Optimising stable retroviral transduction of primary human synovial fibroblasts.
Fibroblast like synoviocytes are the main resident cells in normal joints and are known to play a major role in the pathogenesis of rheumatoid arthritis. Efficient gene targeting of fibroblast like synoviocytes (FLS) is a major goal of current ex vivo gene therapy approaches for the treatment of rheumatoid arthritis. However, there is a need to improve viral systems capable of delivering genes to human rheumatoid fibroblasts and attempts have been made to develop a protocol for high efficiency, reproducible gene transfer using a replication-defective retrovirus vector. The effects of different experimental conditions were examined as well as those related to cellular and viral features on the efficiency of transducing the retroviral-driven expression of enhanced green fluorescent protein (EGFP) to FLS harvested from patients with rheumatoid arthritis. The optimal method established involved a double round of infection by centrifugation with a resting period of 4h between rounds. This approach led to the transduction of 30-70% of FLS obtained from nine patients with rheumatoid arthritis. Consistent transduction efficiencies were achieved in repeat assays such that it could be inferred that the variations observed were attributable to the specific characteristics of each cell line. This simple protocol renders a consistent and reproducible efficiency of rheumatoid fibroblast transduction and makes stable gene targeting using non-replicating retrovirus derived vectors an affordable option for the treatment of rheumatoid arthritis.
Paya
Monica
M
Unidad de Investigación, Laboratorio de Reumatologia, Hospital 12 de Octubre, Avda. Cordoba S/N, Madrid 28049, Spain.
Segovia
Jose C
JC
Santiago
Begoña
B
Galindo
Maria
M
del Rio
Paola
P
Pablos
Jose L
JL
Ramírez
Juan C
JC
D001172:Arthritis, Rheumatoid; D002478:Cells, Cultured; D005347:Fibroblasts / Q000821:virology*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics*; D013582:Synovial Fluid / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods*
D049452:Green Fluorescent Proteins