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Journal Cover Molecular Therapy - Methods & Clinical Development
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  This is an Open Access Journal Open Access journal
   ISSN (Online) 2329-0501
   Published by Elsevier Homepage  [3049 journals]
  • DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular
           Dystrophy 2B

    • Authors: Julia Ma; Christophe Pichavant; Haley du Bois; Mital Bhakta; Michele P. Calos
      Abstract: Publication date: 15 December 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 7
      Author(s): Julia Ma, Christophe Pichavant, Haley du Bois, Mital Bhakta, Michele P. Calos
      Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan’s blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan’s blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.

      PubDate: 2017-11-26T10:25:19Z
      DOI: 10.1016/j.omtm.2017.10.005
      Issue No: Vol. 7 (2017)
  • Atg5flox-Derived Autophagy-Deficient Model of Pompe Disease: Does It Tell
           the Whole Story'

    • Authors: Jeong-A Lim; Hossein Zare; Rosa Puertollano; Nina Raben
      Pages: 11 - 14
      Abstract: Publication date: 15 December 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 7
      Author(s): Jeong-A Lim, Hossein Zare, Rosa Puertollano, Nina Raben

      PubDate: 2017-09-26T20:16:31Z
      DOI: 10.1016/j.omtm.2017.08.002
      Issue No: Vol. 7 (2017)
  • Accelerating Patients’ Access to Advanced Therapies in the EU

    • Authors: Ahmed Elsanhoury; Ralf Sanzenbacher; Petra Reinke; Mohamed Abou-El-Enein
      Pages: 15 - 19
      Abstract: Publication date: 15 December 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 7
      Author(s): Ahmed Elsanhoury, Ralf Sanzenbacher, Petra Reinke, Mohamed Abou-El-Enein

      PubDate: 2017-09-26T20:16:31Z
      DOI: 10.1016/j.omtm.2017.08.005
      Issue No: Vol. 7 (2017)
  • Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for
           Diabetes: 8-Year Follow-Up in Dogs

    • Authors: Maria Luisa Jaén; Laia Vilà; Ivet Elias; Veronica Jimenez; Jordi Rodó; Luca Maggioni; Rafael Ruiz-de Gopegui; Miguel Garcia; Sergio Muñoz; David Callejas; Eduard Ayuso; Tura Ferré; Iris Grifoll; Anna Andaluz; Jesus Ruberte; Virginia Haurigot; Fatima Bosch
      Pages: 1 - 7
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Maria Luisa Jaén, Laia Vilà, Ivet Elias, Veronica Jimenez, Jordi Rodó, Luca Maggioni, Rafael Ruiz-de Gopegui, Miguel Garcia, Sergio Muñoz, David Callejas, Eduard Ayuso, Tura Ferré, Iris Grifoll, Anna Andaluz, Jesus Ruberte, Virginia Haurigot, Fatima Bosch
      Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.008
      Issue No: Vol. 6 (2017)
  • Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with
           Stem Cell Gene Therapy

    • Authors: Yuka Igarashi; Toru Uchiyama; Tomoko Minegishi; Sirirat Takahashi; Nobuyuki Watanabe; Toshinao Kawai; Masafumi Yamada; Tadashi Ariga; Masafumi Onodera
      Pages: 8 - 16
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Yuka Igarashi, Toru Uchiyama, Tomoko Minegishi, Sirirat Takahashi, Nobuyuki Watanabe, Toshinao Kawai, Masafumi Yamada, Tadashi Ariga, Masafumi Onodera
      Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34+ cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.005
      Issue No: Vol. 6 (2017)
  • In Vivo Murine-Matured Human CD3+ Cells as a Preclinical Model for T
           Cell-Based Immunotherapies

    • Authors: Kevin G. Haworth; Christina Ironside; Zachary K. Norgaard; Willimark M. Obenza; Jennifer E. Adair; Hans-Peter Kiem
      Pages: 17 - 30
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Kevin G. Haworth, Christina Ironside, Zachary K. Norgaard, Willimark M. Obenza, Jennifer E. Adair, Hans-Peter Kiem
      Adoptive cellular immunotherapy is a promising and powerful method for the treatment of a broad range of malignant and infectious diseases. Although the concept of cellular immunotherapy was originally proposed in the 1990s, it has not seen successful clinical application until recent years. Despite significant progress in creating engineered receptors against both malignant and viral epitopes, no efficient preclinical animal models exist for rapidly testing and directly comparing these engineered receptors. The use of matured human T cells in mice usually leads to graft-versus-host disease (GvHD), which severely limits the effectiveness of such studies. Alternatively, adult apheresis CD34+ cells engraft in neonatal non-obese diabetic (NOD)-severe combined immunodeficiency (SCID)-common γ chain–/– (NSG) mice and lead to the development of CD3+ T cells in peripheral circulation. We demonstrate that these in vivo murine-matured autologous CD3+ T cells from humans (MATCH) can be collected from the mice, engineered with lentiviral vectors, reinfused into the mice, and detected in multiple lymphoid compartments at stable levels over 50 days after injection. Unlike autologous CD3+ cells collected from human donors, these MATCH mice did not exhibit GvHD after T cell administration. This novel mouse model offers the opportunity to screen different immunotherapy-based treatments in a preclinical setting.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.004
      Issue No: Vol. 6 (2017)
  • Anti-high Mobility Group Box 1 Antibody Ameliorates Albuminuria in MRL/lpr
           Lupus-Prone Mice

    • Authors: Haruki Watanabe; Katsue S. Watanabe; Keyue Liu; Sumie Hiramatsu; Sonia Zeggar; Eri Katsuyama; Noriko Tatebe; Akiya Akahoshi; Fumiaki Takenaka; Takahisa Hanada; Masaru Akehi; Takanori Sasaki; Ken-ei Sada; Eiji Matsuura; Masahiro Nishibori; Jun Wada
      Pages: 31 - 39
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Haruki Watanabe, Katsue S. Watanabe, Keyue Liu, Sumie Hiramatsu, Sonia Zeggar, Eri Katsuyama, Noriko Tatebe, Akiya Akahoshi, Fumiaki Takenaka, Takahisa Hanada, Masaru Akehi, Takanori Sasaki, Ken-ei Sada, Eiji Matsuura, Masahiro Nishibori, Jun Wada
      We evaluated the efficacy of a neutralizing anti-high mobility group box 1 (HMGB1) monoclonal antibody in MRL/lpr lupus-prone mice. The anti-HMGB1 monoclonal antibody (5 mg/kg weight) or class-matched control immunoglobulin G2a (IgG2a) was administered intravenously twice a week for 4–15 weeks. Urine albumin was monitored, and histological evaluation of the kidneys was conducted at 16 weeks. Lymphadenopathies were evaluated by 1-(2′-deoxy-2′-[18F]fluoro-β-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed tomography (PET/CT) at 12 weeks. Following 4-week treatment, [18F]FAC-PET/CT showed similar accumulation in cervical and axillary lymph nodes at 12 weeks of age. However, anti-HMGB1 monoclonal antibody sufficiently inhibited the increase in albuminuria compared to an isotype control following 15-week treatment. Complement deposition was also improved; however, there were no significant differences in IgG deposition and renal pathological scores between the two groups. Anti-double-stranded DNA (dsDNA) antibody titers and cytokine and chemokine levels were also unaltered. Although there were no significant differences in glomerular macrophage infiltration, neutrophil infiltration was significantly decreased by the anti-HMGB1 monoclonal antibody. Antagonizing HMGB1 treatment suppressed HMGB1 translocation from nuclei in the kidney and suppressed neutrophil extracellular traps. The anti-HMGB1 monoclonal antibody demonstrated therapeutic potential against albuminuria in lupus nephritis by inhibiting neutrophil recruitment and neutrophil extracellular traps.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.006
      Issue No: Vol. 6 (2017)
  • Clearance of Heparan Sulfate and Attenuation of CNS Pathology by
           Intracerebroventricular BMN 250 in Sanfilippo Type B Mice

    • Authors: Mika Aoyagi-Scharber; Danielle Crippen-Harmon; Roger Lawrence; Jon Vincelette; Gouri Yogalingam; Heather Prill; Bryan K. Yip; Brian Baridon; Catherine Vitelli; Amanda Lee; Olivia Gorostiza; Evan G. Adintori; Wesley C. Minto; Jeremy L. Van Vleet; Bridget Yates; Sara Rigney; Terri M. Christianson; Pascale M.N. Tiger; Melanie J. Lo; John Holtzinger; Paul A. Fitzpatrick; Jonathan H. LeBowitz; Sherry Bullens; Brett E. Crawford; Stuart Bunting
      Pages: 43 - 53
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Mika Aoyagi-Scharber, Danielle Crippen-Harmon, Roger Lawrence, Jon Vincelette, Gouri Yogalingam, Heather Prill, Bryan K. Yip, Brian Baridon, Catherine Vitelli, Amanda Lee, Olivia Gorostiza, Evan G. Adintori, Wesley C. Minto, Jeremy L. Van Vleet, Bridget Yates, Sara Rigney, Terri M. Christianson, Pascale M.N. Tiger, Melanie J. Lo, John Holtzinger, Paul A. Fitzpatrick, Jonathan H. LeBowitz, Sherry Bullens, Brett E. Crawford, Stuart Bunting
      Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), caused by inherited deficiency of α-N-acetylglucosaminidase (NAGLU), required for lysosomal degradation of heparan sulfate (HS), is a pediatric neurodegenerative disorder with no approved treatment. Intracerebroventricular (ICV) delivery of a modified recombinant NAGLU, consisting of human NAGLU fused with insulin-like growth factor 2 (IGF2) for enhanced lysosomal targeting, was previously shown to result in marked enzyme uptake and clearance of HS storage in the Naglu −/− mouse brain. To further evaluate regional, cell type-specific, and dose-dependent biodistribution of NAGLU-IGF2 (BMN 250) and its effects on biochemical and histological pathology, Naglu −/− mice were treated with 1–100 μg ICV doses (four times over 2 weeks). 1 day after the last dose, BMN 250 (100 μg doses) resulted in above-normal NAGLU activity levels, broad biodistribution, and uptake in all cell types, with NAGLU predominantly localized to neurons in the Naglu −/− mouse brain. This led to complete clearance of disease-specific HS and reduction of secondary lysosomal defects and neuropathology across various brain regions lasting for at least 28 days after the last dose. The substantial brain uptake of NAGLU attainable by this highest ICV dosage was required for nearly complete attenuation of disease-driven storage accumulations and neuropathology throughout the Naglu −/− mouse brain.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.009
      Issue No: Vol. 6 (2017)
  • Lentiviral Fluorescent Genetic Barcoding for Multiplex Fate Tracking of
           Leukemic Cells

    • Authors: Tobias Maetzig; Jens Ruschmann; Lea Sanchez Milde; Courteney K. Lai; Niklas von Krosigk; R. Keith Humphries
      Pages: 54 - 65
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Tobias Maetzig, Jens Ruschmann, Lea Sanchez Milde, Courteney K. Lai, Niklas von Krosigk, R. Keith Humphries
      Tracking the behavior of leukemic samples both in vitro and in vivo plays an increasingly large role in efforts to better understand the leukemogenic processes and the effects of potential new therapies. Such work can be accelerated and made more efficient by methodologies enabling the characterization of leukemia samples in multiplex assays. We recently developed three sets of lentiviral fluorescent genetic barcoding (FGB) vectors that create 26, 14, and 6 unique immunophenotyping-compatible color codes from GFP-, yellow fluorescent protein (YFP)-, and monomeric kusabira orange 2 (mKO2)-derived fluorescent proteins. These vectors allow for labeling and tracking of individual color-coded cell populations in mixed samples by real-time flow cytometry. Using the prototypical Hoxa9/Meis1 murine model of acute myeloid leukemia, we describe the application of the 6xFGB vector system for assessing leukemic cell characteristics in multiplex assays. By transplanting color-coded cell mixes, we investigated the competitive growth behavior of individual color-coded populations, determined leukemia-initiating cell frequencies, and assessed the dose-dependent potential of cells exposed to the histone deacetylase inhibitor Entinostat for bone marrow homing. Thus, FGB provides a useful tool for the multiplex characterization of leukemia samples in a wide variety of applications with a concomitant reduction in workload, processing times, and mouse utilization.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.007
      Issue No: Vol. 6 (2017)
  • In Silico Restriction Enzyme Digests to Minimize Mapping Bias in Genomic

    • Authors: Jason Roszik; György Fenyőfalvi; László Halász; Zsolt Karányi; Lóránt Székvölgyi
      Pages: 66 - 67
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Jason Roszik, György Fenyőfalvi, László Halász, Zsolt Karányi, Lóránt Székvölgyi

      PubDate: 2017-07-01T14:31:40Z
      DOI: 10.1016/j.omtm.2017.06.003
      Issue No: Vol. 6 (2017)
  • Emerging issues in AAV-mediated in vivo gene therapy

    • Authors: Pasqualina Colella; Giuseppe Ronzitti; Federico Mingozzi
      Abstract: Publication date: Available online 1 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Pasqualina Colella, Giuseppe Ronzitti, Federico Mingozzi
      In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, have established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective “therapeutic products” is, however, a long process that involves several cycles of iterations from bench-to-bedside that are required to address issues encountered during drug developments. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from pre-clinical and clinical studies.

      PubDate: 2017-12-07T13:54:19Z
      DOI: 10.1016/j.omtm.2017.11.007
  • Title: Modeling anti-HIV-1 HSPC based gene therapy in humanized mice
           previously infected with HIV-1.

    • Authors: Wannisa Khamaikawin; Saki Shimizu; Masakazu Kamata; Ruth Cortado; Yujin Jung; Jennifer Lam; Jing Wen; Patrick Kim; Yiming Xie; Sanggu Kim; Hubert Arokium; Angela P. Presson; Irvin SY. Chen; Dong Sung An
      Abstract: Publication date: Available online 1 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Wannisa Khamaikawin, Saki Shimizu, Masakazu Kamata, Ruth Cortado, Yujin Jung, Jennifer Lam, Jing Wen, Patrick Kim, Yiming Xie, Sanggu Kim, Hubert Arokium, Angela P. Presson, Irvin SY. Chen, Dong Sung An
      Investigations of anti-HIV-1 human hematopoietic stem/progenitor cells (HSPC) based gene therapy have been performed by an HIV-1 challenge after the engraftment of gene modified HSPC in humanized mouse models. However, the clinical application of gene therapy is to treat HIV-1 infected patients. Here, we developed a new method to investigate an anti-HIV-1 HSPC based gene therapy in humanized mice previously infected with HIV-1. First, humanized mice were infected with HIV-1. When plasma viremia reached at >107 copies/ml after 3 weeks post HIV-1 infection, the mice were myeloablated with busulfan and transplanted with anti-HIV-1 gene modified CD34+ HSPC transduced with a lentiviral vector expressing two short hairpin RNAs (shRNA) against CCR5 and HIV-1 long terminal repeat (LTR) along with human thymus tissue under the kidney capsule. Anti-HIV-1 vector modified human CD34+ HSPC successfully repopulated peripheral blood and lymphoid tissues in HIV-1 previously infected humanized mice. Anti-HIV-1 shRNA vector modified CD4+ T lymphocytes showed selective advantage in HIV-1 previously infected humanized mice. This new method will be useful for investigations of anti-HIV-1 gene therapy to test in a more clinically relevant experimental setting.

      PubDate: 2017-12-07T13:54:19Z
      DOI: 10.1016/j.omtm.2017.11.008
  • Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in
           Normal and Pancreatic Cancer Mice

    • Authors: Kayla Quirin; Jason Kwon Arafat Alioufi Tricia Factora Constance Temm
      Abstract: Publication date: 16 March 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 8
      Author(s): Kayla A. Quirin, Jason J. Kwon, Arafat Alioufi, Tricia Factora, Constance J. Temm, Max Jacobsen, George E. Sandusky, Kim Shontz, Louis G. Chicoine, K. Reed Clark, Joshua T. Mendell, Murray Korc, Janaiah Kota
      Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

      PubDate: 2017-11-26T10:25:19Z
  • A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem
           Cell Gene Editing Strategies for β-hemoglobinopathies

    • Authors: Olivier Humbert; Christopher W. Peterson; Zachary K. Norgaard; Stefan Radtke; Hans-Peter Kiem
      Abstract: Publication date: Available online 21 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Olivier Humbert, Christopher W. Peterson, Zachary K. Norgaard, Stefan Radtke, Hans-Peter Kiem
      Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting Bcl11a coding sequence. In vivo gene editing levels were low, but some Bcl11a deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cells staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof of concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the Bcl11a locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

      PubDate: 2017-11-26T10:25:19Z
      DOI: 10.1016/j.omtm.2017.11.005
  • Engineering PTEN-L for cell-mediated delivery

    • Authors: Sylvie J. Lavictoire; Alexander Gont; Lisa M. Julian; William L. Stanford; Caitlyn Vlasschaert; Douglas A. Gray; Danny Jomaa; Ian AJ. Lorimer
      Abstract: Publication date: Available online 21 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Sylvie J. Lavictoire, Alexander Gont, Lisa M. Julian, William L. Stanford, Caitlyn Vlasschaert, Douglas A. Gray, Danny Jomaa, Ian AJ. Lorimer
      The tumour suppressor PTEN is frequently inactivated in glioblastoma. PTEN-L is a long form of PTEN produced by translation from an alternate upstream start codon. Unlike PTEN, PTEN-L has a signal sequence and a tract of six arginine residues that allow PTEN-L to be secreted from cells and be taken up by neighbouring cells. This suggests that PTEN-L could be used as a therapeutic to restore PTEN activity. However effective delivery of therapeutic proteins to treat central nervous system cancers such as glioblastoma is challenging. One method under evaluation is cell-mediated therapy, where cells with tumour-homing abilities such as neural stem cells are genetically modified to express a therapeutic protein. Here we have developed a version of PTEN-L that is engineered for enhanced cell-mediated delivery. This was accomplished by replacement of the native leader sequence of PTEN-L with a leader sequence from human light chain IgG. This version of PTEN-L showed increased secretion and an increased ability to transfer to neighbouring cells. Neural stem cells derived from human fibroblasts could be modified to express this version of PTEN-L and were able to deliver catalytically-active lclPTEN-L to neighbouring glioblastoma cells.

      PubDate: 2017-11-26T10:25:19Z
      DOI: 10.1016/j.omtm.2017.11.006
  • A combined in vivo HSC transduction/selection approach results in
           efficient and stable transgene expression in peripheral blood cells in

    • Authors: Hongjie Wang; Maximilian Richter Nikoletta Psatha Chang Jiho Kim Jing
      Abstract: Publication date: Available online 10 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Hongjie Wang, Maximilian Richter, Nikoletta Psatha, Chang Li, Jiho Kim, Jing Liu, Anja Ehrhardt, Susan K. Nilsson, Benjamin Cao, Donna Palmer, Philip Ng, Zsuzsanna Izsvák, Kevin Harworth, Hans-Peter Kiem, Thalia Papayannopoulou, André Lieber
      We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of GCSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve a more selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs, however no increased marking in peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates a basis for a simpler HSC gene therapy.

      PubDate: 2017-11-26T10:25:19Z
  • Delivery of CR2-fH using AAV vector therapy as treatment strategy in the
           mouse model of choroidal neovascularization

    • Authors: Gloriane Schnabolk; Nathaniel Parsons; Elisabeth Obert; Balasubramaniam Annamalai; Cecile Nasarre; Stephen M. Tomlinson; Alfred Lewin; Bärbel Rohrer
      Abstract: Publication date: Available online 10 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Gloriane Schnabolk, Nathaniel Parsons, Elisabeth Obert, Balasubramaniam Annamalai, Cecile Nasarre, Stephen M. Tomlinson, Alfred Lewin, Bärbel Rohrer
      Complement activation plays a significant role in age-related macular degeneration (AMD) pathogenesis, and polymorphisms interfering with factor H (fH) function, a complement alternative pathway (AP) inhibitor, are associated with increased AMD risk. We have previously validated an AP inhibitor, a fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of fH (CR2-fH) as an efficacious treatment for choroidal neovascularization (CNV) when delivered intravenously. Here we tested an alternative approach of AAV-mediated delivery (AAV5-VMD2-CR2-fH, AAV5-VMD2-mCherry) using subretinal delivery in C57BL/6J mice. Secretion of CR2-fH was confirmed in polarized retinal pigment epithelium (RPE) cells. A safe concentration of AAV5-VMD2-CR2-fH was identified using electroretinography, optical coherence tomography (OCT), RPE morphology and antibody profiling. One month after gene delivery, CNV was induced using argon laser photocoagulation. OCT assessment demonstrated reduced CNV with AAV5-VMD2-CR2-fH administration. Bioavailability studies revealed that gene-therapy delivered similar levels of CR2-fH to the RPE/choroid as treatment by intravenous injections, and C3a ELISA verified reduced CNV-associated ocular C3a production. These results contribute to existing data illustrating the importance of the AP of complement in CNV development and its potential role in AMD treatment. Importantly, demonstration of AAV-vector efficacy opens new avenues for the development of treatment strategies.

      PubDate: 2017-11-26T10:25:19Z
      DOI: 10.1016/j.omtm.2017.11.003
  • A robust system for production of superabundant VP1 recombinant AAV

    • Authors: Qizhao Wang; Zhongren Junping Zhang Jenni Firrman Hongying Wei Zhengjing
      Abstract: Publication date: Available online 7 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Qizhao Wang, Zhongren Wu, Junping Zhang, Jenni Firrman, Hongying Wei, Zhengjing Zhuang, LinShu Liu, Linqing Miao, Yang Hu, Dong Li, Yong Diao, Weidong Xiao
      Recombinant adeno-associated viral (rAAV) vectors have been widely used in human gene therapy. One major impediment to its broad application is the inability to produce high quality vectors in mass quantity. Here, an efficient and scalable suspension cell culture system for the production of rAAV vectors is described. In this system, the AAV trans factors, Rep78, Rep52, VP1, VP2, and VP3, were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons between genes with identical amino acids, and the cis rAAV genome was carried by an E1/E3 gene-deleted adenovirus. Infection of an improved, E1 integrated, suspension cultured cells with these two viral vectors, resulted in the robust production of rAAV vectors. The newly enhanced system can consistently produce ∼1 × 1015 genome containing rAAV vectors per liter of suspension cells. Moreover, the capsid composition of rAAV vectors produced by this system is markedly different from those produced using the traditional system in that the VP1 protein is more abundant than the VP2 protein (19:1 vs 1:1). The unique VP1 superabundant rAAV vectors produced in this new system exhibited improved transduction in vivo after intravitreal injection.

      PubDate: 2017-11-26T10:25:19Z
  • Improved Expansion and In Vivo Function of Patient T Cells by a Serum-Free

    • Authors: Andrew Medvec; Christopher Ecker Hong Kong Emily Winters Joshua Glover
      Abstract: Publication date: Available online 7 November 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Andrew R. Medvec, Christopher Ecker, Hong Kong, Emily A. Winters, Joshua Glover, Angel Varela-Rohena, James L. Riley
      Improvements to T cell culture systems that promote long-term engraftment and function of adoptively transferred T cells will likely result in superior clinical benefit to more individuals. To this end, we recently developed a chemically-defined cell culture medium that robustly expands all T cell subsets in absence of human serum. Using a humanized mouse model, we observed that T cells expanded in the absence of human serum provided durable control of tumors, whereas T cells expanded in medium supplemented with human serum only mediated transient control of tumor growth. Importantly, our new medium effectively expanded more differentiated T cells from multiple myeloma patients in the absence of serum. These patient-derived T cells were also able to provide durable control of B cell tumors in vivo, and this long-term control of cancer was lost when T cells were expanded in the presence of serum. Thus, engineered T cells expanded in an optimized medium in the absence of serum may have improved therapeutic potential.

      PubDate: 2017-11-26T10:25:19Z
  • Production and Purification of High Titer Newcastle Disease Virus for use
           in Preclinical Mouse Models of Cancer

    • Authors: Lisa A. Santry; Thomas M. McAusland; Leonardo Susta; Geoffrey A. Wood; Pierre P. Major; Jim J. Petrik; Byram W. Bridle; Sarah K. Wootton
      Abstract: Publication date: Available online 16 October 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Lisa A. Santry, Thomas M. McAusland, Leonardo Susta, Geoffrey A. Wood, Pierre P. Major, Jim J. Petrik, Byram W. Bridle, Sarah K. Wootton
      Newcastle disease virus (NDV) is a single-stranded, negative-sense, RNA virus in the Paramyxoviridae family. Although primarily an avian pathogen, NDV is a potent oncolytic virus that has been shown to be safe and effective in a variety of preclinical cancer models and human clinical trials. To produce virus for oncolytic trials, NDV is commonly amplified in embryonated chicken eggs and purified from the allantoic fluid. Conventional methods for purifying virus from allantoic fluid often result in relatively low-titer preparations containing high levels of impurities, including immunogenic chicken host cell proteins from allantoic fluid. However, to administer oncolytic NDV systemically to mice, large quantities of virus need to be delivered intravenously. This route of administration requires virus preparations that are both highly concentrated (to enable delivery of small volumes) and highly pure (to limit toxic effects from contaminants). Given the accumulation of promising preclinical and clinical data demonstrating the efficacy of NDV as an oncolytic agent, strategies for increasing the titer and purity of NDV preparations are sorely needed to allow for effective intravenous administration in mice. Here we describe an optimized protocol for the rescue, production, and purification of high titer in vivo grade NDV for preclinical studies in mouse models.

      PubDate: 2017-11-26T10:25:19Z
      DOI: 10.1016/j.omtm.2017.10.004
  • A humoral immune response alters the distribution of enzyme replacement
           therapy in murine mucopolysaccharidosis type I

    • Authors: Steven Q. Le; Shih-hsin Kan; Don Clarke; Valentina Sanghez; Martin Egeland; Kristen N. Vondrak; Terence M. Doherty; Moin U. Vera; Michelina Iacovino; Jonathan D. Cooper; Mark S. Sands; Patricia I. Dickson
      Abstract: Publication date: Available online 5 October 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Steven Q. Le, Shih-hsin Kan, Don Clarke, Valentina Sanghez, Martin Egeland, Kristen N. Vondrak, Terence M. Doherty, Moin U. Vera, Michelina Iacovino, Jonathan D. Cooper, Mark S. Sands, Patricia I. Dickson
      Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase. Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by 40% compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naïve mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than hepatocytes in the antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.

      PubDate: 2017-10-14T10:48:25Z
      DOI: 10.1016/j.omtm.2017.09.008
  • Targeted cell-to-cell delivery of protein payloads via the
           granzyme-perforin pathway

    • Authors: Daniel Woodsworth; Lisa Dreolini Libin Abraham Robert Holt
      Abstract: Publication date: Available online 10 October 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Daniel J. Woodsworth, Lisa Dreolini, Libin Abraham, Robert A. Holt
      There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the colocalization of the chaperones with lytic granules and the chaperone mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

      PubDate: 2017-10-11T06:55:21Z
  • Interactions Between Retroviruses and the Host Cell genome

    • Authors: Valentina Poletti; Fulvio Mavilio
      Abstract: Publication date: Available online 5 October 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Valentina Poletti, Fulvio Mavilio
      Replication-defective retroviral vectors have been used for over 25 years as a tool for efficient and stable insertion of therapeutic transgenes in human cells. Patients suffering from severe genetic diseases have been successfully treated by transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with retroviral vectors, and the first of this class of therapies – Strimvelis® - has recently received market authorization in Europe. Some clinical trials, however, resulted in severe adverse events caused by vector-induced proto-oncogene activation, which showed that retroviral vectors may retain a genotoxic potential associated to proviral integration in the human genome. The adverse events sparked a renewed interest in the biology of retroviruses, which led in a few years to a remarkable understanding of the molecular mechanisms underlying retroviral integration site selection within mammalian genomes. This review summarizes the current knowledge on retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which different retroviruses, and their related gene transfer vectors, integrate in, and interact with, the human genome. This knowledge provides the basis for the development of safer and more efficacious retroviral vectors for human gene therapy.

      PubDate: 2017-10-11T06:55:21Z
      DOI: 10.1016/j.omtm.2017.10.001
  • Analyzing the genotoxicity of retroviral vectors in hematopoietic cell
           gene therapy

    • Authors: Luca Biasco; Michael Rothe; Hildegard Büning; Axel Schambach
      Abstract: Publication date: Available online 5 October 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Luca Biasco, Michael Rothe, Hildegard Büning, Axel Schambach
      Retroviral vectors, including those derived from gammaretroviruses and lentiviruses, have found their way into the clinical arena and demonstrated remarkable efficacy for the treatment of immunodeficiencies, leukodystrophies and globinopathies. Despite these successes, gene therapy unfortunately also had to face severe adverse events in form of leukemias and myelodysplastic syndromes, related to the semi-random vector integration into the host cell genome that caused deregulation of neighboring proto-oncogenes. Although improvements in vector design clearly lowered the risk of this insertional mutagenesis, analysis of potential genotoxicity and consequences of vector integration remain important parameters for basic as well as translational research and most importantly for the clinic. In this article, we review current assays to analyze biodistribution and genotoxicity in the preclinical setting and describe tools to monitor vector integration sites in vector-treated patients as a biosafety readout.

      PubDate: 2017-10-11T06:55:21Z
      DOI: 10.1016/j.omtm.2017.10.002
  • Rapid Generation of Multiple Loci-Engineered Marker-Free Vaccinia Viruses
           and In Vitro Characterization of Clinical-Grade Oncolytic Poxvirus

    • Authors: Zong Sheng Guo; Zuqiang Liu; Magesh Sathaiah; Jiahu Wang; Roshni Ravindranathan; Eun Kim; Shaohua Huang; Thomas W. Kenniston; John C. Bell; Herbert J. Zeh; Lisa H. Butterfield; Andrea Gambotto; David L. Bartlett
      Abstract: Publication date: Available online 30 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Zong Sheng Guo, Zuqiang Liu, Magesh Sathaiah, Jiahu Wang, Roshni Ravindranathan, Eun Kim, Shaohua Huang, Thomas W. Kenniston, John C. Bell, Herbert J. Zeh, Lisa H. Butterfield, Andrea Gambotto, David L. Bartlett
      Recombinant poxviruses, utilized as vaccine vectors and oncolytic viruses, often require manipulation at multiple genetic loci in the viral genome. It is essential for viral vectors to possess no adventitious mutations and no (antibiotic) selection marker in the final product for human patients in order to comply with the guidance from the regulatory agencies. Rintoul et al have previously developed a selectable and excisable marker (SEM) system for the rapid generation of recombinant vaccinia virus. In the current study, we describe an improved methodology for rapid creation and selection of recombinant poxviruses with multiple genetic manipulations solely based on expression of a fluorescent protein and with no requirement for drug selection drug that can lead to cellular stress and the risk of adventitious mutations throughout the viral genome. Using this improved procedure combined with the SEM system, we have constructed multiple marker-free oncolytic poxviruses expressing different cytokines and other therapeutic genes. The high fidelity of inserted DNA sequences validates the utility of this improved procedure for generation of therapeutic viruses for human patients. We have created an oncolytic poxvirus expressing human chemokine CCL5, designated as vvDD-A34R-hCCL5, with manipulations at two genetic loci in a single virus. Finally, we have produced and purified this virus in clinical grade for it use in a phase I clinical trial and presented data on initial in vitro characterization of the virus.

      PubDate: 2017-10-03T20:46:49Z
      DOI: 10.1016/j.omtm.2017.09.007
  • Molecular Therapy of Melanocortin-4-Receptor Obesity by an autoregulatory
           BDNF vector

    • Authors: Jason J. Siu; Nicholas J. Queen; Xianglan Liu; Wei Huang; Travis McMurphy; Lei Cao
      Abstract: Publication date: Available online 29 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Jason J. Siu, Nicholas J. Queen, Xianglan Liu, Wei Huang, Travis McMurphy, Lei Cao
      Mutations in the melanocortin-4-receptor (MC4R) comprise the most common monogenic form of severe early-onset obesity, and conventional treatments are either ineffective long-term or contraindicated. Immediately downstream of MC4R- in the pathway for regulating energy balance- is brain-derived neurotrophic factor (BDNF). Our previous studies show that AAV-mediated hypothalamic BDNF gene transfer alleviates obesity and diabetes in both diet-induced and genetic models. To facilitate clinical translation we developed a built-in autoregulatory system to control therapeutic gene expression mimicking the body’s natural feedback systems. This autoregulatory approach leads to a sustainable plateau of body weight after substantial weight loss is achieved. Here we examined the efficacy and safety of autoregulatory BDNF gene therapy in Mc4r heterozygous mice, which best resemble MC4R obese patients. Mc4r heterozygous mice were treated with either autoregulatory BDNF vector or YFP control and monitored for 30 weeks. BDNF gene therapy prevented the development of obesity and metabolic syndromes characterized by: decreasing body weight and adiposity, suppressing food intake, alleviating hyperleptinemia and hyperinsulinemia, improving glucose and insulin tolerance, and increasing energy expenditure without adverse cardiovascular function or behavioral disturbances. These safety and efficacy data provide preclinical evidence that BDNF gene therapy is a compelling treatment option for MC4R-deficient obese patients.
      Graphical abstract image

      PubDate: 2017-10-03T20:46:49Z
      DOI: 10.1016/j.omtm.2017.09.005
  • A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene
           Therapy for Vein Graft Atherosclerosis

    • Authors: Lianxiang Bi; Bradley K. Wacker; Emma Bueren; Ervin Ham; Nagadhara Dronadula; David A. Dichek
      Abstract: Publication date: Available online 28 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Lianxiang Bi, Bradley K. Wacker, Emma Bueren, Ervin Ham, Nagadhara Dronadula, David A. Dichek
      Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

      PubDate: 2017-10-03T20:46:49Z
      DOI: 10.1016/j.omtm.2017.09.004
  • 13C/31P MRS metabolic biomarkers of disease progression and response to
           recombinant AAV delivery of hGAA in a mouse model of Pompe disease

    • Authors: Celine Baligand; Adrian G. Todd; Brittany Lee-McMullen; Ravneet S. Vohra; Barry J. Byrne; Darin J. Falk; Glenn A. Walter
      Abstract: Publication date: Available online 8 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Celine Baligand, Adrian G. Todd, Brittany Lee-McMullen, Ravneet S. Vohra, Barry J. Byrne, Darin J. Falk, Glenn A. Walter
      The development of therapeutic clinical trials for glycogen storage disorders, including Pompe disease, has called for non-invasive and objective biomarkers. Glycogen accumulation can be measured in vivo with 13C MRS. However, clinical implementation remains challenging due to low signal-to-noise. On the other hand, the build-up of glycolytic intermediates may be detected with 31P MRS. We sought to identify new biomarkers of disease progression in muscle using 13C/31P MRS and 1H HR-MAS in a mouse model of Pompe disease (Gaa-/-). We evaluated the sensitivity of these MR biomarkers in vivo after treatment using an adeno-associated virus vector 2/9 encoding hGAA driven by the desmin promotor. 31P MRS showed significantly elevated phosphomonoesters (PME) in Gaa-/- compared to control at 2 (0.06 ± 0.02 vs. 0.03 ± 0.01; p=0.003), 6, 12 and 18 months of age. Correlative 1H HR-MAS measures in intact gastrocnemius muscles revealed high glucose-6-phosphate (G-6-P). After intramuscular AAV injections, glycogen, PME and G-6-P were decreased within normal range. The changes in PME levels likely partly resulted from changes in G-6-P, one of the overlapping phosphomonoesters in the 31P MR spectra in vivo. Because 31P MRS is inherently more sensitive than 13C MRS, PME levels have greater potential as a clinical biomarker and should be considered as a complementary approach for future studies in Pompe patients.
      Graphical abstract image

      PubDate: 2017-09-15T06:24:30Z
      DOI: 10.1016/j.omtm.2017.09.002
  • Clodronate improves acute survival of transplanted Hoxb8 myeloid
           progenitor cells expressing constitutively active GMCSFR in
           immunocompetent mice

    • Authors: Simon Lee; Saul Kivimäe; Francis C. Szoka
      Abstract: Publication date: Available online 7 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Simon Lee, Saul Kivimäe, Francis C. Szoka
      New methods to produce large numbers of myeloid progenitor cells, precursors to macrophages (MΦ), by maintaining Hoxb8 transcription factor activity1 has reinvigorated interest in MΦ cell therapies. We generated Hoxb8-dependent myeloid progenitors (HDP) by transducing lineage negative bone marrow cells with a constitutively expressed Hoxb8 flanked by loxP. HDP proliferate indefinitely and differentiate into MΦ when Hoxb8 is removed by a tamoxifen-inducible Cre. We genetically modified HDP with a constitutively active GMCSF receptor and tamoxifen-induced IRF8, which we have termed HDP-on. HDP-on proliferate without GMCSF and differentiate into MΦ upon exposure to tamoxifen and ruxolitinib (GMCSF inhibitor via JAK1/2 blockade). We quantified the biodistribution of HDP transplanted via intraperitoneal injection into immunodeficient NCG mice with a luciferase reporter; HDP are detected for 14 days in the peritoneal cavity, liver, spleen, kidney, bone marrow, brain, lung, heart and blood. In immunocompetent BALB/c mice, HDP-on, but not HDP, are detected 1 day post-transplantation in the peritoneal cavity. Pretreatment of BALB/c mice with liposomal clodronate significantly enhances survival at day 7 for HDP and HDP-on in the peritoneal cavity, spleen and liver, but cells are undetectable at day 14. Short-term post-transplantation survival of HDP is significantly improved using HDP-on and liposomal clodronate, opening a path for MΦ-based therapeutics.

      PubDate: 2017-09-08T23:52:12Z
      DOI: 10.1016/j.omtm.2017.08.007
  • One-Step Fabrication of Bone Morphogenetic Protein-2 Gene Activated,
           Porous Poly-L-Lactide Scaffold for Bone Induction

    • Authors: Jingwen Xue; Hang Lin; Allison Bean; Ying Tang; Jian Tan; Rocky S. Tuan; Bing Wang
      Abstract: Publication date: Available online 7 September 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Jingwen Xue, Hang Lin, Allison Bean, Ying Tang, Jian Tan, Rocky S. Tuan, Bing Wang
      Bone morphogenetic protein 2 (BMP2) is an efficacious inducer for the osteogenesis of mesenchymal stem cells (MSCs). Conventional applications of BMP2 have involved either the direct incorporation of BMP2 protein or ex vivo BMP2 gene transfer into stem cells prior to their transplantation. These approaches are able to promote bone formation to some extent, however are hampered either by the lack of stability and sustainability of BMP2 protein or by the time-consuming and cost-prohibitive in vitro cell culture procedure. To overcome these limitations, we have developed a gene-activated poly-L-lactide acid (PLLA) scaffold with the encapsulation of recombinant adeno-associated viral (AAV) vector encoding a full-length cDNA of human BMP2, using an ice-based microparticle porogenization method that was recently developed. Results showed continuous release of AAV particles from the micropores of scaffolds for up to 1 week, subsequently transducing embedded human MSCs and producing functional BMP2. MSCs within scaffolds underwent efficacious osteogenesis, on the basis of osteoinductive gene expression and osteogenic differentiation, which resulted in robust new bone formation in vivo at 4 weeks. These findings show the potential of the technology towards developing clinical applications of a rapid, cost-effective and potentially point-of-care approach for the repair of bone defects.

      PubDate: 2017-09-08T23:52:12Z
      DOI: 10.1016/j.omtm.2017.08.008
  • In-situ liver expression of HBsAg/CD3-bispecific antibodies for HBV

    • Authors: Robert L. Kruse; Thomas Shum; Xavier Legras; Mercedes Barzi; Frank P. Pankowicz; Stephen Gottschalk; Karl-Dimiter Bissig
      Abstract: Publication date: Available online 31 August 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Robert L. Kruse, Thomas Shum, Xavier Legras, Mercedes Barzi, Frank P. Pankowicz, Stephen Gottschalk, Karl-Dimiter Bissig
      Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T-cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplant can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold) in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T-cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which mimics closely the transcriptional template for HBV, the covalently closed circular (ccc)DNA. We found the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg IgG antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecifc antibodies represents a promising therapeutic strategy for patients with HBV.

      PubDate: 2017-09-04T07:17:15Z
      DOI: 10.1016/j.omtm.2017.08.006
  • DNA-loaded cationic liposomes efficiently function as a vaccine against
           malarial proteins

    • Authors: Wesley L. Fotoran; Rachele Santangelo; Beatriz N.M. de Miranda; Darrell J. Irvine; Gerhard Wunderlich
      Abstract: Publication date: Available online 23 August 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Wesley L. Fotoran, Rachele Santangelo, Beatriz N.M. de Miranda, Darrell J. Irvine, Gerhard Wunderlich
      The delivery of antigens as DNA vaccines is an efficient alternative to induce immune responses against antigens which are difficult to produce in recombinant form. However, the delivery of naked DNA is ineffective or relies on sophisticated ballistic devices. Here we show a combination of liposome application and naked DNA vaccine which successfully overcomes these problems. Upon entrapment of plasmids encoding different antigens in cationic particles, transfection efficiencies similar to commercial kits were achieved in in vitro cell cultures. The liposome-based approach provided strong humoral responses against three malarial antigens, namely the Circumsporozoite protein and the C-terminus of merozoite surface protein 1 from Plasmodium vivax (titers 104 or 103-104, respectively) and P. falciparum Rhoptry antigen 5 from Plasmodium falciparum (titers 103-104). When employed in P. falciparum growth inhibition assays, antibodies demonstrated consistent reinvasion blocking activities which were dose-dependent. Liposome-formulated DNA vaccines may prove useful when targets cannot be produced as recombinant proteins and when conformation-dependent and highly specific antibodies are mandatory.
      Graphical abstract image

      PubDate: 2017-08-25T00:22:54Z
      DOI: 10.1016/j.omtm.2017.08.004
  • Safety and efficacy of the complement inhibitor AMY-101 (Cp40) in a
           natural model of periodontitis in non-human primates

    • Authors: Tetsuhiro Kajikawa; Ruel A. Briones; Ranillo R.G. Resuello; Joel V. Tuplano; Edimara S. Reis; Evlambia Hajishengallis; Cristina A.G. Garcia; Despina Yancopoulou; John D. Lambris; George Hajishengallis
      Abstract: Publication date: Available online 18 August 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Tetsuhiro Kajikawa, Ruel A. Briones, Ranillo R.G. Resuello, Joel V. Tuplano, Edimara S. Reis, Evlambia Hajishengallis, Cristina A.G. Garcia, Despina Yancopoulou, John D. Lambris, George Hajishengallis
      Periodontitis is a chronic inflammatory disease associated with overactivation of the complement system. Recent preclinical studies suggest that host-modulation therapies may contribute to effective treatment of human periodontitis, which – if untreated – may lead to loss of teeth and function. We have previoulsy shown that locally administered AMY-101 (Cp40), a peptidic inhibitor of the central complement component C3, can inhibit naturally occurring periodontitis in non-human primates (NHPs) when given once a week. This study was undertaken to determine the local safety of increasing doses of the drug as well as its efficacy when given at reduced frequency or after systemic administration. Our findings have determined a local dose of AMY-101 (0.1 mg/site) that is free of local irritation and effective when given once every three weeks. Moreover, a daily subcutaneous dose of AMY-101 (4 mg/kg bodyweight) was protective against NHP periodontitis, suggesting that patients treated for systemic disorders (e.g., paroxysmal nocturnal hemoglobinuria) can additionally benefit in terms of improved periodontal condition. In summary, AMY-101 appears to be a promising candidate drug for the adjunctive treatment of human periodontitis, a notion that merits investigation in human clinical trials.

      PubDate: 2017-08-25T00:22:54Z
      DOI: 10.1016/j.omtm.2017.08.001
  • Profiling the Targets of Protective CD8+ T Cell Responses to Infection

    • Authors: Joseph T. Bruder; Ping Chen; Greg Ekberg; Emily C. Smith; Christopher A. Lazarski; Bennett A. Myers; Jessica Bolton; Martha Sedegah; Eileen Villasante; Thomas L. Richie; C. Richter King; Joao C. Aguiar; Denise L. Doolan; Douglas E. Brough
      Abstract: Publication date: Available online 18 August 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Joseph T. Bruder, Ping Chen, Greg Ekberg, Emily C. Smith, Christopher A. Lazarski, Bennett A. Myers, Jessica Bolton, Martha Sedegah, Eileen Villasante, Thomas L. Richie, C. Richter King, Joao C. Aguiar, Denise L. Doolan, Douglas E. Brough
      T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging, and for tuberculosis and malaria many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof-of-principle for this approach in identifying antigens for vaccine development.

      PubDate: 2017-08-25T00:22:54Z
      DOI: 10.1016/j.omtm.2017.08.003
  • Minimal Purkinje cell-specific PCP2/L7 promoter virally available for
           rodents and non-human primates

    • Authors: Keisuke Nitta; Yasunori Matsuzaki; Ayumu Konno; Hirokazu Hirai
      Abstract: Publication date: Available online 27 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Keisuke Nitta, Yasunori Matsuzaki, Ayumu Konno, Hirokazu Hirai
      Cell type-specific promoters in combination with viral vectors and gene editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood brain barrier confirmed the Purkinje cell specificity of the L7-6 in the central nervous system. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell-specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.

      PubDate: 2017-08-05T00:20:14Z
      DOI: 10.1016/j.omtm.2017.07.006
  • A Five-Repeat Micro-Dystrophin Gene Ameliorated Dystrophic Phenotype in
           the Severe DBA/2J-mdx Model of Duchenne Muscular Dystrophy

    • Authors: Chady H. Hakim; Nalinda B. Wasala; Xiufang Pan; Kasun Kodippili; Yongping Yue; Keqing Zhang; Gang Yao; Brittney Haffner; Sean X. Duan; Julian Ramos; Joel S. Schneider; N. Nora Yang; Jeffrey S. Chamberlain; Dongsheng Duan
      Abstract: Publication date: Available online 27 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Chady H. Hakim, Nalinda B. Wasala, Xiufang Pan, Kasun Kodippili, Yongping Yue, Keqing Zhang, Gang Yao, Brittney Haffner, Sean X. Duan, Julian Ramos, Joel S. Schneider, N. Nora Yang, Jeffrey S. Chamberlain, Dongsheng Duan
      Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 1013 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.

      PubDate: 2017-08-05T00:20:14Z
      DOI: 10.1016/j.omtm.2017.06.006
  • Lipidomic evaluation of feline neurologic disease after AAV gene therapy

    • Authors: H.L. Gray-Edwards; X. Jiang; A.N. Randle; A.R. Taylor; T.L. Voss; A.K. Johnson; V.J. McCurdy; M. Sena-Esteves; D.S. Ory; D.R. Martin
      Abstract: Publication date: Available online 26 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): H.L. Gray-Edwards, X. Jiang, A.N. Randle, A.R. Taylor, T.L. Voss, A.K. Johnson, V.J. McCurdy, M. Sena-Esteves, D.S. Ory, D.R. Martin
      GM1 gangliosidosis is a fatal lysosomal disorder for which there is no effective treatment. AAV gene therapy in GM1 cats has resulted in a greater than six-fold increase in lifespan, with many cats remaining alive at >5.7 years of age with minimal clinical signs. Glycolipids are the principal storage product in GM1 gangliosidosis, whose pathogenic mechanism is not completely understood. Targeted lipidomics analysis was performed to better define disease mechanisms and to identify markers of disease progression for upcoming clinical trials in humans. Thirty-six sphingolipids and subspecies associated with ganglioside biosynthesis were tested in cerebrospinal fluid of untreated GM1 cats at humane endpoint (∼8 months), AAV-treated GM1 cats (∼ 5 years old) and normal adult controls. In untreated GM1 cats, significant alterations were noted in 16 sphingolipid species including gangliosides (GM1, GM3), lactosylceramides, ceramides, sphingomyelins, monohexosylceramides and sulfatides. Variable degrees of correction in many lipid metabolites reflected the efficacy of AAV gene therapy. Sphingolipid levels were highly predictive of neurologic disease progression, with 11 metabolites having a coefficient of determination (R2) >0.75. Also, a specific detergent additive significantly increased the recovery of certain lipid species in cerebrospinal fluid samples. This report demonstrates the methodology and utility of targeted lipidomics to examine the pathophysiology of lipid storage disorders.

      PubDate: 2017-07-26T14:12:57Z
      DOI: 10.1016/j.omtm.2017.07.005
  • Thermal stability as a determinant of AAV serotype identity

    • Authors: Antonette Bennett; Saajan Patel; Mario Mietzsch; Ariana Jose; Bridget Lins-Austin; Jennifer C. Yu; Brian Bothner; Robert McKenna; Mavis Agbandje-McKenna
      Abstract: Publication date: Available online 24 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Antonette Bennett, Saajan Patel, Mario Mietzsch, Ariana Jose, Bridget Lins-Austin, Jennifer C. Yu, Brian Bothner, Robert McKenna, Mavis Agbandje-McKenna
      Currently there are over 150 ongoing clinical trials utilizing Adeno-Associated viruses (AAV) to target various genetic diseases, including hemophilia (AAV2, AAV8), congenital heart failure (AAV1, AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm), for AAV serotype identification. This is a simple, rapid, cost effective, and robust method, utilizing small amounts of purified AAV capsids (∼25 μl of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, while AAV5, the most stable, has a narrow Tm range, in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically the ratio of basic/acidic amino acids, and independent of VP1 and VP2 content or genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.

      PubDate: 2017-07-26T14:12:57Z
      DOI: 10.1016/j.omtm.2017.07.003
  • Non clinical safety and efficacy of a recombinant AAV2/8 vector

    • Authors: Rita Ferla; Marialuisa Alliegro; Jean-Brice Marteau; Margherita Dell'Anno; Edoardo Nusco; Severine Pouillot; Stefania Galimberti; Maria Grazia Valsecchi; Vincent Zuliani; Alberto Auricchio
      Abstract: Publication date: Available online 24 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Rita Ferla, Marialuisa Alliegro, Jean-Brice Marteau, Margherita Dell'Anno, Edoardo Nusco, Severine Pouillot, Stefania Galimberti, Maria Grazia Valsecchi, Vincent Zuliani, Alberto Auricchio
      In vivo gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. We recently demonstrated that AAV8-mediated liver gene transfer is effective in animal models of mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disease due to arylsulfatase B (ARSB) deficiency. In preparing for a first-in-human, we performed non-clinical studies to assess the safety of intravenous administrations of AAV2/8.TBG.hARSB produced under good manufacturing practice-like conditions. No toxicity was observed in AAV-treated mice, except for a transient increase in alanine aminotransferase in females, and thyroid epithelial hypertrophy. AAV2/8.TBG.hARSB biodistribution and expression confirmed liver as the main site of both infection and transduction. Shedding and breeding studies suggest that the risk of both horizontal and germline transmission is minimal. An AAV-dose-response study in MPS VI mice was performed to define the range of doses to be used in the clinical study. Overall these data support the non clinical safety and the efficacy of AAV2/8.TBG.hARSB and pave the way for a phase I/II clinical trial based on intravascular infusions of AAV8 in MPS VI patients.

      PubDate: 2017-07-26T14:12:57Z
      DOI: 10.1016/j.omtm.2017.07.004
  • Global Screening of Antiviral Genes that Suppress Baculovirus Transgene
           Expression in Mammalian Cells

    • Authors: Chia-Hung Wang; Nenavath Gopal Naik; Lin-Li Liao; Sung-Chan Wei; Yu-Chan Chao
      Abstract: Publication date: Available online 18 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Chia-Hung Wang, Nenavath Gopal Naik, Lin-Li Liao, Sung-Chan Wei, Yu-Chan Chao
      Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, alias RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)—an inhibitor of RIP1 kinase activity—dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study has presented an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus". In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

      PubDate: 2017-07-19T21:20:50Z
      DOI: 10.1016/j.omtm.2017.07.002
  • Neonatal gene therapy for hemophilia B by a novel adenovirus vector
           showing sustained transgene expression via a reduction in the leaky
           expression of viral genes

    • Authors: Shunsuke Iizuka; Fuminori Sakurai; Masashi Tachibana; Kazuo Ohashi; Hiroyuki Mizuguchi
      Abstract: Publication date: Available online 8 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Shunsuke Iizuka, Fuminori Sakurai, Masashi Tachibana, Kazuo Ohashi, Hiroyuki Mizuguchi
      Gene therapy during neonatal/infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal/infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and completely rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal/infant gene therapy for hemophilia B.

      PubDate: 2017-07-10T19:45:48Z
      DOI: 10.1016/j.omtm.2017.07.001
  • Traceless targeting and isolation of gene edited immortalised
           keratinocytes from epidermolysis bullosa simplex patients

    • Authors: Magomet Aushev; Ulrich Koller; Claudio Mussolino; Toni Cathomen; Julia Reichelt
      Abstract: Publication date: Available online 5 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Magomet Aushev, Ulrich Koller, Claudio Mussolino, Toni Cathomen, Julia Reichelt
      Epidermolysis bullosa simplex (EBS) is a blistering skin disease due to dominant-negative mutations in either KRT5 or KRT14 resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events, and devise a strategy, which allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here, we report on TALENs, which efficiently disrupt the KRT5 locus in immortalised patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

      PubDate: 2017-07-10T19:45:48Z
      DOI: 10.1016/j.omtm.2017.06.008
  • Generation of a Vero-based packaging cell line for the production of SV40
           gene delivery vector particles for use in clinical gene therapy studies

    • Authors: Miguel G. Toscano; Jeroen van der Velden; Sybrand van der Werf; Machteld Odijk; Ana Roque; Rafael J. Camacho-Garcia; Irene Herrera-Gomez; Irene Mancini; Peter de Haan
      Abstract: Publication date: Available online 5 July 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Miguel G. Toscano, Jeroen van der Velden, Sybrand van der Werf, Machteld Odijk, Ana Roque, Rafael J. Camacho-Garcia, Irene Herrera-Gomez, Irene Mancini, Peter de Haan
      Replication-defective (RD) recombinant Simian Virus 40 (SV40)-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC) free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

      PubDate: 2017-07-10T19:45:48Z
      DOI: 10.1016/j.omtm.2017.06.007
  • Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote
           Neuroprotection in Experimental Acute Ischemic Stroke

    • Authors: Masataka Nakajima; Chikako Nito; Kota Sowa; Satoshi Suda; Yasuhiro Nishiyama; Aki Nakamura-Takahashi; Yuko Nitahara-Kasahara; Kiwamu Imagawa; Tohru Hirato; Masayuki Ueda; Kazumi Kimura; Takashi Okada
      Abstract: Publication date: Available online 23 June 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Masataka Nakajima, Chikako Nito, Kota Sowa, Satoshi Suda, Yasuhiro Nishiyama, Aki Nakamura-Takahashi, Yuko Nitahara-Kasahara, Kiwamu Imagawa, Tohru Hirato, Masayuki Ueda, Kazumi Kimura, Takashi Okada
      Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90-minute MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hours after ischemia-reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hour transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hour transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hours and 7days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.

      PubDate: 2017-07-01T14:31:40Z
      DOI: 10.1016/j.omtm.2017.06.005
  • Public Attitudes toward Gene Therapy in China

    • Authors: Jiang-Hui Wang; Rong Wang Jia Hui Lee Tiara W.U. Iao
      Abstract: Publication date: 15 September 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 6
      Author(s): Jiang-Hui Wang, Rong Wang, Jia Hui Lee, Tiara W.U. Iao, Xiao Hu, Yu-Meng Wang, Lei-Lei Tu, Yi Mou, Wen-Li Zhu, Ai-Yong He, Shen-Yu Zhu, Di Cao, Lei Yang, Xiao-Bo Tan, Qing Zhang, Guan-Lu Liang, Shu-Min Tang, Ye-Di Zhou, Li-Jun Feng, Li-Jun Zhan, Nan-Nan Tian, Ming-Jie Tang, Ya-Ping Yang, Moeen Riaz, Peter van Wijngaarden, Gregory J. Dusting, Guei-Sheung Liu, Yan He

      PubDate: 2017-06-22T14:03:38Z
  • Rgulatable Transgene Expression for Prevention of Chemotherapy-Induced
           Peripheral Neuropathy

    • Authors: Daisuke kawata; Zetang
      Abstract: Publication date: Available online 21 June 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Daisuke kawata, Zetang Wu
      Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer. For which there are no effective strategies of prevention or treatment. In this study we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin 10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency associated promoter 2 (LAP-2) and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.

      PubDate: 2017-06-22T14:03:38Z
  • Targeting Visceral Fat by Intraperitoneal Delivery of Novel Recombinant
           Adeno-associated Viral Vectors Restricting Off-target Transduction in

    • Authors: Wei Huang; Xianglan Liu; Nicholas Queen; Lei Cao
      Abstract: Publication date: Available online 19 June 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Wei Huang, Xianglan Liu, Nicholas Queen, Lei Cao
      It is challenging to genetically manipulate fat in adults. We demonstrate that intraperitoneal injection of an engineered adeno-associated virus (AAV) serotype Rec2 leads to high transduction of multiple visceral fat depots at a dose 1-2 orders lower than commonly used doses for systemic gene delivery. To target adipose tissue, we develop a single AAV vector harboring two expression cassettes, one using CBA promoter to drive transgene expression and the other using liver specific albumin promoter to drive a microRNA targeting WPRE sequence that only exists in this AAV vector. This dual-cassette vector achieves highly selective transduction of visceral fat while severely restricting off-target transduction of liver. As proof of efficacy, intraperitoneal administration of adipose-targeting Rec2 vector harboring leptin gene corrects leptin deficiency, obesity and metabolic syndromes of ob/ob mice. This study provides a powerful tool to genetically manipulate fat for basic research and gene therapies of genetic and acquired diseases.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.06.002
  • The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ
           Bound by a Heparinoid Pentasaccharide

    • Authors: Qing Xie; John Spear Alex Noble Duncan Sousa Nancy Meyer
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Qing Xie, John M. Spear, Alex J. Noble, Duncan R. Sousa, Nancy L. Meyer, Omar Davulcu, Fuming Zhang, Robert J. Linhardt, Scott M. Stagg, Michael S. Chapman
      Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ’s directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
  • Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV,
           Adenovirus, CMV, and EBV from Umbilical Cord Blood

    • Authors: Hema Dave; Min Luo J.W. Blaney Shabnum Patel Cecilia Barese
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Hema Dave, Min Luo, J.W. Blaney, Shabnum Patel, Cecilia Barese, Conrad Russell Cruz, Elizabeth J. Shpall, Catherine M. Bollard, Patrick J. Hanley
      Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party “off the shelf” treatment for viral infections following transplantation.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
  • Deletion of the virion host shut-off gene enhances neuronal-selective
           transgene expression from an HSV vector lacking functional IE genes

    • Authors: Yoshitaka Miyagawa; Gianluca Verlengia; Bonnie Reinhart; Fang Han; Hiroaki Uchida; Silvia Zucchini; William F. Goins; Michele Simonato; Justus B. Cohen; Joseph C. Glorioso
      Abstract: Publication date: Available online 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Yoshitaka Miyagawa, Gianluca Verlengia, Bonnie Reinhart, Fang Han, Hiroaki Uchida, Silvia Zucchini, William F. Goins, Michele Simonato, Justus B. Cohen, Joseph C. Glorioso
      The ability of herpes simplex virus (HSV) to establish life-long latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.06.001
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