Increased Viral Titer Through Concentration of Viral Harvests from Retroviral Packaging Lines

Abstract

Dependent on the viral vector and the specific assay used, viral titers produced from commonly used retroviral packaging cell lines have an upper limit in the range of 10(5) to 10(7) infectious units/ml. We have developed a generally applicable method, using hollow-fiber filtration technology, which allows for the concentration of infectious virus derived from packaging lines. This method resulted in a reproducible 10- to 30-fold increase in viral titer and can readily be scaled to accommodate larger input volumes. Over 80% of the input virus is recovered in an infectious form in the concentrate. Concentrated virus containing media was seen to produce higher infection frequencies in Jurkat T cells as compared to unconcentrated virus containing media; however, this was not proportional to the differences in viral titer observed by limiting dilution analysis on NIH-3T3 cells. These results are discussed in relation to the importance of factors other than viral titer in determining transduction frequencies.

Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene

“…Other approaches include: (1) the development of optimized conditions of ome of target cells makes them effective gene transfer vehicles for the introduction of foreign genes into a broad retroviral production and transduction; 9,10 (2) concentration of retrovirus particles from cell-free supernatant range of cell lines. The delivery of therapeutic gene(s) into target cells using retroviral vectors has been achieusing hollow-fiber filtration technology; 11 and (3) screening of retroviral packaging cell lines using high selection ved in cultured cells and more recently ex vivo in clinical settings. 1 The efficiency with which genes can be intropressure.…”

A preclinical model of hepatocyte gene transfer: the in vivo, in situ perfused rat liver

“…Efforts to concentrate retroviruses using centrifugation 28 and tangential filtration 29,30 showed that target cell transduction rate increases proportionally with the virus titer. Kitten et al 31 described a high-producer cell line (10 8 ffu/ml) transducing 46 ± 17% of rat hepatocytes with retrovirus bolus-injected through the portal vein, without any concentration step.…”

Enhanced retroviral gene delivery in ultrasonic standing wave fields

“…In fact, diminution of retroviral infectivity during the process of concentration (ie, ultracentrifugation) and purification (ie, ultrafiltration) has been reported. 25,26 This is because the association of gp70 (binding to cellular receptor) and p15E (fusion with plasma membrane) subunits of type C retroviral envelope protein is a labile disulfide linkage, which is not robust under shearing forces. 27,28 Owing to the loss of gp70 subunit (or SU domain), the retrovirus cannot enter the target cell via receptor-mediated endocytosis.…”