FIG. 5.
Optimization of vector production for targeted transduction in vivo. (A) Optimization of ratios of pCG-HcΔ18-AA-IL-13 and pCG-FcΔ30 plasmids. Samples containing 0.53 μg of pNL(CMV)EGFP/CMV/WPREΔU3, 0.32 μg of pCD/NL-BHΔ1, 0.76 μg of pCMV-rev, and various amounts of pCG-FcΔ30 and pCG-HcΔ18-AA-IL-13 plasmid DNAs were prepared to transfect HEK 293T cells in 6-well plates. The combined amounts of pCG-FcΔ30 and pCG-HcΔ18-AA-IL-13 plasmid DNAs used were 0.18 μg; this amount was kept the same for each sample. Sixty hours posttransfection, vector-containing supernatants were harvested and used to transduce U251 cells. Transduced cells were analyzed by FACS 48 hr posttransduction. Vector titers were calculated on the basis of the percentage of EGFP-positive cells. (B) Optimization of vector/helper to Env glycoprotein plasmid ratios. The ratio of the vector to the helper plasmids (1.5:1) was the same for each sample; the ratio of pCG-FcΔ30 to pCG-HcΔ18-AA-IL-13 Env glycoprotein plasmid (2:1) was kept the same; the total amount of plasmid DNA in each sample (1.08 μg) was kept the same but the ratios of the combined amounts of vector and helper plasmids to the Env glycoprotein plasmid varied as indicated.