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Large‐scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols
Author(s) -
Farson Deborah,
McGuinness Ryan,
Dull Tom,
Limoli Kay,
Lazar Richard,
Jalali Sayeh,
Reddy Sridhar,
PennathurDas Rukmini,
Broad David,
Finer Mitchell
Publication year - 1999
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/(sici)1521-2254(199905/06)1:3<195::aid-jgm31>3.0.co;2-#
Subject(s) - retrovirus , immunotherapy , computer science , virology , biology , immunology , virus , immune system
Background The use of gene modified T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an efficient, safe ex vivo method for modification of these cells at manufacturing scale. Since retroviruses have been shown to be a moderately effective means of stably integrating therapeutic genes into T lymphocytes, we wanted to create packaging and producer cell lines that would produce replication competent retrovirus (RCR)‐free supernatants, at large scale (>200 l), and transduce with high efficiency. Methods cDNA expression plasmids containing only coding sequences for gagpol or env were built and sequentially transfected into human 293 cells. Packaging and producer clones were characterized for stability, titer and RCR. A producer clone delivering chimeric immune receptors was scaled‐up and supernatants used to transduce patient T lymphocytes for clinical studies. PCR and RT‐PCR assays were utilized to evaluate the transmission of HERV‐H sequences. Relative infectivity of producer clones pseudotyped with different envelopes was determined by transduction and RT assays. Results RCR‐free, human 293 split‐genome packaging lines, pseudotyped with amphotropic, xenotropic, or 10A1 envelopes, were created. A CC49 ζ producer clone was scaled‐up to 5×54 l lots and supernatants used to safely and efficiently transduce patient T lymphocytes with minimal ex vivo manipulation. While 293 cells express HERV‐H mRNA, the transmission frequency in our packaging clones was less than 1 HERV‐H sequence per 5×10 5 proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone. Conclusion These packaging lines represent the safest configuration for the large‐scale production of retroviral vectors, and are capable of producing high titer, RCR‐free retroviral vector for large scale clinical use. While all three clones efficiently transduce human T lymphocytes, the 10A1 clone has the highest infectivity. These packaging cell lines will be valuable for use in human gene therapy protocols. Copyright © 1999 John Wiley & Sons, Ltd.