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Chimeric RNA͞DNA oligonucleotides (''chimeraplasts'') have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated ''MDX1'') designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were ''revertant fibers.'' Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. M uscular dystrophies are hereditary, degenerative disorders of muscle that result from defects in genes that encode a diverse group of proteins (1). The most common form of muscular dystrophy in humans is Duchenne muscular dystrophy (DMD), which occurs when a defect in the dystrophin gene results in a deficiency of dystrophin protein in skeletal muscle (2). The absence of dystrophin leads to muscle cell death and progressive muscle degeneration, although the pathogenetic mechanisms remain a mystery (3) and are the subject of active study (4, 5). As there are no effective long-term treatments for the muscular dystrophies, there has been much interest in gene therapy approaches to these disorders. Currently, the most actively studied methods involve viral-mediated delivery of normal genes to skeletal muscle, although all current viral vectors have limitations and͞or adverse effects.Recently, several groups have used a strategy to induce single base pair changes in genomic DNA in both mammalian cells (6 -11) and plant cells (12,13). The strategy involves the use of chimeric RNA͞DNA oligonucleotides (''chimeraplasts''), each of which contains a stretch of oligonucleotides homologous to a sequence in the genome except for a single mismatched base. When the sequence in the chimeraplast aligns with that in the genomic DNA, the single base mismatch induces endogenous repair mechanisms to ''correct'' the base in the targeted gene (14 -16). Chimeric oligonucleotides have been used to induce single nucleotide changes in different mammalian cell types both in vitro (6 -10) and in vivo (9, 11). Whereas the efficiency of chimeraplast-mediated gene conversion has varied widely depending on the particular cell type and the experimental co...

Because of its prominent expression in central nervous system inflammatory pathology by astrocytes, we examined the mechanism of human IP-10 (hIP-10) gene induction by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in astrocytoma cells. When present together, IFN-gamma and TNF-alpha induced robust accumulation of hIP-10 mRNA, but hIP-10 mRNA was minimally induced when astrocytoma cells were treated with individual cytokines. This pattern of expression resembled that previously described for murine IP-10 (mIP-10) gene induction in fibroblasts and in rat astroglia. Nuclear run-on experiments showed that the synergistic effect of the cytokines resulted from an increased rate of IP-10 transcriptional initiation. Functional analysis of the hIP-10 promoter after deletion and substitution mutagenesis indicated that an interferon-stimulated response element (ISRE) governed both simple response to IFN-gamma and synergy with TNF-alpha. Synergistic induction of hIP-10 also required an ISRE-proximal nuclear factor kappa-B (NFkappaB) binding site. TNF-alpha-induced NFkappaB binding activity at this site was composed of RelA (p65) homodimers. Our results document that cis-elements through which cytokines mediate synergistic induction of IP-10 in mouse and human are strictly conserved despite divergence elsewhere within the proximal 5'-flanking region.

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Human IFN-γ-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-γ alone, and synergistically induced by TNF-α and IFN-γ. Upstream regions of the human and murine genes contain conserved regulatory motifs, including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-γ. Trans-acting factors mediating the IFN-γ response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-γ, with or without TNF-α, was demonstrated using p48-deficient U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed IFN-γ/TNF-α-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1. Induction of hIP-10 by TNF-α/IFN-γ also required NFκB binding sites, which were protected in vivo and bound p65 homodimeric NFκB in vitro. These results documented the essential role of p48 (complexed with STAT-1α) for induction and sustained transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory cytokines.