734. Efficiency of Preloading of Retroviral Vectors without Fibronectin Peptide CH-296 Is Packing Cell Clone Dependent
Authors: Claus S. Sondergaard, Christa Haldrup, Lene Pedersen
Journal: Molecular Therapy (2004)

Retroviral vectors derived from murine fibroblastic cell lines are widely used for introducing heterologous DNA into a variety of target cells in pre-clinical and clinical gene therapy approaches. A number of target cells including human hematopoietic stem cells are, however, difficult to transduce. One of the most important methods for improving the transduction efficiency has been the coating of culture plates with the fibronectin-derived peptide CH-296, which is thought to co-localize vector particles and target cells and preserve the repopulating potential of primitive hematopoietic cells by engaging cell surface receptors. However, in a recent remarkable report, Kuhlcke et al (Mol Ther 5: 473, 2002) found that efficient vector preloading of culture dishes (non-tissue culture treated and tissue culture treated) could be obtained without the presence of CH-296 when applying low speed centrifugation at 1000g of vector containing media (VCM) from the gibbon ape leukemia virus (GALV) pseudotype-encoding PG13 packaging cell line; the observed phenomenon was suggested to be due to soluble fibronectin present in the VCM. Due to the possibility of reducing costs and handling time and improving transduction efficiencies, we further evaluated the importance of CH-296 and centrifugation on the vector preloading efficiency. Three vector pseudotypes and packaging cell lines often employed in clinical and pre-clinical gene transfer studies were analyzed, namely one PG13-derived producer clone, two PA17-derived producer clones (amphotropic murine leukemia (A-MLV) pseudotypes), and one PT67-derived producer clone (10A1 MLV pseudotypes). VCMs were harvested in serum-free X-VIVO 15. The transduction efficiencies obtained with the VCMs subjected to preloading with or without centrifugation at 1000g onto uncoated or CH-296 coated culture plates were compared using a cell line susceptible to all three vector pseudotypes. Moreover, the effects of extending sedimentation and centrifugation preloading times were examined. Although centrifugation generally increased viral titers of all clones tested, we found that the preloading efficiency was highly clone dependent and that centrifugation could not generally replace CH-296 coating. Thus, preloading by centrifugation on uncoated plates yielded lower viral titers as compared to sedimentation onto CH-296 coated plates for one of the A-MLV clones and for the 10A1 clone examined, whereas the other A-MLV clone and the GALV clone were both efficiently preloaded by centrifugation on uncoated plates. Moreover, the centrifugation related increase in viral titer could be ascribed to a soluble factor secreted by the packaging cell line, possibly fibronectin, as it was possible to transfer the centrifugation related effect from one viral supernatant to another by mixing the two prior to preloading. Finally, we found that extending preloading times from 20 to 60 minutes significantly reduced the transduction efficiency when preloading by centrifugation on CH-296 coated plates. Our results show 1) that every producer clone needs testing for efficient gene transfer without the presence of CH-296, 2) if prior testing is not performed inclusion of CH-296 is preferable, since it ensures high transduction efficiency.