Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors
The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral ti...
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Elsevier
2024-12-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253124002762 |
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| author | Denise Klatt Lucia Sereni Boya Liu Pietro Genovese Axel Schambach Els Verhoeyen David A. Williams Christian Brendel |
| author_facet | Denise Klatt Lucia Sereni Boya Liu Pietro Genovese Axel Schambach Els Verhoeyen David A. Williams Christian Brendel |
| author_sort | Denise Klatt |
| collection | DOAJ |
| description | The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral titers compared with wild-type BaEV, but vector production is impaired by syncytia formation and cell death of the HEK293T cells due to the high fusogenic activity of the glycoprotein. This lowers viral titers, leads to increased batch-to-batch variability, and impedes the establishment of stable packaging cell lines essential for the economical production of viral supernatants. Here, we show that knockout of the entry receptor ASCT2 in HEK293T producer cells eliminates syncytia formation, resulting in a 2-fold increase in viral titers, reduced toxicity of viral supernatants, and enables the generation of stable packaging cell lines. In successive steps, we stably integrated BaEVRLess and α-retroviral a.Gag/Pol expression cassettes and isolated clones supporting titers up to 108 to 109 infectious particles/mL, a 10-fold increase in concentrated viral titers. The additional overexpression of CD47 and knockout of β2-microglobulin in the packaging cell line are tailored for future use in in vivo gene therapy applications by reducing non-specific uptake by macrophages and the immunogenicity of viral particles. |
| format | Article |
| id | doaj-art-8e2e01e0ba9340b482b868e06259719f |
| institution | OA Journals |
| issn | 2162-2531 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
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| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-8e2e01e0ba9340b482b868e06259719f2025-08-20T02:27:42ZengElsevierMolecular Therapy: Nucleic Acids2162-25312024-12-0135410238910.1016/j.omtn.2024.102389Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectorsDenise Klatt0Lucia Sereni1Boya Liu2Pietro Genovese3Axel Schambach4Els Verhoeyen5David A. Williams6Christian Brendel7Gene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USAGene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USADivision of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USAGene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USADivision of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, GermanyCentre International de Recherche en Infectiologie (CIRI), Université Lyon, Université Claude Bernard Lyon 1, INSERM, U1111, CNRS, UMR 5308, Ecole Normale Supérieure de Lyon, 69007 Lyon, France; Université Côte d'Azur, INSERM U1065, Centre Méditerranéen de Médecine Moléculaire, 06200 Nice, FranceGene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USAGene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; Corresponding author: Christian Brendel, Gene Therapy Program, Dana Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 02115, USA.The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral titers compared with wild-type BaEV, but vector production is impaired by syncytia formation and cell death of the HEK293T cells due to the high fusogenic activity of the glycoprotein. This lowers viral titers, leads to increased batch-to-batch variability, and impedes the establishment of stable packaging cell lines essential for the economical production of viral supernatants. Here, we show that knockout of the entry receptor ASCT2 in HEK293T producer cells eliminates syncytia formation, resulting in a 2-fold increase in viral titers, reduced toxicity of viral supernatants, and enables the generation of stable packaging cell lines. In successive steps, we stably integrated BaEVRLess and α-retroviral a.Gag/Pol expression cassettes and isolated clones supporting titers up to 108 to 109 infectious particles/mL, a 10-fold increase in concentrated viral titers. The additional overexpression of CD47 and knockout of β2-microglobulin in the packaging cell line are tailored for future use in in vivo gene therapy applications by reducing non-specific uptake by macrophages and the immunogenicity of viral particles.http://www.sciencedirect.com/science/article/pii/S2162253124002762MT: Delivery Strategiespackaging cell lineBaEVRLess envelopeASCT2 knockoutα-retroviral vectorsCD47 overexpression |
| spellingShingle | Denise Klatt Lucia Sereni Boya Liu Pietro Genovese Axel Schambach Els Verhoeyen David A. Williams Christian Brendel Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors Molecular Therapy: Nucleic Acids MT: Delivery Strategies packaging cell line BaEVRLess envelope ASCT2 knockout α-retroviral vectors CD47 overexpression |
| title | Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors |
| title_full | Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors |
| title_fullStr | Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors |
| title_full_unstemmed | Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors |
| title_short | Engineered packaging cell line for the enhanced production of baboon-enveloped retroviral vectors |
| title_sort | engineered packaging cell line for the enhanced production of baboon enveloped retroviral vectors |
| topic | MT: Delivery Strategies packaging cell line BaEVRLess envelope ASCT2 knockout α-retroviral vectors CD47 overexpression |
| url | http://www.sciencedirect.com/science/article/pii/S2162253124002762 |
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