We tested the ability of recombinant hMutS␣ (hMSH2͞hMSH6) and hMutS␤ (hMSH2͞hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base͞base mispairs and insertion-deletion loops was restored by hMutS␣, only the latter substrates were addressed in extracts supplemented with hMutS␤. hMutS␣ was also able to complement a defect in the repair of base͞base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutS␣ and MutS␤. As a rule, MSH2 is primarily complexed with MSH6. MutS␣ is thus relatively abundant in mammalian cell extracts, whereas MutS␤ levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutS␤. This leads to degradation of the partnerless MSH6 and depletion of MutS␣. CHO R and HL60R cells therefore lack correction of base͞base mispairs, whereas loop repair is maintained by MutS␤. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al.

Interleukin‐6 (IL‐6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post‐menopausal osteoporosis. IL‐6, a four‐helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low‐affinity IL‐6 receptor (IL‐6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL‐6R alpha. These mutants behave as full and selective IL‐6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL‐6 super‐antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL‐6R alpha. The IL‐6 super‐antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild‐type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL‐6 suggests that it could be possible to design super‐antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-␣). However, systemic delivery of r-hIFN-␣ is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-␣ antiviral efficacy, we have explored the therapeutic potential of murine IFN-␣2 (mIFN␣2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-␣2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-␣2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-␣2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-␣2.Interferon (IFN) was discovered by Isaac and Lindenmann in 1957 (18), and recently the U.S. Food and Drug Administration has approved recombinant human IFN-␣ (r-hIFN-␣) for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. IFN-␣ acts on target cells to confer a state of resistance to viral infectivity at one or more stages of virus entry or replication. These biological effects require binding to the type I IFN receptor complex, which is composed of two subunits, ␣ and ␤ (12). Both subunits undergo rapid ligand-dependent tyrosine phosphorylation, and the ␣ subunit itself acts as a species-specific transducer for type I IFN action (7). At least 30 genes are known to be transcriptionally induced by type I IFNs, including 2Ј,5Ј-oligoadenylate synthetase (2Ј5ЈOAS), the double-stranded RNA-activated protein kinase, and the IFN-I response factor I (8, 10). 2Ј5ЈOAS is important for antiviral response, and its activity is required by cells to activate the endonuclease RNase L, which degrades RNA (31).The currently available treatment for HCV with r-hIFN-␣ results in clearance of the virus in only 20% of patients. However, recent clinical trials have shown that a combination of r-hIFN-␣ and the antiviral drug ribavirin can increase the percentage of recovery up to 40% (9, 30). Although these results appear to be very promising, systemic injection of r-hIFN-␣ is associated with severe side effects, which worsen in combination with ribavirin, causing the withdrawal of 20% of patients from therapy.It is not clear why r-hIFN-␣ treatment is effective in only a minority of patients. One possible explanation has been postulated on the basis of association of specific HCV genotype and lack of sustained response. HCV proteins may block IFN-␣-induced antiviral polypeptides, t...

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