title: Protein impurities from cell culture dramatically impact transduction efficiency of polymer/virus hybrid vectors.
abstract: Polyethylenimine (PEI) was used recently with murine leukemia virus-like particles (MLV-VLPs) to produce a hybrid vector that possesses advantages over the native virus; the transduction efficiency of this vector, however, was less than the transduction efficiency of the native virus. The cause of the reduced efficiency was hypothesized to be related to the involvement of proteins in PEI/MLV-VLP complex formation and overall complex size. To test the hypothesis and potentially improve the efficiency of the hybrid vector, ultracentrifugation and size exclusion chromatography were used to purify MLV-VLP and to study the effect of proteins in cell culture medium on complex formation. Based on dynamic light scattering and electron microscopy, complexes formed from the purified MLV-VLPs were smaller, but surprisingly, less efficient than complexes formed from unpurified MLV-VLPs. The addition of protein to purified MLV-VLPs showed that the initial efficiency could be restored and that the purification strategy was not inactivating the MLV-VLPs. Further, by optimizing the amount of protein added to the purified MLV-VLPs, the level of transduction by PEI/MLV-VLP improved 1.6-fold. Particle characterization showed a correlation between the size of the PEI/MLV-VLP complex and the transduction efficiency, which is likely a result of greater sedimentation and cell contact during in vitro studies.
authors.affiliation: School of Chemical Engineering, Oklahoma State University, Stillwater, OK, USA.
authors.forename: Sravanthi
authors.initials: S
authors.lastname: Vupputuri
authors.forename: Shishir
authors.initials: S
authors.lastname: Karode
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Neely
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Ramsey
mesh_terms: D018929:Cell Culture Techniques; D002460:Cell Line; D002850:Chromatography, Gel; D003470:Culture Media / Q000737:chemistry*; D005822:Genetic Vectors / Q000302:isolation & purification; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000302:isolation & purification; D011506:Proteins / Q000378:metabolism*; D014161:Transduction, Genetic; D014461:Ultracentrifugation
chemical_list: D003470:Culture Media; D011506:Proteins