Effects of APC De-Targeting and GAr Modification on the Duration of Luciferase Expression from Plasmid DNA Delivered to Skeletal Muscle

Author(s):

Maria C. Subang, Rewas Fatah, Ying Wu, Drew Hannaman, Jason Rice, Claire F. Evans, Yuti Chernajovsky and David GouldPages 3-14 (12)

Abstract:

Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3’UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.

Keywords:

Gene therapy, luciferase, microRNA, plasmid DNA, skeletal muscle, tissue-specific promoter, transgene immunogenicity.

Affiliation:

Bone & Joint Research Unit, Queen Mary University of London, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK.

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Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease

Author(s):

Olivier Negre, Cynthia Bartholomae, Yves Beuzard, Marina Cavazzana, Lauryn Christiansen, Celine Courne, Annette Deichmann, Maria Denaro, Edouard de Dreuzy, Mitchell Finer, Raffaele Fronza, Beatrix Gillet-Legrand, Christophe Joubert, Robert Kutner, Philippe Leboulch, Leila Maouche, Anais Paulard, Francis J. Pierciey, Michael Rothe, Byoung Ryu, Manfred Schmidt, Christof von Kalle, Emmanuel Payen and Gabor VeresPages 64-81 (18)

Abstract:

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

Keywords:

β-hemoglobinopathy, β-thalassemia, gene therapy, lentiviral vector, mouse model.

Affiliation:

bluebird bio, 150 Second Street, Cambridge, MA 02141, USA.

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Preclinical Evaluation of Efficacy and Safety of an Improved Lentiviral Vector for the Treatment of β-Thalassemia and Sickle Cell Disease

Author(s):

Olivier Negre, Cynthia Bartholomae, Yves Beuzard, Marina Cavazzana, Lauryn Christiansen, Celine Courne, Annette Deichmann, Maria Denaro, Edouard de Dreuzy, Mitchell Finer, Raffaele Fronza, Beatrix Gillet-Legrand, Christophe Joubert, Robert Kutner, Philippe Leboulch, Leila Maouche, Anais Paulard, Francis J. Pierciey, Michael Rothe, Byoung Ryu, Manfred Schmidt, Christof von Kalle, Emmanuel Payen and Gabor VeresPages 64-81 (18)

Abstract:

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

Keywords:

β-hemoglobinopathy, β-thalassemia, gene therapy, lentiviral vector, mouse model.

Affiliation:

bluebird bio, 150 Second Street, Cambridge, MA 02141, USA.

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A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors

Author(s):

Carmen Maria Jimenez-Moreno, Irene de Gracia Herrera-Gomez, Livia Lopez-Noriega, Petra Isabel Lorenzo, Nadia Cobo-Vuilleumier, Esther Fuente-Martin, Jose Manuel Mellado-Gil, Geraldine Parnaud, Domenico Bosco, Benoit Raymond Gauthier and Alejandro Martin-MontalvoPages 436-446 (11)

Abstract:

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

Keywords:

Diabetes Mellitus, Gene transfer, Infection, Lentivirus, Pancreatic islet, Transduction.

Affiliation:

Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, Avenida Americo Vespucio, Parque Científico y Tecnologico Cartuja 93, 41092 Sevilla, Spain.

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Building A New Treatment For Heart Failure-Transplantation of Induced Pluripotent Stem Cell-derived Cells into the Heart

Author(s):

Shigeru Miyagawa, Satsuki Fukushima, Yukiko Imanishi, Takuji Kawamura, Noriko Mochizuki- Oda, Shigeo Masuda and Yoshiki SawaPages 5-13 (9)

Abstract:

Advanced cardiac failure is a progressive intractable disease and is the main cause of mortality and morbidity worldwide. Since this pathology is represented by a definite decrease in cardiomyocyte number, supplementation of functional cardiomyocytes into the heart would hypothetically be an ideal therapeutic option. Recently, unlimited in vitro production of human functional cardiomyocytes was established by using induced pluripotent stem cell (iPSC) technology, which avoids the use of human embryos. A number of basic studies including ours have shown that transplantation of iPSCderived cardiomyocytes (iPSC-CMs) into the damaged heart leads to recovery of cardiac function, thereby establishing “proof-of-concept” of this iPSC-transplantation therapy. However, considering clinical application of this therapy, its feasibility, safety, and therapeutic efficacy need to be further investigated in the pre-clinical stage. This review summarizes up-to-date important topics related to safety and efficacy of iPSC-CMs transplantation therapy for cardiac disease and discusses the prospects for this treatment in clinical studies.

Keywords:

iPSC, Cardiomyocytes, Transplantation, Heart failure, Cardiomyoplasty, Immunogenicity.

Affiliation:

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

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Gene Electrotransfer: A Mechanistic Perspective

Author(s):

Christelle Rosazza, Sasa Haberl Meglic, Andreas Zumbusch, Marie-Pierre Rols and Damijan MiklavcicPages 98-129 (32)

Abstract:

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

Keywords:

Electric field, Electroporation, Gene electrotransfer, Plasmid DNA, Gene therapy, DNA vaccination.

Affiliation:

Institute of Pharmacology and Structural Biology (IPBS), CNRS UMR5089, 205 route de Narbonne, 31077 Toulouse, France.

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