A Recombinant E1-Deleted Canine Adenoviral Vector Capable of Transduction and Expression of a Transgene in Human-Derived Cells andIn Vivo

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Abstract
Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSV βgal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSV βgal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos. There are over 100 members of the adenovirus group identified, with 49 belonging to the human type. Human adenoviruses have been the most extensively characterized and were unfortunately chosen as backbones for gene transfer vectors for precisely this reason. For gene transfer in humans, non-human adenoviral vectors eliminate several innate disadvantages of human-derived vectors while retaining most, if not all, of the well-characterized advantages of adenoviral vectors. Our initial efforts with CAV-2 have shown that this non-human vector can infect, and is replication defective in, human-derived cell. An E1-deleted canine adenovirus type 2 vector containing lacZ was able to direct expression of the transgene in human cells and non-human cells in vitro. In addition, ~104 transduction units of CAVRSV βgal were able to transduce a wide variety of cell types in 2-day-old chick embryos.