Regions downstream from the WW domain of dystrophin are important for binding to postsynaptic densities in the brain.

In order to investigate the mechanism of dystrophin localization in the central nervous system (CNS), we generated adenovirus vectors that contained minidystrophin or truncated minidystrophin cDNA. We infected a primary neuronal culture derived from mdx mouse hippocampus with these viruses. Minidystrophin was observed along the plasma membrane as punctate dots or very short segments. In double immunofluorescence staining with anti-dystrophin and anti-postsynaptic density-95 antibodies, we observed that these proteins entirely colocalized. On the other hand, the truncated minidystrophin, which has deleted WW, cysteine-rich and C-terminal domains, was homogenously expressed in cytoplasm, neurites and axons. These findings suggest that a binding site to postsynaptic densities exists in the region extending from the WW domain to the C-terminal domain of dystrophin and that this site is necessary for binding to membrane.

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin.

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.

Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging.

Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination.

Adenovirus-mediated murine interferon-gamma receptor transfer enhances the efficacy of IFN-gamma in vivo.

Interferon gamma (IFN-gamma) plays an important role in immune response, apoptosis, and anti-tumor activity. Its biological activity depends on expression of IFN-gamma receptor (IFN-gammaR). To address whether increased expression of IFN-gammaR is associated in vivo with a higher biological response by IFN-gamma, we constructed an adenovirus vector including murine IFN-gammaR (Ad-mIFN-gammaR). We confirmed the appropriate function of mIFN-gammaR derived from Ad-mIFN-gammaR based on the observation of signal transduction and transcription. We also found that elevated expression of mIFN-gammaR increases sensitivity to recombinant murine IFN-gamma (rmIFN-gamma) in vitro in target cells. Furthermore, we demonstrated that the growth rate of tumors transfected with Ad-mIFN-gammaR is suppressed in response to rmIFN-gamma in vivo and that such growth suppression is partly due to apoptosis. To our knowledge, this is the first report of adenovirus-mediated IFN-gammaR gene transfer being effective in augmenting the biological activity of IFN-gamma, and the strategy employed in the present study will be useful in studying other kinds of cytokine receptors and applications to gene therapy for cancer and infectious diseases.