New tools for the generation of E1- and/or E3-substituted adenoviral vectors.
(9/94)
We have designed new vectors for the construction of recombinant adenoviruses containing expression cassettes in the E1 and/or E3 regions. Using a versatile set of restriction enzymes, the cassettes are cloned into small bacterial vectors and subsequently introduced into large plasmids containing the adenoviral sequences. Two positive selection markers facilitate the recovery of a cosmid containing a copy of the sequence of the recombinant adenovirus. The resulting cosmid is transfected into 293 or 911 cells in order to rescue the virus. Importantly, the method does not require any recombination event, either in E. coli or in mammalian cells. The entire procedure can generate viral plaques in 12 days. Gene Therapy (2000) 7, 80-87. (+info)
Growth inhibition of prostate cancer by an adenovirus expressing a novel tumor suppressor gene, pHyde.
(10/94)
It has been estimated that there will be > 180,400 new cases of prostate cancer and 31,900 prostate cancer deaths in the United States this year. New therapeutic strategies against locally advanced prostate cancer are desperately needed. A novel gene (pHyde) was identified by an improved cDNA competition hybridization technique for Dunning rat prostate cancer cell lines. A recombinant replication-deficient E1/E3-deleted adenovirus type 5 containing a pHyde gene under the control of a truncated Rous sarcoma virus (RSV) promoter (AdRSVpHyde) was generated. In vitro, AdRSVpHyde significantly inhibited growth of human prostate cancer cell lines DU145 and LNCaP in culture. In vivo, a single injection of AdRSVpHyde (5 x 10(9) plaque-forming units) reduced DU145 tumors in nude mice remarkably compared with untreated control or viral control-treated DU145 tumors. Moreover, AdRSVpHyde induced apoptosis and stimulated p53 expression. These results together suggest that pHyde is a tumor suppressor gene that inhibits growth of prostate cancer and that this inhibition is at least in part due to the induction of apoptosis. (+info)
A new vector system with inducible E2a cell line for production of higher titer and safer adenoviral vectors.
(11/94)
Adenoviral vectors have been used in gene therapy and for vaccination. The major concerns with using adenoviral vectors are the pathogenic potential of the virus backbone and the generation of replication-competent adenovirus that may replicate in an uncontrolled manner, especially in immunocompromised patients. It is important to develop new vectors that are safer for clinical trials while maintaining high titer and efficient transduction. A new adenovirus vector production system was developed, which includes several vector backbone plasmids deleted for E2a and a new cell line expressing both E1 and E2a. The new cell line with the tTA-inducible E2a expression cassette can significantly increase the titer of E1/E2a-deleted vectors by four to five orders of magnitude upon withdrawal of tetracycline. Furthermore, there is no sequence overlap between the vector and the cellular DNA within the E2a open reading frame and downstream, making the generation of virus with wild-type E2a through homologous recombination substantially less likely. The new vector system may improve the safety of vectors for vaccination and cancer therapy and may also provide safer backbones for further vector development, such as helper-dependent and hybrid vectors. (+info)
Downregulation of CXCR4 gene expression in primary human endothelial cells following infection with E1(-)E4(+) adenovirus gene transfer vectors.
(12/94)
Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant. (+info)
An immunomodulatory procedure that stabilizes transgene expression and permits readministration of E1-deleted adenovirus vectors.
(13/94)
Immune responses against E1-deleted adenovirus vectors and/or their transgene products result in the rapid elimination of vector-transduced cells and the generation of neutralizing antibodies. Different strategies of immunomodulation to stabilize transgene expression at therapeutic levels and to permit productive vector readministration have been examined. Our previous studies have shown that depletion of macrophages from spleen and liver decreases hepatic inflammation, significantly prolongs transgene expression, and delays the onset of humoral immune responses after systemic administration of an E1-deleted adenovirus vector. In the present study, we have examined the effects of macrophage depletion in combination with temporary blockade of CD40 ligation on E1-deleted adenovirus vector-mediated gene transfer. Alone, each of these treatments significantly inhibited the humoral immune response against the transgene product and prolonged its expression. Together, these treatments completely stabilized transgene expression and inhibited the production of neutralizing anti-adenovirus antibodies, permitting successful vector readministration. Animals rendered immunologically unresponsive to vector and transgene antigens regained their ability to mount productive immune responses against the vector after recovery of immune function, but remained unresponsive to the transgene product. These experiments demonstrate that this treatment is transient and antigen-specific. (+info)
Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII.
(14/94)
The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced. (+info)
Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice.
(15/94)
Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols. (+info)
A Cre-expressing cell line and an E1/E2a double-deleted virus for preparation of helper-dependent adenovirus vector.
(16/94)
Adenoviral vectors are attractive for the delivery of transgenes into mammalian cells because of their efficient transduction, high titer, and stability. The major concerns with using E1-deleted adenoviral vectors in gene therapy are the pathogenic potential of the virus backbone and the leaky viral protein synthesis that leads to host immune responses and a short duration of transgene expression. Helper-dependent (HD) adenoviral vectors that are devoid of all viral protein-coding sequences have significantly increased the safety and reduced the immunogenicity of these vectors. Currently available HD vectors depend on an E1-deleted adenovirus as a helper to provide viral proteins in trans. As a consequence, contamination with helper virus cannot be avoided in the HD vector preparation though it can be decreased to 0.01% using a Cre/loxP mechanism. Since the presence of E1-deleted helper virus may have substantial unwanted effects, we have developed a new Cre-expressing cell line based on an E1- and E2a-complementing cell. This new cell line can efficiently cleave the packaging region in the helper virus genome. We have also developed an E1 and E2a double-deleted helper virus. By using the CreE cell with the helper virus deleted in both the E1 and the E2a genes it may be possible to further improve the safety of the vectors. (+info)