ESGCT 30th Annual Congress In collaboration with SFTCG and NVGCT Brussels, Belgium October 24–27, 2023 Abstracts

Human Gene TherapyAhead of Print AbstractsFree AccessESGCT 30th Annual Congress In collaboration with SFTCG and NVGCT Brussels, Belgium October 24–27, 2023 AbstractsPublished Online:22 Jan 2024https://doi.org/10.1089/hum.2023.29258.abstractsAboutSectionsPDF/EPUB Permissions & CitationsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookXLinked InRedditEmail Oral PresentationsOR01 Identification of key regulatory factors driving CAR‐T cell dysfunction in MM by single cell multiomicsL Jordana‐Urriza1 G Serrano2 M E Calleja‐Cervantes12 P San Martin‐Uriz1 T Lozano3 A Vilas‐Zornoza113 A Ullate‐Agote1 A Lopez14 A Zabaleta1 D Alignani14 A Oliver‐Caldes5 M Español‐Rego6 M Pascal6 V Cabañas7 A Navarro‐Bailon8 M Juan6 A Urbano‐Ispizua5 J L Reguera9 J A Perez‐Simon9 J M Moraleda7 M V Mateos813 F Sanchez‐Guijo813 A Alfonso1013 J Rifon1013 P Rodriguez‐Otero101213 B Paiva141213 S Inoges101113 A Lopez‐Diaz de Cerio101113 J J Lasarte312 C Fernandez de Larrea5 J San‐Miguel1101213 M Hernaez21314 J R Rodriguez‐Madoz113 F Prosper11012131: Hemato‐Oncology Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 2: Computational Biology Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 3: Immunology and Immunotherapy Program. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 4: Flow Cytometry Core. Cima Universidad de Navarra. IdiSNA. Pamplona, Spain. 5: Department of Hematology. Hospital Clinic de Barcelona. IDIBAPS. University of Barcelona. Barcelona, Spain. 6: Department of Immunology. Hospital Clinic de Barcelona. IDIBAPS. University of Barcelona. Barcelona, Spain. 7: Department of Hematology, IMIB‐Virgen de la Arrixaca University Hospital. University of Murcia. Murcia, Spain. 8: Hematology Department, IBSAL‐University Hospital of Salamanca. University of Salamanca. Salamanca, Spain. 9: Department of Hematology, University Hospital Virgen del Rocio‐IBIS. Universidad de Sevilla. Sevilla, Spain. 10: Hematology and Cell Therapy Department. Clinica Universidad de Navarra, IdiSNA. Pamplona, Spain. 11: Immunology and Immunotherapy Department. Clinica Universidad de Navarra. Pamplona, Spain. 12: Cancer Center Clinica Universidad de Navarra (CCUN). Pamplona, Spain. 13: Centro de Investigacion Biomedica en Red de Cancer (CIBERONC). Madrid, Spain. 14: Data Science and Artificial Intelligence Institute (DATAI). Universidad de Navarra. Pamplona, Spain.CAR‐T cells have revolutionized cancer immunotherapy, representing a promising option for relapsed/refractory Multiple Myeloma (MM) patients. Nevertheless, despite the high remission rates observed after BCMA CAR‐T therapy, a significant number of patients still relapse. However, knowledge of the molecular mechanisms governing CAR‐T cell function in MM is very limited. To shed some light on specific transcriptomic programs activated after CAR‐T cell administration, we interrogated longitudinal samples of CAR‐T cells collected from patients enrolled in ARI0002h clinical trial. In this work we characterized more than 50.000 CAR‐T cells from 11 different samples, including infusion products (IP), as well as CAR‐T cells isolated from bone marrow (BM) and peripheral blood (PB), at one and three months after infusion. Single‐cell RNA and TCR sequencing (scRNAseq, scTRCseq) coupled with SimiC analysis, a novel machine learning algorithm that infers gene regulatory networks (GRNs), were applied to all samples.scRNAseq revealed that although CAR‐T cells from IP presented similar profiles, with highly proliferative CD4+ and CD8+ memory cells, CAR‐T cells remaining after infusion were mainly non‐proliferating CD8+ cells, with effector/effector‐memory phenotypes. Interestingly, transcriptomic profile of CAR‐T cells differed among patients, with increased presence of terminally differentiated effector cells presenting an exhausted signature in patients with partial response. In contrast, complete responders presented CAR‐T cells in transition to central memory or effector memory phenotype. In addition, CAR‐T cells infiltrating BM presented increased expression of cytotoxic (GZMA, PRF1) and exhaustion (LAG3, HAVCR2, TIGIT) markers compared to their PB counterparts. GRN analysis with SimiC identified several regulons, such as PRDM1 and ARID4B, with increased activity in the CAR‐T cells from BM, which could be responsible for these differences. PRDM1 has been already associated with CAR‐T cell exhaustion and its depletion promotes TCF7‐dependent CAR‐T cell stemness and proliferation. ARID4B, a chromatin remodeler TF, could be acting as an epigenetic regulator of CAR‐T cell function.The combination of scTCRseq and scRNAseq allowed the identification of a hyperexpanded CAR‐T clone, with immunosuppressor features, mainly present in the BM of a patient with partial response. Deeper characterization showed that this clone had higher expression of cytotoxicity and activation markers, as well as an increased expression of IL10. Further analysis with SimiC showed association of IL10 with transcription factors related to exhausted CD8+ T cells, like CREM, BHLHE40 and again PRDM1, which is also implicated in the production of IL10 in Treg. Additional in vitro studies suggested that subsequent activation of endogenous TCR after CAR T cell activation led to IL10 production, and functional validations corroborated that IL10 reduces CAR‐T cell functionality.Overall, our analysis combining scRNAseq and scTCRseq with novel machine learning models, allowed us not only to characterize transcriptional differences observed between patients and CAR‐T localization, but also to identify regulatory mechanisms that could promote CAR‐T cell dysfunction and would represent a potential target to be modulated for the development of improved CAR‐T therapies for MM.OR02 CAR ProTcell, towards well‐tolerated and persistent off‐the‐shelf allogeneic CAR‐T cellsS Soheili1 H Sadek1 P Rault1 J Paillet12 R Moirangthem1 M Martin‐Corredera12 A Consalus16 F Pflumio6 M Rivera Franco5 I Andre2 M Cavazzana134 E Gluckman5 A Hannart7O Nègre11: Smart Immune, Paris, France 2: Human Lymphohematopoiesis Laboratory, Imagine Institute, INSERM UMR 1163, Université Paris Cité, Paris, France 3: Department of Biotherapy, Hôpital Universitaire Necker‐Enfants Malades, Groupe Hospitalier Paris Centre, Assistance Publique‐Hôpitaux de Paris, Paris, France 4: Imagine Institute, Université Paris Cité, Paris, France 5: Eurocord, Hôpital Saint Louis and IUH University Paris VII, Paris, France 6: Université de Paris, Inserm, CEA, Stabilité Génétique Cellules Souches et Radiations, Fontenay‐aux‐Roses, F‐92260, France 7: Université McGill, Montréal, CanadaAllogeneic chimeric antigen receptor (CAR) T cells pave the way towards off‐the‐shelf products immediately available for patients and manufactured with cells from selected healthy donors. To avoid graft versus host disease (GvHD) in case of partial human leukocyte antigen (HLA) mismatch between patient and donor, T cell receptor (TCR) expression is knocked down in existing allogenic CAR‐T cell therapies. However, it was shown that co‐expression of endogenous TCR and CAR leads to superior persistence of T cells and significantly prolonged leukemia control in vivo. Smart Immune is developing a new allogeneic T cell therapy platform based on T cell progenitors, called ProTcell Platform. Human CD34+ cells are cultured for seven days in a clinical grade and feeder‐free process using an immobilized fusion protein DLL4‐Fc and a cytokine cocktail. In organoids, human ProTcells can differentiate into mature T cells with a naïve & Tscm phenotype. A detailed analysis of ProTcells demonstrated the expression of markers known to be involved in thymic homing such as CXCR4, CCR9. Infused intravenously, ProTcells seed the thymus of NSG mice and differentiate into double positive (CD4+ CD8+) thymocytes and then to CD3+ CD4+ or CD3+ CD8+ naïve T cells. Despite the presence of TCR, no GvHD was observed after infusion of ProTcells in xenogeneic mouse models and in the first patients treated in phase I trials, thanks to thymic education. Within seven days, CD34+ cells have also been efficiently transduced (>50%) with a lentiviral vector coding for an inducible CAR. Transduced ProTcells can differentiate into mature T cells. CAR T cells obtained from CAR ProTcells can kill target cells in a cytotoxcity assay. We also conduct donor selection algorithm studies as a way of providing HLA match for a reasonable percentage of the target population with a limited number of cell banks. Without the need for any TCR knock down, ProTcells pave the way toward a new generation of off‐the‐shelf allogeneic CAR T cells, avoiding the risk of GvHD thanks to thymic selection while harboring a functional TCR with the potential to fight infection and cancer relapse. Our algorithm for donor selection will reduce the risk of rejection and increase the likelihood of persistence of this new generation of allogeneic CAR‐T cells.OR04 Significant clinical benefit and enhanced T‐cell responses with repeated administration of PRGN‐2012, a novel gorilla adenoviral vector based immunotherapy, in adult patients with severe recurrent respiratory papillomatosisS M Norberg2 K Bai3 M Kenyon3 A Lankford1 R Semnani1 R R Shah1 D E Brough1 H Sabzevari1 C T Allen231: Precigen, Inc. 2: Center for Immune‐Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health 3: Surgical Oncology Program, Center for Cancer Research, National Cancer Institute, National Institutes of HealthRecurrent respiratory papillomatosis (RRP) is a rare, neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11. RRP is characterized by growth of papillomas in the upper aerodigestive tract, leading to significant morbidity due to airway obstruction and voice change and infrequently mortality or malignant transformation. There are no approved therapeutics and the current standard‐of‐care for RRP is frequent ablative surgeries that lead to irreversible laryngotracheal scarring and disability, highlighting the need for medical strategies that address the underlying infection.PRGN‐2012 is a novel, gorilla adenovirus based immunotherapy designed to enhance HPV6/11‐specific T cell immunity upon repeated administrations. This is a first‐in‐human, Phase 1/2 study of PRGN‐2012 in patients with severe RRP (NCT04724980). In the Phase 1 portion of the study, patients received 4 subcutaneous injections of PRGN‐2012 at dose level 1 (1x1011 Particle Units (PU) per injection; n = 3) or dose level 2 (5x1011 PU; n = 12) over 12 weeks. Enrolled patients had severe RRP requiring frequent surgery, with a median total lifetime surgeries of 32 (range 9‐300+) and median of 6 (range 3‐10) surgeries in the 12‐months prior to treatment. PRGN‐2012 was well‐tolerated at both dose levels, with only mild treatment‐related adverse events (TRAEs) which reduced in frequency over the treatment interval. The majority of TRAEs (97%) were Grade 1, and the most common were injection site reaction, fever, chills and fatigue. There were no TRAEs >Grade 2. Based on this safety profile, dose level 2 was selected as the recommended phase 2 dose (RP2D) and was further evaluated in the dose expansion cohort. Treatment at the RP2D resulted in clinically significant benefit with 50% (6/12) of patients experiencing a Complete Response (CR), defined as no interventions required in the 12‐months post‐treatment with PRGN‐2012. All complete responders remain surgery‐free as of the data cutoff (follow‐up range 440‐600 days). 83% (10/12) patients experienced a decrease in the number of interventions in the 12‐months post‐treatment compared to the 12‐months prior to the start of treatment. PRGN‐2012 at the RP2D significantly (p < 0.01) reduced papilloma burden as quantified by anatomic Derkay scores and significantly (p < 0.01) improved vocal function as quantified using the Vocal Handicap Index‐10, at 24‐week follow‐up. PRGN‐2012 treatment enhanced polyclonal HPV 6/11‐specific T‐cell immunity in responders whereas anti‐drug neutralizing antibodies did not increase with subsequent injections. Additional correlative studies identified features of the papilloma microenvironment related to HPV gene expression, chemokine expression and immune subset infiltration/activity that appear to govern clinical response.These data demonstrate the overall favorable safety profile and significant clinical benefit of PRGN‐2012 in adult patients with severe RRP, with 50% of patients requiring no surgery for a minimum of 12‐months post‐treatment. There was significant reduction in papilloma burden and concurrent improvement in vocal function post‐treatment which was correlated with induction of HPV6/11‐specific T‐cell immune response. Based on these findings, PRGN‐2012 has been granted FDA's Breakthrough Therapy Designation for the treatment of RRP. The Phase 2 portion of the study has completed enrollment and treatment at the RP2D (n = 23), and interim data will be presented.OR05 Global phase 1 study results of lentiviral mediated gene therapy for severe pyruvate kinase deficiencyJ Sevilla12 A J Shah345 J L L Lorenzo67 S Navarro278 L Lanos67 B P de Camino Gaisse67 S Sanchez67 J Zubicaray1 B Glader45 M Chien45 O Q Bustamante278 M Zeini9 G Choi 9 E Nicoletti9 G R Rao9 M G Roncarolo345 J A Bueren 278 J D Schwartz9 J C Segovia2781: Hematología y Hemoterapia, Fundación para la investigación Biomédica, Hospital Infantil Universitario Niño Jesús (HIUNJ), Madrid, Spain 2: Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain 3: Center for Definitive and Curative Medicine, Stanford University, Stanford, CA 4: Div. of Pediatric Hematology/Oncology/Stem Cell Transplant and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 5: Lucile Packard Children's Hospital, Palo Alto, CA 6: Hospital Universitario Fundación Jiménez Díaz, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD), Madrid, Spain 7: Unidad Mixta de Terapias Avanzadas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS‐FJD), Madrid, Spain 8: Unidad de Innovación Biomédica, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain 9: Rocket Pharmaceuticals, Inc., Cranbury, NJPyruvate kinase deficiency (PKD) is a rare inherited hemolytic anemia caused by PKLR gene mutations. Manifestations include anemia, splenomegaly and iron overload, which may be life‐threatening. Currently available treatments are limited to a recently‐approved enzyme activator or palliative therapies such as chronic blood transfusions, iron chelation therapy and splenectomy which are associated with significant side effects. A global Phase 1 clinical trial RP‐L301‐0119 (N° EudraCT 2019‐001656‐1) is underway to evaluate lentiviral mediated hematopoietic stem and progenitor cell (HSPC)‐targeted gene therapy for adults and children with severe PKD. Splenectomized patients with severe and/or transfusion‐dependent anemia are eligible. Following apheresis, HSPCs are transduced with lentiviral vector carrying the PKLR gene and cryopreserved. Myeloablative therapeutic drug monitoring‐guided busulfan (target area under the curve [AUC]: 73.1 mg/L*hr) is administered and the gene therapy product (RP‐L301) is thawed and infused. Patients are followed for safety assessments (including insertion site analysis [ISA]), and efficacy (genetic correction, decrease in transfusion requirements, significant improvement in anemia and reduction of hemolysis) for 2 years post‐infusion. As of May 3, 2023, 2 adult patients and one pediatric patient have received RP‐L301. Patient 1 (adult) received 3.9x106 CD34+ cells/kg with mean vector copy number (VCN) of 2.73. Patient 2 (adult) received 2.4x106 CD34+ cells/kg with mean VCN of 2.08. Despite baseline hemoglobin (Hb) levels in the 7.0‐7.5 g/dL range, both adult patients had normal‐range hemoglobin (13.2 g/dL at 24 months post infusion and 14.7 g/dL at 30 months post infusion, respectively), and no red blood cell transfusion requirements post‐engraftment. Other parameters of hemolysis and anemia (lactate dehydrogenase [LDH], bilirubin, erythropoietin) are improved. Peripheral blood mononuclear cell (PBMC) vector copy numbers (VCNs) were 1.75 and 1.43 at 24‐months. Both patients reported improved quality of life (QOL), also demonstrated by increases in both FACT‐An and SF‐36 scores, with marked improvement in SF‐36 energy/fatigue, physical functioning, and general health components. No serious adverse events (SAEs) have been attributed to RP‐L301. Hematopoietic reconstitution occurred within 2 weeks of administration. ISA in PB and BM for both adult patients up to 24 months following therapy demonstrate highly polyclonal patterns and no evidence of insertional mutagenesis; longitudinal results delineating clonal diversity will be presented. Early pediatric data suggests similar clinical efficacy as seen in adult cohort long‐term efficacy data. Infusion was well‐tolerated in the initial pediatric patient with engraftment achieved at day +15. There were no red blood cell transfusion requirements following engraftment. Clinical efficacy and safety data indicate that RP‐L301 is a potential treatment for patients with severe PKD, including those who did not derive benefit from available therapies. Updated safety and efficacy including additional pediatric data will be presented.OR06 Lentiviralex vivoautologous HSC gene therapy as a tool to deliver therapeutic antibodies beyond the blood brain barrierM Masdeu1 C Whiting1 S Acors1 A Luiz1 K Piponi1 G Crawford1 P Sagoo1 B Gaspar1 F Mavilio1C Recchi11: Orchard TherapeuticsHematopoietic stem cell (HSC) gene therapy allows the permanent reconstitution of the immune system with cells that have been genetically modified ex vivo by lentiviral transduction to express a specific therapeutic transgene. Upon proper myeloablative conditioning, cells derived from transplanted HSCs are also able to migrate and engraft, among other organs, in the brain, where they differentiate into microglia‐like cells. These cells can then express locally in the central nervous system (CNS) the therapeutic transgene they have been transduced with. A powerful example of this is the prevention of severe neurologic defects in devastating diseases such as metachromatic leukodystrophy (MLD), where microglia replacement with gene modified HSC‐derived microglia‐like cells is a highly successful approach to deliver therapeutic molecules beyond the blood brain barrier (BBB) directly in the CNS, while this is unachievable by enzyme replace therapies (ERTs) due to the impermeability of the BBB itself. Antibodies are effective therapeutic agents for a variety of pathologies. However, when delivered systemically, antibodies only minimally penetrate the BBB, and therefore they have very limited efficacy in the treatment of CNS disorders. Here, we sought to investigate whether ex vivo autologous HSC GT could represent an effective approach to deliver antibodies directly in the brain via engrafted gene‐modified microglia‐like cells.First, we demonstrated that microglia cells can be gene modified in vitro to efficiently express and secrete a model single chain variable fragment (scFv) antibody, which then correctly binds to its target antigen when immobilised on a synthetic surface or expressed on the plasma membrane of HEK cells. Subsequently, we transduced HSCs with lentiviral particles carrying the scFv construct and optimised protocols to differentiate them into microglia cells in vitro to investigate the ability of HSC‐derived microglia to efficiently secrete a functional antibody. The same HSCs were also transplanted into conditioned mice to analyse antibody production in the brain upon HSC engraftment and differentiation into microglia‐like cells in vivo.Microglia cells play a central role in neuroinflammation and they are particularly enriched in certain pathologies. The ability to harness their localisation for the target delivery of therapeutic antibodies could dramatically improve the prognosis of serious neurological conditions.OR07 Acquisition of somatic mutations after hematopoietic stem cell gene therapy varies among cell lineages and is modulated by vector genotoxicity and the activity of key cellular senescence geneF Gazzo12 D Cesana1 P Gallina1 L Rudilosso1 G Spinozzi1 G Pais1 M Masseroli2 A Calabria1 E Montini11: San Raffaele Telethon Institute for Gene Therapy (SR‐Tiget) 2: Department of Electronics, Information and Bioengineering, Politecnico di MilanoThe hematopoietic system of patients undergoing Hematopoietic Stem and Progenitor Cell (HSPC) Gene Therapy (GT) is fully restored when autologous engineered HSPCs are reinfused into the patient. During this process, HSPCs go through a high level of proliferation until the hematopoietic reconstitution is complete. The impact of proliferation in HSPCs on cellular fitness and safety remains an open question. Moreover, the accumulation of somatic mutations in vivo could show differences in different hematopoietic lineages depending on their susceptibility to the negative effects elicited by the DNA damage response. Furthermore, oncogene activation in human HSPCs has been shown to trigger a chronic inflammatory response leading to hematopoietic decay.Here we studied the clonality and the accumulation of somatic mutations in different hematopoietic lineages and during hematopoietic reconstitution in mice subjected to HSPC‐GT. Indeed, wild type C57 mice were transplanted with bone marrow‐derived lineage negative (Lin‐) cells from WT mice or tumor‐prone Cdkn2a‐/‐ mice which lack p16INK4A and p19ARF proteins and thus have no barriers against proto‐oncogene activation. Moreover, to evaluate if genotoxic integrations may increase the probability of acquiring somatic mutation upon oncogene activation, Lin‐ cells were transduced with a genotoxic LV harboring the strong retroviral enhancer/promoter Spleen Focus Forming Virus in the LTR (LV.SF.LTR) or the safer GT‐like non‐genotoxic LV (SIN.LV.PGK).Mice receiving WT Lin‐ cells treated with LV.SF.LTR (N = 25) or SIN.LV.PGK (N = 24) did not develop tumors, while mice transplanted with Cdkn2a/LV.SF.LTR‐marked cells (N = 24) developed tumors significantly earlier compared to mock (N = 20, p < 0.0001) and mice receiving Cdkn2a/SIN.LV.PGK‐treated cells (N = 23, p < 0.0001). To evaluate the clonal dynamics of hematopoietic reconstitution, vector integration sites (IS) were identified by by Sonication Mediated Integration Site (SLiM) PCR from peripheral blood, lymphoid (B and T) and myeloid cells collected every 4 weeks post‐transplantation. Somatic mutations were identified by analyzing the mouse genomic portion flanking each IS using VarScan2. Overall, we detected >200,000 IS, corresponding to more than 135 Mb of genomic sequence information. We introduced a new Mutation Index (MI), which normalizes the number of mutations by clones and coverage to assess mutation accumulation rates. By this approach, we found that the MI increased over time in LV.SF.LTR‐treated mice and was significantly higher when compared to SIN.LV.PGK‐treated mice (p < 0.001). Notably, myeloid clones exhibited a higher frequency of mutation accumulation compared to T and B cell lineages. This phenomenon was further exacerbated in Cdkn2a/LV.SF.LTR‐marked cells, indicating that the absence of barriers to proto‐oncogene activation and the presence of genotoxic insertions result in progressive somatic mutation accumulation and insertional mutagenesis.These results demonstrate for the first time that by combining the assessment of acquired mutations with IS analysis at the single clone level we can identify differential accumulations of somatic mutations in different hematopoietic lineages in vivo which depend on the genotoxic potential of the vector used and the ability of the genetically modified cells to sense and react to genotoxic lesions.OR08 In vivohematopoietic stem cell gene therapy using BaEVRLess‐pseudotyped retroviral vectorsD Klatt12 B Liu2 A Mucci12 M Peschers1 G Schiroli3 A Schambach24 C Harris2 D Pellin1 J Manis5 M Armant6 D T Scadden3 E Verhoeyen78 D A Williams12C Brendel121: Dana Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA 2: Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 3: Regenerative Medicine, Massachusetts General Hospital, Boston, MA 4: Experimental Hematology, Hannover Medical School, Germany 5: Transfusion Medicine, Boston Children's Hospital, Boston, MA 6: TransLab, Boston Children's Hospital, Boston, MA 7: Centre International de Recherche en Infectiologie, Université Lyon, Lyon, France 8: Université Côte d'Azur, Centre Méditerranéen de Médecine Moléculaire, Nice, FranceIn vivo hematopoietic stem cell (HSC) gene therapy has several potential advantages over ex vivo gene therapy, such as eliminating the need for stem cell harvest, ex vivo cell manipulation and conditioning of the patient. The VSVg envelope glycoprotein is commonly used for the pseudotyping of retroviral vectors but is not well suited for in vivo application due to its serum sensitivity and poor ability to mediate gene transfer into quiescent HSCs. In contrast, the baboon endogenous retrovirus (BaEV) glycoprotein, and its derivative BaEVRLess, mediate efficient gene transfer into resting HSCs and are serum resistant. Here, we explore the potential of BaEVRLess‐pseudotyped retroviral particles for in vivo HSC gene therapy. Initially, to overcome problems during virus production related to high fusogenic activity of the BaEVRLess envelope, we generated a stable BaEVRLess‐packaging cell line carrying knockout of the viral entry receptor ASCT2. This resulted in a 2‐fold increase in titers (108‐109 infectious particles/mL) and a 3‐fold reduction in cytotoxicity. To reduce phagocytosis and immune responses in vivo, CD47 was overexpressed and beta‐2‐microglobulin was knocked out in the packaging cell line, respectively. Next, we tested BaEVRLess‐pseudotyped lenti‐ and alpha‐retroviral vectors in competitive in vivo gene transfer experiments, which revealed that both vectors transduce CD34+ cells at similar rates. To enhance gene transfer, we first optimized the mobilization of HSCs in humanized NBSGW mice. The combination of AMD3100 and Gro‐beta efficiently mobilizes HSCs within 60 minutes of drug application in a range of 3‐112 hCD34+ cells/μL of blood. 0.5‐1 x 108 viral particles were injected intravenously into animals after mobilization. Initial gene marking reached up to 1.9 ± 0.9% of hCD45+ cells seven days post injection. To enrich gene‐marked cells, we overexpressed MGMT‐P140K in transduced cells, which mediates resistance to the alkylating agent BCNU. After three cycles of BCNU treatment, we observed a 20‐fold (0.7% to 21.1% GFP+) or 144‐fold (0.38 to 54.6% GFP+) enrichment of gene marked cells in the peripheral blood using either a low escalating or a high dose regimen, respectively. In bone marrow CD34+ cells, gene‐marking averaged 18.1 ± 9.1%. Barcode analysis revealed the presence of 300‐640 unique barcodes and a polyclonal distribution in transplanted mice. To confirm transduction and selection of HSCs, secondary transplantations were performed, which revealed 39.3‐48.1% transduced hCD45+ cells in the bone marrow of secondary recipients. In proof‐of‐concept experiments, we targeted the transcription factors BCL11A and ZNF410 using miRNA‐embedded shRNAs (shmiR) containing vectors. Downregulation of BCL11A and ZNF410 in erythroid cells leads to sustained reactivation of gamma‐globin and induction of fetal hemoglobin, which largely attenuates the hematologic effects of sickle cell disease. Following in vivo gene transfer and BCNU selection in humanized NBSGW mice, we observed a significant upregulation of gamma‐globin (35.6%) and fetal hemoglobin (25.1%) in erythroid differentiated cells. In summary, we demonstrate the proof‐of‐principle that BaEVRLess‐pseudotyped retroviral particles can be applied for in vivo gene therapy to treat sickle cell disease.OR09 Zinc finger activators restore normal gene and protein expression in a mouse model of SCN2A haploinsufficiencyJ Hodges1 Y Santiago1 A Olin1 J Eshleman1 L Bogart2 J Hsiao2 M Gulino2 M Riley2 S Hromadka2 S Petit2 P Dunn1 J Lee1 D Chung1 G Cisbani3 C Melis3 F Peters3 T Parman1 E Hudry2 K Worringer2 B Zeitler1 G Davis1 A Pooler11: Sangamo BioSciences, Inc. 2: Novartis Pharma AG 3: EvotecSCN2A haploinsufficiency is caused by de novo loss‐of‐function mutations in the SCN2A gene resulting in insufficient gene expression and abnormally low levels of its encoded protein, neuronal voltage‐gated sodium channel Nav1.2. Patients with mutations causing SCN2A haploinsufficiency suffer developmental delays commonly associated with autism spectrum disorder (ASD) and intellectual disability (ID) for which no effective treatment exists. A potential therapeutic approach to treating genetic haploinsufficiency disorders involves transcriptional upregulation of the healthy allele to restore normal protein levels. We utilized our proprietary zinc finger transcriptional activator (ZF‐A) platform to upregulate Scn2a expression in vitro and in vivo in wildtype (WT) and Scn2a+/‐ mice. Several hundred ZF‐As targeting multiple transcription start sites were designed and screened to identify hot spots for gene activation. We identified several ZF‐As targeting the Scn2a promoter that upregulated the target gene up to two‐fold with high specificity. Selective upregulation of gene expression by ZF‐As subsequently increased and restored Nav1.2 protein to normal levels in vitro in both WT and Scn2a+/‐ mouse cortical neurons. These results translated in vivo in WT adult mice treated intravenously with AAV ZF‐A whereby a 1.5‐fold incr