Historical Perspective

In theory, the easiest way to completely eliminate the toxicity associated with viral gene expression is to delete all the viral coding sequences from the vector. Adenoviruses with large deletions of viral sequences were among the very first vectors reported. For example, in 1970, Lewis and Rowe (43) described a recombinant Ad in which essential viral genes were replaced with SV40 DNA. This replacement rendered the hybrids replication defective and they could only be propagated in the presence of a coinfecting wild-type helper Ad, which provided trans-complementation. A recombinant Ad was also described in 1981 by Deuring et al. (44), following repeated passages at high multiplicity infection of wild-type Ad serotype 12 through human KB cells. These hybrids were found to contain symmetrically duplicated human chromosomal DNA flanked by approximately 1 kb of DNA from the left end of the Ad genome. As in the case of the Ad-SV40 hybrids, these symmetrical recombinants (SYRECs) were replication defective and could only be propagated in the presence of a coinfect-ing helper Ad12 by trans-complementation. The SYRECs were maintained for years in this manner and could be partially purified from the helper virus by cesium chloride (CsCl) density equilibrium centrifugation, owing to differences in their genome sizes that bestowed different buoyant densities.

The defective helper virus-dependent Ad-SV40 and SYREC hybrids demonstrated the possibility of generating vectors completely devoid of Ad coding sequences. In principle, only ~500 bp of cis-acting Ad sequences necessary for DNA replication (ITR) and encapsidation are required for propagation of these HDAds in the presence of a coinfecting helper virus. The advantages of HDAds are considerable: like first-generation (FG) and multiply deleted Ads, HDAds would retain the ability to efficiently transduce a wide variety of cell types from numerous species in a cell-cycle-independent manner. However, unlike FG and multiply deleted Ads, deletion of all viral coding sequences would drastically reduce vector-mediated toxicity and significantly prolong the duration of transgene expression as described in detail below.

0 0

Post a comment