BABOON ENVELOPE PSEUDOTYPED LENTIVIRAL VECTORS: A HIGHLY EFFICIENT NEW TOOL TO GENETICALLY MANIPULATE T-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA INITIATING CELLS
(Abstract release date: 05/19/16)
EHA Library. Tesio M. 06/10/16; 133152; P164
Disclosure(s): I have no disclosures
Dr. Melania Tesio
Contributions
Contributions
Abstract
Abstract: P164
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-Acute Lymphoblastic Leukaemia (T-ALL) is a heterogeneous and aggressive hematologic cancer arising from the malignant proliferation of T-cell progenitor cells, arrested at different stages of development. Although recently developed multi-agent chemotherapy protocols significantly improved the clinical outcome of this disease, the prognosis of patients undergoing relapse or therapy resistance remains poor. Since T-ALL chemo-resistance is linked to the activation of pathways that promote and sustain the self-renewal of leukemic initiating cells (LICs), it becomes imperative to develop novel therapies to eradicate these cells. To accomplish this objective it is necessary to perform pre-clinical, functional studies with primary T-ALL patient samples, a challenging task given that tools enabling an efficient genetic manipulation of primary T-ALL cells are still lacking.
Aims
To develop novel strategies to genetically manipulate T-ALL leukaemia initiating cells.
Methods
Fresh or thawed primary T-ALL samples were transduced with lentiviral vectors pseudotyped with a baboon envelop chmeric glycoprotein (BaEV). Two days post-transduction, 500.000-700.000 transduced cells were injected into cohorts of primary NSG(NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ) mice to verify stable gene expression and T-ALL development from the transduced clones. The leukemic engraftment of the transduced cells was than evaluated by FACS analysis, based on GFP, CD45 and CD7 expression in the bone marrow, spleen and peripheral blood. To further assess the leukemic initiating ability of the transduced cells, 500.000-700.000 bone marrow cells issued from primary animals were injected into serial cohorts of NSG mice.
Results
Our work revealed that lentiviral vectors pseudotyped with a Baboon retroviral envelope glycoprotein (BaEV-LVs) are excellent tools to genetically modify T-ALL leukaemia initiating cells. These lentiviral vectors enabled high-level transduction rates (40-80%) at low multiplicity of infection (MOI=10) as they entered primary T-ALL blasts using ASCT1 and ASCT2, two aminoacid transporters that were highly expressed on all T-ALL subtypes. The transduced blasts engrafted cohorts of primary, secondary and tertiary mice, where they infiltrated bone marrow, spleen and peripheral blood, thus demonstrating that BaEV-LVs target leukemic-initiating cells. Importantly, this high performance was not linked to particular oncogenic features of the primary blasts not to a specific stage of maturation arrest. Competitive xenograft experiments, moreover, demonstrated the transduced clones did not acquire a selective growth advantage nor were outcompeted by the not-transduced clones, as evidenced by the relative ratio between GFP positive and GFP negative cells, which remained stable in all the mice serially transplanted. Hence, BaEV-LV mediated transduction does not alter LICs self-renewing pathways. This feature is an essential pre-requisite to functionally investigate the signalling cascades involved in T-ALL initiation and development.
Conclusion
BaEV-LV vectors are essential and highly efficient tools to manipulate leukemic-initiating cells and to functionally investigate the molecular pathways involved in T-ALL development and clonal evolution.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Lentiviral transduction, Leukemic stem cell
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-Acute Lymphoblastic Leukaemia (T-ALL) is a heterogeneous and aggressive hematologic cancer arising from the malignant proliferation of T-cell progenitor cells, arrested at different stages of development. Although recently developed multi-agent chemotherapy protocols significantly improved the clinical outcome of this disease, the prognosis of patients undergoing relapse or therapy resistance remains poor. Since T-ALL chemo-resistance is linked to the activation of pathways that promote and sustain the self-renewal of leukemic initiating cells (LICs), it becomes imperative to develop novel therapies to eradicate these cells. To accomplish this objective it is necessary to perform pre-clinical, functional studies with primary T-ALL patient samples, a challenging task given that tools enabling an efficient genetic manipulation of primary T-ALL cells are still lacking.
Aims
To develop novel strategies to genetically manipulate T-ALL leukaemia initiating cells.
Methods
Fresh or thawed primary T-ALL samples were transduced with lentiviral vectors pseudotyped with a baboon envelop chmeric glycoprotein (BaEV). Two days post-transduction, 500.000-700.000 transduced cells were injected into cohorts of primary NSG(NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ) mice to verify stable gene expression and T-ALL development from the transduced clones. The leukemic engraftment of the transduced cells was than evaluated by FACS analysis, based on GFP, CD45 and CD7 expression in the bone marrow, spleen and peripheral blood. To further assess the leukemic initiating ability of the transduced cells, 500.000-700.000 bone marrow cells issued from primary animals were injected into serial cohorts of NSG mice.
Results
Our work revealed that lentiviral vectors pseudotyped with a Baboon retroviral envelope glycoprotein (BaEV-LVs) are excellent tools to genetically modify T-ALL leukaemia initiating cells. These lentiviral vectors enabled high-level transduction rates (40-80%) at low multiplicity of infection (MOI=10) as they entered primary T-ALL blasts using ASCT1 and ASCT2, two aminoacid transporters that were highly expressed on all T-ALL subtypes. The transduced blasts engrafted cohorts of primary, secondary and tertiary mice, where they infiltrated bone marrow, spleen and peripheral blood, thus demonstrating that BaEV-LVs target leukemic-initiating cells. Importantly, this high performance was not linked to particular oncogenic features of the primary blasts not to a specific stage of maturation arrest. Competitive xenograft experiments, moreover, demonstrated the transduced clones did not acquire a selective growth advantage nor were outcompeted by the not-transduced clones, as evidenced by the relative ratio between GFP positive and GFP negative cells, which remained stable in all the mice serially transplanted. Hence, BaEV-LV mediated transduction does not alter LICs self-renewing pathways. This feature is an essential pre-requisite to functionally investigate the signalling cascades involved in T-ALL initiation and development.
Conclusion
BaEV-LV vectors are essential and highly efficient tools to manipulate leukemic-initiating cells and to functionally investigate the molecular pathways involved in T-ALL development and clonal evolution.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Lentiviral transduction, Leukemic stem cell
Abstract: P164
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-Acute Lymphoblastic Leukaemia (T-ALL) is a heterogeneous and aggressive hematologic cancer arising from the malignant proliferation of T-cell progenitor cells, arrested at different stages of development. Although recently developed multi-agent chemotherapy protocols significantly improved the clinical outcome of this disease, the prognosis of patients undergoing relapse or therapy resistance remains poor. Since T-ALL chemo-resistance is linked to the activation of pathways that promote and sustain the self-renewal of leukemic initiating cells (LICs), it becomes imperative to develop novel therapies to eradicate these cells. To accomplish this objective it is necessary to perform pre-clinical, functional studies with primary T-ALL patient samples, a challenging task given that tools enabling an efficient genetic manipulation of primary T-ALL cells are still lacking.
Aims
To develop novel strategies to genetically manipulate T-ALL leukaemia initiating cells.
Methods
Fresh or thawed primary T-ALL samples were transduced with lentiviral vectors pseudotyped with a baboon envelop chmeric glycoprotein (BaEV). Two days post-transduction, 500.000-700.000 transduced cells were injected into cohorts of primary NSG(NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ) mice to verify stable gene expression and T-ALL development from the transduced clones. The leukemic engraftment of the transduced cells was than evaluated by FACS analysis, based on GFP, CD45 and CD7 expression in the bone marrow, spleen and peripheral blood. To further assess the leukemic initiating ability of the transduced cells, 500.000-700.000 bone marrow cells issued from primary animals were injected into serial cohorts of NSG mice.
Results
Our work revealed that lentiviral vectors pseudotyped with a Baboon retroviral envelope glycoprotein (BaEV-LVs) are excellent tools to genetically modify T-ALL leukaemia initiating cells. These lentiviral vectors enabled high-level transduction rates (40-80%) at low multiplicity of infection (MOI=10) as they entered primary T-ALL blasts using ASCT1 and ASCT2, two aminoacid transporters that were highly expressed on all T-ALL subtypes. The transduced blasts engrafted cohorts of primary, secondary and tertiary mice, where they infiltrated bone marrow, spleen and peripheral blood, thus demonstrating that BaEV-LVs target leukemic-initiating cells. Importantly, this high performance was not linked to particular oncogenic features of the primary blasts not to a specific stage of maturation arrest. Competitive xenograft experiments, moreover, demonstrated the transduced clones did not acquire a selective growth advantage nor were outcompeted by the not-transduced clones, as evidenced by the relative ratio between GFP positive and GFP negative cells, which remained stable in all the mice serially transplanted. Hence, BaEV-LV mediated transduction does not alter LICs self-renewing pathways. This feature is an essential pre-requisite to functionally investigate the signalling cascades involved in T-ALL initiation and development.
Conclusion
BaEV-LV vectors are essential and highly efficient tools to manipulate leukemic-initiating cells and to functionally investigate the molecular pathways involved in T-ALL development and clonal evolution.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Lentiviral transduction, Leukemic stem cell
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
T-Acute Lymphoblastic Leukaemia (T-ALL) is a heterogeneous and aggressive hematologic cancer arising from the malignant proliferation of T-cell progenitor cells, arrested at different stages of development. Although recently developed multi-agent chemotherapy protocols significantly improved the clinical outcome of this disease, the prognosis of patients undergoing relapse or therapy resistance remains poor. Since T-ALL chemo-resistance is linked to the activation of pathways that promote and sustain the self-renewal of leukemic initiating cells (LICs), it becomes imperative to develop novel therapies to eradicate these cells. To accomplish this objective it is necessary to perform pre-clinical, functional studies with primary T-ALL patient samples, a challenging task given that tools enabling an efficient genetic manipulation of primary T-ALL cells are still lacking.
Aims
To develop novel strategies to genetically manipulate T-ALL leukaemia initiating cells.
Methods
Fresh or thawed primary T-ALL samples were transduced with lentiviral vectors pseudotyped with a baboon envelop chmeric glycoprotein (BaEV). Two days post-transduction, 500.000-700.000 transduced cells were injected into cohorts of primary NSG(NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ) mice to verify stable gene expression and T-ALL development from the transduced clones. The leukemic engraftment of the transduced cells was than evaluated by FACS analysis, based on GFP, CD45 and CD7 expression in the bone marrow, spleen and peripheral blood. To further assess the leukemic initiating ability of the transduced cells, 500.000-700.000 bone marrow cells issued from primary animals were injected into serial cohorts of NSG mice.
Results
Our work revealed that lentiviral vectors pseudotyped with a Baboon retroviral envelope glycoprotein (BaEV-LVs) are excellent tools to genetically modify T-ALL leukaemia initiating cells. These lentiviral vectors enabled high-level transduction rates (40-80%) at low multiplicity of infection (MOI=10) as they entered primary T-ALL blasts using ASCT1 and ASCT2, two aminoacid transporters that were highly expressed on all T-ALL subtypes. The transduced blasts engrafted cohorts of primary, secondary and tertiary mice, where they infiltrated bone marrow, spleen and peripheral blood, thus demonstrating that BaEV-LVs target leukemic-initiating cells. Importantly, this high performance was not linked to particular oncogenic features of the primary blasts not to a specific stage of maturation arrest. Competitive xenograft experiments, moreover, demonstrated the transduced clones did not acquire a selective growth advantage nor were outcompeted by the not-transduced clones, as evidenced by the relative ratio between GFP positive and GFP negative cells, which remained stable in all the mice serially transplanted. Hence, BaEV-LV mediated transduction does not alter LICs self-renewing pathways. This feature is an essential pre-requisite to functionally investigate the signalling cascades involved in T-ALL initiation and development.
Conclusion
BaEV-LV vectors are essential and highly efficient tools to manipulate leukemic-initiating cells and to functionally investigate the molecular pathways involved in T-ALL development and clonal evolution.
Session topic: Acute lymphoblastic leukemia - Biology 2
Keyword(s): Acute lymphoblastic leukemia, Lentiviral transduction, Leukemic stem cell
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