878. Toward ‘Pseudotyping’ Adenovirus Vectors: Efficient Generation of pIX-Expressing Helper Cell Lines

Specificity of gene delivery is a key prerequisite for gene therapy. We have previously demonstrated that modification of the minor capsid protein IX (pIX) can be used to modify cell type specificity of human adenovirus type-5 (HAdV-5) particles. The 14.3 kDa hexon-associated pIX is strongly conserved between the different members of the mastadenoviruses. Protein IX is believed to function as a capsid cement by linking the hexons in groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine-zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains the putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. Further analysis of these mutants revealed that deletion of the putative leucine-zipper domain does not affect capsid stabilization by pIX, demonstrating that trimerization is not necessary for the heat-stability phenotype1. To facilitate pseudotyping adenoviral vectors with pIX variants we have set up an efficient system for the generation of pIX-expressing helper cell lines. With an optimized pIX-expression cassette, we generated monoclonal and polyclonal helper cell lines that express wt as well as modified pIX genes to levels equivalent as in a wt HAdV-5 infected cells. Immune-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein incorporated in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX amounts in the helper cells are sufficient to generate heat-stable particles. Finally, the ratio between the pIX protein and other capsid proteins is identical to those found in wt particles. The cell lines are very stable: cell lines grown for two months in the absence of selection contain pIX in amounts identical to the parental line. This demonstrates that pIX is not toxic to cells. These data, together with our previous demonstration that alpha-helical spacers can be fused to pIX to expose new ligands on the outer surface of particles2, establishes that modification of pIX is feasible for pseudotyping adenovirus vectors.