title: Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency.
abstract: Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 104to 1.0 × 106infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimizedgag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1-2 × 109IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
authors.forename: Md
authors.initials: M
authors.lastname: Nasimuzzaman
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Lynn
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Ernst
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Michele
authors.initials: M
authors.lastname: Beuerlein
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Smith
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
authors.forename: Archana
authors.initials: A
authors.lastname: Shrestha
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Cross
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Link
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; University of Cincinnati College of Medicine, Cincinnati, Ohio, USA; Division of Regenerative Medicine and Cellular Therapies, Hoxworth Blood Center, University of Cincinnati, Cincinnati, Ohio, USA.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Lutzko
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio, USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Nordling
authors.affiliation: Division of Hematology, University of Washington , Seattle, Washington, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Russell
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland, USA.
authors.forename: Andre
authors.initials: A
authors.lastname: Larochelle
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
authors.forename: Punam
authors.initials: P
authors.lastname: Malik
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA; University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
authors.forename: Johannes C M
authors.initials: JC
authors.lastname: Van der Loo