Contributions
Abstract: S846
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 12:30 - 12:45
Location: Room K2
Background
Ineffective erythropoiesis in β-thalassemia occurs primarily as a result of an early erythroid progenitor apoptosis, due to precipitation of the excess of α-chains at the polychromatic normoblast stage, becoming more profound upon enhanced activation of the autophagy pathway [Ann Hematol 90:7, 2011]. We have previously demonstrated that the self-inactivating γ-globin lentiviral vector GGHI, can significantly increase HbF (α2γ2) in erythroid cells of thalassemia patients and improve the disease phenotype in vitro, by reducing apoptosis and restoring thalassemic erythropoiesis [Hum Gene Ther 23:1, 2012]. Furthermore, we have recently shown that the alternative baboon envelope glycoprotein BaEVRless specifically targets hCD34+ cells and increases transduction at low multiplicity of infection (MOI) [Blood 119:5, 2012; Blood 124:8, 2014].
Aims
To this end, we generated a novel, improved, γ-globin vector designated as GGHI-mB-3D by 1) incorporating two regulatory novel elements, i.e the 3D HPFH-1 enhancer and the 3' β-globin UTR and 2) pseudotyping with the BaEVRless envelope glycoprotein, and assessed its potency to specifically target hCD34+ cells from normal and thalassemic donors and its ability for stable and high transgene expression at low MOIs.
Methods
Both VSVG and BaEVRless envelope glycoproteins were used for pseudotyping γ-globin and GFP lentiviral vectors, while titration was carried out in 293T cells, using FACS and/or qPCR. Peripheral blood CD34+ cells from normal and thalassemic donors were isolated, transduced at low MOIs and cultured for up to 14 days in liquid and methylcellulose cultures. Assessment of HbF expression at both protein and mRNA levels was carried out at the terminal differentiation stage. Improvement of erythropoiesis was evaluated by both Annexin/7AAD staining and orthochromatic erythroblast determination, while ATG5 expression at the mRNA level was determined using qPCR.
Results
We documented high and stable HbF expression in thalassemic cells for the novel GGHI-mB-3D/BaEVRless vector, exhibiting increased transduction efficiency, compared to the conventional GGHI-mB-3D/VSVG, with a concomitant 119% mean HbF increase (p=0.029, n=9), a mean VCN/cell of 0.86 and a mean transduction efficiency of 56.4%. Transduced populations also exhibited a trend towards late-erythroid, orthochromatic differentiation and reduced apoptosis, a further indication of a successful gene therapy treatment. Monitoring expression of ATG5, a key link between autophagy and apoptosis, showed that gene correction correlated with reduction of enhanced autophagy activation, a typical feature of thalassemic polychromatophilic normoblasts.
Conclusion
The novel GGHI-mB-3D regulatory elements, coupled with BaEVRless pseudotyping, can lead to efficient transduction and potentially therapeutic HbF levels, which can have significant implications for the clinical efficiency of the currently ongoing clinical trials.
Session topic: 28. Thalassemias
Keyword(s): Beta thalassemia, Erythropoieisis, Gamma globin, Gene therapy
Abstract: S846
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 12:30 - 12:45
Location: Room K2
Background
Ineffective erythropoiesis in β-thalassemia occurs primarily as a result of an early erythroid progenitor apoptosis, due to precipitation of the excess of α-chains at the polychromatic normoblast stage, becoming more profound upon enhanced activation of the autophagy pathway [Ann Hematol 90:7, 2011]. We have previously demonstrated that the self-inactivating γ-globin lentiviral vector GGHI, can significantly increase HbF (α2γ2) in erythroid cells of thalassemia patients and improve the disease phenotype in vitro, by reducing apoptosis and restoring thalassemic erythropoiesis [Hum Gene Ther 23:1, 2012]. Furthermore, we have recently shown that the alternative baboon envelope glycoprotein BaEVRless specifically targets hCD34+ cells and increases transduction at low multiplicity of infection (MOI) [Blood 119:5, 2012; Blood 124:8, 2014].
Aims
To this end, we generated a novel, improved, γ-globin vector designated as GGHI-mB-3D by 1) incorporating two regulatory novel elements, i.e the 3D HPFH-1 enhancer and the 3' β-globin UTR and 2) pseudotyping with the BaEVRless envelope glycoprotein, and assessed its potency to specifically target hCD34+ cells from normal and thalassemic donors and its ability for stable and high transgene expression at low MOIs.
Methods
Both VSVG and BaEVRless envelope glycoproteins were used for pseudotyping γ-globin and GFP lentiviral vectors, while titration was carried out in 293T cells, using FACS and/or qPCR. Peripheral blood CD34+ cells from normal and thalassemic donors were isolated, transduced at low MOIs and cultured for up to 14 days in liquid and methylcellulose cultures. Assessment of HbF expression at both protein and mRNA levels was carried out at the terminal differentiation stage. Improvement of erythropoiesis was evaluated by both Annexin/7AAD staining and orthochromatic erythroblast determination, while ATG5 expression at the mRNA level was determined using qPCR.
Results
We documented high and stable HbF expression in thalassemic cells for the novel GGHI-mB-3D/BaEVRless vector, exhibiting increased transduction efficiency, compared to the conventional GGHI-mB-3D/VSVG, with a concomitant 119% mean HbF increase (p=0.029, n=9), a mean VCN/cell of 0.86 and a mean transduction efficiency of 56.4%. Transduced populations also exhibited a trend towards late-erythroid, orthochromatic differentiation and reduced apoptosis, a further indication of a successful gene therapy treatment. Monitoring expression of ATG5, a key link between autophagy and apoptosis, showed that gene correction correlated with reduction of enhanced autophagy activation, a typical feature of thalassemic polychromatophilic normoblasts.
Conclusion
The novel GGHI-mB-3D regulatory elements, coupled with BaEVRless pseudotyping, can lead to efficient transduction and potentially therapeutic HbF levels, which can have significant implications for the clinical efficiency of the currently ongoing clinical trials.
Session topic: 28. Thalassemias
Keyword(s): Beta thalassemia, Erythropoieisis, Gamma globin, Gene therapy