| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1996: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Research Abstract |
The replication-defective adenovirus vector was used for gene therapy in US but there are problems such as inflammation due to the CTL induction and short duration of expression due to immunological elimination of the expressed cells. One possible reason is weak expression of E2A gene on the adenovirus vector but E2A gene product is essential for virus propagation in 293 cells. If a general method for preparing virus lacking genes essential for its propagation can be developed, such problem could be resolved. From this point of view, we tried to develop a method for constructing recombinant adenovirus (rAd) specifically lacking E2A/L3 region of the viral genome. The sitespecific recombinase Cre was used for this method. The enzyme recognizes 34-nucleotide specific target sequence, loxP,and excises the DNA flanked with a pair of loxP sites as a circular molecule. First, we constructed a target rAd containing a pair of loxP sites at both sides of the E2A/L3 region. The loxP insertion site near the E2A gene was a new insertion site not previously described. By coinfection of the target virus with the Cre-expressing rAd, we succeeded in generating rAd lacking the E2A/L3 region. Because the deletion virus has a shorter genome and lower density than those of the"parent" target virus, these virus particles can be separated from each other by CsCl density gradient. Therefore, a new method for constructing rAd lacking E2A gene that causes the CTL response is now established. Moreover, the strategy will be useful for a general application to prepare a DNA virus lacking any essential gene.
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