We have compared the in vitro and in vivo behaviors of a set of isogenic E1-and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded ␤-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

Fifteen children with advanced neuroblastoma according to Evans' classification (1 with stage III and 14 with stage IV) were treated with high-dose melphalan (HDM) followed by autologous bone marrow transplantation. Before HDM, all patients had been extensively treated with multimodality therapy for a median duration of 9 months. At the time of HDM, seven children were in partial remission (PR) with measurable residual tumor and 8 were in complete remission (CR) or good partial remission (GPR). No reduction in measurable tumor size was observed in any of the PR patients. However, when HDM was used as consolidation therapy (CR and GPR patients) survival appeared encouraging, since five of eight patients are alive with no evidence of disease at (NED) 29+ to 54+ months after HDM. Tolerance of this high-dose chemotherapy was satisfactory; gastrointestinal toxicity appeared to be the most important limiting factor. These results suggest that chemotherapy including high-dose melphalan is promising when used as consolidation therapy in patients who have already attained CR with conventional therapies.

A critical issue for E1-deleted adenoviral vectors manufactured from 293 cells is the emergence of replication-competent adenovirus (RCA). These contaminants arise through homologous recombination between identical sequences framing the E1 locus displayed by 293 cells, and the vector backbones. Modified recombinogenic sequences (syngen) were thus introduced within the vector backbone, and virus viability and RCA emergence were assessed. Syngen#1 is a synthetic sequence displaying silent point mutations in the pIX and IVa2 coding regions. A side by side comparison of Ad5CMV/p53 (E1-deleted adenovirus expressing the p53 tumor suppressor gene) and AV⌬E1#1CMV/p53 (with syngen#1 in place of wild-type sequences) demonstrated a Keywords: replicative competent adenovirus (RCA); silent mutations; synthetic sequences; Ad5 pIX region IntroductionSubgroup C-based adenoviral vectors are being used in a growing number of clinical trials for acquired (eg cancer) and monogenic hereditary disorders. Because these serotypes induce a lytic productive infection in a wide range of human cell types, first-generation gene therapy vectors have been disabled by deleting the E1 regulatory genes from the viral chromosome.1 Accordingly, first-generation vectors must be amplified in human cells capable of efficiently transcomplementing for the missing E1 genes. Most of clinical grade preparations of E1-deleted recombinant vectors are carried out in 293, a cell line of human embryonic origin that displays the Ad5 E1 locus and flanking sequences.2 These cells harbor the left 12% of the Ad5 genome (up to nt position 4344), thereby encompassing the entire E1 locus. Unfortunately, identical/recombinogenic sequences on both sides of the E1 genes allow the vector to reacquire the E1 genes by homologous recombination during virus growth. [4][5][6] Contamination of clinical lots with E1+ replication-competent adenovirus (RCA) raises specific safety issues due to the lytic nature of Ad5. Also, exacerbation of host inflammatory responses with significant tissue damage and pathogenicity has been assigned to such autonomous contaminants. 4,5,7 In practice, a significant proportion of clinical batches cannot be used because RCA contamination exceeds levels specified by the Food and Drug Administration. Cell-or vector-based solutions have been proposed to decrease the occurrence of homologous recombination which triggers the emergence of RCA during amplification of E1-deleted recombinants. 5,9 In particular, human embryonic retinoblasts have been engineered that harbor minimal E1 sequences (nt Ad5 position 459 to 3510) integrated in the cellular chromosome. Importantly, PERC6 cells can efficiently rescue E1-deleted (first-generation) adenoviral vectors with no detectable RCA contamination.9 Ideally, the PERC6 strategy requires extended E1 deletions within the vector backbone.9 Also, this cell line is not appropriate to produce second-generation adenoviral vectors harboring a second lethal deletion. 10Another approach relies on modifying the overall archit...

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1␣ (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of Ͼ10 5 infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10-to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.