A novel human suspension culture packaging cell line for production of high-titre retroviral vectors.
Retroviruses are currently the most widely used vectors in clinical trials for gene therapy. These vectors are, however, limited by low titres partly due to the restrictive nature of monolayer cell culture. We have developed a stable suspension producer cell line derived from human lymphoblastoid cells (WIL-2) by electroporating these cells with the necessary trans components required for production of defective retrovirus particles which encode a nuclear localising beta-galactosidase gene. We show that this anchorage-independent cell line generates viruses at a titre of 7 x 10(5) iu/ml on NIH3T3 indicator cells which remains constant after at least 2 months in culture. The producer cells can be cultured at a density of 6 x 10(6) cells/ml with consistent virus titre production. WIL-2 can also be grown as single cells by rotation culture while maintaining virus production. By treating the cells with the transcriptional activator sodium butyrate titres above 1 x 10(6) i.u./ml are achieved. Concentrating viral supernatants by ultrafiltration can further increase virus titre to 5 x 10(8) i.u./ml. Even at these high titres no replication-competent virus was detected. Virus titre fell only slightly when cells were placed in serum-free media before harvest. The generation of this novel cell line provides proof-of-principle that large-scale production of retroviral vectors in serum-free growth conditions can be safely generated for use in gene therapy.
Chan
L M
LM
Cystic Fibrosis Gene Therapy Research Group, Division of Biomedical Sciences, Imperial College School of Medicine, London, UK.
Coutelle
C
C
Themis
M
M
D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002452:Cell Count; D002460:Cell Line; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects; D014161:Transduction, Genetic; D014462:Ultrafiltration; D001616:beta-Galactosidase / Q000235:genetics
D002087:Butyrates; D016895:Culture Media, Serum-Free; D001616:beta-Galactosidase