AbstractBook-37-ESGCT 2019 Abstract Book

TH ESGCT 27 ANNUAL CONGRESS IN COLLABORATION WITH SETGYC 22-25 OCTOBER 2019 BARCELONA INTERNATIONAL CONVENTION CENTRE Invited Speaker Abstracts INV016 Adenovirus and AAV vectors – Zooming in immunogenicity, vaccination and targeting K Benihoud H Büning 1: CNRS UMR 8203 Vectorology and antitumor therapeutics, Villejuif, France; University Paris-Sud, Faculté des Sciences d’Orsay, Orsay, France 2: Hannover Medical School, Institute of Experiment Hematology, Hannover, Germany Vectors derived from adenoviruses (Ad) and adeno-associated viruses (AAV) are the most commonly applied DNA-based delivery tools While Ad have become in particular popular in the fields of tumor virotherapy and vaccination, AAV vectors have become famous as tools for in vivo gene therapy resulting already in three marketing approvals After recalling the main properties of both kinds of vectors, the presentation will highlight recent advances in the understanding of the molecular bases of their immunogenicity In particular, we will present the different innate immune pathways activated following the recognition of viral components by specific sensors Then, we will discuss genetic engineering of the capsid of both vectors and its use for vaccination or targeting purpose INV017 Delivery of Genome Editing Reagents M Porteus 1: Stanford University While genome editing provides a precise “hit and run” approach to gene therapy, the components for genome editing still must be successfully delivered efficiently and in a non-toxic manner to the desired cell type In this educational lecture, I will discuss some of the barriers to successful delivery and approaches that overcome some of these barriers INV018 Precise engineering of mammalian genomes M Güell 1: Pompeu Fabra University, Barcelona Over the last decade, our capacity to engineering genomes has increased significantly impacting biomedical research and medicine Despite important progress, mammalian genome engineering still faces important challenges such as limited efficacy, precision and the difficulty to efficiently generate large edits I will present an overview of new gene editing technologies based on programmable nucleases (CRISPR, TALEN) which are revolutionizing the new generations of advanced therapies opening a large number of therapeutic possibilities (gene correction, epigenetic editing, RNA modification, multiplex modifications, ) INV019 Understanding cellular proliferation and differentiation using single-cell transcriptomics M Plass 1: Centre de Regulació Genịmica Single-cell transcriptomics has revolutionized the way we can study the dynamics of gene regulation and its impact in cell proliferation and differentiation In a single-cell transcriptomics experiment, we sequence the gene repertoire of thousands of cells simultaneously By comparing the transcriptomic profiles of all these cells computationally, we can capture the dynamics of any given cellular process and understand its dynamics We have used this technology to understand the differentiation and regeneration of the flatworm Schmidtea mediterranea, a popular animal model to study adult stem cells in vivo Our results showed for the first time how stem cells give rise to all possible cell types in an adult animal and identified sets of genes likely involved in regulating this process More recently, we have used a similar approach to understand the subtle differences that exist at the transcriptomic level among cells during cell cycle progression Our preliminary results show that many oscillating genes, some known cell cycle regulators, use specific 3’ isoforms in different cell cycle phases These results suggest that the choice of 3’UTR could be related to the observed changes in expression levels of these genes INV020 CARs, TRUCKs, and beyond: the next generation CAR T cells H Abken 1: Regensburg Center for Interventional Immunology (RCI), Chair Gene-Immunotherapy, University Hospital Regensburg, D-93053 Regensburg, Germany Adoptive therapy with chimeric antigen receptor (CAR) redirected T cells achieved spectacular remissions of refractory leukemia/lymphoma, the treatment of solid tumors remains so far challenging In new developments, CAR T cells are used as “living factories” to deposit immune modulating cytokines in the targeted tumor tissue aiming at converting the immune cell environment into a more favorite one to sustain a productive anti-tumor response Such TRUCKs (T cells redirected for unrestricted cytokine release and killing) releasing IL-12 or IL-18 upon CAR engagement of antigen in the CAR targeted tumor lesion are superior in attracting and activating the innate immune response in the tumor lesion In a further development a blocking anti-CD30 antibody is integrated into the extracellular CAR domain to prevent CD30L engagement T cells engineered with an anti-CEA and CD30 blocking CAR showed an improved response against CEA+ CD30-negative solid tumors This new CAR design aims at targeting tumor cells by one scFv and blocking the CD30/CD30L interaction on the T cell by the other scFv The strategy thereby combines tumor targeting with preventing repression in order to prolong the anti-tumor response INV021 Lentiviral gene therapy and gene editing for the treatment of metabolic diseases V Nieto S Fañanas-Baquero O Quintana-Bustamante A Molinos S Lopez-Manzaneda A Garcia-Torralba R Sanchez-Dominguez O Alberquilla S Navarro J A Bueren M GarciaBravo J C Segovia 1: CIEMAT/CIBERER 2: IIS-Fundación Jimenez Diaz Mutations affecting genes involved in metabolic cellular processes are responsible for an important number of rare diseases Gene therapy can treat some of these inherited metabolic diseases, mainly those caused by alterations in a single gene We will review different aspects of metabolic diseases to take into account for the designing of a successful gene therapy approach for these diseases The metabolic disease can be cell autonomous, in which only genetically corrected cells function properly, or produced by proteins/enzymes that are secreted, meaning that a relatively small number of modified cells could restore the activity of many others Different cell and gene therapy strategies have been explored to correct metabolic deficiencies, like providing a functional version of the mutated gene, transplanting functionally active cells or reducing the substrate of the deficient enzyme to enhance alternative metabolic pathways that could compensate the deficiency Moreover, different viral vectors can be used depending on the biology of the target organ AAV vectors are being the choice for non-, slow-dividing tissues In proliferating tissues, integrative vectors are required In the presentation, examples of liver and hematopoietic diseases will be presented, reviewing the different possibilities that are been explored nowadays INV022 Neuronopathic lysosomal storage diseases: cellular and animal models to test therapeutic approaches D Grinberg 1: University of Barcelona, CIBERER, IBUB, IRSJD Lysosomal storage disorders (LSD) are a group of rare, inherited diseases caused by the dysfunction of lysosomal proteins leading to accumulation of specific substrates Approximately 70% of LSDs present as progressive neurodegenerative diseases Two examples of our research, focused on models and treatment options for LSD, will be presented The first one refers to the identification of a deep intronic mutation in a Niemann-Pick C patient that generates a pseudoexon, the treatment of fibroblasts with an antisense oligonucleotide and the generation of a mouse model for the disease with this mutation (with the help of the Addi and Cassi Fund) We are currently studying the use of AONs as a therapeutic tool disease in these mice, before the treatment could be applied to patients The second example is on Sanfilippo C disease In this case, we reprogrammed fibroblasts from two patients and one healthy donor to produce induced pluripotent cells (iPSC) that were differentiated to neurons Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease Additionally, we generated Sanfillippo C neurons by editing the HGSNAT gene in wildtype iPSC using the CRISPR/Cas9 system, with the advantage that wild-type iPSC can be used as isogenic controls We are also using this approach for Sanfilippo B editing the NAGLU gene We are currently using the Sanfilippo C neuronal models with siRNAs to downregulate the EXTL2 gene, which is involved in heparan sulfate synthesis, as a substrate reduction therapeutic approach INV023 Cell Therapy for Parkinson’s disease: use of Carotid Body tissue as a source of GDNF J Villadiego J J Toledo-Aral 1: Biomedical Institute of Seville-IBiS, Univ Hosp VR/CSIC/University of Seville Seville, 41013, Spain 2: Department of Medical Physiology and Biophysics University of Seville Seville, 41009, Spain Intrastriatal carotid body (CB) grafts produce trophic protection and restoration of the dopaminergic nigrostriatal pathway in rodent and primate models of Parkinson’s disease (PD), which is mediated by high levels of glial cell linederived neurotrophic factor (GDNF) produced by CB implants Phase I/II open trials showed that CB autotrasplantation improve motor symptoms in PD patients However, the efficiency of CB cell therapy observed in clinical trials is lower than in experimental models, being patient age one of the factors influencing the clinical outcome To explore limiting factors that affect the efficacy of human CB transplants, we have studied how aging and chronic hypoxia present in intracerebral grafts can modify CB GDNF expression Chronic hypoxia induced an upregulation of CB GDNF expression in young mice, while the same treatment in aged mice decreased CB GDNF expression This age-related differential regulation of GDNF is also present in the intrastriatal graft and affects the efficacy of mice antiparkinsonian CB cell therapy Moreover, human CB xenografts from young (≤40 years) donors induced an important protection of the nigrostriatal dopaminergic neurons of parkinsonian mice, while CB implants from aged (≥60 years) donors failed to produce a significant effect Finally, we performed a study of the methylation status of human and murine GDNF promoter from young and aged CBs, identifying hypoxia-related regions that could explain the differential regulation of GDNF expression These findings provide a molecular explanation of the outcome of previous clinical trials and offer insights for the design of new antiparkinsonian cell therapy treatments This study was supported by grants from the Spanish Government (Red TerCel ISCIII RD16/0011/0025; RTC-2015-3309-1) INV024 Reprogramming benign tumours of the peripheral nervous system associated to Neurofibromatosis Type as a model system E Serra 1: IGTP, Barcelona Neurofibromatosis type (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene There is a great variability in the clinical expressivity of the disease, but the development of different tumors of the peripheral nervous system, such as cutaneous neurofibromas (CNFs), plexiform neurofibromas (PNFs) or, less frequently, malignant peripheral nerve sheath tumors (MPNSTs), constitute one of the hallmarks of the disease PNFs are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma There is a lack of non-perishable model systems to investigate PNF development We generated and characterized different PNF-derived NF1(-/-) induced pluripotent stem cell (iPSC) lines and set up culture conditions to differentiate iPSCs into NCs and further to SCs PNF-derived NF1(-/-) iPSCs tend to form spheroids when differentiating towards SCs Cells within these spheroids recapitulate the expression markers of PNF-derived primary SCs This model is helping us to understand the role of NF1 loss in SC biology, tumor formation and cellular composition We took advantage of the higher proliferation capacity of NF1(-/-) iPSC differentiating SCs and their tendency to form spheres, to further develop a multiplexed 3D model which will allow us to investigate tumor formation, progression and therapy, when combined with DNA editing techniques INV025 The lysosome: main regulator of cell metabolism and attractive therapeutic target A Ballabio 1: Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 2: Neurological Research Institute, Baylor college of Medicine, Houston Texas, USA 3: Casma Therapeutics, Boston, MA, USA The lysosome is the main mediator of cellular degradation and recycling processes Several diseases are due to lysosomal malfunction We have identified a lysosomal gene network and a master gene, TFEB, that controls lysosomal biogenesis and autophagy The activity of TFEB is regulated by the mTORC1 kinase complex through a lysosomal signaling pathway that enables lysosomal function to adapt to environmental cues, such as nutrient availability Thus, the lysosome acts as a signaling hub to control cell homeostasis and the switch between anabolism and catabolism Targeting lysosomemediated signaling pathways is an attractive therapeutic strategy for a variety of disease conditions INV026 The benefits and risks of iPSC-derived retinal cell therapy M Takahashi 1: Riken, Kobe The first in man application of iPS-derived cells started in September 2014, targeted retinal disease called age-related macular degeneration (AMD) AMD is caused by the senescence of retinal pigment epithelium (RPE), so that we aimed to replace damaged RPE with normal, young RPE made from iPS cells The grafted autologous RPE cell sheet was not rejected nor made tumor after three years The patient’s visual acuity stabilized after the surgery whereas it deteriorated before surgery in spite of 13 times injection of anti-VEGF in the eye With the safety results of this patient, we started the second clinical research using HLA loci homozygous iPSCs from Feb 2017 In this study, HLA loci matched iPSC-derived RPE was transplanted to see if we could manage the immune reaction without systemic immune suppression Another cell type in the retina; photoreceptor transplantation is a promising treatment to restore visual function to photoreceptor degenerated retinas such as retinitis pigmentosa The sensory retinal sheet transplantation, which supplies photoreceptors and secondary retinal neurons, has been shown able to reintroduce visual function in mice with end-stage retinal degeneration Transplanted retina sheets survive for a longer period than suspended cells To see the functional recovery, we developed new disease mice models and functional tests to confirm the efficacy With those, synaptic contact between graft photoreceptors and host bipolar cells was confirmed by immunohistochemistry MEA recording showed that grafted cells could elicit light responses in the host ganglion cells Now we have the tools for retinal cell therapy, however, we have to consider benefits and risks before making reatment INV027 Site specific genome editing in human T, B cells and HSCs using baboon envelope gp pseudotyped “Nanoblades” loaded with Cas9/sgRNA and specific knock-in in HSCs when combined with an AAV-6 encoding donor DNA A Gutierrez M J Abrey Recalde P E Mangeot C Costa O Bernadin F Fusil G Forment F Martin K Benabdellah E P Ricci E Ayuso F L Cosset E Verhoeyen 15 1: EVIR/CIRI/ INSERM U1111; Lyon, France 2: GENYO, Pfizer/University of Granada, Spain 3: INSERM UMR1089, University of Nantes, CHU, Nantes, Franc 4: CIRI; Inserm U1111 5: C3M, INSERM U1065, Nice, France Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms Here, we have designed “Nanoblades”, a new technology that will deliver a genomic cleaving agent into cells These are modified Murine Leukemia Virus (MLV) or HIVderived virus like particle (VLP), in which the viral structural protein Gag has been fused to Cas9 These VLPs are thus loaded with Cas9 protein together with the guide RNAs Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells (Mangeot et al., Nature Com 2019) Now we showed that nanoblades were remarkably efficient for entry into human T, B and HSCs thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelopes A brief nanoblade incubation of human T and B cells resulted in 40% and 20% gene editing HSCs treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target Finally, we also treated HSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus Currently, we are evaluating these gene-modified HSCs for their long-term reconstitution of NOD/SCIDgc-/- mice Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy INV028 “One size fits all" strategy for T cell correction, selection and depletion as new treatment for HIGM1 syndrome V Vavassori E Mercuri G Marcovecchio G Schiroli L Albano M C Castiello A Annoni C Margulies C Cotta-Ramusino A Villa L Naldini P Genovese 57 1: San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) 2: Vita-Salute San Raffaele University 3: Milano Bicocca University 4: Editas Medicine 5: Dana-Farber&Boston Children's Cancer and Blood Disorder Center 6: Equal contribution 7: Co-senior author X-linked hyper-IgM syndrome (HIGM1) is caused by mutations of CD40LG, whose absence in CD4 T-cells impairs their helper signaling for B-cell activation/immunoglobulin class-switching Since its unregulated expression caused lymphoproliferation/lymphomas, we aimed to correct CD40LG while preserving its physiologic regulation Corrected autologous T-cells could provide immediate therapeutic benefit by resolving pre-existing infections and bridge towards a definitive cure by HematopoieticStem/Progenitor-Cell (HSPC) transplant To validate this strategy, we infused wild-type Tcells into HIGM1 mice pre-conditioned with different lymphodepleting regimens, reaching long-term, stable T-cell engraftment and rescue of antigen-specific IgG response upon vaccination Thus, we optimized a CRISPR/Cas9based protocol to insert a corrective cDNA into CD40LG of human T-cells and obtained ~40% of correction while preserving the long-termrepopulating T-stem-memory cells CD40L expression and physiologic regulation was restored on edited CD4+ T-cells from both healthy donors and HIGM1 patients, which provided contact-dependent activation of B-cell on in-vitro proliferation, class-switching and IgG secretion assays To increase the yield of edited T-cell, we coupled the corrective cDNA with an optimized, truncated version of EGFR gene This strategy allows selection, tracking and depletion, in case of adverse events, of edited cells with a pharmacological-grade monoclonal-antibody and, surprisingly increased also the level of CD40LG expression We then adapted our strategy for targeting human HSPC and obtained a stable ~30% CD40LG editing after xenotransplantion in NSG mice Competitive HSPC transplants in HIGM1 mice indicate that this threshold could restore serologic immunity Our work establishes the rationale and guiding principles for clinical translation of CD40LG correction for treating HIGM1 patients INV029 Pancreatic cancer: from target discovery to innovative therapies C Fillat 1: Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS) 2: CIBERER Pancreatic cancer, in its most common form of pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a very dismal prognosis that has shown very modest improvement in survival for decades The molecular pathology of PDAC is evolving a framework for classification into different molecular subtypes that generate biological insights and can guide therapeutic development This talk will present evidences of DYRK1A as a novel therapeutic target and discuss on PDAC subtype association The inherent complexity of PDAC tumors with a significant number of genetic alterations elicits massive reprogramming of cellular gene expression, creating a novel context that impacts on the activity of oncolytic viral therapies This talk will assess how the miRNome deregulation in cancer cells not necessarily favors viral replication and propagation within the tumor, but can guide for strategies to enhance adenoviral oncolysis Furthermore, we will present a patient-derived preclinical model that can help to predict treatment efficacy INV030 Genome editing of memory stem T cells for cancer immunotherapy C Bonini 1: Università Vita-Salute San Raffaele, Milano, Italy 2: Ospedale San Raffaele Scientific institute, Milano, Italy Adoptive T cell therapy that relies on the ability of T lymphocytes to recognize and destroy specific targets on microbes and tumors through their T cell receptors (TCR) The priming of a naïve T cells, namely the first encounter of a naïve T cells with the target antigen in inflammatory conditions, leads to T cell activation and differentiation in an effector T cell, highly efficient in killing antigen bearing targets, and in a memory T cell, able to persist and provide longterm protection against diseases Adoptive T cell therapy exploits these major characteristics of T lymphocytes for cancer treatment To be effective adoptively transferred T cells: Specific for cancer antigens, Able to expand and persist long-term, Able to counteract the immunosuppressive signals mediated by cancer cells and by the tumor microenvironment Gene transfer and genome editing technologies allow to generate such potent anti-tumor living drugs The transfer of genes encoding for chimeric antigen receptors (CAR) has clearly shown high efficacy in selected diseases However, CAR-T cells target only antigens expressed on the surface of cancer cells On the contrary, TCRs recognize antigen-derived peptides processed and presented on HLA molecules, thus allowing to largely increase the array of potential targets The simple transfer of tumor specific TCR genes into T cells is affected by other limitations: genetically modified T cells shall express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on lymphocyte surface, thus limiting the efficacy of the therapeutic cellular products To overcome these issues, we developed the TCR gene editing protocol, based on the genetic disruption of the endogenous TCR genes (Provasi et al, Nature Medicine 2102; Mastaglio et al., Blood 2017) followed by lentiviral mediated transfer of a tumor-specific TCR TCR gene edited lymphocytes, proved safer and more effective than conventional TCR gene transferred cells in vitro and in animal models of acute myeloid leukemia and multiple myeloma Today, the multiplex potential of the Crispr/Cas9 system allows to simultaneously disrupting several genes, and enforcing integration of selected genes in specific genome sites, thus creating a wide array of opportunities for adoptive T cell therapy Early differentiated T cells, such as memory stem T cells and central memory lymphocytes, cells endowed with long term persistence capacity, can be engineered by TCR gene editing, thus allowing to produce long-lasting living drugs, with the ultimate aim of eliminating cancer cells and patrol the organism for tumor recurrence Challenges and opportunities of genome editing of memory T cells will be discussed INV031 GAIA-102: A novel natural killer cell-like phenotype that can eliminate solid tumors Y Yonemitsu 1: Kyushu University Clinical trials of genetically modified Tlymphocytes with chimeric antigen receptor (CAR-Ts) targeted to solid tumor fail to show apparent antitumor activity, suggesting possible critical factors in the tumor microenvironment that impede the current CAR-T strategies We here demonstrated that GAIA-102 cells, a novel CD3–/CD56bright/CD57– immature phenotype of natural killer (NK)-like cells generated using a novel culture method, shown efficient accumulation, retention, and elimination of multiple tumor spheroids depending on the expression of CCR5 and CCR6 Surprisingly, the gene expression pattern of GAIA-102 was much closer to that of HER-2 scFv-CD28-CD3z-CAR-T cells than to that of NK cells Furthermore, the sphere-destroying activity of GAIA-102 was not affected by myeloid-derived suppressor cells or regulatory T-cells These findings indicate that GAIA-102, that not require any genetic modification, have a great potential to become an upward-compatible modality over CAR-T strategy, and a new and promising candidate for adoptive immunotherapy against solid tumors INV032 The “new stem cell”: macrophages for disease modeling and cell base N Lachmann 1: Hannover Medical School, Institute of Experiment Hematology, Hannover, Germany Hematopoietic stem cell gene therapy has been proven to be effective for a variety of different hematopoietic disorders Hematopoietic stem cells (HSCs) have the ability for self-renewal and differentiation towards all blood cells and transplantation of genetically corrected HSCs represents a long-lasting treatment approach In contrast to HSCs, the view on macrophages has recently changed dramatically Nowadays, macrophages are understood as a unique cell type of the hematopoietic system with high plasticity and regenerative potential Given these specific functions, the talk will provide recent insights into the therapeutic use of macrophages, which can be derived from various stem cell sources Introducing a new HSC gene therapy approach for IFNgR1-deficient “Mendelian Susceptibility to Mycobacterial Disease “(MSMD), the therapeutic action of macrophages will be highlighted In addition, the talk will also introduce the scalable generation of hematopoietic cells from pluripotent stem cells Given the potential of iPSCs to differentiate also into cells of the hematopoietic lineage, the talk will highlight a recently developed, continuous hematopoietic differentiation process, which is able to produce different hematopoietic cell subsets Using this technology, the presentation will cover the use of iPSCs and iPSCmacrophages for disease modelling and cellbased therapies For future clinical translational of iPSC-derived cell subsets, the talk will further provide an overview on upscaling of macrophage production into industry compatible bioreactor systems and will shed light into the therapeutic use of generated cell types for rare and common diseases INV033 Engineering human pluripotent stem cells for organoid applications in regenerative medicine N Montserrat 1: Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain 2: Pluripotency for Organ Regeneration, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Technology (BIST), Barcelona, Spain 3: Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Madrid, Spain The generation of human pluripotent stem cells (hPSCs) derived organoids is one of the biggest scientific advances in regenerative medicine Recently, we have demonstrated that by lengthening the time that hPSCs are exposed to a three-dimensional microenvironment in the presence of defined renal inductive signals we are able to generate kidney organoids that transcriptomically match second-trimester human fetal kidneys Furthermore, we have recently developed a transplantation method that utilizes the chick chorioallantoic membrane (CAM) In our hands, this approach created a soft in vivo microenvironment that promotes the growth and differentiation of implanted kidney organoids, as well as providing a vascular component Through bioengineering we have mimicked the stiffness of the chick CAM by fabricating compliant hydrogels This approach resulted in the acceleration of kidney organoid formation proving that mechanical cues are determinant for the generation of hPSC-renal progenitor cells and kidney organoids Overall, we will discuss how these preliminary findings are advancing our research towards the application of different bioengineering strategies (i.e., including 3D bioprinting and tissue engineering) for kidney organoid generation and human disease modeling INV034 Gastruloids: an ESC based model for mammalian gastrulation and axial organization A Martinez Arias 1: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK When small, specified numbers of mESCs are placed in defined culture conditions they aggregate and initiate a sequence of pattern forming events that mimic the events that take place in the embryo: they undergo symmetry breaking, gastrulation like movements, axial specification and germ layer organization We can culture them for up to seven days to reach a stage comparable to E9.0 in the mouse embryo and exhibit a similar organization including two orthogonal axes and bilateral asymmetries This experimental system can be used to gain insights into the process of gastrulation and axial organization I shall be discussing specific examples and the implications these have for the theoretical and practical understanding of developmental events in mammals References: Turner, D et al (2017) Anteroposterior polarity and elongation in the absence of extraembryonic tissues and spatialy organized signaling in Gastruloids, mammalian embryonic organoids Development 144, 3894-3906 van den Brink, S et al (2014) Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse ES cells Development 141, 4231-4242 Beccari et al (2018) Multiaxial self organization properties of mouse embryonic stem cells gastruloids Nature 562, 272-276 INV035 Viral vector manufacturing and testing R O Snyder 1: Thermo Fisher Scientific The manufacture of viral gene transfer vectors for in vivo and ex vivo applications has largely been in support of early phase clinical trials, but as product candidates move to later development stages, demand is rapidly increasing for commercial grade vectors at a variety of scales Decisions regarding vector design, manufacturing platform, product configuration, and regulatory strategy have an impact on timelines and resources, raw materials sourcing, and analytical testing Developing a strategy that supports an efficient path to commercialization while reducing risk helps to bring these cutting edge cell and gene therapies to patients in need INV036 AAV manufacturing: Critical parameters influencing vector quality attributes M Hebben 1: LogicBio Therapeutics With the recent clinical successes of gene therapy, the need for viral vector manufacturing has increased dramatically for the last years Currently, the demand exceeds the worldwide production capacities which are technically limited by low process yield, thus resulting in a substantial queueing in specialized CDMOs Although several GMP-compliant production platforms coexist for AAV production, as well as a variety of purification methods, vector titers and recovery are often considered insufficient and not cost effective New promising technologies are emerging for both upstream and downstream processes but it will probably take several years before proving their efficiency and robustness As a matter of fact, despite the apparent simplicity of AAV vectors, many of their quality attributes are still not totally understood or controlled, so even the well-established production platforms may lead to disappointing titers or poorly potent vectors This presentation will review the existing production technologies to highlight the raw materials and the critical parameters in upstream and downstream processes that have an impact on major quality attributes such as full/empty capsids ratio, genome integrity, encapsidated residual DNA and vector potency INV037 Translational research of AAV expressing VEGFB: large scale manufacturing process development, analytical development and preclinical studies in ischemic porcine model L Galibert H Leinonen E Lipponen I Oruetxebarria A Valkama V Turkki T Nieminen H Hynynen K J Airenne T Heikura S YläHerttuala H P Lesch 1: Kuopio Center for Gene and Cell Therapy, Kuopio Finland 2: A.I.Virtanen Institute 3: University of Eastern Finland Coronary artery disease is still a significant public health problem and there is a need for new therapies Therapeutic revascularization through the delivery of vascular endothelial growth factors (VEGF-s) have shown their promise in many preclinical applications Adenoviral vector mediated VEGF-B gene transfer have induced efficiently angiogenesis in the myocardium resulting in an increased myocardial perfusion in ischemic pig heart Compared to adenoviruses, AAV vectors can better deliver genes into cardiomyocytes and was chosen for next generation VEGF-B deliver tool In the translation stage of AAV2-VEGF-B, the scalable, disposable and controlled manufacturing process was developed using fixed-bed bioreactor iCELLis and process was scaled up into iCELLis500 scale and 333m2 culture area Downstream development relies on filtration and affinity chromatographic methods Authorities are highlighting the importance of proper analytics and product understanding AAV functional and particle titering assays, and AAV2-VEGF-B product specific expression and potency assays were developed The effects of the gene transfer were tested in the bottleneck stent model of chronic myocardial ischemia in the domestic pig Altogether, this translational work allows the further development of the product towards clinical trials INV038 Process intensification for manufacturing of viral vectors for cell and gene therapy A A Kamen 1: McGill University The last decade has seen a rapid expansion in the use of viral gene transfer vectors, with approved therapies and late stage clinical trials underway for the treatment of genetic disorders, and multiple forms of cancer In December 2017, critical milestones were reached in the United States (US) with the first in vivo gene therapy receiving FDA approval of LuxturnaTM In August of the same year, the FDA approved the first exvivo gene therapies with KymriahTM, followed shortly after by the approval for YescartaTM Importantly, the analysis of the clinical trial pipeline (clinicaltrial.gov) indicates a much broader use of viral vectors in the coming years; chimeric antigen receptor (CAR) T-cell therapies for cancers, and treatments for genetic disorders are in late phase II and entering III clinical trials With this increasing interest in the widespread adoption of viral vectors from clinicians and industry, it is essential to engineer safer and more efficacious vectors The amount of vector necessary for an effective therapy can be considerable, particularly when administered systemically Trials for AAV-vectored therapies for Hemophilia B use doses as high as ~ 1012 viruses per kilogram of patient bodyweight (Ledgerwood et al., 2017) Developing scalable, cost-effective, and robust production platforms for viral gene transfer vectors is therefore critical to sustain the development of the field and enable late phase clinical trials This presentation with focus on major innovations in viral vector design and production systems for three of the most widely used viral vectors: Adeno-Associated Virus, and Lentivirus INV039 20 years journey of viral vector manufacturing and cell engeneering L Alberici 1: MolMed S.p.A MolMed is a biotech company focused on innovative cell and gene therapies that can meet the therapeutic needs in the treatment of tumors and rare diseases, by combining scientific and research excellence with a clear and strong industrial project MolMed is now structured on a dual business model, combining the development of our onco-haematology proprietary pipeline with contract develop and manufacturing organization (CDMO), offering world class services and technologies to our clients Manufacture and manipulate Retroviral and Lentiviral vectors or/and Hematopoietic Stem Cells and T-Lymphocytes is today MolMed’s every-day life However, the MolMed journey started more than 20 years ago, when the company was founded in 1996 as a spin-off of an Italian academic environment, with the aim of being a pioneer in developing new therapies MolMed will present its evolutions throughout these years, remarking the scientific and technical challenges for scaling up and introducing automation to its viral vector cell engeneering processes A case-study of a cell&gene pioneer which is operating and improving in such a new a continuously evolving field, focusing on challenges and mistakes that dramatically improved our experience as product developer ... 2018 Höfig, J Gene Med, 2012 Heffner, Mol Ther 2018 Masiuk, Mol Ther, Methods Clin Dev, 2019 Morgan, Mol Ther, 2019 10 Hu, Gene Ther, 2018 INV064 Intrathecal AAV9 as a platform approach to treat... editing was obtained in cell lines, IPS and primary mouse and human cells (Mangeot et al., Nature Com 2019) Now we showed that nanoblades were remarkably efficient for entry into human T, B and HSCs... systemic, single-dose administration of AT132 in XLMTM patients £5 years of age As of the August 2019, seven patients were enrolled in Cohort (1x1014 vg/kg) and five in Cohort (3x1014 vg/kg) AT132