Evaluation of antitumor effects of two vine stalk oligomers of resveratrol on a panel of lymphoid and myeloid cell lines: comparison with resveratrol.

This study aims to evaluate and compare the antiproliferative and proapoptotic effects of resveratrol (trans-3,4',5-trihydoxystilbene) with two of its naturally occurring oligomers, epsilon-viniferin (a dimer) and miyabenol C (a trimer). Proliferation assays performed on myeloid and lymphoid cell lines show that the three compounds inhibit cell growth of all cell types tested, with miyabenol C being the most efficient (IC50 ranging from 10.8 to 29.4 muM). Further analysis performed on the multiple myeloma cell line U266 shows that all compounds modify cell cycle distribution probably via actions on different targets. Whereas cells treated with resveratrol accumulate in S phase, cells treated with epsilon-viniferin and miyabenol C accumulate in G2/M and G0/G1, respectively. Miyabenol C is also the most efficient at inducing cell death in U266 cells. All compounds induce apoptosis of U266 cells via mechanisms entirely dependent on caspase activation and associated with mitochondrial membrane potential disruption. Compounds do not act directly on the mitochondrial membrane, but could induce activation of upstream caspases such as caspase 8 and/or caspase 2, depending on the compound. In no case did upstream caspase 8 activation involve Fas/FasL interaction. Taken together, these results show that epsilon-viniferin and, more importantly, miyabenol C represent potent antitumor agents that require further investigation, either alone or in combination with resveratrol.

Efficient rescue of gutted adenovirus genomes allows rapid production of concentrated stocks without negative selection.

Gutted adenoviral (Ad) vectors have a greater cloning capacity and elicit less immune response than conventional Ad vectors. Unfortunately, clinical use of gutted vectors has been slowed by production difficulties, including low yield and a tendency for recombinant virus to emerge. These two problems are related, because expansion of dilute vector stocks requires selective pressure against helper virus. The ability to rescue gutted virus at high titer would lessen the requirement for selective pressure, thereby limiting the advantage afforded to undesirable recombinants. We tested gutted virus rescue from plasmids and from synthetic terminal protein (TP)-DNA complexes by transfection/infection or cotransfection with various forms of helper viral DNA. Optimal rescue required cotransfection of gutted and helper genomes with identical origins of replication. Transfection/infection, which introduces unequal origins, was 30 times less efficient than cotransfection of genomes that had been released from plasmid DNA and bore identical origins. Cotransfection of TP-linked genomes was several times more efficient than that of unlinked genomes, yielding average gutted viral titers above 10(7) transducing units (TU)/ml. In addition, we found that limited expression of Cre recombinase doubled the yield of gutted virus. Using these techniques, gutted viruses can be rescued at titers greater than 3 x 10(7) TU/ml, about 100 times higher than is usually achieved. Finally, we found that high-titer lysates could be serially passaged on Cre-negative cells without loss of titer, further reducing selective pressure. These methods allow large-scale production of gutted virus in three or four serial passages, while minimizing exposure to Cre recombinase.

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.

BACKGROUND: Helper-dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high-titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo. METHODS: Replication-defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7-cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre-terminal protein, and with a cre-recombinase plasmid. RESULTS: We show that C7-cre cells allow efficient production of gutted Ad using deltaE1 + deltaE2b + deltaE3 helper viruses whose growth can be limited by cre-loxP-mediated excision of the packaging signal. Gutted Ad vectors carrying approximately 28 kb cassettes expressing full-length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%. CONCLUSIONS: These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b-deleted vectors display a profound reduction in viral gene expression.

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin.

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25-30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.

Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector.

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.