Improvement of Gene Therapy by Use of Aritificial Virus

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In order to improve the integration of artificial vital DNA into host chromosome, we have studied the components of preintegration complex (PIC) of molony murine leukemia virus as a model system. We found that integrase which is one of the main components of the PIC, physically interacted with a transcription factor YY1. We also found that Bmi-1 which is a subunit of polycomb group repressive complex, interacted with INI-1(integrase interacter-1). INI-1 associates with integrase and is also a main component of PIC. To clarify the interaction between integrase and YY-1, first, we analyzed the interaction by GST-pull down assay. We found that the N-terminal region of YY-1 interacted with the integrase. Because N-terminal region of YY-1 is known as an activation domain and the C-terminal region is a domain for DNA interaction, we assumed that binary binding trough integrase and YY-1 to virus cDNA may affect the integration reaction. Integrase from HIV-1 also interacted with YY-1 by the GST-pull down assay.In order to improve the viral vector production, we applied a transient viral production system, so-calle d Q-vector system, to molony leukemia virus based vector system. So far now, high titer viral vector preparation has been prepared by so-called packaging cell line method. But the establishment of packaging cell line is laborious and it take longer time. On the other hand, virus vector can be produced within 2-3 days by the Q-vector system. We found that the virus titer produced by the Q-vector was largely dependent on vector size but the titer was comparable to the original packaging cell method if the transgene size was around 2 kb. When the transgene has a cytotoxic effect on animal cells, the Q-vector system gave a higher titer comparing to the packaging cell method.