Abstract
Retroviral vector-mediated gene transfer has been used successfully in clinical gene therapy. Cells of the hematopoietic lineages, however, remain difficult to transduce, although precoating of culture vessels with the fibronectin fragment CH-296 may improve transduction efficiency. Alternatively, low-speed centrifugation of vector-containing supernatant onto culture vessels may improve transduction efficiency in the absence of CH-296 preloading. Using the NIH/3T3-derived Moloney murine leukemia virus-based packaging cell lines PG13, PA317, and PT67, we here show that preloading by low-speed centrifugation improves transduction efficiency in a packaging cell subclone-dependent manner. Preloading by centrifugation, however, cannot generally replace CH-296 and we obtained the overall highest transduction levels when combining centrifugation and CH-296 precoating. We found, moreover, that the factor responsible for high susceptibility to preloading in our PG13-derived vector supernatant was transferable to a PA317-derived vector supernatant with low susceptibility to preloading. Furthermore, our PA317, PG13, and PT67 subclones shed into their supernatants variable amounts of fibronectin. This soluble fibronectin formed aggregates of various sizes and generated complexes with vector particles. The fibronectin–vector complexes readily sedimented onto culture vessels and copurified after fibronectin-specific affinity purification of vector-containing supernatants. Finally, vector supernatant from 293T cells, which barely produce fibronectin, was not susceptible to preloading. The susceptibility to preloading by centrifugation thus appears to be dependent both on the specific packaging cell line and on the association of vector particles and packaging cell-produced fibronectin. Rigorous screening of individual vector-containing supernatants is therefore required to identify optimal transduction conditions for retroviral gene transfer.
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