Four different methods of retroviral transduction of rheumatoid synovium were compared: A) one static transduction at passage 1; B) one static transduction at passage 2; C) transduction at passage 1 during centrifugation; D) two static transductions made 24 h apart during passage 1 (Fig. ).
Figure 1 IL-1Ra production by human synoviocytes after transduction by MFG-IRAP under various conditions. Monolayer cultures of synoviocytes from five different patients with RA were transduced with standard-titer MFG-IRAP under the following conditions: A) first (more ...)
Cells transduced once at passage 1 by static infection with standard-titer MFG-IRAP secreted 14.2 ± 2.8 ng IL-1Ra/106 cells per 48 h. None of the cultures secreted the ≥ 30 ng IL-1Ra/106 cells per 48 h required by the clinical protocol. Similar results were obtained with second passage cells. Static transduction twice within a passage resulted in an average secretion of 33.8 ± 8.7 ng IL-1Ra/106 cells per 48 h, but only two of the five cultures secreted more than 30 ng/106 cells per 48 h. First passage cells transduced once while being centrifuged secreted 110 ± 8.0 ng IL-1Ra/106 cells per 48 h (P < 0.0001 vs one or two static transductions) and all five cultures exceeded the cutoff threshold of 30 ng/106cells per 48 h. Routine static transductions with MFG-LacZ vector, carried out in parallel with these experiments, resulted in approximately 30% LacZ+ cells, whereas transductions carried out during centrifugation resulted in nearly 100% transduction.
Lowering the temperature from 37 to 32°C or increasing the volume of the retroviral supernatant from 1 to 3 ml per well produced small (25% or less; statistically insignificant) improvements in transgene expression (data not shown). Increasing the time of transduction beyond 24 h raised the transduction efficiency considerably (Fig. ). With transduction periods of 48 and 72 h, all cultures produced at least 30 ng IL-1Ra/106 cells per 48 h.
Figure 2 IL-1Ra production by human synoviocytes after static transduction for extended periods. Monolayer cultures of synoviocytes from five different patients with RA were transduced statically in duplicate with standard titer MFG-IRAP for the indicated lengths (more ...)
In another series of experiments, high titer MFG-IRAP, flow-through transduction [11
], and DOGS [12
] instead of Polybrene were evaluated for their effects on transduction efficiency and gene expression (Table ). Qualitatively equivalent results were obtained with two different patients in triplicate experiments. Transduction was significantly more efficient with high titer than with low titer virus, regardless of the polycation used (P
< 0.05) or the transduction method (static or flow-through). The flow-through transduction method was significantly better (P
< 0.05) than static transduction when Polybrene was used. In the presence of DOGS, flow-through transduction was significantly better than static transduction if low titer virus was used (P
< 0.05). Overall, DOGS did not consistently provide significantly better transduction efficiency than Polybrene.
Effect of flow-through, high retroviral titer and DOGS on the transduction of human synoviocytes.