Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application.
An artificial capillary culture/transduction technique has been developed for application in a phase I gene therapy clinical trial for HIV. The trial protocol involves isolation of CD4+ T-lymphocytes from a genetically matched HIV negative twin, retroviral transduction of equal numbers of cells with the ribozyme therapeutic and control genes, and expansion in Cellmax artificial capillary modules. Preclinical studies showed transduction efficiencies in the range of 3-30%, with preferential expansion of CD4+ lymphocytes over a culture period of 10-14 days. Over this time period, an average yield of 1.7 x 10(9) lymphocytes was readily attainable from 5 x 10(7) CD8-depleted lymphocytes. In addition, a sensitive and reliable quantitative competitive PCR method was developed to assess the levels of transduction before infusion into the recipient. The transduction data suggest that the efficiency of retroviral transduction was affected by the presence of inhibitory factors present in the virus preparations or generated as a result of the transduction process itself. It is hypothesised that the method of transduction could significantly affect the extent of this inhibition, and thus impact on clinical efficacy of retrovirus mediated gene therapy.
Knop
A E
AE
Johnson & Johnson Research Laboratories, New South Wales, Australia.
Arndt
A J
AJ
Raponi
M
M
Boyd
M P
MP
Ely
J A
JA
Symonds
G
G
D015496:CD4-Positive T-Lymphocytes; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods*