The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.
Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals f lanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an Ϸ10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of Ϸ10 10 transducing virions͞ml, with <0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.
Extensive analyses of mice carrying null mutations in paired box 7 (Pax7) have confirmed the progressive loss of the satellite cell lineage in skeletal muscle, resulting in severe muscle atrophy and death. A recent study using floxed alleles and tamoxifen-induced inactivation concluded that after 3 wk of age, Pax7 was entirely dispensable for satellite cell function. Here, we demonstrate that Pax7 is an absolute requirement for satellite cell function in adult skeletal muscle. Following Pax7 deletion, satellite cells and myoblasts exhibit cell-cycle arrest and dysregulation of myogenic regulatory factors. Maintenance of Pax7 deletion through continuous tamoxifen administration prevented regrowth of Pax7-expressing satellite cells and a profound muscle regeneration deficit that resembles the phenotype of skeletal muscle following genetically engineered ablation of satellite cells. Therefore, we conclude that Pax7 is essential for regulating the expansion and differentiation of satellite cells during both neonatal and adult myogenesis.CreERT2 | stem cell
Many applications for human gene therapy would be facilitated by high levels and long duration of physiologic gene expression. Adenoviral vectors are frequently used for gene transfer because of their high cellular transduction efficiency in vitro and in vivo. Expression of viral proteins and the low capacity for foreign DNA limits the clinical application of first- and second-generation adenoviral vectors. Adenoviral vectors with all viral coding sequences deleted offer the prospect of decreased host immune responses to viral proteins, decreased cellular toxicity of viral proteins and increased capacity to accommodate large regulatory DNA regions. Currently most vectors used in vivo for preclinical and clinical studies express cDNAs under the control of heterologous eukaryotic or viral promoters. Using an adenoviral vector with all viral coding sequences deleted and containing the complete human alpha1-antitrypsin (PI) locus, we observed tissue-specific transcriptional regulation in cell culture and in vivo; intravenous injection in mice resulted in high levels of very stable expression for more than ten months and decreased acute and chronic toxicity. These results indicate significant advantages of regulated gene expression using genomic DNA for gene transfer and of adenoviral gene transfer vectors devoid of all viral coding sequences.
The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human ␣1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3-5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helperdependent vectors with sequential delivery of vectors of different serotypes.
Optimal gene therapy for many disorders will require efficient transfer to cells in vivo, high-level and long-term expression, and tissue-specific regulation, all in the absence of significant toxicity or inflammatory responses. While recombinant adenoviral vectors are efficient for gene transfer to hepatocytes, their usefulness is limited by short duration of expression related, at least in part, to immune responses to viral proteins and by a low capacity for foreign DNA. A number of systems have been developed for producing adenoviral vectors devoid of all viral coding sequences. Using AdSTK109, a vector lacking all viral coding sequences and carrying the complete human alpha1-antitrypsin (hAAT) genomic DNA locus, we have demonstrated sustained expression for longer than 10 months in mice. Utilizing high doses of this vector for hepatic gene transfer in mice, we find that supraphysiological levels of hAAT can be achieved without hepatotoxicity.
Helper-dependent adenovirus (HD-Ad
To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.
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