Induced transgene expression for the treatment of solid tumors by hematopoietic stem cell-based gene therapy

Abstract

Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-b receptor II (TbRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TbRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.

Stress-Induced Gene Expression Sensing Intracellular Heating Triggered by Magnetic Hyperthermia

“…Cell-free lentiviral supernatants were generated by transient cotransfection of 293T packaging cell line with one of the two transfer vector used in this study (CMV/EGFP or HSP/EGFP, kindly provided by F. Noyan et al 22 ), together with the packaging constructs: the Gag/Polexpressing plasmid (pMDLg/pRRE), the Rev-expressing plasmid (pRSV-REV) and the envelope plasmid (pCMV-VSVG), as previously described 23 . Viral titers were determined on A549 cells in the presence of 1mg ml -1 geneticin for 2 weeks, yielding vector titers of 10 6 -10 8 TU (transducing units) ml -1 .…”

Naive Tumour‐Specific CD4+ T Cells were Efficiently Primed in Acute Lymphoblastic Leukaemia

“…Production of viral particles and generation of transgenic tumour cells by lentiviral transduction. Lentiviral particles were generated by transient cotransfection of 293T packaging cells with transfer vector encoding for GFP or GFP and OVA together with packaging plasmid pMDL-gp-rre, rev-expressing plasmid pRSV-rev and the vsv-g-encoding plasmid as previously described [18]. Viral titres were determined on A549 cells, yielding vector titres up to 10 7 transducing units.…”

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

“…Lentiviral particles were generated by transient co-transfection of 293T packaging cells with transfer vector encoding for GFP and luciferase together with packaging plasmid pMDL-gp-rre, rev expressing plasmid pRSV-rev and the vsv-g encoding plasmid as previously described [38]. Viral titers were determined on A549 cells, yielding vector titers up to 10 7 transducing units.…”

“…L23 and BM185 cells were transduced with lentiviral particles as previously described [38]. In brief, cells were spin infected with generated lentiviral cell-free particles at a multiplicity of infection of five.…”