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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  [3043 journals]
  • 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
           Sequencing

    • 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)
       
  • Vectofusin-1 Promotes RD114-TR-Pseudotyped Lentiviral Vector Transduction
           of Human HSPCs and T Lymphocytes

    • Authors: Claudia Piovan; Virna Marin; Cinzia Scavullo; Stefano Corna; Erica Giuliani; Sergio Bossi; Anne Galy; David Fenard; Claudio Bordignon; Gian Paolo Rizzardi; Chiara Bovolenta
      Pages: 22 - 30
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Claudia Piovan, Virna Marin, Cinzia Scavullo, Stefano Corna, Erica Giuliani, Sergio Bossi, Anne Galy, David Fenard, Claudio Bordignon, Gian Paolo Rizzardi, Chiara Bovolenta
      Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.02.003
      Issue No: Vol. 5 (2017)
       
  • Efficacy of Gene Therapy Is Dependent on Disease Progression in Dystrophic
           Mice with Mutations in the FKRP Gene

    • Authors: Charles Harvey Vannoy; Will Xiao; Peijuan Lu; Xiao Xiao; Qi Long Lu
      Pages: 31 - 42
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Charles Harvey Vannoy, Will Xiao, Peijuan Lu, Xiao Xiao, Qi Long Lu
      Loss-of-function mutations in the Fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) and other forms of congenital muscular dystrophy-dystroglycanopathy that are associated with glycosylation defects in the α-dystroglycan (α-DG) protein. Systemic administration of a single dose of recombinant adeno-associated virus serotype 9 (AAV9) vector expressing human FKRP to a mouse model of LGMD2I at various stages of disease progression was evaluated. The results demonstrate rescue of functional glycosylation of α-DG and muscle function, along with improvements in muscle structure at all disease stages versus age-matched untreated cohorts. Nevertheless, mice treated in the latter stages of disease progression revealed a decrease in beneficial effects of the treatment. The results provide a proof of concept for future clinical trials in patients with FKRP-related muscular dystrophy and demonstrate that AAV-mediated gene therapy can potentially benefit patients at all stages of disease progression, but earlier intervention would be highly preferred.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.02.002
      Issue No: Vol. 5 (2017)
       
  • A One-Step PCR-Based Assay to Evaluate the Efficiency and Precision of
           Genomic DNA-Editing Tools

    • Authors: Diego Germini; Yara Bou Saada; Tatiana Tsfasman; Kristina Osina; Chloé Robin; Nikolay Lomov; Mikhail Rubtsov; Nikolajs Sjakste; Mar≿ Lipinski; Yegor Vassetzky
      Pages: 43 - 50
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Diego Germini, Yara Bou Saada, Tatiana Tsfasman, Kristina Osina, Chloé Robin, Nikolay Lomov, Mikhail Rubtsov, Nikolajs Sjakste, Mar≿ Lipinski, Yegor Vassetzky
      Despite rapid progress, many problems and limitations persist and limit the applicability of gene-editing techniques. Making use of meganucleases, TALENs, or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time consuming and material consuming. Here we propose a simple, cheap, reliable, time-saving, and highly sensitive method to evaluate a given gene-editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique, designated engineered nuclease-induced translocations (ENIT), a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, then specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced, thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs, and CRISPR/Cas9-based editing tools.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.001
      Issue No: Vol. 5 (2017)
       
  • Viral Vector-Based Innovative Approaches to Directly Abolishing
           Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine

    • Authors: Kaoru Mitsui; Kanako Ide; Tomoyuki Takahashi; Ken-ichiro Kosai
      Pages: 51 - 58
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Kaoru Mitsui, Kanako Ide, Tomoyuki Takahashi, Ken-ichiro Kosai
      Human pluripotent stem cells (hPSCs) are a promising source of regenerative material for clinical applications. However, hPSC transplant therapies pose the risk of teratoma formation and malignant transformation of undifferentiated remnants. These problems underscore the importance of developing technologies that completely prevent tumorigenesis to ensure safe clinical application. Research to date has contributed to establishing safe hPSC lines, improving the efficiency of differentiation induction, and indirectly ensuring the safety of products. Despite such efforts, guaranteeing the clinical safety of regenerative medicine products remains a key challenge. Given the intrinsic genome instability of hPSCs, selective growth advantage of cancer cells, and lessons learned through failures in previous attempts at hematopoietic stem cell gene therapy, conventional strategies are unlikely to completely overcome issues related to hPSC tumorigenesis. Researchers have recently embarked on studies aimed at locating and directly treating hPSC-derived tumorigenic cells. In particular, novel approaches to directly killing tumorigenic cells by transduction of suicide genes and oncolytic viruses are expected to improve the safety of hPSC-based therapy. This article discusses the current status and future perspectives of methods aimed at directly eradicating undifferentiated tumorigenic hPSCs, with a focus on viral vector transduction.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.002
      Issue No: Vol. 5 (2017)
       
  • Engineering of GlcNAc-1-Phosphotransferase for Production of Highly
           Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy

    • Authors: Lin Liu; Wang-Sik Lee; Balraj Doray; Stuart Kornfeld
      Pages: 59 - 65
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Lin Liu, Wang-Sik Lee, Balraj Doray, Stuart Kornfeld
      Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.006
      Issue No: Vol. 5 (2017)
       
  • Dendritic Cell Therapies for Hematologic Malignancies

    • Authors: Matthew Weinstock; Jacalyn Rosenblatt; David Avigan
      Pages: 66 - 75
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Matthew Weinstock, Jacalyn Rosenblatt, David Avigan
      Dendritic cells (DCs) are potent antigen-presenting cells that constitute a major component of the immune system’s role in the recognition, elimination, and tolerance of cancer. The unique immunologic capabilities of DCs have recently been harnessed for therapeutic use with the creation of DC-based anti-tumor vaccines, several of which have moved into testing in clinical trials for hematologic malignancies. This review summarizes how treatment strategies using DC-based anti-tumor vaccines are advancing immunotherapeutic options for these diseases.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.004
      Issue No: Vol. 5 (2017)
       
  • Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted
           Lentiviral Gene Transfer to Primary B Cells

    • Authors: Xiaomei Wang; Roland W. Herzog; Barry J. Byrne; Sandeep R.P. Kumar; Qi Zhou; Christian J. Buchholz; Moanaro Biswas
      Pages: 76 - 82
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Xiaomei Wang, Roland W. Herzog, Barry J. Byrne, Sandeep R.P. Kumar, Qi Zhou, Christian J. Buchholz, Moanaro Biswas
      Gene-modified B cells expressing immunoglobulin G (IgG) fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX) lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20). Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV)-lentiviral vector (LV) and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB). In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.005
      Issue No: Vol. 5 (2017)
       
  • Toward Personalized Gene Therapy: Characterizing the Host Genetic Control
           of Lentiviral-Vector-Mediated Hepatic Gene Delivery

    • Authors: Thipparat Suwanmanee; Martin T. Ferris; Peirong Hu; Tong Gui; Stephanie A. Montgomery; Fernando Pardo-Manuel de Villena; Tal Kafri
      Pages: 83 - 92
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Thipparat Suwanmanee, Martin T. Ferris, Peirong Hu, Tong Gui, Stephanie A. Montgomery, Fernando Pardo-Manuel de Villena, Tal Kafri
      The success of lentiviral vectors in curing fatal genetic and acquired diseases has opened a new era in human gene therapy. However, variability in the efficacy and safety of this therapeutic approach has been reported in human patients. Consequently, lentiviral-vector-based gene therapy is limited to incurable human diseases, with little understanding of the underlying causes of adverse effects and poor efficacy. To assess the role that host genetic variation has on efficacy of gene therapy, we characterized lentiviral-vector gene therapy within a set of 12 collaborative cross mouse strains. Lentiviral vectors carrying the firefly luciferase cDNA under the control of a liver-specific promoter were administered to female mice, with total-body and hepatic luciferase expression periodically monitored through 41 weeks post-vector administration. Vector copy number per diploid genome in mouse liver and spleen was determined at the end of this study. We identified major strain-specific contributions to overall success of transduction, vector biodistribution, maximum luciferase expression, and the kinetics of luciferase expression throughout the study. Our results highlight the importance of genetic variation on gene-therapeutic efficacy; provide new models with which to more rigorously assess gene therapy approaches; and suggest that redesigning preclinical studies of gene-therapy methodologies might be appropriate.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.009
      Issue No: Vol. 5 (2017)
       
  • The Clonal Fate of Live Cells

    • Authors: Wei Wang; Raffaele Fronza; Manfred Schmidt
      Pages: 93 - 95
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Wei Wang, Raffaele Fronza, Manfred Schmidt


      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.007
      Issue No: Vol. 5 (2017)
       
  • Genetic Engineering and Manufacturing of Hematopoietic Stem Cells

    • Authors: Xiuyan Wang; Isabelle Rivière
      Pages: 96 - 105
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Xiuyan Wang, Isabelle Rivière
      The marketing approval of genetically engineered hematopoietic stem cells (HSCs) as the first-line therapy for the treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a tribute to the substantial progress that has been made regarding HSC engineering in the past decade. Reproducible manufacturing of high-quality, clinical-grade, genetically engineered HSCs is the foundation for broadening the application of this technology. Herein, the current state-of-the-art manufacturing platforms to genetically engineer HSCs as well as the challenges pertaining to production standardization and product characterization are addressed in the context of primary immunodeficiency diseases (PIDs) and other monogenic disorders.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.03.003
      Issue No: Vol. 5 (2017)
       
  • Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice
           without Apparent Toxicity after Intracisternal Delivery

    • Authors: Sarah E. Sinnett; Ralph D. Hector; Kamal K.E. Gadalla; Clifford Heindel; Daphne Chen; Violeta Zaric; Mark E.S. Bailey; Stuart R. Cobb; Steven J. Gray
      Pages: 106 - 115
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Sarah E. Sinnett, Ralph D. Hector, Kamal K.E. Gadalla, Clifford Heindel, Daphne Chen, Violeta Zaric, Mark E.S. Bailey, Stuart R. Cobb, Steven J. Gray
      Intravenous administration of adeno-associated virus serotype 9 (AAV9)/hMECP2 has been shown to extend the lifespan of Mecp2 −/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 × 1012 viral genomes [vg]; ICM) extended Mecp2 −/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2 −/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2 −/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2 −/y survival, without apparent toxicity.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.006
      Issue No: Vol. 5 (2017)
       
  • STRIP2 Is Indispensable for the Onset of Embryonic Stem Cell
           Differentiation

    • Authors: Davood Sabour; Sureshkumar Perumal Srinivasan; Susan Rohani; Vilas Wagh; John Antonydas Gaspar; Darius Panek; Mostafa Abootorabi Ardestani; Michael Xavier Doss; Nicole Riet; Hinrich Abken; Jürgen Hescheler; Symeon Papadopoulos; Agapios Sachinidis
      Pages: 116 - 129
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Davood Sabour, Sureshkumar Perumal Srinivasan, Susan Rohani, Vilas Wagh, John Antonydas Gaspar, Darius Panek, Mostafa Abootorabi Ardestani, Michael Xavier Doss, Nicole Riet, Hinrich Abken, Jürgen Hescheler, Symeon Papadopoulos, Agapios Sachinidis
      The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.001
      Issue No: Vol. 5 (2017)
       
  • Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter
           in Retinal Ganglion Cell Injury

    • Authors: Kosuke Fujita; Koji M. Nishiguchi; Yukihiro Shiga; Toru Nakazawa
      Pages: 130 - 141
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Kosuke Fujita, Koji M. Nishiguchi, Yukihiro Shiga, Toru Nakazawa
      Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter-driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.003
      Issue No: Vol. 5 (2017)
       
  • An Immune-Competent Murine Model to Study Elimination of AAV-Transduced
           Hepatocytes by Capsid-Specific CD8+ T Cells

    • Authors: Brett Palaschak; Damien Marsic; Roland W. Herzog; Sergei Zolotukhin; David M. Markusic
      Pages: 142 - 152
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Brett Palaschak, Damien Marsic, Roland W. Herzog, Sergei Zolotukhin, David M. Markusic
      Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.004
      Issue No: Vol. 5 (2017)
       
  • Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly
           Efficient CRISPR/Cas9-Mediated Gene Editing

    • Authors: Pavel I. Ortinski; Bernadette O’Donovan; Xiaoyu Dong; Boris Kantor
      Pages: 153 - 164
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Pavel I. Ortinski, Bernadette O’Donovan, Xiaoyu Dong, Boris Kantor
      The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs) present an attractive means for delivery of CRISPR/Cas9 components because: (1) they are capable of transducing a broad range of cells and tissues, (2) have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors), and (3) they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T) cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.002
      Issue No: Vol. 5 (2017)
       
  • Efficient Production of Papillomavirus Gene Delivery Vectors in Defined
           In Vitro Reactions

    • Authors: Carla Cerqueira; Cynthia D. Thompson; Patricia M. Day; Yuk-Ying S. Pang; Douglas R. Lowy; John T. Schiller
      Pages: 165 - 179
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Carla Cerqueira, Cynthia D. Thompson, Patricia M. Day, Yuk-Ying S. Pang, Douglas R. Lowy, John T. Schiller
      Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 1011 infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.005
      Issue No: Vol. 5 (2017)
       
  • Development of a Novel AAV Gene Therapy Cassette with Improved Safety
           Features and Efficacy in a Mouse Model of Rett Syndrome

    • Authors: Kamal K.E. Gadalla; Thishnapha Vudhironarit; Ralph D. Hector; Sarah Sinnett; Noha G. Bahey; Mark E.S. Bailey; Steven J. Gray; Stuart R. Cobb
      Pages: 180 - 190
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Kamal K.E. Gadalla, Thishnapha Vudhironarit, Ralph D. Hector, Sarah Sinnett, Noha G. Bahey, Mark E.S. Bailey, Steven J. Gray, Stuart R. Cobb
      Rett syndrome (RTT), caused by loss-of-function mutations in the MECP2 gene, is a neurological disorder characterized by severe impairment of motor and cognitive functions. The aim of this study was to investigate the impact of vector design, dosage, and delivery route on the efficacy and safety of gene augmentation therapy in mouse models of RTT. Our results show that AAV-mediated delivery of MECP2 to Mecp2 null mice by systemic administration, and utilizing a minimal endogenous promoter, was associated with a narrow therapeutic window and resulted in liver toxicity at higher doses. Lower doses of this vector significantly extended the survival of mice lacking MeCP2 or expressing a mutant T158M allele but had no impact on RTT-like neurological phenotypes. Modifying vector design by incorporating an extended Mecp2 promoter and additional regulatory 3′ UTR elements significantly reduced hepatic toxicity after systemic administration. Moreover, direct cerebroventricular injection of this vector into neonatal Mecp2-null mice resulted in high brain transduction efficiency, increased survival and body weight, and an amelioration of RTT-like phenotypes. Our results show that controlling levels of MeCP2 expression in the liver is achievable through modification of the expression cassette. However, it also highlights the importance of achieving high brain transduction to impact the RTT-like phenotypes.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.007
      Issue No: Vol. 5 (2017)
       
  • Targeting Wnt/β-Catenin Activated Cells with Dominant-Negative N-cadherin
           to Reduce Neointima Formation

    • Authors: Sarah Hulin-Curtis; Helen Williams; Kerry S. Wadey; Graciela B. Sala-Newby; Sarah J. George
      Pages: 191 - 199
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Sarah Hulin-Curtis, Helen Williams, Kerry S. Wadey, Graciela B. Sala-Newby, Sarah J. George
      Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/β-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/β-catenin/TCF pathway. Infection with Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis (3 ± 0.2% versus 9 ± 0.7%; p < 0.05, n = 6) and significantly inhibited VSMC migration by 83 ± 15% (p < 0.05, n = 6), but did not affect VSMC proliferation (p > 0.05, n = 5). In an ex vivo human saphenous vein organ culture model, luminal delivery of Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis after 7 days of culture (4 ± 1.4% versus 9 ± 1.6%; p < 0.01, n = 6) and suppressed intimal thickening by 75 ± 7% (p < 0.05, n = 5), without a detrimental effect on endothelial cell coverage. In vivo, Ad-TOP-dn-N-cadherin significantly reduced intimal thickening at day 21 (n = 10) in comparison to the Ad-β-galactosidase (Ad-β-gal) control virus (n = 12, p < 0.05) in the mouse carotid artery ligation model. In summary, we have developed a novel approach to selectively reduce intimal thickening, which may be beneficial in reducing late vein graft failure.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.009
      Issue No: Vol. 5 (2017)
       
  • Neuroglobin Can Prevent or Reverse Glaucomatous Progression in DBA/2J Mice

    • Authors: Hélène Cwerman-Thibault; Christophe Lechauve; Sébastien Augustin; Delphine Roussel; Élodie Reboussin; Ammara Mohammad; Julie Degardin-Chicaud; Manuel Simonutti; Hong Liang; Françoise Brignole-Baudouin; Anne Maron; Thomas Debeir; Marisol Corral-Debrinski
      Pages: 200 - 220
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Hélène Cwerman-Thibault, Christophe Lechauve, Sébastien Augustin, Delphine Roussel, Élodie Reboussin, Ammara Mohammad, Julie Degardin-Chicaud, Manuel Simonutti, Hong Liang, Françoise Brignole-Baudouin, Anne Maron, Thomas Debeir, Marisol Corral-Debrinski
      Mitochondrial dysfunction is responsible for hereditary optic neuropathies. We wished to determine whether preserving mitochondrial bioenergetics could prevent optic neuropathy in a reliable model of glaucoma. DBA/2J mice exhibit elevated intraocular pressure, progressive degeneration of their retinal ganglion cells, and optic neuropathy that resembles glaucoma. We established that glaucoma in these mice is directly associated with mitochondrial dysfunction: respiratory chain activity was compromised in optic nerves 5 months before neuronal loss began, and the amounts of some mitochondrial proteins were reduced in retinas of glaucomatous mice. One of these proteins is neuroglobin, which has a neuroprotective function. Therefore, we investigated whether gene therapy aimed at restoring neuroglobin levels in the retina via ocular administration of an adeno-associated viral vector could reduce neuronal degeneration. The approach of treating 2-month-old mice impeded glaucoma development: few neurons died and respiratory chain activity and visual cortex activity were comparable to those in young, asymptomatic mice. When the treatment was performed in 8-month-old mice, the surviving neurons acquired new morphologic and functional properties, leading to the preservation of visual cortex activity and respiratory chain activity. The beneficial effects of neuroglobin in DBA/2J retinas confirm this protein to be a promising candidate for treating glaucoma.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.008
      Issue No: Vol. 5 (2017)
       
  • Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial
           Cell-Penetrating Peptide and a Glutathione Analog Peptide

    • Authors: Carmine Pasquale Cerrato; Ülo Langel
      Pages: 221 - 231
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Carmine Pasquale Cerrato, Ülo Langel
      Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs) superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs), exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP) internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.04.010
      Issue No: Vol. 5 (2017)
       
  • Cell Therapy in Myology: Dynamics of Muscle Precursor Cell Death after
           Intramuscular Administration in Non-human Primates

    • Authors: Daniel Skuk; Jacques P. Tremblay
      Pages: 232 - 240
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Daniel Skuk, Jacques P. Tremblay
      Cell therapy could be useful for the treatment of myopathies. A problem observed in mice, with different results and interpretations, is a significant death among the transplanted cells. We analyzed this problem in non-human primates, the animal model more similar to humans. Autologous or allogeneic myoblasts (with or without a reporter gene) were proliferated in vitro, labeled with [14C]thymidine, and intramuscularly injected in macaques. Some monkeys were immunosuppressed for long-term follow-up. Cell-grafted regions were biopsied at different intervals and analyzed by radiolabel quantification and histology. Most radiolabel was lost during the first week after injection, regardless of whether the cells were allogeneic or autologous, the culture conditions, and the use or not of immunosuppression. There was no significant difference between 1 hr and 1 day post-transplantation, a significant decrease between days 1 and 3 (45% to 83%), a significant decrease between days 3 and 7 (80% to 92%), and no significant differences between 7 days and 3 weeks. Our results confirmed in non-human primates a progressive and significant death of the grafted myoblasts during the first week after administration, relatively similar to some observations in mice but with different kinetics.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.002
      Issue No: Vol. 5 (2017)
       
  • Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing:
           Targeting Mobilized CD34+ Cells for Correction of Fabry Disease

    • Authors: Ju Huang; Aneal Khan; Bryan C. Au; Dwayne L. Barber; Lucía López-Vásquez; Nicole L. Prokopishyn; Michel Boutin; Michael Rothe; Jack W. Rip; Mona Abaoui; Murtaza S. Nagree; Shaalee Dworski; Axel Schambach; Armand Keating; Michael L. West; John Klassen; Patricia V. Turner; Sandra Sirrs; C. Anthony Rupar; Christiane Auray-Blais; Ronan Foley; Jeffrey A. Medin
      Pages: 241 - 258
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Ju Huang, Aneal Khan, Bryan C. Au, Dwayne L. Barber, Lucía López-Vásquez, Nicole L. Prokopishyn, Michel Boutin, Michael Rothe, Jack W. Rip, Mona Abaoui, Murtaza S. Nagree, Shaalee Dworski, Axel Schambach, Armand Keating, Michael L. West, John Klassen, Patricia V. Turner, Sandra Sirrs, C. Anthony Rupar, Christiane Auray-Blais, Ronan Foley, Jeffrey A. Medin
      Fabry disease is a rare lysosomal storage disorder (LSD). We designed multiple recombinant lentivirus vectors (LVs) and tested their ability to engineer expression of human α-galactosidase A (α-gal A) in transduced Fabry patient CD34+ hematopoietic cells. We further investigated the safety and efficacy of a clinically directed vector, LV/AGA, in both ex vivo cell culture studies and animal models. Fabry mice transplanted with LV/AGA-transduced hematopoietic cells demonstrated α-gal A activity increases and lipid reductions in multiple tissues at 6 months after transplantation. Next we found that LV/AGA-transduced Fabry patient CD34+ hematopoietic cells produced even higher levels of α-gal A activity than normal CD34+ hematopoietic cells. We successfully transduced Fabry patient CD34+ hematopoietic cells with “near-clinical grade” LV/AGA in small-scale cultures and then validated a clinically directed scale-up transduction process in a GMP-compliant cell processing facility. LV-transduced Fabry patient CD34+ hematopoietic cells were subsequently infused into NOD/SCID/Fabry (NSF) mice; α-gal A activity corrections and lipid reductions were observed in several tissues 12 weeks after the xenotransplantation. Additional toxicology studies employing NSF mice xenotransplanted with the therapeutic cell product demonstrated minimal untoward effects. These data supported our successful clinical trial application (CTA) to Health Canada and opening of a “first-in-the-world” gene therapy trial for Fabry disease.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.003
      Issue No: Vol. 5 (2017)
       
  • Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

    • Authors: Karine Cambon; Virginie Zimmer; Sylvain Martineau; Marie-Claude Gaillard; Margot Jarrige; Aurore Bugi; Jana Miniarikova; Maria Rey; Raymonde Hassig; Noelle Dufour; Gwenaelle Auregan; Philippe Hantraye; Anselme L. Perrier; Nicole Déglon
      Pages: 259 - 276
      Abstract: Publication date: 16 June 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 5
      Author(s): Karine Cambon, Virginie Zimmer, Sylvain Martineau, Marie-Claude Gaillard, Margot Jarrige, Aurore Bugi, Jana Miniarikova, Maria Rey, Raymonde Hassig, Noelle Dufour, Gwenaelle Auregan, Philippe Hantraye, Anselme L. Perrier, Nicole Déglon
      Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT) protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA), vectorized in an HIV-1-derived lentiviral vector (LV), that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL) backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.001
      Issue No: Vol. 5 (2017)
       
  • Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using
           an Insulated Lentiviral Vector

    • Authors: Swati Singh; Iram Khan; Socheath Khim; Brenda Seymour; Karen Sommer; Matthew Wielgosz; Zachary Norgaard; Hans-Peter Kiem; Jennifer Adair; Denny Liggitt; Arthur Nienhuis; David J. Rawlings
      Pages: 1 - 16
      Abstract: Publication date: 17 March 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 4
      Author(s): Swati Singh, Iram Khan, Socheath Khim, Brenda Seymour, Karen Sommer, Matthew Wielgosz, Zachary Norgaard, Hans-Peter Kiem, Jennifer Adair, Denny Liggitt, Arthur Nienhuis, David J. Rawlings
      Wiskott-Aldrich syndrome (WAS) is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp) expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN) lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS) promoter driving WASp expression. Used as a gene therapeutic in Was −/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression) demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2016.11.001
      Issue No: Vol. 4 (2017)
       
  • INSPIIRED: Quantification and Visualization Tools for Analyzing
           Integration Site Distributions

    • Authors: Charles C. Berry; Christopher Nobles; Emmanuelle Six; Yinghua Wu; Nirav Malani; Eric Sherman; Anatoly Dryga; John K. Everett; Frances Male; Aubrey Bailey; Kyle Bittinger; Mary J. Drake; Laure Caccavelli; Paul Bates; Salima Hacein-Bey-Abina; Marina Cavazzana; Frederic D. Bushman
      Pages: 17 - 26
      Abstract: Publication date: 17 March 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 4
      Author(s): Charles C. Berry, Christopher Nobles, Emmanuelle Six, Yinghua Wu, Nirav Malani, Eric Sherman, Anatoly Dryga, John K. Everett, Frances Male, Aubrey Bailey, Kyle Bittinger, Mary J. Drake, Laure Caccavelli, Paul Bates, Salima Hacein-Bey-Abina, Marina Cavazzana, Frederic D. Bushman
      Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2016.11.003
      Issue No: Vol. 4 (2017)
       
  • Efficient Presentation of Multiple Endogenous Epitopes to Both CD4+ and
           CD8+ Diabetogenic T Cells for Tolerance

    • Authors: Shamael R. Dastagir; Jorge Postigo-Fernandez; Chunliang Xu; James H. Stoeckle; Rebuma Firdessa-Fite; Rémi J. Creusot
      Pages: 27 - 38
      Abstract: Publication date: 17 March 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 4
      Author(s): Shamael R. Dastagir, Jorge Postigo-Fernandez, Chunliang Xu, James H. Stoeckle, Rebuma Firdessa-Fite, Rémi J. Creusot
      Antigen-specific immunotherapy of type 1 diabetes, typically via delivery of a single native β cell antigen, has had little clinical benefit to date. With increasing evidence that diabetogenic T cells react against multiple β cell antigens, including previously unappreciated neo-antigens that can be emulated by mimotopes, a shift from protein- to epitope-based therapy is warranted. To this end, we aimed to achieve efficient co-presentation of multiple major epitopes targeting both CD4+ and CD8+ diabetogenic T cells. We have compared native epitopes versus mimotopes as well as various targeting signals in an effort to optimize recognition by both types of T cells in vitro. Optimal engagement of all T cells was achieved with segregation of CD8 and CD4 epitopes, the latter containing mimotopes and driven by endosome-targeting signals, after delivery into either dendritic or stromal cells. The CD4+ T cell responses elicited by the endogenously delivered epitopes were comparable with high concentrations of soluble peptide and included functional regulatory T cells. This work has important implications for the improvement of antigen-specific therapies using an epitope-based approach to restore tolerance in type 1 diabetes and in a variety of other diseases requiring concomitant targeting of CD4+ and CD8+ T cells.
      Graphical abstract image

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2016.12.002
      Issue No: Vol. 4 (2017)
       
  • INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA
           Integration in Cellular Genomes

    • Authors: Eric Sherman; Christopher Nobles; Charles C. Berry; Emmanuelle Six; Yinghua Wu; Anatoly Dryga; Nirav Malani; Frances Male; Shantan Reddy; Aubrey Bailey; Kyle Bittinger; John K. Everett; Laure Caccavelli; Mary J. Drake; Paul Bates; Salima Hacein-Bey-Abina; Marina Cavazzana; Frederic D. Bushman
      Pages: 39 - 49
      Abstract: Publication date: 17 March 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 4
      Author(s): Eric Sherman, Christopher Nobles, Charles C. Berry, Emmanuelle Six, Yinghua Wu, Anatoly Dryga, Nirav Malani, Frances Male, Shantan Reddy, Aubrey Bailey, Kyle Bittinger, John K. Everett, Laure Caccavelli, Mary J. Drake, Paul Bates, Salima Hacein-Bey-Abina, Marina Cavazzana, Frederic D. Bushman
      Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2016.11.002
      Issue No: Vol. 4 (2017)
       
  • 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
           Liver

    • 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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