Abstract
Objectives
To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins.
Results
AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed “CELiD” DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with “CELiD” DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %.
Conclusions
The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.
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Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (No. 81371670), the Natural Science Foundation of Jiangsu Province (Grant No. BK20140381) and the Science and Technology Program of Suzhou (Grant No. ZXY201432).
Supporting information
Supplementary Methods: Cells and culture conditions, construction of CD19-specific CARs and plasmids, western blotting.
Supplementary Material: Sequences of AAVS1 probe and NeoR probe for Southern blotting.
Supplementary Fig. 1—CAR-T cells activation assay.
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Zhang, Y., Liu, X., Zhang, J. et al. Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering. Biotechnol Lett 38, 1423–1431 (2016). https://doi.org/10.1007/s10529-016-2139-7
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DOI: https://doi.org/10.1007/s10529-016-2139-7