Study on the production and purification of a new generation adenovirus vector (gutless adenovirus) and its clinical application

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We successfully produced and purified a new generation adenovirus vector (gutless adenovirus) aiming for gene therapy by using Cre/loxP system. This new generation adenovirus is helper-dependent adenovirus and lack virtually almost all of the wild adenoviral DNA.Instead of the deletion it was replaced to non-viral DNA longer than 30kb. After the repetitive propagations viral titer increased. Then propagated vectors could be condensed by CsCl buoyant density centrifugation to 1000-fold higher than that of crude lysate.And we optimized the structure of packaging DNA and the structure of helper virus for a robust system capable of producing high-titer gutless adenovirus stocks with low helper contamination.Furthermore, we constructed a gutless adenovirus containing the expression units of 16.5kb-long mouse full-length dystrophin and 2kb-long human Coxsackievirus and adenovirus receptor (CAR). By using this vector we could transduced the expression of dystrophin in tibialis anterior skeletal muscles of mdx-mouse in vivo.As this vector is very promising for gene therapy, long-term gene expression and activation of immunity must be carefully studied for safe clinical application.