Presentation Information
[1ACCE-02]Production of adeno-associated virus vectors in baculovirus-free insect cell system
○Hideki Yamaji1 (1. Kobe University (Japan))
Keywords:
Adeno-associated virus,Virus vector,Insect cell
[Purpose]
Adeno-associated virus (AAV) vectors have been used in clinical applications, mainly in in vivo gene therapy for genetic diseases, with remarkable therapeutic results. AAV vectors can be produced in relatively large amounts using the baculovirus–insect cell system. However, this method still has some problems such as the need to remove and inactivate newly proliferated and budded recombinant baculoviruses. In the present study, we investigated the production of AAV vectors in insect cells without the use of baculoviruses.
[Methods]
When a therapeutic gene inserted between two ITRs (inverted terminal repeats) of AAV and the genes for the AAV regulatory protein Rep and capsid protein Cap are introduced and expressed in host cells, a non-replicating recombinant AAV vector can be generated. Rep and Cap genes together with a reporter gene inserted between two ITRs were transfected and transiently expressed in Trichoplusia ni BTI-TN-5B1-4 (High Five) cells using plasmid vectors instead of baculoviruses. To regulate the expression of Rep and Cap genes, we used promoters that function independently in insect cells without relying on baculovirus.
[Results]
Depending on the promoters used, the expression levels of Rep and Cap proteins and the titer of AAV vector produced varied. We confirmed that the AAV vectors produced were able to infect mammalian cells and express the reporter gene.
[Conclusion]
By using appropriate promoters, adeno-associated virus vectors that infect mammalian cells and express a reporter gene can be produced using lepidopteran insect cells as hosts without the use of baculoviruses.
Adeno-associated virus (AAV) vectors have been used in clinical applications, mainly in in vivo gene therapy for genetic diseases, with remarkable therapeutic results. AAV vectors can be produced in relatively large amounts using the baculovirus–insect cell system. However, this method still has some problems such as the need to remove and inactivate newly proliferated and budded recombinant baculoviruses. In the present study, we investigated the production of AAV vectors in insect cells without the use of baculoviruses.
[Methods]
When a therapeutic gene inserted between two ITRs (inverted terminal repeats) of AAV and the genes for the AAV regulatory protein Rep and capsid protein Cap are introduced and expressed in host cells, a non-replicating recombinant AAV vector can be generated. Rep and Cap genes together with a reporter gene inserted between two ITRs were transfected and transiently expressed in Trichoplusia ni BTI-TN-5B1-4 (High Five) cells using plasmid vectors instead of baculoviruses. To regulate the expression of Rep and Cap genes, we used promoters that function independently in insect cells without relying on baculovirus.
[Results]
Depending on the promoters used, the expression levels of Rep and Cap proteins and the titer of AAV vector produced varied. We confirmed that the AAV vectors produced were able to infect mammalian cells and express the reporter gene.
[Conclusion]
By using appropriate promoters, adeno-associated virus vectors that infect mammalian cells and express a reporter gene can be produced using lepidopteran insect cells as hosts without the use of baculoviruses.
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