759. Reduction of HLH-like Manifestations in Murine Model of Munc13-4 Deficiency Following Lentiviral Gene Transfer into Hematopoietic Stem and Progenitor Cells

Hematologic & Immunologic Diseases II targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcl11a-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcl11a-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.

759. Reduction of HLH-like Manifestations in Murine Model of Munc13-4 Deficiency Following Lentiviral Gene Transfer into Hematopoietic Stem and Progenitor Cells

Tayebeh-Shabi Soheili1, Fernando Sepulveda1, Amandine Durand1, Julie Rivière1, Samia Martin2, Geneviève de Saint Basile1, Marina Cavazzana3, Isabelle André-Schmutz1 1 U1163, Inserm, Imagine Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 2Genethon, Evry Cedex, France, 3U1163 Inserm, Département de Biothérapie, AP-HP, Hôpital Universitaire Necker – Enfants Malades, Paris, France Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system which represents 25% of all FHLs. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and it’s defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. In S300

this study, we took advantages from a murine model of FHL3, the Jinx mice, to investigate the feasibility of HSPC gene therapy for this pathology. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus (LCMV). We generated and used a self-inactivated lentiviral vector to complement HSC from Unc13d -/- (Jinx) mice and transplanted them back into the irradiated Jinx recipients. This transplantation led to the complete reconstitution of the immune system at levels comparable to that of control mice. The recipients were then challenged with LCMV. While Jinx mice reconstituted with GFP expressing HSPC developed leukopenia, anemia and body weight loss, characteristic of HLH in this murine model, gene corrected Jinx recipients developed only mild or no HLH manifestations. This reduction in HLH manifestation correlated with a significant reduction of virus titer in the liver and serum level of IFN-g and inflammatory cytokines. All these ameliorations might be explained by the restoration of cytotoxic function of CTLs as demonstrated in an in-vitro degranulation assay. Overall, this study provides data supporting the potential of HSC gene therapy in a FHL immune dysregulation such as UNC13D deficiency.

760. Optimized AAV-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice and Cynomolgus Macaques

Jenny A. Greig1, Qiang Wang1, Amanda L. Reicherter1, Erin Bote1, Deirdre McMenamin1, Christine Draper1, Shu-Jen Chen1, Alexandra L. Hanlon2, Tamara Goode1, K. Reed Clark3, Samuel Wadsworth3, Lili Wang1, James M. Wilson1 1 Gene Therapy Program, Department of Medicine, University of Pennsylvania, Philadelphia, PA, 2School of Nursing, University of Pennsylvania, Philadelphia, PA, 3Dimension Therapeutics, Cambridge, MA In an effort to optimize expression of human coagulation VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed combining liver-specific promoter and enhancer elements with a codon-optimized human B-domain-deleted hFVIII transgene. Due to the large size of the FVIII coding sequence, there is a strong requirement for gene expression control elements to be as short as possible while retaining hepatocyte-restricted transcription. Several strong liver-specific promoters were shortened and combined, with combinations of up to three liver-specific enhancer sequences, to generate 42 enhancer/promoter combinations. These 42 liver regulatory gene cassettes were packaged into the AAVrh10 capsid and were tested in FVIII KO mice. Following intravenous (IV) administration of 1010 genome copies (GC), mice were bled every two weeks to follow hFVIII activity and antibody generation to the transgene. At week 2 post-injection, mice showed a range in hFVIII activity from 0.12-2.12 IU/ml. FVIII KO mice developed antibodies to hFVIII at week 4, and by week 8, mice in most of the 42 vector groups had detectable anti-hFVIII IgG levels. Based on the FVIII KO mouse studies and a small pilot rhesus macaque study, two of the original 42 enhancer/promoter combinations were selected for further evaluation in cynomolgus macaques, using two different Clade E capsids for expression. Each of the four vector combinations were administered IV at a dose of 1.2x1013 GC/kg into five macaques per group. With one capsid plus enhancer/promoter combination, peak expression of 37% of normal FVIII levels was seen at week 2 post-vector administration, which then plateaued at 20% of normal. While antibodies to the hFVIII were detected in the majority of macaques by week 8, antibodies remained undetectable in two animals at week 30 post-vector administration.

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