203 Extended Duration of INFRADURE Biopump Secreting IFNα in Mice Shows Potential for Prolonged Therapeutic Effect in Patients Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Amer[.]
IMMUNE RESPONSES TO CELL AND GENE THERAPY carrying OTC gene mutations had higher transaminase elevations and loss of transgene in the presence of systemic inflammation However, in the absence TLR9 ligands, OTC gene mutations had no significant impact on the loss of vector genome copies Thus, OTC gene mutations could predispose a gene therapy trial subject to activation of a destructive cytotoxic T lymphocyte response in the presence of systemic inflammation 201 Factors Involved in the Induction of Ag-Specific Immune Tolerance By HepatocytesDirected LV.ET.mir142T Gene Transfer Andrea Annoni,1 Mahzad Akbarpour,1,2 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2 San Raffaele Telethon Institute for Gene Therapy (HSR-Tiget), Milan, Italy; 2Vita-Salute San Raffaele University, Milan, Italy We developed a lentiviral vector (LV) platform for hepatocytedirected gene expression, which combines two layers of transgene regulation: hepatocyte-specific promoter (ET), and miR-142-targets (142T) LV.ET.142T platform induces active tolerance towards the encoded-antigen (Ag) via de novo generation of Ag-specific FoxP3+ peripheral T regulatory cells (pTregs), and partial deletion of Agspecific CD8+ effector T cells Gene transfer mediated by LV.ET.142T led to stable gene replacement and suppression of FIX neutralizing response in Hemophilia B mice In addition, inhibition of type-1 diabetes development in NOD mice was observed after gene transfer with LV.ET.142T encoding for an insulin peptide We investigated the cellular and molecular mediators underlying the induction of pTregs and the deletion effector T cells, which led to Ag-specific tolerance in mice treated with the LV.ET.142T platform Differentiation of Ag-specific pTreg requires Ag-presentation in the presence of transforming growth factor-beta (TGF-beta) In vitro stimulation of CD4+ T cells revealed that all the studied hepatic nonparenchymal cell subsets were capable to perform Ag-presentation TGF-beta was up-regulated in the serum of the LV-tolerized mice compared to LV-immunized or untreated controls, and gene expression analyses indicated hepatocytes as the only hepatic cell subset up-regulating tgf-beta transcription days after LV.ET.142T in vivo gene transfer Therefore, hepatocytes may represent the source of TGF-beta in the liver microenvironment at the time of Ag presentation, promoting pTreg differentiation In addition to tgf-beta, hepatocytes also significantly up-regulated the expression of pdl-1 (Programmed cell death ligand 1), cIIta (class II trans-activator), and h2ab1 (class II major histocompatibility complex, MHC-II) To identify promoting factors for these genes, primary hepatocytes were kept in culture for 48 hours with cytokine related to immune response to LV, such as IL6, IFN-alpha and IFNgamma, alone or in combination with LV Results revealed that IFN-gamma per se can up-regulate transcription of those genes IFN-gamma-induced expression of PDL-1 was confirmed at protein level Therefore, PDL-1-PD1 co-stimulatory pathway was blocked in Balb.c mice by anti-PDL-1 mAb after LV.ET.GFP.142T Three weeks post LV injection an increase in GFP-specific CD8+ T effector cells and a significant reduction in GFP expressing hepatocytes was observed These results indicate that PDL-1-PD1 co-stimulatory pathway plays a role in promoting the contraction of transgene specific CD8+ T cells response Overall these data indicate that the tolerogenic program induced by hepatocyte-targeted gene transfer requires partial activation of the effector CD8+ T cells These effector cells incompletely primed by hepatocytes release IFN-gamma, which up-regulates PDL-1, dampening the effector response Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy 202 Nucleic Acid-Binding Polymers Regulate Inflammatory Disorders Without Causing Overall Immune Suppression Eda Holl,1 Kara Shumansky,1 Bruce Sullenger.1 Department of Surgery, Duke University, Durham, NC Toll-like receptor (TLR) family members, TLR 3, and are key components in initiation and progression of autoimmune disorders and along with their downstream signaling pathways, they have become one of the main drug targets for disease amelioration During autoimmune disease progression, such as in the case of systemic lupus erythematosus (SLE), these receptors recognize self nucleic acid complexes and contribute to inflammatory cytokine production and subsequent enhancement of serum autoantibody levels We have recently discovered a new class of nucleic-acid scavenging agents that can neutralize the proinflammatory effects of nucleic acids on a variety of immune cells implicated in autoimmune disease development We have shown that these nucleic acid-scavenging polymers can inhibit TLR activation and subsequent cytokine production by both dendritic cells (DCs) and B cells of wild type and lupus prone mice Moreover, several of these agents can block nucleic acid-driven plasma cell differentiation and antibody production In vivo, we have shown that nucleic-acid binding polymers can prevent skin lesions following mechanical injury in lupus prone animal models Stimulation of immune cells by encapsulated viral particles is unaffected in the presence of polymers These findings are further supported by an in vivo model of murine influenza Infected animals treated with polymer undergo a normal disease progression, and their immune response to said infection remains fully intact Thus showing that the effects observed in the initiation and maintenance of the immune response in the presence of polymers is specific to nucleic acid stimulation and not overall immune suppression These findings provide a new avenue in drug development as these agents can potentially be utilized to block overt autoimmune disorders without compromising normal immune responses 203 Extended Duration of INFRADURE Biopump Secreting IFNα in Mice Shows Potential for Prolonged Therapeutic Effect in Patients Reem Miari,1 Tamar Shatil,1 Yael Grimpel,1 Osnat Tal,1 Atar Liron,1 Mati Metzuyanim,1 Simcha Krispel,1 Philip Ng,2 Nir Shapir.1 Medgenics Medical Israel Ltd, Misgav, Israel; 2Dept of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Autologous Biopumps are processed from dermis needle biopsies, with typical dimensions of 30 mm by mm diameter These Biopumps harvested from the patient’s skin under local anesthesia, are transduced ex-vivo with Helper Dependent Adenoviral vector (HDAd) containing target protein expression cassette This process enables the Biopumps to continuously secrete the desired patient’s deficient protein Previously we have reported months of sustained elevated hemoglobin levels from a single subcutaneous implantation of EPODURE Biopumps secreting erythropoietin (EPO) in anemic patients with renal failure in the absence of exogenous EPO injections However, protein expression from implanted Biopumps could be limited post-implantation by various factors, including local inflammation, reducing metabolic function, loss of gene copies, or expression cassette instability To minimize these factors and prolonging the expression duration, two improvements were made: i) anti-inflammatory agent, Depo-Medrol (DM), was applied along with the Biopump implantation and ii) new HDAd gene expression cassette was designed to increase stability of expression In this study we report the in-vivo and in-vitro performance of INFRADURE Biopumps containing the HDAd-CAG-opt-hIFNα (control) or the CpG free stabilized expression cassette: HDAd-MARS77 IMMUNE RESPONSES TO CELL AND GENE THERAPY EF1α-opt-hIFNα Biopumps transduced with viral vectors containing each of the expression cassettes were implanted subcutaneously into SCID mice, with or without administration of DM at the implantation site Without DM, human Interferon-α (hIFNα) serum levels of mice implanted with Biopumps transduced with the stabilized vector remained to secrete 6.3% of initial peak at about 100 days post implantation, whereas only about 1% of the initial peak levels was observed in the control vector group, measured less than two months post implantation When DM was applied, hIFNα serum levels were improved to 19% of initial peak at about 100 days post implantation with the stabilized vector, whereas no major improvement in duration of secretion was observed with the control vector Since our clinical studies to date have used the control vector without DM, these results suggest that Biopumps transduced with the combination of stabilized expression cassette and DM may secrete sustained therapeutic levels of target proteins and could prolong and enhance the therapeutic effect in patients 204 Research on Molecular Chimeric Cells Infusion To Reduce Spleen T Lymphocyte Response To Allogeneic T Cells Stimulation Weidong Li,1 Weihua Fu,1 Li Lu,1 Ningbo Liu,1 Jiangong Cui,1 Liwei Zhu.1 General Surgery Department, Tianjin Medical University General Hospital, Tianjin, China Objective: To construct the mouse bone marrow hematopoietic stem cells (BHSCs) and Pre-T cells with chimeric MHC-I gene, and to explore the mechanism of them reducing spleen T lymphocyte response to allogeneic mouse T cells Methods: The mouse (BALB/c) BHSCs and Pre-T cells were collected The identified BHSCs and Pre-T cells were transfected by the constructed eukaryotic expression vector of C57BL/6 mouse MHC-I (pIRES-H-2Db and pIRES-H-2Kb) Then the molecular chimeric cells were transfused back BALB/c mouse After days, the ability of molecular chimeric cells inducing spleen T lymphocyte response to allogeneic T cells was observed through mixed lymphocyte culture (MLC) Results: The BALB/c mouse BHSCs and Pre-T cells were successfully separated and cultured Flow cytometry analysis indicated that H-2Db and H-2Kb protein expression were (25.20±0.88)% and (34.20±0.46)% in BHSCs group, and in Pre-T cells group, H-2Db and H-2Kb protein expression were (14.90±0.56)% and (14.20±0.63)% respectively S78 The result of MLC demonstrated that the stimulation index (SI) of T lymphocyte were significantly decreased in molecular chimeric cells group(P