Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease

Valentina Poletti; Fabrizia Urbinati; Sabine Charrier; Guillaume Corre; Roger P Hollis; Beatriz Campo Fernandez; Samia Martin; Michael Rothe; Axel Schambach; Donald B Kohn; Fulvio Mavilio

doi:10.1016/j.omtm.2018.10.014

Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease

Mol Ther Methods Clin Dev. 2018 Nov 1:11:167-179. doi: 10.1016/j.omtm.2018.10.014. eCollection 2018 Dec 14.

Authors

Affiliations

¹ Genethon, Evry, France.
² Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, CA, USA.
³ Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
⁴ Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
⁵ Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
⁶ Paris Descartes University, Imagine Institute, Paris, France.

Abstract

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%-80% of mobilized CD34⁺ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12-14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.

Keywords: NSG mice; gene therapy; genotoxicity; hematopoietic stem-progenitor cells; integration; lentiviral vector; sickle cell disease; stem cell clonality; xenograft.