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Journal Cover Molecular Therapy - Methods & Clinical Development
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  This is an Open Access Journal Open Access journal
   ISSN (Online) 2329-0501
   Published by Elsevier Homepage  [3175 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)
       
  • 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)
       
  • 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
           hemoglobinopathies

    • 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 (https://clinicaltrials.gov/ct2/show/NCT02362438). 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
           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
       
  • 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
       
  • 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)
       
  • 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
       
  • 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
       
  • 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
       
  • 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
       
 
 
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