230. Designing High-Titer Lentiviral Vectors for Gene Therapy of Sickle-Cell Disease

Hematologic & Immunologic Diseases I cells transduced with a VSVG-pseudotyped lentiviral vector in the presence of cytokines and 10 uM PGE2 yielded a vector copy number per cell (VCN) approximately 2-fold higher than CD34+ cells transduced in the absence of PGE2. This effect was seen consistently in 16 of 16 tested normal human donors in vitro, as well as primary CD34+ cells from both thalassemia and sickle cell disease patients. Importantly, PGE2 was observed to improve transduction of prospectively-isolated CD34+CD38- hematopoietic stem cells - a sub-population thought to be enriched for the long term repopulating stem cell. Transduction improvements were not associated with increased viral entry, but were associated with elevated expression of cAMP genes, supporting a post-entry mechanism of action that involves cAMP signaling downstream of prostaglandin receptors. Lastly, in a mouse xenotransplantation model of hematopoietic stem cell transplant, transduction of PB CD34+ cells in the presence of PGE2 improved VCN levels in engrafted human CD45+ cells 4-5 months post-transplant by ~2-fold without adversely affecting overall human cell engraftment. These data suggest that PGE2-mediated improvements in lentiviral transduction of human CD34+ cells could result in higher transduction efficiency and provide potential benefit in clinical gene therapy applications.

230. Designing High-Titer Lentiviral Vectors for Gene Therapy of Sickle-Cell Disease

Fabrizia Urbinati1, Valentina Poletti2, Beatriz Campo Fernandez1, Roger P. Hollis1, Colin Koziol1, Michael L. Kaufman1, Devin Brown1, Annarita Miccio3, Michael Antoniou4, Donald B. Kohn1, Fulvio Mavilio2 1 UCLA, Los Angeles, CA, 2Genethon, Evry, France, 3Imagine Institute, Paris, France, 4King’s College London, London, United Kingdom

Sickle cell disease (SCD, OMIM 603903) is one the most common inherited blood disorders worldwide, caused by a single amino acid substitution (E6V) in the β-globin chain. SCD is associated with abnormal red cell morphology, hemolytic anemia, vascular occlusion, severe pain, progressive organ damage and reduced life expectancy. Gene therapy by transplantation of autologous, genetically corrected hematopoietic stem cells could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Recent attempts to treat β-thalassemia and SCD with gene therapy showed encouraging results as well as potential limits of a technology based on complex lentiviral vectors for stem cell transduction due to the vector size and complexity, and limited globin synthesis. Designing an efficient vector that combines highlevel globin expression with high titer and infectivity remains a formidable challenge. The GLOBE vector carries a β-globin gene under the control of its promoter and a reduced version of the β-globin locus control region (LCR); it was designed to reduce the size and complexity of the LCR while maintaining its enhancer and chromatin opening activity (Miccio et al., PNAS 2008). Based on the GLOBE design, we developed novel LVs for SCD gene therapy, carrying a modified transgene encoding an anti-sickling β-globin gene with 3 amino acid substitutions (AS3), alternative β-globin promoters and a short sequence (FB) with insulating and enhancer-blocking activity in the LTR. The vectors are produced at high titer and reproducibly transduce 60-70% of human BM CD34+ cell-derived clonogenic progenitors at an average VCN of 3. The GLOBE vectors were compared to the Lenti/βAS3-FB vector (Romero, Urbinati et al. JCI 2013), previously developed for a gene therapy clinical trial of SCD. We will present data comparing the different vectors for transduction efficiency in BM CD34+ cells from healthy donors and SCD patients, βAS3-globin transgene expression and anti-sickling activity, in vivo repopulation activity in NSG mice and vector integration profiles in repopulating cells before and after transplantation. This study provides a comprehensive analysis of different LV vectors, useful to S90

determine the optimal candidate for gene therapy of SCD that can provide high transduction of Hematopoietic Stem and Progenitor Cells (HSPCs) as well as maintaining an adequate expression of the anti-sickling globin gene.

231. Mixed Chimerism After Allogeneic Hematopoietic Stem Cell Transplantation in Sickle Cell Disease: Preliminary Results on Peripheral Blood Sorted Subpopulations and Erythroid Progenitors

Alessandra Magnani1, Laure Caccavelli1, Mathieu Fusaro1, Jeremy Magalon1, Elisa Magrin1, Emmanuelle Six2, Fabien Touzot1, Chloé Couzin1, Jean Antoine Ribeil1, Cécile Arnaud3, Corinne Pondarré3, Annie Kamdem3, Françoise Bernaudin3, Jean Paul Vernant4, Eliane Gluckman5, Dominique Bories6, Marina Cavazzana1 1 Biotherapy Department, Necker-Enfants Malades Hospital, Paris, France, 2INSERM Unité Mixte de Recherche 1163, Laboratory of Human Lymphohematopoiesis, Institut Imagine, Paris, France, 3 Centre de Référence de Drépanocytose, CHIC Centre Hospitalier Intercommunal de Créteil, Créteil, France, 4Hematology Department, Pitié-Salpêtrière Hospital, Paris, France, 5Hôpital Saint Louis, Paris, France, 6Hématologie Moléculaire, Hôpital Henri Mondor, UPEC, Créteil, France Introduction: Patients with sickle cell disease (SCD) may develop a persistent mixed chimerism (MC) after hematopoietic stem cell transplantation (HSCT) associated with clinical control of the disease. In order to further investigate this condition we analyzed the chimerism in sorted myeloid and lymphoid subpopulations, and erythroid progenitors. Methods: Between 1990 and 2013, 112 sickle cell disease (SCD) patients underwent allogeneic HSCT after myeloablative conditioning. Among the 107 patients with available chimerism at 2 years, 55.1% had a MC, i.e. <95% of cells from donor origin on white blood cells (WBCs). Myeloid (CD14+, CD15+) and lymphoid (CD3+, CD19+) subpopulations, erythroid burst-forming units (BFU-E) and granulocyte monocyte colony forming units (CFU-GM) were analyzed in patients presenting with MC, follow-up ≥1 year and the hemoglobin (Hb) profile comparable to their donors’. High performance liquid chromatography (HPLC) analysis was performed on whole blood. Results: Thirty patients were included. Preliminary results from 13 patients with clinical control of the disease reveal heterogeneous patterns of subpopulation chimerism (table). A significant discrepancy in chimerism measured in WBCs compared to the other populations is observed. Patients were divided into two groups according to the difference in the donor chimerism between the CD3+ and in the myeloid compartment. This difference in donor chimerism in CD3+ cells might be explained by the expansion of recipient’s memory T cells after transplantation and/or a specific T-cell progenitor engraftment in these patients. Interestingly, a trend to a lower percentage of donor chimerism in the BFU-E than in the peripheral erythroid compartment is observed. Conclusions: These data show that the chimerism analysis on total WBCs is a poor indicator of the myeloid and erythroid engraftment in SCD patients. Chimerism study on sorted subsets and erythroid progenitors allows a more exhaustive investigation. Further analyses on T cells compartment are necessary to complete this information. Results in BFU-E as compared to the peripheral erythroid compartment suggest a selective genetically modified CD34+ cells.

Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy