Generation Adenoviral Vectors Research Articles

Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo.

An E1a-deleted adenovirus vector constitutively expressing native adenovirus E3-gp19K (Ad.RSV-gp19K) was constructed in order to determine whether or not E3-gp19K mediated interference with antigen presentation would result in prolonged transgene expression in vivo. Cultured fibroblasts infected with Ad.RSV-gp19K produced a native size gp19K protein and had decreased cell surface levels of MHC I as shown by immunoprecipitation and flow cytometry. The congenic mouse strains Balb/b (H-2b MHC I with high gp19K affinity), Balb/k (H-2k MHC I with no gp19K affinity), and Balb/c (H-2d MHC I with moderate gp19K affinity) were chosen for in vivo experiments because of their range of gp19K affinities. Following transduction of mice form each strain with Ad.RSV-gp19K and AD/RSV-hAAT (a reporter adenovirus), or Ad/RSV-cFIX (control adenovirus) and Ad/RSV-hAAT, the level and duration of serum hAAT protein were unrelated to gp19K protein expression. Evaluation of MHC I abundance on hepatocytes following in vivo transduction demonstrated that recombinant adenovirus rapidly increased the abundance of surface MHC I molecules on hepatocytes, and surface MHC I molecules were reduced earlier and to a greater extent following wild-type adenovirus infection compared with hepatocytes transduced with control or Ad.RSV-gp19K recombinant adenovirus. This difference in surface MHC I down-regulation may be related to the different promoters (RSV-LTR versus the native E3 promoter) and will be an important consideration in the development of newer generation adenovirus vectors designed to evade host immune responses.

Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: implications for gene therapy.

Current generation adenovirus (Ad) vectors are deleted for the E1 region of genes and require propagation in E1 expressing 293 cells. Expression of genes delivered by Ad vectors into immunocompetent hosts is generally transient since the current vectors are not completely replication defective. Viral proteins expressed by Ad vectors, in part, induce a rapid, T cell-mediated loss of the transduced cells. Introduction of temperature-sensitive point mutation into new Ad vectors may be of limited usefulness in prolonging transduced gene expression in vivo. Isolation of new Ad vectors deleted for genes required for normal Ad growth may further prevent Ad protein expression. These new vectors will need to be grown in 293 cells capable of coexpressing other Ad genes. Unfortunately, many of the Ad genes are toxic when coexpressed in 293 cells. We describe the isolation of E1 expressing 293 cells which also express both the Ad polymerase and preterminal proteins, both of which are essential to normal Ad growth. The isolation of new Ad vectors deleted for the E1, polymerase and preterminal proteins are predicted to have many advantageous properties, including the prolongation of transduced foreign gene expression in vivo.

Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that Ad-LacZ, a prototype first generation adenoviral vector containing an expression cassette for the reporter gene LacZ, directs transgene expression to mature and immature olfactory neurons and to sustentacular cells. The technique to apply the vector to the nasal cavity and the amount of viral vector per mouse are important variables that determine the success of viral vector-mediated gene transfer to the mouse olfactory neuroepithelium. Slow infusion of the viral vector solution in fully anaesthetized mice yields the best result in terms of the number of epithelial cells transduced. Infection of the olfactory neuroepithelium with a moderate amount of viral vector (10 9 plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 8–12 days, followed by a decline in transduced cells at 25 days postinstillation of the virus. This decrement in transgene expression is consistent with the natural turnover process that occurs in the epithelium throughout adulthood. At high viral loads (1.3 × 10 10 PFU) extinction of transgene expression occurs as early as 8 days postinjection and is accompanied by epithelial degeneration indicating that the vector dose used should be carefully chosen. Taken together, the current observations demonstrate that adenoviral vectors are effective tools to genetically modify the adult mouse olfactory neuroepithelium in vivo.

Gene therapy using adenoviral vectors.

Growing interest in adenoviral gene-transfer vectors, stimulated by efforts to develop in vivo gene therapy for cystic fibrosis, has led to an evaluation of their use in many other applications of in vivo gene therapy. Studies are beginning to define strategies for the efficient, albeit transient, expression of the transferred gene and have further identified and partially characterized important host responses to in vivo gene transfer that modulate the duration of expression of the transgene. Ongoing work is actively exploring these issues, with a view to the design of the next generation of adenoviral vectors.