombination. Isolating the wild type adenoviral DNA, then cut by the restriction enzyme ClaI can carry this out. The digested viral DNA is then ligated with the adenoviral DNA vector containing the gene of interest, which has been treated with the same enzyme ClaI. Both the adenoviral DNA vector and the viral DNA component are introduced into a packaging cell line. These DNA are recombined in the cell producing the vector. (Stratford- Perricaudet, et al 1992, Wolff, 1994,Rosenfeld, et al 1990 and Mettal, et al 1993).