81. Modification of HEK 293 Cell Integrin Expression Profile Allows Convenient Large-Scale Roller Bottle Production of Lentiviral Vectors

Abstract

Human embryonic kidney cells (HEK 293) are the most widely used cell type for production of viral gene transfer vectors. They are highly transfectable with plasmid DNAs encoding viral proteins using a number of transfection reagents and protocols to transiently express recombinant viruses. A variety of stable 293-based packaging cell lines have also been generated for virus production. We have developed a gene transfer system based on the non-primate lentivirus equine infectious anemia virus (EIAV), that utilizes a stable 293 based inducible packaging cell line (BiG 45) for high-titer virus production. Large-scale production of vectors using 293 cells is often difficult since they easily detach from regular tissue culture plates. This can be overcome by adapting the cells to grow in suspension cultures, however this can result in a significant loss in viral titer. Alternately, cultures can be grown in vessels that are treated with cell attachment substrates such as fibronectin or poly-lysine. Both strategies, however, significantly increase the cost of large-scale vector production. In this study, we tested a new strategy of increasing the adhesion properties of 293 cells by modifying their integrin expression profile. First, we tested the effects of over-expressing the integrin beta-3 subunit alone or in combination with its known binding partners, alpha-v or alpha-IIb. The cell adhesion assay involved transiently expressing integrin sub-units in 293-based EIAV packaging cells expressing green fluorescent protein. The cells were plated at a density of 2–4×105 cells/well on untreated multi-well plates. The plating efficiency of the cells was determined 1 hr after plating by measuring the fluorescence of the cells before and after washing the cells. We found that over-expression of the beta-3 integrin subunit in 293 cells resulted in a significant increase in adhesion of the cells to untreated tissue culture plates as compared to unmodified cells. Co-expression of alpha-v, but not alpha-IIb further increased 293 cell adherence. We next investigated production of both HIV and EIAV-based lentiviral vectors from integrin-modified 293 cells. Vector titers and reporter gene expression in target cells were not compromised as a result of this modification and this led us to scale up vector production in roller bottles for generating vectors for in vivo experiments. In summary, over-expression of alpha-v and beta-3 in 293 cells increases adhesion to regular untreated tissue culture dishes without compromising viral vector production. This modification should be of use in other applications that require the use of poorly adherent cell lines.