In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

Abstract

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Recent advances in genetic modification of adenovirus vectors for cancer treatment

“…(8) To suppress leaky adenovirus genes, E2AÀ, E4À, and/or pIX-deleted adenovirus vectors have been used, and this approach was shown to result in a decreased CTL response and diminished hepatotoxicity. (30,31) A helperdependent adenovirus vector that lacks all viral coding regions resulted in a reduced inflammatory response in the organs. (32,33) However, the development of a production system for such mutant adenovirus vectors has been hampered by technical complexity and low titers.…”

Adenoviruses as vaccine vectors

“…E1-and E2A-deleted vectors have been constructed but resulted in 30-to 40-fold lower yields compared to E2A-containing constructs [45]. Vectors deleted of E1, E2A, E4, and E3 [46,47] and vectors lacking all viral genes have been developed for gene therapy. The latter type of constructs, commonly referred to as gutted vectors, can be propagated only in the presence of helper viruses, which commonly contaminate the vector batches [48].…”

“…Alternatively, construction of other serotypes of adenoviral vectors, in which the endogenous E4 ORF6 (which binds to E1B) is replaced by that of AdHu5 virus, may allow for their propagation on available cell lines that provide the E1 of AdHu5 virus in trans [51]. Propagation of adenoviral vectors with additional deletion in either E4 or E2 requires that the deleted genes be provided by the packaging cell lines [46,47] or by a helper virus [48].…”

Regulated and prolonged expression of mIFNα in immunocompetent mice mediated by a helper-dependent adenovirus vector

“…These vectors have been modified by introducing deletions in the early genes to increase cloning capacity and reduce cellular toxicity. 2 However, these modifications have not lead to a prolonged transgene expression in rodents and nonhuman primates, indicating that residual low level expression of Ad genes is responsible, at least in part, for the short-term persistence. The 'gutless' or helper-dependent adenovirus vector (HD) allowed transgene expression to persist for almost the entire lifetime of mouse as shown in liver, brain and muscle.…”