title: Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.
abstract: Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Olivier
authors.initials: O
authors.lastname: Humbert
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Don W
authors.initials: DW
authors.lastname: Gisch
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Wohlfahrt
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Amie B
authors.initials: AB
authors.lastname: Adams
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.|Clinical Research Division, Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.|Department of Immunology, University of Washington, Seattle, Washington, USA.|Department of Medicine, Division of Medical Oncology, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Phil D
authors.initials: PD
authors.lastname: Greenberg
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Tom M
authors.initials: TM
authors.lastname: Schmitt
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Spokane, Washington, USA.|School of Molecular Biosciences, Washington State University, Pullman, Washington, USA.
authors.forename: Grant D
authors.initials: GD
authors.lastname: Trobridge
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.|Department of Medicine, University of Washington, Seattle, Washington, USA.|Department of Pathology, University of Washington, Seattle, Washington, USA.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
mesh_terms: D018929:Cell Culture Techniques; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic; D019076:Transgenes; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D011973:Receptors, LDL; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; C114950:glycoprotein G, Cocal virus