ABSTRACT
Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.
Subject(s)
Adenoviridae/genetics , Baculoviridae/genetics , Gene Deletion , Genes, Viral , Genetic Engineering , Genetic Vectors , Blotting, Southern , Cell Line , Gene Expression , Humans , Transduction, Genetic/methods , beta-Galactosidase/geneticsABSTRACT
BACKGROUND: A major limitation of adenovirus-mediated gene therapy for metabolic and inherited diseases is the instability of transgene expression in vivo. This instability results, at least in part, from the inability of the vector genome to maintain the transgene through replication or integration. In this study we evaluated the possibility of stabilization of an adenovirus-delivered transgene by non-adenovirus replicative elements. METHODS: We have developed a novel system for the maintenance of transgenes delivered by adenovirus vectors through extrachromosomal replication. In its initial configuration, this system combines the Epstein-Barr virus (EBV) replicative elements, a tetracycline (Tc)-inducible expression system, and the Cre-lox recombination system in the context of a single E1/E3/E4-deleted adenovirus vector. Induction of Cre expression initiates a Cre-mediated recombination, resulting in the excision of a fragment of the vector genome and its circularization into an EBV-based episome. RESULTS: In vitro studies have demonstrated that excision of the circular episome can occur in a cell-free system as well as in cultured cells transfected with plasmid DNA or transduced by a virus vector carrying the episome-excising cassette. PCR studies have shown that in proliferating, non-permissive, cultured primate cells the episome generated from the adenovirus vector is maintained much more stably than the genome of the parent vector. This episome was also able to replicate in mammalian cells. CONCLUSION: Together these studies demonstrate the feasibility of this approach for the stabilization of transgenes delivered to dividing cells by adenovirus vectors.
Subject(s)
Adenoviridae/genetics , DNA Replication , Genetic Vectors , Transgenes , Base Sequence , DNA Primers , Polymerase Chain Reaction , Recombination, GeneticABSTRACT
A new system for hammerhead ribozyme (Rz) expression was examined in which fowl adenovirus type 1 (CELO) virus-associated RNA (CELO VA RNA) was used as a vector for the incorporation of Rz to target the mRNA of secreted alkaline phosphatase (SEAP) both in vitro and in vivo. The Rz gene was integrated into the CELO VA RNA between the internal promoter boxes A and B; apparently this did not interfere with its transcription. Rz integrated into CELO VA RNA and, lacking the viral sequences, exhibited the same activity in vitro, Consequently, CELO VA RNA sequences did not inhibit the integrated Rz activity in vitro. In vivo experiments were carried out with human 293 cells by co-transfection with plasmids containing Rz and SEAP. Inhibition of enzyme activity was 50% in 48 h. We conclude that CELO VA RNA may be used for effective expression of hammerhead Rz.
Subject(s)
Aviadenovirus/genetics , Genetic Vectors , RNA, Catalytic/genetics , RNA, Viral/genetics , Alkaline Phosphatase/genetics , Aviadenovirus/enzymology , Base Sequence , Cell Line , Gene Expression , Genes, Viral , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids/genetics , RNA, Viral/chemistry , TransfectionABSTRACT
A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.
