title: Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.
abstract: Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Labenski
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Julia D
authors.initials: JD
authors.lastname: Suerth
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Barczak
authors.affiliation: Department of Pediatric Hematology & Oncology, Hannover Medical School, Hannover, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Heckl
authors.affiliation: CIRI, EVIR Team, Inserm, U1111, CNRS, UMR5308, Université de Lyon-1, ENS de Lyon, Lyon, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Levy
authors.affiliation: CIRI, EVIR Team, Inserm, U1111, CNRS, UMR5308, Université de Lyon-1, ENS de Lyon, Lyon, France.
authors.forename: Ornellie
authors.initials: O
authors.lastname: Bernadin
authors.affiliation: Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Charpentier
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.affiliation: Research Department Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.affiliation: CIRI, EVIR Team, Inserm, U1111, CNRS, UMR5308, Université de Lyon-1, ENS de Lyon, Lyon, France; Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe "contrôle métabolique des morts cellulaires", Nice, France.
authors.forename: Els
authors.initials: E
authors.lastname: Verhoeyen
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. Electronic address: schambach.axel@mh-hannover.de.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
mesh_terms: D001355:Alpharetrovirus / Q000378:metabolism*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics; D002999:Clone Cells; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014161:Transduction, Genetic; D019076:Transgenes; D019065:Virus Assembly
keywords: Alpharetroviral SIN vector; CRISPR-Cas9; Immunotherapy; Stable packaging cell line; Superinfection; TK007