title: Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements.
abstract: Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.
authors.affiliation: Bone Marrow Transplantation, University Hospital Eppendorf, Hamburg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Ayuk
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: K
authors.initials: K
authors.lastname: Kühlcke
authors.forename: C
authors.initials: C
authors.lastname: Lindemann
authors.forename: U
authors.initials: U
authors.lastname: Schade
authors.forename: H G
authors.initials: HG
authors.lastname: Eckert
authors.forename: A
authors.initials: A
authors.lastname: Zander
authors.forename: B
authors.initials: B
authors.lastname: Fehse
mesh_terms: D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy*; D017252:CD3 Complex; D002478:Cells, Cultured; D002498:Centrifugation; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D020800:Receptor, Nerve Growth Factor / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D016511:Severe Combined Immunodeficiency / Q000628:therapy*; D018709:Statistics, Nonparametric; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D017252:CD3 Complex; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D020800:Receptor, Nerve Growth Factor