Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols
✍ Scribed by Deborah Farson; Ryan McGuinness; Tom Dull; Kay Limoli; Richard Lazar; Sayeh Jalali; Sridhar Reddy; Rukmini Pennathur-Das; David Broad; Mitchell Finer
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 399 KB
- Volume
- 1
- Category
- Article
- ISSN
- 1099-498X
No coin nor oath required. For personal study only.
✦ Synopsis
Background The use of gene modi®ed T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an ef®cient, safe ex vivo method for modi®cation 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 (w200 l), and transduce with high ef®ciency.
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 CC49f producer clone was scaled-up to 5654 l lots and supernatants used to safely and ef®ciently 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 5610 5 proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone.
Conclusion
These packaging lines represent the safest con®guration 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 ef®ciently 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.