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Molecular Therapy - Methods & Clinical Development
Journal Prestige (SJR): 1.311
Citation Impact (citeScore): 3
Number of Followers: 0  

  This is an Open Access Journal Open Access journal
ISSN (Online) 2329-0501
Published by Elsevier Homepage  [3163 journals]
  • Immunomonitoring of MSC-Treated GvHD Patients Reveals Only Moderate
           Potential for Response Prediction but Indicates Treatment Safety

    • Authors: Joni Keto; Tanja Kaartinen; Urpu Salmenniemi; Johanna Castrén; Jukka Partanen; Arno Hänninen; Matti Korhonen; Kaarina Lähteenmäki; Maija Itälä-Remes; Johanna Nystedt
      Pages: 109 - 118
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Joni Keto, Tanja Kaartinen, Urpu Salmenniemi, Johanna Castrén, Jukka Partanen, Arno Hänninen, Matti Korhonen, Kaarina Lähteenmäki, Maija Itälä-Remes, Johanna Nystedt
      Mesenchymal stromal cells (MSCs) are used as salvage therapy to treat steroid-refractory acute graft-versus-host disease (aGvHD). We studied the immunological response to MSC treatment in 16 aGvHD patients by assessing lymphocyte profiles and three proposed aGvHD serum markers during the MSC treatment. Surprisingly, there were no obvious differences in the lymphocyte profiles between the responders and non-responders. The numbers of T, B, and NK cells were below the normal reference interval in all patients. CD4+ T helper (Th) cell levels remained particularly low throughout the follow-up period. The relative proportion of Th1 cells decreased, while regulatory T cells remained unaltered, and only very few Th2 and Th17 cells could be detected. Serum concentrations of regenerating islet-derived protein 3-alpha, cytokeratin-18 fragments (CK18F), and elafin were significantly elevated in patient samples compared with healthy controls, but only CK18F showed any potential in the prediction of patients’ response to MSCs. No obvious markers for MSC therapy response were revealed in this study, but the results suggest that allogeneic MSCs do not provoke overt T cell-mediated immune responses at least in immunosuppressed aGvHD patients. The results advocate for the safety of MSC therapy and bring new insights in MSC immunomodulation mechanisms.
      Graphical abstract image

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.001
      Issue No: Vol. 9 (2018)
  • 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
      Pages: 181 - 191
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      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, large quantities of virus need to be delivered intravenously to administer oncolytic NDV systemically to mice. 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: 2018-03-18T08:01:00Z
      DOI: 10.1016/j.omtm.2017.10.004
      Issue No: Vol. 9 (2018)
  • Serum-free Erythroid Differentiation for Efficient Genetic Modification
           and High-Level Adult Hemoglobin Production

    • Authors: Naoya Uchida; Selami Demirci; Juan J. Haro-Mora; Atsushi Fujita; Lydia N. Raines; Matthew M. Hsieh; John F. Tisdale
      Pages: 247 - 256
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Naoya Uchida, Selami Demirci, Juan J. Haro-Mora, Atsushi Fujita, Lydia N. Raines, Matthew M. Hsieh, John F. Tisdale
      In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro. To model therapeutic strategies, we transduced human CD34+ cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%–93%) in serum-free media than serum-containing media (20%–69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34+ cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.03.007
      Issue No: Vol. 9 (2018)
  • An Assay that Predicts In Vivo Efficacy for DNA Aptamers that Stimulate
           Remyelination in a Mouse Model of Multiple Sclerosis

    • Authors: Robin M. Heider; John A. Smestad; Hernan Nicolas Lemus; Brandon Wilbanks; Arthur E. Warrington; Justin P. Peters; Moses Rodriguez; L. James Maher
      Pages: 270 - 277
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Robin M. Heider, John A. Smestad, Hernan Nicolas Lemus, Brandon Wilbanks, Arthur E. Warrington, Justin P. Peters, Moses Rodriguez, L. James Maher
      Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler’s murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo, as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.03.005
      Issue No: Vol. 9 (2018)
  • Enhanced Production of Exosome-Associated AAV by Overexpression of the
           Tetraspanin CD9

    • Authors: Lara Timantra Schiller; Nicolás Lemus-Diaz; Rafael Rinaldi Ferreira; Kai Oliver Böker; Jens Gruber
      Pages: 278 - 287
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Lara Timantra Schiller, Nicolás Lemus-Diaz, Rafael Rinaldi Ferreira, Kai Oliver Böker, Jens Gruber
      Research on cell-free vesicles revealed a multitude of characteristics, in particular of microvesicles and exosomes, that range from their potential as biomarkers to a function in horizontal transfer of genetic information from cell to cell and also include supportive functions in viral infection. Exosome-associated adeno-associated viruses (exo-AAVs) are of particular interest for the past couple of years, because they introduced a new source of highly potent recombinant AAVs with improved features, including accelerated transduction rates and more efficient immune escape. However, key factors like the mode of action, efficiency of production, or engineering of exo-AAVs remain elusive to a large extent. Here, we used the established system of CD9 overexpression to boost the exosome output of AAV producing HEK-AAV cells. The CD9-powered high-exosome environment was established during exo-AAV1 production, and we could demonstrate that the yield of exo-AAVs dramatically increased when compared to standard exo-AAVs. Furthermore, we report that exo-AAV-CD9GFP was more efficient in transduction of cells in the same titer ranges as standard exo-AAVs. Our results provide a technological approach for the generation of exo-AAVs with superior performance.
      Graphical abstract image

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.03.008
      Issue No: Vol. 9 (2018)
  • The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can
           Be Measured by In Vitro Prenylation of RAB6A

    • Authors: Maria I. Patrício; Alun R. Barnard; Christopher I. Cox; Clare Blue; Robert E. MacLaren
      Pages: 288 - 295
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Maria I. Patrício, Alun R. Barnard, Christopher I. Cox, Clare Blue, Robert E. MacLaren
      Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.03.009
      Issue No: Vol. 9 (2018)
  • A New Method for Reactivating and Expanding T Cells Specific for Rhizopus

    • Authors: Paul Castillo; Kaylor E. Wright; Dimitrios P. Kontoyiannis; Thomas Walsh; Shabnum Patel; Elizabeth Chorvinsky; Swaroop Bose; Yasmin Hazrat; Bilal Omer; Nathaniel Albert; Ann M. Leen; Cliona M. Rooney; Catherine M. Bollard; Conrad Russell Y. Cruz
      Pages: 305 - 312
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Paul Castillo, Kaylor E. Wright, Dimitrios P. Kontoyiannis, Thomas Walsh, Shabnum Patel, Elizabeth Chorvinsky, Swaroop Bose, Yasmin Hazrat, Bilal Omer, Nathaniel Albert, Ann M. Leen, Cliona M. Rooney, Catherine M. Bollard, Conrad Russell Y. Cruz
      Mucormycosis is responsible for an increasing proportion of deaths after allogeneic bone marrow transplantation. Because this disease is associated with severe immunodeficiency and has shown resistance to even the newest antifungal agents, we determined the feasibility of reactivating and expanding Rhizopus oryzae-specific T cells for use as adoptive immunotherapy in transplant recipients. R. oryzae extract-pulsed monocytes were used to stimulate peripheral blood mononuclear cells from healthy donors, in the presence of different cytokine combinations. The generated R. oryzae-specific T cell products were phenotyped after the third stimulation and further characterized by the use of antibodies that block class I/II molecules, as well as pattern recognition receptors. Despite the very low frequency of R. oryzae-specific T cells of healthy donors, we found that stimulation with interleukin-2 (IL-2)/IL-7 cytokine combination could expand these rare cells. The expanded populations included 17%–83% CD4+ T cells that were specific for R. oryzae antigens. Besides interferon-γ (IFN-γ), these cells secreted IL-5, IL-10, IL-13, and tumor necrosis factor alpha (TNF-α), and recognized fungal antigens presented by HLA-II molecules rather than through nonspecific signaling. The method described herein is robust and reproducible, and could be used to generate adequate quantities of activated R. oryzae-specific T cells for clinical testing of safety and antifungal efficacy in patients with mucormycosis.

      PubDate: 2018-04-24T19:55:40Z
      DOI: 10.1016/j.omtm.2018.03.003
      Issue No: Vol. 9 (2018)
  • An Integrated Approach toward the Biomanufacturing of Engineered Cell
           Therapy Products in a Stirred-Suspension Bioreactor

    • Authors: Charlie Y.M. Hsu; Tylor Walsh; Breanna S. Borys; Michael S. Kallos; Derrick E. Rancourt
      Pages: 376 - 389
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Charlie Y.M. Hsu, Tylor Walsh, Breanna S. Borys, Michael S. Kallos, Derrick E. Rancourt
      Recent advances in stem cell biology have accelerated the pre-clinical development of cell-based therapies for degenerative and chronic diseases. The success of this growing area hinges upon the concomitant development of scalable manufacturing platforms that can produce clinically relevant quantities of cells for thousands of patients. Current biomanufacturing practices for cell therapy products are built on a model previously optimized for biologics, wherein stable cell lines are established first, followed by large-scale production in the bioreactor. This “two-step” approach can be costly, labor-intensive, and time-consuming, particularly for cell therapy products that must be individually sourced from patients or compatible donors. In this report, we describe a “one-step” integrated approach toward the biomanufacturing of engineered cell therapy products by direct transfection of primary human fibroblast in a continuous stirred-suspension bioreactor. We optimized the transfection efficiency by testing rate-limiting factors, including cell seeding density, agitation rate, oxygen saturation, microcarrier type, and serum concentration. By combining the genetic modification step with the large-scale expansion step, this not only removes the need for manual handing of cells in planar culture dishes, but also enables the biomanufacturing process to be streamlined and automated in one fully enclosed bioreactor.
      Graphical abstract image

      PubDate: 2018-06-01T09:57:31Z
      DOI: 10.1016/j.omtm.2018.04.007
      Issue No: Vol. 9 (2018)
  • HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases
           CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells

    • Authors: Chang Li; Nikoletta Psatha; Sucheol Gil; Hongjie Wang; Thalia Papayannopoulou; André Lieber
      Pages: 390 - 401
      Abstract: Publication date: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Chang Li, Nikoletta Psatha, Sucheol Gil, Hongjie Wang, Thalia Papayannopoulou, André Lieber
      We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr) peptides (AcrII4 and AcrII2) capable of binding to the CRISPR/Cas9 complex (HDAd-Acr). CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

      PubDate: 2018-06-01T09:57:31Z
      DOI: 10.1016/j.omtm.2018.04.008
      Issue No: Vol. 9 (2018)
  • 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: 15 June 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 9
      Author(s): Lindsey M. Skrdlant, Randall J. Armstrong, Brett M. Keidaisch, Mario F. Lorente, David L. DiGiusto

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2017.09.001
      Issue No: Vol. 8 (2018)
  • Helper-free Production of Laboratory Grade AAV and Purification by
           Iodixanol Density Gradient Centrifugation

    • Authors: Sean M. Crosson; Peter Dib; J. Kennon Smith; Sergei Zolotukhin
      Pages: 1 - 7
      Abstract: Publication date: 21 September 2018
      Source:Molecular Therapy - Methods & Clinical Development, Volume 10
      Author(s): Sean M. Crosson, Peter Dib, J. Kennon Smith, Sergei Zolotukhin
      Adeno-associated virus (AAV) is one of the most promising gene therapy vectors and is widely used as a gene delivery vehicle for basic research. As AAV continues to become the vector of choice, it is increasingly important for new researchers to have access to a simplified production and purification protocol for laboratory grade recombinant AAV. Here we report a detailed protocol for serotype independent production of AAV using a helper-free HEK293 cell system followed by iodixanol gradient purification, a method described earlier. 1 While the core principals of this mammalian AAV production system are unchanged, there have been significant advancements in the production and purification procedure that serve to boost yield, maximize efficiency, and increase the purity of AAV preps. Using this protocol, we are able to constantly obtain high quantities of laboratory grade AAV particles (>5 × 1012 vg) in a week’s time, largely independent of serotype.

      PubDate: 2018-06-01T09:57:31Z
      DOI: 10.1016/j.omtm.2018.05.001
      Issue No: Vol. 10 (2018)
  • Treatment of bone defects by transplantation of genetically modified
           mesenchymal stem cell spheroids

    • Authors: Kayoko Yanagihara; Satoshi Uchida; Shinsuke Ohba; Kazunori Kataoka; Keiji Itaka
      Abstract: Publication date: Available online 18 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Kayoko Yanagihara, Satoshi Uchida, Shinsuke Ohba, Kazunori Kataoka, Keiji Itaka
      Cell transplantation is promising for regenerative medicine. A combination of a three-dimensional spheroid culture system with gene transfection was developed to enhance the therapeutic effects of mesenchymal stem cell (MSC) transplantation. The spheroid cell culture system is based on micropatterned substrates composed of a regular array of 100-μm-diameter cell-adhesion areas coated with a temperature-responsive polymer, poly (N-isopropylacrylamide-co-methacrylic acid), which allows for spheroid detachment by simply cooling the plates. In this study, MSC spheroids were transfected with plasmid DNA encoding runt-related transcription factor 2 (Runx2) and tested for their ability to enhance bone regeneration. In vitro analyses revealed that osteogenic differentiation of the MSCs was enhanced by forming spheroids, and was further promoted by Runx2 expression. The enhanced osteogenic differentiation was maintained under pathological conditions such as hypoxia and inflammation. Transplanting Runx2-transfected MSC spheroids into bone defects on rat femurs induced significantly faster bone regeneration compared with nontransfected MSC spheroids or genetically modified MSCs from conventional monolayer culture. MSC migration into the bone defect area was enhanced by upregulation of cell migration–related genes. In conclusion, genetically modified MSC spheroids are effective for enhancing bone regeneration, providing a promising option for cell transplantation therapy in the fields of regenerative medicine.
      Graphical abstract image

      PubDate: 2018-04-24T19:55:40Z
      DOI: 10.1016/j.omtm.2018.04.006
  • A novel triple mutant AAV6 capsid induces rapid and potent transgene
           expression in the muscle and respiratory tract of mice

    • Authors: Laura P. van Lieshout; Jakob M. Domm; Tara N. Rindler; Kathy L. Frost; Debra L. Sorensen; Sarah J. Medina; Stephanie A. Booth; James P. Bridges; Sarah K. Wootton
      Abstract: Publication date: Available online 14 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Laura P. van Lieshout, Jakob M. Domm, Tara N. Rindler, Kathy L. Frost, Debra L. Sorensen, Sarah J. Medina, Stephanie A. Booth, James P. Bridges, Sarah K. Wootton
      Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types and longevity of transgene expression, will render extension of such successes to many other human diseases. Engineering of AAV capsids continues to evolve the specificity and efficiency of AAV-mediated gene transfer. Here we describe a triple AAV6 mutant, termed AAV6.2FF, containing F129L, Y445F and Y731F mutations. AAV6.2FF yielded 10-fold greater transgene expression in lung than AAV6 after 21 days. Additionally, this novel capsid demonstrated 101-fold and 49-fold increased transgene expression in the muscle and lungs, respectively, 24 hours post vector delivery when compared to the parental AAV6. Furthermore, AAV6.2FF retains heparin sulfate binding capacity and displays a 10-fold increase in resistance to pooled immunoglobulin neutralization in vitro. The rapid and potent expression mediated by AAV6.2FF is ideally suited to applications such as vectored immunoprophylaxis, in which rapid transgene expression is vital for use during an outbreak response scenario.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.04.005
  • Liposome lipid-based formulation has least influence on rAAV transduction
           compared to other transfection agents

    • Authors: Pengpeng Guo; Chenghui Yu; Qingxin Wang; Ruirong Zhang; Xianze Meng; Yinglu Feng
      Abstract: Publication date: Available online 12 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Pengpeng Guo, Chenghui Yu, Qingxin Wang, Ruirong Zhang, Xianze Meng, Yinglu Feng
      Recombinant adeno-associated virus (rAAV) vectors are considered ideal vehicles for human gene therapy. Meanwhile, non-viral strategies, such as transfection agents (TA), have also shown promise to deliver genetic materials, such as siRNA. Transduction with the rAAV vector is performed concurrently with transfection with plasmid DNA or RNA. In the present study, we report that various TAs inhibited rAAV-mediated transgene expression at diverse levels. Overall, cationic polymers and dendrimers dramatically blocked rAAV transduction, while lipid-based liposomes displayed the least effect. The inhibitory effect was dependent on the dose of TAs and the timing of infection, suggesting that early stage of viral infection was involved. In addition, the present results indicate that the transgene expression of rAAV vectors was significantly increased by liposome-mediated transfection with adenoviral helper genes. At the same time, this was dramatically inhibited by liposome-mediated transfection with trichosanthin gene encoding a type I ribosome inactivating protein isolated from traditional Chinese medicine. Furthermore, liposomes also have little effect on rAAV-mediated transgene expression in vivo. Taken together, these findings suggest liposome as the best choice of TAs, which should be used in combination with rAAV-mediated gene therapy.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.04.004
  • Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after
           Intracardiac Injection One Week Post-Myocardial Infarction in Swine

    • Authors: Leif Carlsson; Jonathan C. Clarke; Christopher Yen; Francine Gregoire; Tamsin Albery; Martin Billger; Ann-Charlotte Egnell; Li-Ming Gan; Karin Jennbacken; Edvin Johansson; Gunilla Linhardt; Sofia Martinsson; Muhammad Waqas Sadiq; Nevin Witman; Qing-Dong Wang; Chien-Hsi Chen; Yu-Ping Wang; Susan Lin; Barry Ticho; Patrick Hsieh; Kenneth R. Chien; Regina Fritsche-Danielson
      Abstract: Publication date: Available online 10 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Leif Carlsson, Jonathan C. Clarke, Christopher Yen, Francine Gregoire, Tamsin Albery, Martin Billger, Ann-Charlotte Egnell, Li-Ming Gan, Karin Jennbacken, Edvin Johansson, Gunilla Linhardt, Sofia Martinsson, Muhammad Waqas Sadiq, Nevin Witman, Qing-Dong Wang, Chien-Hsi Chen, Yu-Ping Wang, Susan Lin, Barry Ticho, Patrick Hsieh, Kenneth R. Chien, Regina Fritsche-Danielson
      Messenger RNA (mRNA) can direct dose-dependent protein expression in cardiac muscle without genome integration, but to date has not been shown to improve cardiac function in a safe, clinically applicable way. Herein, we report that a purified and optimized mRNA in a biocompatible citrate-saline formulation is tissue specific, long-acting, and does not stimulate an immune response. In small and large animal, permanent occlusion myocardial infarction models VEGF-A 165 mRNA improves systolic ventricular function and limits myocardial damage. Following a single administration a week post infarction in mini-pigs, left ventricular ejection fraction, inotropy, and ventricular compliance improved, border zone arteriolar and capillary density increased, and myocardial fibrosis decreased at two months post-treatment. Purified VEGF-A mRNA establishes the feasibility of improving cardiac function in the sub-acute therapeutic window and may represent a new class of therapies for ischemic injury.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.04.003
  • Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human
           Hematopoietic Stem and Progenitor Cells

    • Authors: Gretchen Lewis; Lauryn Christiansen; Jessica McKenzie; Min Luo; Eli Pasackow; Yegor Smurnyy; Sean Harrington; Philip Gregory; Gabor Veres; Olivier Negre; Melissa Bonner
      Abstract: Publication date: Available online 5 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Gretchen Lewis, Lauryn Christiansen, Jessica McKenzie, Min Luo, Eli Pasackow, Yegor Smurnyy, Sean Harrington, Philip Gregory, Gabor Veres, Olivier Negre, Melissa Bonner
      Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.04.001
  • Efficient enrichment of gene-modified primary T-cells via CCR5-targeted
           integration of mutant dihydrofolate reductase

    • Authors: Biswajit Paul; Guillermo Romano Ibarra; Nicholas Hubbard; Teresa Einhaus; Alexander Astrakhan; David J. Rawlings; Hans-Peter Kiem; Christopher W. Peterson
      Abstract: Publication date: Available online 5 April 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Biswajit Paul, Guillermo Romano Ibarra, Nicholas Hubbard, Teresa Einhaus, Alexander Astrakhan, David J. Rawlings, Hans-Peter Kiem, Christopher W. Peterson
      Targeted gene therapy strategies utilizing homology driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression– significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4+ T cells, and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than five-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T-cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.04.002
  • An adeno-associated viral vector capable of penetrating the mucus barrier
           to inhaled gene therapy

    • Authors: Gregg A. Duncan; Namho Kim; Yanerys Colon-Cortes; Jason Rodriguez; Marina Mazur; Susan E. Birket; Steven M. Rowe; Natalie E. West; Alessandra Livraghi-Butrico; Richard C. Boucher; Justin Hanes; George Aslanidi; Jung Soo Suk
      Abstract: Publication date: Available online 22 March 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Gregg A. Duncan, Namho Kim, Yanerys Colon-Cortes, Jason Rodriguez, Marina Mazur, Susan E. Birket, Steven M. Rowe, Natalie E. West, Alessandra Livraghi-Butrico, Richard C. Boucher, Justin Hanes, George Aslanidi, Jung Soo Suk
      Diffusion of the viral vectors evaluated in inhaled gene therapy clinical trials to date are largely hindered within airway mucus, which limits their access to, and transduction of, the underlying airway epithelium prior to clearance from the lung. Here, we discovered that adeno-associated virus (AAV) serotype 6 was able to rapidly diffuse through mucus collected from cystic fibrosis (CF) patients, unlike previously tested AAV serotypes. A point mutation of the AAV6 capsid suggests a potential mechanism by which AAV6 avoids adhesion to the mucus mesh. Significantly greater transgene expression was achieved with AAV6 compared to a mucoadhesive serotype, AAV1, in air-liquid interface cultures of human CF bronchial epithelium with naturally secreted mucus or induced mucus hypersecretion. In addition, AAV6 achieved superior distribution and overall level of transgene expression compared to AAV1 in the airways and whole lungs, respectively, of transgenic mice with airway mucus obstruction. Our findings motivate further evaluation and clinical development of AAV6 for inhaled gene therapy.

      PubDate: 2018-04-15T07:34:07Z
      DOI: 10.1016/j.omtm.2018.03.006
  • A Rationally Engineered Capsid Variant of AAV9 For Peripheral
           Tissue-Detargeted and CNS-Directed Systemic Gene Delivery in Neonatal Mice

    • Authors: Dan Wang; Shaoyong Li; Dominic J. Gessler; Jun Xie; Li Zhong; Jia Li; Karen Tran; Kim Van Vliet; Lingzhi Ren; Qin Su; Ran He; Jason E. Goetzmann; Terence R. Flotte; Mavis Agbandje-McKenna; Guangping Gao
      Abstract: Publication date: Available online 16 March 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Dan Wang, Shaoyong Li, Dominic J. Gessler, Jun Xie, Li Zhong, Jia Li, Karen Tran, Kim Van Vliet, Lingzhi Ren, Qin Su, Ran He, Jason E. Goetzmann, Terence R. Flotte, Mavis Agbandje-McKenna, Guangping Gao
      Adeno-associated virus (AAV) has provided the gene therapy field with the most powerful in vivo gene delivery vector to realize safe, efficacious, and sustainable therapeutic gene expression. Because many clinically relevant properties of AAV-based vectors are governed by the capsid, much research effort has been devoted to the development of AAV capsids for desired features. Here, we combine AAV capsid discovery from nature and rational engineering, to report an AAV9 capsid variant, designated as AAV9.HR, which retains AAV9’s capability to traverse the blood-brain barrier and transduce neurons. This variant shows reduced transduction in peripheral tissues when delivered through intravascular (I.V.) injection into neonatal mice. Therefore, when I.V. AAV delivery is used to treat central nervous system (CNS) diseases, AAV9.HR has the advantage of mitigating potential off-target effects in peripheral tissues compared to AAV9. We also demonstrate that AAV9.HR is suitable for peripheral tissue-detargeted CNS-directed gene therapy in a mouse model of a fatal pediatric leukodystrophy. In light of recent success with profiling diversified natural AAV capsid repertoires, and the understanding of AAV capsid sequence-structure-function relationship, such a combinatory approach to AAV capsid development is expected to further improve vector targeting and expand the vector toolbox for therapeutic gene delivery.

      PubDate: 2018-03-18T08:01:00Z
      DOI: 10.1016/j.omtm.2018.03.004
  • Preclinical Development of a Lentiviral Vector for Gene Therapy of
           X-linked Severe Combined Immunodeficiency

    • Authors: Valentina Poletti; Sabine Charrier; Guillaume Corre; Bernard Gjata; Alban Vignaud; Fang Zhang; Michael Rothe; Axel Schambach; H. Bobby Gaspar; Adrian J. Thrasher; Fulvio Mavilio
      Abstract: Publication date: Available online 10 March 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Valentina Poletti, Sabine Charrier, Guillaume Corre, Bernard Gjata, Alban Vignaud, Fang Zhang, Michael Rothe, Axel Schambach, H. Bobby Gaspar, Adrian J. Thrasher, Fulvio Mavilio
      X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor γ chain gene (IL2RG), and is characterized by profound defects in T-, B- and NK-cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early MLV-derived gammaretroviral vectors showed restoration of T-cell immunity in patients but resulted in vector-induced insertional oncogenesis. We developed a SIN lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1α short promoter (EFS-IL2RG) and tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin- HSPCs in an Il2rg -/-/Rag2 -/- mouse model. The study showed restoration of T, B and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile and showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase-I/II clinical trial aimed at restoring both T- and B-cell immunity in SCID-X1 children upon non-myeloablative conditioning.

      PubDate: 2018-03-18T08:01:00Z
      DOI: 10.1016/j.omtm.2018.03.002
  • Seizure-suppressant and neuroprotective effects of encapsulated
           BDNF-producing cells in a rat model of temporal lobe epilepsy

    • Authors: Chiara Falcicchia; Giovanna Paolone; Dwaine F. Emerich; Francesca Lovisari; William J. Bell; Tracie Fradet; Lars U. Wahlberg; Michele Simonato
      Abstract: Publication date: Available online 9 March 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Chiara Falcicchia, Giovanna Paolone, Dwaine F. Emerich, Francesca Lovisari, William J. Bell, Tracie Fradet, Lars U. Wahlberg, Michele Simonato
      Brain-derived neurotrophic factor (BDNF) may represent a therapeutic for chronic epilepsy, but evaluating its potential is complicated by difficulties in its delivery to the brain. Here, we describe the effects on epileptic seizures of encapsulated cell biodelivery (ECB) devices filled with genetically modified human cells engineered to release BDNF. These devices, implanted into the hippocampus of pilocarpine-treated rats, highly decreased the frequency of spontaneous seizures by more than 80%. These benefits were associated with improved cognitive performance, as epileptic rats treated with BDNF performed significantly better on a novel object recognition test. Importantly, long-term BDNF delivery did not alter normal behaviors such as general activity or sleep/wake patterns. Detailed immunohistochemical analyses revealed that the neurological benefits of BDNF were associated with several anatomical changes, including reduction in degenerating cells and normalization of hippocampal volume, neuronal counts (including parvalbumin positive interneurons), and neurogenesis. In conclusion, the present data suggest that BDNF, when continuously released in the epileptic hippocampus, reduces the frequency of generalized seizures, improves cognitive performance, and reverts many histological alterations associated with chronic epilepsy. Thus, ECB device-mediated long-term supplementation of BDNF in the epileptic tissue may represent a valid therapeutic strategy against epilepsy and some of its co-morbidities.

      PubDate: 2018-03-18T08:01:00Z
      DOI: 10.1016/j.omtm.2018.03.001
  • Cre recombinase mediated removal of bacterial backbone to efficiently
           generate rSV40

    • Authors: Xiaoxia Shi; Matthew Ryan Ykema; Jaco Hazenoot; Lysbeth ten Bloemendaal; Irene Mancini; Machteld Odijk; Peter de Haan; Piter J. Bosma
      Abstract: Publication date: Available online 27 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Xiaoxia Shi, Matthew Ryan Ykema, Jaco Hazenoot, Lysbeth ten Bloemendaal, Irene Mancini, Machteld Odijk, Peter de Haan, Piter J. Bosma
      Gene therapy has been shown to be a feasible approach to treat inherited disorders in vivo. Among the currently used viral vector systems, adeno-associated viral (AAV) vectors are the most advanced and have been applied in patients successfully. An important drawback of non-integrating AAV vectors is their loss of expression upon cell division while repeating systemic administration lacks efficacy due to the induction of neutralizing antibodies. In addition, a significant percentage of the general population is not eligible to AAV mediated gene therapy due to pre-existing immunity. Development of additional viral vectors may overcome this hurdle. Simian Virus 40 (SV40) derived vectors have been reported to transduce different tissues including the liver and prevalence of neutralizing antibodies in the general population is very low. This renders recombinant SV40 (rSV40) vector an interesting candidate for effective (re)-administration. Clinical use of SV40 vectors is in part hampered by less advanced production methods compared to AAV. To optimize the production of rSV40 and make it better suitable for clinical practice we developed a production system that relies on Cre recombinase mediated removal of the bacterial plasmid backbone.

      PubDate: 2018-03-07T03:25:28Z
      DOI: 10.1016/j.omtm.2018.02.010
  • Neurturin gene therapy protects parasympathetic function to prevent
           irradiation-induced murine salivary gland hypofunction.

    • Authors: Joao Nuno Ferreira; Changyu Zheng; Isabelle M.A. Lombaert; Corinne M. Goldsmith; Ana P. Cotrim; Jennifer M. Symonds; Vaishali N. Patel; Matthew P. Hoffman
      Abstract: Publication date: Available online 23 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Joao Nuno Ferreira, Changyu Zheng, Isabelle M.A. Lombaert, Corinne M. Goldsmith, Ana P. Cotrim, Jennifer M. Symonds, Vaishali N. Patel, Matthew P. Hoffman
      Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus to recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 h before irradiation and compared to a control adenovirus. The control-treated glands have ∼ 50% reduction in salivary flow 60 days post-irradiation whereas, neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.008
  • A non-integrating lentiviral approach overcomes Cas9-induced immune
           rejection to establish an immunocompetent murine model of metastatic renal
           cell carcinoma

    • Authors: Junhui Hu; Shiruyeh Schokrpur; Maani Archang; Kip Hermann; Allison C. Sharrow; Prateek Khanna; Jesse Novak; Sabina Signoretti; Rupal S. Bhatt; Beatrice S. Knudsen; Hua Xu; Lily Wu
      Abstract: Publication date: Available online 23 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Junhui Hu, Shiruyeh Schokrpur, Maani Archang, Kip Hermann, Allison C. Sharrow, Prateek Khanna, Jesse Novak, Sabina Signoretti, Rupal S. Bhatt, Beatrice S. Knudsen, Hua Xu, Lily Wu
      The clustered regularly interspaced short palindromic repeats (CRISPR) based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (VHL) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.009
  • Integrating HDAd5/35++ vectors as a new platform for HSC gene therapy of

    • Authors: Chang Li; Nikoletta Psatha; Hongjie Wang; Manvendra Singh; Himanshu Bhusan Samal; Wenli Zhang; Anja Ehrhardt; Zsuzsanna Izsvák; Thalia Papayannopoulou; André Lieber
      Abstract: Publication date: Available online 15 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Chang Li, Nikoletta Psatha, Hongjie Wang, Manvendra Singh, Himanshu Bhusan Samal, Wenli Zhang, Anja Ehrhardt, Zsuzsanna Izsvák, Thalia Papayannopoulou, André Lieber
      We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12kb transgene cassette included a β-globin LCR/promoter driven human γ-globin gene and a EF1α-mgmtP140K expression cassette which allows for drug-controlled increase of γ-globin expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10-20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. Ten weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.004
  • Development of Intrathecal AAV9 Gene Therapy for Giant Axonal Neuropathy

    • Authors: Rachel M. Bailey; Diane Armao; Sahana Nagabhushan Kalburgi; Steven J. Gray
      Abstract: Publication date: Available online 15 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Rachel M. Bailey, Diane Armao, Sahana Nagabhushan Kalburgi, Steven J. Gray
      An NIH-sponsored Phase I clinical trial is underway to test a potential treatment for Giant Axonal Neuropathy (GAN) using viral-mediated GAN gene replacement ( This trial marks the first instance of intrathecal (IT) adeno-associated viral (AAV) gene transfer in humans. GAN is a rare pediatric neurodegenerative disorder caused by autosomal recessive loss-of-function mutations in the GAN gene, which encodes the gigaxonin protein. Gigaxonin is involved in the regulation, turnover and degradation of intermediate filaments (IFs). The pathologic signature of GAN is giant axonal swellings filled with disorganized accumulations of IFs. Herein we describe the development and characterization of the AAV vector carrying a normal copy of the human GAN transgene (AAV9/JeT-GAN) currently employed in the clinical trial. Treatment with AAV/JeT-GAN restored the normal configuration of IFs in patient fibroblasts within days in cell culture and by four weeks in GAN KO mice. IT delivery of AAV9/JeT-GAN in aged GAN KO mice preserved sciatic nerve ultrastructure, reduced neuronal IF accumulations and attenuated rotarod dysfunction. This strategy conferred sustained wild type gigaxonin expression across the PNS and CNS for at least one year in mice. These results support the clinical evaluation of AAV9/JeT-GAN for potential therapeutic outcomes and treatment for GAN patients.

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.005
  • Isoliquiritigenin inhibits interleukin-1β-induced production of matrix
           metalloproteinase in articular chondrocytes in vivo and in vitro through
           NF-κB pathway

    • Authors: Lei Zhang; Shiyun Ma; Hang Su; Jiaxiang Cheng
      Abstract: Publication date: Available online 15 February 2018
      Source:Molecular Therapy - Methods & Clinical Development
      Author(s): Lei Zhang, Shiyun Ma, Hang Su, Jiaxiang Cheng
      Osteoarthritis (OA) is a major joint disease in which inflammatory cytokine IL-1β and matrix metalloproteinases (MMPs) play a pivotal role. Isoliquiritigenin has been reported to have anti-inflammation activity. In this study, the effect of isoliquiritigenin on IL-1β-induced production of matrix metalloproteinase and NF-κB activation was analyzed. We treated primary cultured articular chondrocytes with isoliquiritigenin and the expressions of MMPs were analyzed on mRNA and protein level. The phosphorylation of IκBa and p65 was analyzed to detect NF-κB activation. We also used in vivo model by treating mice with isoliquiritigenin and detecting the level of matrix metalloproteinases (MMPs). IL-1β induced NF-κB activation and MMP-1, MMP-3, MMP-9, MMP-13, A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 production on chondrocytes. 10 μM isoliquiritigenin treatment significantly inhibited IL-1β-induced NF-κB activation and these MMPs production on chondrocytes. Injecting isoliquiritigenin into rat knee joint also inhibited IL-1β-induced NF-κB activation and MMPs production in articular cartilage. Isoliquiritigenin treatment inhibited IL-1β-induced MMPs production and NF-κB activation both in vitro and in vivo, suggesting a potential therapeutic role of isoliquiritigenin to treat osteoarthritis.

      PubDate: 2018-02-26T00:54:35Z
      DOI: 10.1016/j.omtm.2018.02.006
  • 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
  • Amelioration of muscle and nerve pathology in LAMA2 muscular dystrophy by

    • 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
  • 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
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