Journal Cover Molecular Therapy - Methods & Clinical Development
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   ISSN (Online) 2329-0501
   Published by Elsevier Homepage  [3123 journals]
  • Adeno-Associated Virus Genome Population Sequencing achieves full vector
           genome resolution and reveals human-vector chimeras

    • Authors: Phillip W.L. Tai; Jun Xie; Kaiyuen Fong; Matthew Seetin; Cheryl Heiner; Qin Su; Michael Weiand; Daniella Wilmot; Maria L. Zapp; Guangping Gao
      Abstract: Publication date: Available online 13 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Phillip W.L. Tai, Jun Xie, Kaiyuen Fong, Matthew Seetin, Cheryl Heiner, Qin Su, Michael Weiand, Daniella Wilmot, Maria L. Zapp, Guangping Gao
      Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines, and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel-electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with ITR-containing vector sequences. We show that vector populations can contain between 1.3 to 2.3% of this type of undesirable genomes. These discoveries redefine quality control standards for viral vector preparations, and highlight the degree of foreign products in rAAV-based therapeutic vectors.

      PubDate: 2018-02-14T21:10:38Z
      DOI: 10.1016/j.omtm.2018.02.002
       
  • Influence of pre-existing anti-capsid neutralizing and binding antibodies
           on AAV vector transduction

    • Authors: Zachary Fitzpatrick; Christian Leborgne; Elena Barbon; Elisa Masat; Giuseppe Ronzitti; Laetitia van Wittenberghe; Alban Vignaud; Fanny Collaud; Séverine Charles; Marcelo Simon-Sola; Fabienne Jouen; Olivier Boyer; Federico Mingozzi
      Abstract: Publication date: Available online 13 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Zachary Fitzpatrick, Christian Leborgne, Elena Barbon, Elisa Masat, Giuseppe Ronzitti, Laetitia van Wittenberghe, Alban Vignaud, Fanny Collaud, Séverine Charles, Marcelo Simon-Sola, Fabienne Jouen, Olivier Boyer, Federico Mingozzi
      Pre-existing immunity to adeno associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then then rapidly cleared, without resulting in transgene expression. In vitro, imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. While no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction.
      Graphical abstract image

      PubDate: 2018-02-14T21:10:38Z
      DOI: 10.1016/j.omtm.2018.02.003
       
  • Platelet-targeted gene therapy for hemophilia

    • Authors: Qizhen Shi
      Abstract: Publication date: Available online 7 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Qizhen Shi
      Gene therapy is an attractive approach for disease treatment. Since platelets are abundant cells circulating in blood with the distinctive abilities of storage and delivery and fundamental roles in hemostasis and immunity, they could be a unique target for gene therapy of diseases. Recent studies have demonstrated that ectopic expression of factor VIII (FVIII) in platelets under control of the platelet-specific promoter results in FVIII storage together with its carrier protein von Willebrand factor (VWF) in α-granules and the phenotypic correction of hemophilia A. Importantly, the storage and sequestration of FVIII in platelets appears to effectively restore hemostasis even in the presence of functional-blocking inhibitory antibodies. This review summarizes studies on platelet-specific gene therapy of hemophilia A as well as hemophilia B.

      PubDate: 2018-02-14T21:10:38Z
      DOI: 10.1016/j.omtm.2018.01.011
       
  • Optimizing EphA2-CAR T cells for the Adoptive Immunotherapy of Glioma

    • Authors: Zhongzhen Yi; Brooke L. Prinzing; Felicia Cao; Stephen Gottschalk; Giedre Krenciute
      Abstract: Publication date: Available online 2 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Zhongzhen Yi, Brooke L. Prinzing, Felicia Cao, Stephen Gottschalk, Giedre Krenciute
      Glioblastoma is the most aggressive primary brain tumor in humans and is virtually incurable with conventional therapies. Chimeric antigen receptor (CAR) T-cell therapy targeting the glioblastoma antigen EphA2 is an attractive approach to improve outcomes because EphA2 is expressed highly in glioblastoma but only at low levels in normal brain tissue. Building upon our previous findings in this area, we generated and evaluated a panel of EphA2-specific CARs. We demonstrate here that T cells expressing CD28.ζ and 41BB.ζ CARs with short spacers had similar effector function, resulting in potent antitumor activity. In addition, incorporating the 41BB signaling domain into CD28.ζ CARs did not improve CAR T-cell function. While we could not determine functional differences between CD28.ζ, 41BB.ζ, and CD28.41BB.ζ CAR T cells, we selected CD28.ζ CAR T cells for further clinical development based on safety consideration.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.009
       
  • Target Cell Directed Bioengineering Approaches for Gene Therapy of
           Hemophilia A

    • Authors: Harrison C. Brown; Philip M. Zakas; Stephan N. George; Ernest T. Parker; H Trent Spencer; Christopher B. Doering
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Harrison C. Brown, Philip M. Zakas, Stephan N. George, Ernest T. Parker, H Trent Spencer, Christopher B. Doering
      Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of messenger RNA to improve translation, and amino acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally-sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146 nucleotide transcription regulatory module, a target cell specific codon optimization algorithm and a high-expression bioengineered fVIII variant. The minimal synthetic promoter allows for the smallest AAV-fVIII vector genome known at 4832 nucleotides, while the tissue-directed codon optimization strategy facilitates increased fVIII transgene product expression in target cell types, e.g. hepatocytes, over traditional genome-level codon optimization strategies. As a tertiary approach, we incorporated ancient and orthologous fVIII sequence elements previously shown to facilitate improved biosynthesis through post-translational mechanisms. Together these technologies contribute to an AAV-fVIII vector that confers sustained, curative levels of fVIII at a minimal dose in hemophilia A mice. Moreover, the first two technologies should be generalizable to all liver-directed gene therapy vector designs.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.004
       
  • A Rapamycin-based GMP Compatible Process for the Isolation and Expansion
           of Regulatory T cells for Clinical Trials

    • Authors: H. Fraser; N. Safinia; N. Grageda; S. Thirkell; K. Lowe; L. Fry; C. Scotta; A. Hope; C. Fisher; R. Hilton; D. Game; P. Harden; A. Bushell; K. Wood; R.I. Lechler; G. Lombardi
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): H. Fraser, N. Safinia, N. Grageda, S. Thirkell, K. Lowe, L. Fry, C. Scotta, A. Hope, C. Fisher, R. Hilton, D. Game, P. Harden, A. Bushell, K. Wood, R.I. Lechler, G. Lombardi
      The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, whilst maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicentre phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.006
       
  • Amelioration of muscle and nerve pathology in LAMA2 muscular dystrophy by
           AAV9-mini-agrin

    • Authors: Chunping Qiao; Yi Dai; Viktoriya D. Nikolova; Quan Jin; Jianbin Li; Bin Xiao; Juan Li; Sheryl S. Moy; Xiao Xiao
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Chunping Qiao, Yi Dai, Viktoriya D. Nikolova, Quan Jin, Jianbin Li, Bin Xiao, Juan Li, Sheryl S. Moy, Xiao Xiao
      LAMA2-related muscular dystrophy (LAMA2 MD) is the most common and fatal form of early onset congenital muscular dystrophies. Due to the large size of the laminin α2 cDNA and heterotrimeric structure of the protein, it is challenging to develop a gene replacement therapy. Our group has developed a novel adeno-associated viral (AAV) vector carrying the mini-agrin, which is a non-homologous functional substitute for the mutated laminin α2. A significant therapeutic effect in skeletal muscle was observed in our previous study using AAV serotype 1 (AAV1). In this investigation, we examined AAV9 vector, which has more widespread transduction than AAV1, to determine if the therapeutic effects could be further improved. As expected, AAV9-mini-agrin treatment offered enhanced therapeutic effects over the previously used AAV1-mini-agrin in extending mouse life span and improvement of muscle pathology. Additionally, over-expression of mini-agrin in peripheral nerves of dy w /dy w mice partially amended nerve pathology as evidenced by: improved motor function and sensorimotor processing, partial restoration of myelination, partial restoration of basement membrane via EM examination, as well as decreased regeneration of Schwann cells. In conclusion, our studies indicate that over-expression of mini-agrin into dy w /dy w mice offers profound therapeutic effects in both skeletal muscle and nervous system.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.005
       
  • The Pharmacology of T cell Therapies

    • Authors: Michael C. Milone; Vijay Bhoj
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Michael C. Milone, Vijay Bhoj
      Adoptive cellular therapy using T cells with tumor specificity derived from either natural T cell receptors (TCRs) or an artificial chimeric antigen receptor (CAR) has reached late phase clinical testing with two CAR T cell therapies achieving regulatory approval within the United States in 2017. The effective use of these therapies depends upon an understanding of their pharmacology, which is quite divergent from traditional small molecule or biologic drugs. We review the different types of T cell therapy under clinical development, the factors affecting cellular kinetics following infusion and the relationship between this cellular kinetics and anti-cancer activity. We also discuss the toxicity associated with T cell therapies with an emphasis on cytokine release syndrome and neurotoxicity, and the gaps in knowledge regarding these frequent and unique adverse effects.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.010
       
  • Transduction Patterns of Adeno-associated Viral Vectors in a Laser-induced
           Choroidal Neovascularization Mouse Model

    • Authors: Si Hyung Lee; Ye Seul Kim; Seung Kwan Nah; Hee Jong Kim; Ha Yan Park; Jin Young Yang; Keerang Park; Tae Kwann Park
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Si Hyung Lee, Ye Seul Kim, Seung Kwan Nah, Hee Jong Kim, Ha Yan Park, Jin Young Yang, Keerang Park, Tae Kwann Park
      Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy, and recently clinical successes to treat choroidal neovascularization (CNV) in the wet type of age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV 2, 5, and 8 vectors in a laser-induced choroidal neovascularization (CNV) mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV type 2, 5 and 8 capsids expressing enhanced green fluorescent protein (EGFP). Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and 8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion cells, Müller cells, INL cells, ONL cells, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, 5, and 8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.008
       
  • Plastin 3 promotes motor neuron axonal growth and extends survival in a
           mouse model of spinal muscular atrophy

    • Authors: Aziza Alrafiah; Evangelia Karyka; Ian Coldicott; Iremonger K; Katherin E. Lewis; Ke Ning; Mimoun Azzouz
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Aziza Alrafiah, Evangelia Karyka, Ian Coldicott, Iremonger K, Katherin E. Lewis, Ke Ning, Mimoun Azzouz
      Spinal muscular atrophy (SMA) is a devastating childhood motor neuron disease. SMA is caused by mutations in the survival motor neuron gene (SMN1), leading to reduced levels of SMN protein in the central nervous system (CNS). The actin-binding protein plastin 3 (PLS3) has been reported as a modifier for SMA, making it a potential therapeutic target. Here we show reduced levels of PLS3 protein in the brain and spinal cord of a mouse model of SMA. Our study also revealed that lentiviral-mediated PLS3 expression restored axonal length in cultured Smn-deficient motor neurons. Delivery of adeno-associated virus serotype 9 (AAV9) harboring Pls3 cDNA via cisterna magna in SMNΔ7 mice, a widely used animal model of SMA, led to high neuronal transduction efficiency. PLS3 treatment allowed a small but significant increase of lifespan by 42%. Although there was no improvement of phenotype, this study has demonstrated the potential use of Pls3 as a target for gene therapy, possibly in combination with other disease modifiers.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.007
       
  • Toxicology and Biodistribution: the Clinical Value of Animal
           Biodistribution Studies

    • Authors: Beatriz Silva Lima; Mafalda Ascensão Videira
      Abstract: Publication date: Available online 31 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Beatriz Silva Lima, Mafalda Ascensão Videira
      Since the human genome decoding, understanding and identification of genetic disturbances behind many diseases including, (but not exclusive to) cancer are intensively increasing. Scientific and technological advances in this area trigger the search for therapeutic (curative) approaches targeting the correction of gene disturbances. Gene therapy medicinal products (GTMPS) emerge in this context bringing new challenges for their characterization. Compared to small molecules, biodistribution shows fundamental to identify target organs, anticipate safety and efficacy, may integrate in safety/pharmacology studies, and may eventually be anticipated based on specificities of vectors/constructs. This chapter will describe/ discuss requirements for nonclinical development and evaluation of GTMPs vs conventional ones, needs and challenges of constructing nonclinical packages reassuring GTMPs human safety, from early development, taking into consideration usefulness and/or limitations of many conventional, preclinical models. The experience gained in the European context is reffered.

      PubDate: 2018-02-04T16:58:32Z
      DOI: 10.1016/j.omtm.2018.01.003
       
  • Preclinical efficacy and safety evaluation of hematopoietic stem cell gene
           therapy in a mouse model of MNGIE

    • Authors: Rana Yadak; Raquel Cabrera-Pérez; Javier Torres-Torronteras; Marianna Bugiani; Joost C. Haeck; Marshall W. Huston; Elly Bogaerts; Steffi Goffart; Edwin H. Jacobs; Merel Stok; Lorena Leonardelli; Luca Biasco; Robert M. Verdijk; Monique R. Bernsen; George Ruijter; Ramon Martí; Gerard Wagemaker; Niek P. van Til; Irenaeus F.M. de Coo
      Abstract: Publication date: Available online 8 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Rana Yadak, Raquel Cabrera-Pérez, Javier Torres-Torronteras, Marianna Bugiani, Joost C. Haeck, Marshall W. Huston, Elly Bogaerts, Steffi Goffart, Edwin H. Jacobs, Merel Stok, Lorena Leonardelli, Luca Biasco, Robert M. Verdijk, Monique R. Bernsen, George Ruijter, Ramon Martí, Gerard Wagemaker, Niek P. van Til, Irenaeus F.M. de Coo
      Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations, but carries disease and transplant related risks. In this study, lentiviral vector based- hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp -/- Upp1 -/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range,0.2-3.6 ). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp -/- Upp1 -/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development towards clinical implementation.

      PubDate: 2018-01-10T06:53:01Z
      DOI: 10.1016/j.omtm.2018.01.001
       
  • Pharmacology of recombinant adeno-associated virus production

    • Authors: Magalie Penaud-Budloo; Achille François; Nathalie Clément; Eduard Ayuso
      Abstract: Publication date: Available online 8 January 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Magalie Penaud-Budloo, Achille François, Nathalie Clément, Eduard Ayuso
      Recombinant adeno-associated viral vectors (rAAV) have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAV are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAV, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). Most commonly found impurities in rAAV stocks include: defective particles (i.e. AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, Herpes simplex virus or baculoviruses) and illegitimate DNA from plasmids, cells or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.

      PubDate: 2018-01-10T06:53:01Z
      DOI: 10.1016/j.omtm.2018.01.002
       
  • Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

    • Authors: Lindsey M. Skrdlant; Randall J. Armstrong; Brett M. Keidaisch; Mario F. Lorente; David L. DiGiusto
      Pages: 1 - 7
      Abstract: Publication date: 16 March 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 8
      Author(s): Lindsey M. Skrdlant, Randall J. Armstrong, Brett M. Keidaisch, Mario F. Lorente, David L. DiGiusto
      Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL) capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA) guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G]) for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.09.001
      Issue No: Vol. 8 (2017)
       
  • DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular
           Dystrophy 2B

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

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

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


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

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


      PubDate: 2017-09-26T20:16:31Z
      DOI: 10.1016/j.omtm.2017.08.005
      Issue No: Vol. 7 (2017)
       
  • In Vivo Production of Monoclonal Antibodies by Gene Transfer via
           Electroporation Protects against Lethal Influenza and Ebola Infections

    • Authors: Chasity D. Andrews; Yang Luo; Ming Sun; Jian Yu; Arthur J. Goff; Pamela J. Glass; Neal N. Padte; Yaoxing Huang; David D. Ho
      Pages: 74 - 82
      Abstract: Publication date: 15 December 2017
      Source:Molecular Therapy - Methods & Clinical Development, Volume 7
      Author(s): Chasity D. Andrews, Yang Luo, Ming Sun, Jian Yu, Arthur J. Goff, Pamela J. Glass, Neal N. Padte, Yaoxing Huang, David D. Ho
      Monoclonal antibodies (mAbs) have wide clinical utility, but global access is limited by high costs and impracticalities associated with repeated passive administration. Here, we describe an optimized electroporation-based DNA gene transfer platform technology that can be utilized for production of functional mAbs in vivo, with the potential to reduce costs and administration burdens. We demonstrate that multiple mAbs can be simultaneously expressed at protective concentrations for a protracted period of time using DNA doses and electroporation conditions that are feasible clinically. The expressed mAbs could also protect mice against lethal influenza or Ebola virus challenges. Our findings suggest that this DNA gene transfer platform technology could be a game-changing advance that expands access to effective mAb therapeutics globally.

      PubDate: 2017-12-18T20:29:18Z
      DOI: 10.1016/j.omtm.2017.09.003
      Issue No: Vol. 7 (2017)
       
  • Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for
           Diabetes: 8-Year Follow-Up in Dogs

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

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

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

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

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

      PubDate: 2017-06-22T14:03:38Z
      DOI: 10.1016/j.omtm.2017.05.004
      Issue No: Vol. 6 (2017)
       
  • Pre-clinical safety and off-target studies to support translation of
           AAV-mediated RNAi therapy for FSHD

    • Authors: Lindsay M. Wallace; Nizar Y. Saad; Nettie K. Pyne; Allison M. Fowler; Jocelyn O. Eidahl; Jacqueline S. Domire; Danielle Griffin; Adam C. Herman; Zarife Sahenk; Louise Rodino-Klapac; Scott Q. Harper
      Abstract: Publication date: Available online 24 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Lindsay M. Wallace, Nizar Y. Saad, Nettie K. Pyne, Allison M. Fowler, Jocelyn O. Eidahl, Jacqueline S. Domire, Danielle Griffin, Adam C. Herman, Zarife Sahenk, Louise Rodino-Klapac, Scott Q. Harper
      RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal dominant disorder, Facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene, DUX4, using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof-of-concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 over-expression and sequence-specific off-target silencing. In this study, we describe improvements in vector design, expansion of our miDUX4 sequence repertoire, and report differential toxicity elicited by two miDUX4 sequences, where one was toxic and the other was not. This study provides important data to help forward our goal of translating RNAi gene therapy for Facioscapulohumeral muscular dystrophy.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.005
       
  • Exploring cytotoxic mRNAs as a novel class of anti-cancer biotherapeutics

    • Authors: Kristin Hirschberger; Anita Jarzebinska; Eva Kessel; Verena Kretzschmann; Manish K. Aneja; Christian Dohmen; Annika Herrmann-Janson; Ernst Wagner; Christian Plank; Carsten Rudolph
      Abstract: Publication date: Available online 24 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Kristin Hirschberger, Anita Jarzebinska, Eva Kessel, Verena Kretzschmann, Manish K. Aneja, Christian Dohmen, Annika Herrmann-Janson, Ernst Wagner, Christian Plank, Carsten Rudolph
      New treatments to overcome the obstacles of conventional anti-cancer therapy are a permanent subject of investigation. One promising approach is the application of toxins linked to cell-specific ligands, so-called immunotoxins. Another attractive option is the employment of toxin-encoding plasmids. However, immunotoxins cause hepatoxicity and DNA therapeutics, among other disadvantages, bear the risk of insertional mutagenesis. As an alternative, this study examined chemically modified mRNAs coding for diphtheria toxin, subtilase cytotoxin and abrin-a for their ability to reduce cancer cell growth both in vitro and in vivo. The plant toxin abrin-a was the most promising candidate among the three tested toxins and was further investigated. Its expression was demonstrated by Western Blot. Experiments with firefly luciferase in reticulocyte lysates and co-transfection experiments with EGFP demonstrated the capability of abrin-a to inhibit protein synthesis. Its cytotoxic effect was quantified employing viability assays and propidium iodide staining. By studying caspase-3/7 activation, Annexin V-binding and chromatin condensation with Hoechst33258 staining, apoptotic cell death could be confirmed. In mice, repeated intratumoral injections of complexed abrin-a mRNA resulted in a significant reduction (89%) of KB tumor size compared to a non-translatable control mRNA.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.006
       
  • Nonclinical safety evaluation of scAAV8-RLBP1 (CPK850) for treatment of
           RLBP1 retinitis pigmentosa

    • Authors: Timothy K. MacLachlan; Mark N. Milton; Oliver Turner; Francis Tukov; Vivian W. Choi; Jan Penraat; Marie Helene Delmotte; Lydia Michaut; Bruce D. Jaffee; Chad E. Bigelow
      Abstract: Publication date: Available online 22 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Timothy K. MacLachlan, Mark N. Milton, Oliver Turner, Francis Tukov, Vivian W. Choi, Jan Penraat, Marie Helene Delmotte, Lydia Michaut, Bruce D. Jaffee, Chad E. Bigelow
      Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850)1. In this communication, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1 -/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3x107 vg in a volume of 1μl was observed. For safety assessment in these and Rlbp1 +/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes with no qualitative difference noted between Rlbp1 +/+ and Rlbp1 -/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time, and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats as well as from satellite animals in the non-human primate study. The vector was largely detected in ocular tissues as well as at low levels in the optic nerve, superior colliculus and lateral geniculate nucleus with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3x107 vg/μL CPK850 could safely be used in clinical trials.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.001
       
  • Universal Method for the Purification of Recombinant AAV Vectors of
           Differing Serotypes

    • Authors: Shelley A. Nass; Maryellen A. Mattingly; Denise A. Woodcock; Brenda L. Burnham; Jeffrey A. Ardinger; Shayla E. Osmond; Amy M. Frederick; Abraham Scaria; Seng H. Cheng; Catherine R. O’Riordan
      Abstract: Publication date: Available online 22 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Shelley A. Nass, Maryellen A. Mattingly, Denise A. Woodcock, Brenda L. Burnham, Jeffrey A. Ardinger, Shayla E. Osmond, Amy M. Frederick, Abraham Scaria, Seng H. Cheng, Catherine R. O’Riordan
      The generation of clinical good manufacturing practices (GMP)-grade adeno-associated virus (AAV) vectors requires purification strategies that support the generation of vectors of high purity, and that exhibit a good safety and efficacy profile. To date, most reported purification schemas are serotype-dependent, requiring method development for each AAV gene therapy product. Here, we describe a platform purification process that is compatible with the purification of multiple AAV serotypes. The method generates vector preparations of high purity that are enriched for capsids with full vector genomes, and that minimizes the fractional content of empty capsids. The two-column purification method, a combination of affinity and ion exchange chromatographies, is compatible with a range of AAV serotypes generated by either the transient triple transfection method or the more scalable producer cell line platform. In summary, the adaptable purification method described can be used for the production of a variety of high quality AAV vectors suitable for preclinical testing in animal models of diseases.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.004
       
  • Efficacy and safety of glycosidic enzymes for improved gene delivery to
           the retina following intravitreal injection in mice

    • Authors: Jasmina Cehajic-Kapetanovic; Nina Milosavljevic; Robert A. Bedford; Robert J. Lucas; Paul N. Bishop
      Abstract: Publication date: Available online 22 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Jasmina Cehajic-Kapetanovic, Nina Milosavljevic, Robert A. Bedford, Robert J. Lucas, Paul N. Bishop
      Viral gene delivery is showing great promise for treating retinal disease. Whilst subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency could be overcome. To this end we investigated the effects of co-injection of glycosaminoglycan degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combining with hyaluronan lyase. In addition, this optimised AAV2-enzyme combination led to a marked improvement in transduction in retinas with an advanced retinal degeneration compared to AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months post enzyme treatment, while pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species such as non-human primates or for clinical applications will have challenges and require further studies.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.002
       
  • Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac
           transposon-engineered T cells

    • Authors: Daisuke Morita; Nobuhiro Nishio; Shoji Saito; Miyuki Tanaka; Nozomu Kawashima; Yusuke Okuno; Satoshi Suzuki; Kazuyuki Matsuda; Maeda Yasuhiro; Matthew H. Wilson; Gianpietro Dotti; Cliona M. Rooney; Yoshiyuki Takahashi; Yozo Nakazawa
      Abstract: Publication date: Available online 22 December 2017
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Daisuke Morita, Nobuhiro Nishio, Shoji Saito, Miyuki Tanaka, Nozomu Kawashima, Yusuke Okuno, Satoshi Suzuki, Kazuyuki Matsuda, Maeda Yasuhiro, Matthew H. Wilson, Gianpietro Dotti, Cliona M. Rooney, Yoshiyuki Takahashi, Yozo Nakazawa
      Adoptive T cell therapy using chimeric antigen receptor (CAR)-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector, and combining irradiated activated T cells (ATCs) as feeder cells and virus-specific T cell receptor (TCR) stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR T-cell therapy.

      PubDate: 2017-12-26T22:39:20Z
      DOI: 10.1016/j.omtm.2017.12.003
       
  • Emerging issues in AAV-mediated in vivo gene therapy

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

      PubDate: 2017-10-11T06:55:21Z
      DOI: 10.1016/j.omtm.2017.10.002
       
  • Rapid Generation of Multiple Loci-Engineered Marker-Free Vaccinia Viruses
           and In Vitro Characterization of Clinical-Grade Oncolytic Poxvirus
           vvDD-A34R-hCCL5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

      PubDate: 2017-08-25T00:22:54Z
      DOI: 10.1016/j.omtm.2017.08.004
       
  • Profiling the Targets of Protective CD8+ T Cell Responses to Infection

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

      PubDate: 2017-08-25T00:22:54Z
      DOI: 10.1016/j.omtm.2017.08.003
       
 
 
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