Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants.

The cloning capacity of currently available E1- and E3-deleted adenovirus (Ad) vectors does not exceed 8 kb. To increase capacity and improve vector safety further, we have explored the possibility that Early Region 4 (E4) and the gene encoding protein IX (pIX) might also be deleted. To generate cell lines expressing sufficient levels of E4 and pIX proteins in trans in addition to E1-encoded proteins to complement mutations in these genes, we transformed 293 cells with constructs containing the E4 transcription unit and pIX coding sequences under the control of inducible mouse mammary tumor virus (MMTV) and metallothionein promoters, respectively. We obtained two lines, VK2-20 and VK10-9, that express both E4 and pIX proteins as well as E1. The lines could be efficiently transfected with DNA, and allowed the rescue and propagation of an adenovirus; recombinant, Ad5dlE3,4, containing a 2.7-kb E3 deletion and a 2.8-kb E4 deletion in addition to an insertion of plasmid DNA sequences in E1A. Because the E4 sequences within VK2-20 and VK10-9 cells do not overlap with the DNA sequence of Ad5dlE3,E4, the probability of regeneration of the wild-type E4 during virus propagation should be very low. Using the cell lines described in this study, it should be possible to generate Ad vectors lacking E1, pIX, E3, and E4. This would not only increase capacity over that of currently available vectors (to approximately 11 kb) but would also result in more severely attenuated vectors than those with deletions only of E1 or of E1 and E3 and, hence, safer for use in gene therapy protocols.

The fowl adenovirus type 1 (CELO) virus-associated RNA-encoding gene: a new ribozyme-expression vector.

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.

Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

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.

DNA sequence of the deletion/insertion in early region 3 of Ad5 dl309.

dl309 is an adenovirus type 5 (Ad5) mutant that has been extensively utilized for construction of Ad5 mutants in early region 1 (E1), in developing vectors for use as viral vaccines, and in development of gene transfer vectors for gene therapy. Ad5 dl309 has been useful for vector construction because of its altered XbaI restriction pattern and lends itself to a variety of strategies for rescuing inserts or mutations into E1. It contains only one XbaI site at 3.7 map units (m.u.) as compared to wt Ad5 which contains 4(3.7, 29.5, 79.5, and 84.8 m.u.). The loss of the sites at 29.5 and 79.5 m.u. is due to deletions of a few bp but the loss of the site at 84.8 m.u. was the result of a deletion from approximately 83 to 85 m.u. and substitution with a fragment of foreign DNA. Because of the widespread use of dl309 and derivatives of this mutant in the construction of Ad5-based vectors and the need to have precise genetic information on the sequences present in vectors to be used as vaccines and in gene therapy, we have sequenced the alterations in dl309 which affect the XbaI sites at 79.5 and 84.8 m.u. and have determined which E3 proteins are expressed by this virus. The deletion that removes the XbaI site at 84.8 m.u. extends from Ad5 bp 30005-30750 and is substituted with 642-bp of heterologous DNA that shows homology to salmon DNA. This alteration deletes all or part of the coding sequences for the E3 14.7K, 14.5K and 10.4K proteins and these proteins are not detected in dl309 infected cells. The loss of the XbaI site at 79.5 m.u. is the result of a 6-bp deletion which removes two internal amino acids (18 and 19) from the E3 6.7K protein. The E3 6.7K protein and other E3 proteins whose coding sequences are unaffected by the alterations in dl309 (gp19K, 12.5K and 11.6K) were expressed in dl309 infected cells.

Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication.

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.

Investigation of promoter function in human and animal cells infected with human recombinant adenovirus expressing rotavirus antigen VP7sc.

Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and beta-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenovirus to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

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.

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.