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kat: a high-efficiency retroviral transduction system for primary human T
lymphocytes
MH Finer, TJ Dull, L Qin, D Farson and MR Roberts
Cell Genesys Inc., Foster City, CA 94404.
We describe a novel retroviral packaging system in which high titer
amphotropic retrovirus was produced without the need to generate stable
producer clones. kat expression vectors, which produce high levels of
retroviral vector transcripts and retroviral packaging functions, were
transfected into 293 cells followed by virus harvest 48 hours
posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of
frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than
transient viral titers reported using 3T3-based retroviral packaging lines.
Cocultivation of primary human CD8+ T lymphocytes after transient
transfection of 293 cells with kat plasmids resulted in transduction
efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation
with a high titer PA317 producer clone and significantly greater than
previously reported results for transduction of primary human T lymphocytes
with retroviral vectors. Virus produced using the kat system was shown to
be free of detectable replication competent retrovirus by an extended
provirus mobilization assay, demonstrating that this system is as safe as
currently available stable packaging lines. The kat virus production system
should be of general use for the rapid production of high titer viral
supernatants, as well as for high-efficiency transduction hematopoietic
cell types refractory to retroviral transduction.
Volume 83,
Issue 1,
pp. 43-50,
01/01/1994
Copyright © 1994 by The American Society of Hematology
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