Novel human gene transfer vectors: evaluation of wild-type and recombinant animal adenoviruses in human-derived cells.

Abstract

Major disadvantages of human adenovirus (hAd) vectors in gene therapy include preexisting or induced immune responses, and possible coreplication of recombinant hAd in the presence of wild-type hAds. These disadvantages may be overcome by using nonhuman, animal adenoviruses (aAds). We evaluated four different aAds for their potential use as viral vectors. The canine adenovirus type 2 (CAV2) and bovine adenovirus type 3 (BAV3) appeared to be suitable systems, as they infect human cells. CAV2, but not BAV3, caused cytotoxicity, and only limited (CAV2) or no (BAV3) production of infectious virus particles was observed after infection of human cell lines. CAV2 showed higher expression of endogenous genes than did BAV3 in the tested human cells. No interference between hAd and CAV2 or BAV3, such as recombination of DNA or cross-activation of virus replication, was observed in up to five passages in double-infected human cells. Transfection of cloned genomic CAV2 or BAV3 DNA into appropriate permissive cell lines rescued infectious virus. Furthermore, we produced a recombinant E1-deleted BAV3, and showed that it could infect and express a reporter gene in various human cell types. The goal was to construct and evaluate recombinant (E1-deleted) animal adenoviruses (aAds) as new vector systems for human gene therapy. The rationale for developing aAds for human use is the potential higher safety and efficiency, as compared with human adenoviruses (hAds). Coreplication and recombination with preexisting hAds should not be possible owing to lack of homology, and preexisting immunity in the general population should be limited. Of the four aAds we evaluated, BAV3 appeared to be the best candidate. It infects human cells without showing growth or cytotoxic effects, viral gene expression was barely detectable, and no trans-activation of either virus was detected in coinfections with hAd5. Rescue of virus in permissive cells, from plasmids containing the CAV2 or BAV3 genome, confirmed our approach. Furthermore, an E1-deleted recombinant BAV3 was constructed and shown to transduce and express the lacZ reporter gene in human cells.