|Budget Amount *help
¥5,800,000 (Direct Cost : ¥5,800,000)
Fiscal Year 1991 : ¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1990 : ¥3,300,000 (Direct Cost : ¥3,300,000)
The human parvovirus adeno-associated virus (AAV) grows in the presence of helper functions provided by helper adenovirus. AAV is not pathogenic to human beings. When AAV infects cells in the absence of a helper virus, it frequently integrates its genome into the host cell genome. AAV has several advantages as virus vectors. To exploit AAV as virus vectors, we have worked on three main projects, (1) reconstruction of AAV genome, (2) reconstruction of helper adenovirus genome, (3) regulation of expression of foreign genes inserted into AAV genome.
(1) To reconstruct AAV genome, we prepared a recombinant plasmid pAAV which contained whole AAV genome. Several foreign gents, were inserted into the envelope protein region of PAAV. Human thymus epithelial cells were immortalized with the plasmid pAAV-SV40 DNA, which contained the SV40 early gene in the envelope region of PAAV. It indicated that the AAV vector was able to be developed from the recombinant plasmid.
(2) We also made a recombinant helper adenovirus, which expressed AAV envelope proteins to complement the defectiveness of recombinant AAV vectors. We have tried to produce recombinant AAV vectors by co-introduction of pAAV-SV40 DNA and the recombinant helper adenovirus into 293 cells. The recombinant AAV virus was obtained but the virus titer was low, because of low efficiency of production of AAV envelope proteins.
(3) To study the regulation of gene expression of foreign genes inserted into AAV genome, we have developed the affinity latex particles to purify DNA-binding transcription factors. By using the particles, we were able to purify multiple members of the ATF/E4TF3 family or two subunits of E4TF1 directly from crude cell extracts. The particles would be useful for studying various transcription factors.
On the basis of this study, we will try further to exploit AAV as useful virus vectors.