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Molecular Therapy. Methods & Clinical Development

Kenneth Cornetta, Lisa Duffy, Steven A Feldman, Crystal L Mackall, Marco L Davila, Kevin J Curran, Richard P Junghans, Jean Yuh Tang, James N Kochenderfer, Roisin O'Cearbhaill, Gary Archer, Hans-Peter Kiem, Nirali N Shah, Cindy Delbrook, Rosie Kaplan, Renier J Brentjens, Isabelle Rivière, Michel Sadelain, Steven A Rosenberg
Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Ryo Ikeue, Aki Nakamura-Takahashi, Yuko Nitahara-Kasahara, Atsushi Watanabe, Takashi Muramatsu, Toru Sato, Takashi Okada
Hypophosphatasia is an inherited disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP), the major symptom of which is hypomineralization of the bones and teeth. We had recently demonstrated that TNALP-deficient ( Akp2 -/- ) mice, which mimic the phenotype of the severe infantile form of hypophosphatasia, can be treated by intramuscular injection of a self-complementary (sc) type 8 recombinant adeno-associated virus (rAAV8) vector expressing bone-targeted TNALP with deca-aspartates at the C terminus (TNALP-D10 ) via the muscle creatine kinase (MCK) promoter...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Monir Shababi, Eric Villalón, Kevin A Kaifer, Vince DeMarco, Christian L Lorson
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal recessive disease caused by the loss of the ubiquitously expressed IGHMBP2 gene. SMARD1 causes degeneration of alpha-motor neurons, resulting in distal muscle weakness, diaphragm paralysis, and respiratory malfunction. We have reported that delivery of a low dose of AAV9- IGHMBP2 to the CNS results in a significant rescue of the SMARD1 mouse model ( nmd ). To examine how a delivery route can impact efficacy, a direct comparison of intravenous (IV) and intracerebroventricular (ICV) delivery of AAV9- IGHMBP2 was performed...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Marianne Delville, Tayebeh Soheili, Florence Bellier, Amandine Durand, Adeline Denis, Chantal Lagresle-Peyrou, Marina Cavazzana, Isabelle Andre-Schmutz, Emmanuelle Six
Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Haiyan Fu, Kim Zaraspe, Naoko Murakami, Aaron S Meadows, Ricardo J Pineda, Douglas M McCarty, Joseph Muenzer
No treatment is available to address the neurological need and reversibility of MPS II. We developed a scAAV9-h IDS vector to deliver the human iduronate-2-sulfatase gene and test it in mouse model. We treated MPS II mice at different disease stages with an intravenous injection of scAAV9-mCMV-h IDS at different doses. The treatments led to rapid and persistent restoration of IDS activity and the reduction of glycosaminoglycans (GAG) throughout the CNS and somatic tissues in all cohorts. Importantly, the vector treatment at up to age 6 months improved behavior performance in the Morris water maze and normalized the survival...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Nikoletta Psatha, Andreas Reik, Susan Phelps, Yuanyue Zhou, Demetri Dalas, Evangelia Yannaki, Dana N Levasseur, Fyodor D Urnov, Michael C Holmes, Thalia Papayannopoulou
In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Maha Tijani, Altar M Munis, Christopher Perry, Khaled Sanber, Marta Ferraresso, Tarit Mukhopadhyay, Michael Themis, Ilaria Nisoli, Giada Mattiuzzo, Mary K Collins, Yasuhiro Takeuchi
Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Jérôme Denard, Jérémy Rouillon, Thibaut Leger, Camille Garcia, Michele P Lambert, Graziella Griffith, Christine Jenny, Jean-Michel Camadro, Luis Garcia, Fedor Svinartchouk
Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Kota Sowa, Chikako Nito, Masataka Nakajima, Satoshi Suda, Yasuhiro Nishiyama, Yuki Sakamoto, Yuko Nitahara-Kasahara, Aki Nakamura-Takahashi, Masayuki Ueda, Kazumi Kimura, Takashi Okada
Hepatocyte growth factor (HGF) has neuroprotective effects against ischemia-induced injuries. Dental pulp stem cell (DPSC) transplantation attenuates tissue injury in the brain of rats with post-transient middle cerebral artery occlusion. We sought to determine whether DPSCs that overexpress HGF can enhance their therapeutic effects on brain damage post-ischemia/reperfusion injury. Treatment with DPSCs overexpressing HGF reduced infarct volumes compared to unmodified DPSC treatment at 3 and 7 days post-transient middle cerebral artery occlusion...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Leslie Weber, Valentina Poletti, Elisa Magrin, Chiara Antoniani, Samia Martin, Charles Bayard, Hanem Sadek, Tristan Felix, Vasco Meneghini, Michael N Antoniou, Wassim El-Nemer, Fulvio Mavilio, Marina Cavazzana, Isabelle Andre-Schmutz, Annarita Miccio
Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene ( HBBAS3 ), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (β-AS3) or HS2, HS3, and HS4 (β-AS3 HS4)...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Junjiang Sun, Wenwei Shao, Xiaojing Chen, Elizabeth P Merricks, Lauren Wimsey, Yasmina L Abajas, Glenn P Niemeyer, Clinton D Lothrop, Paul E Monahan, R Jude Samulski, Timothy C Nichols, Chengwen Li
Adeno-associated virus (AAV) vectors have been successfully applied in hemophilia clinical trials. However, this approach is limited to patients without AAV-neutralizing antibodies (NAbs). In this study, we explored the feasibility of AAV re-administration in hemophilia A dogs treated initially 8 years ago with AAV8.canine FVIII. After the re-administration in two NAb-negative dogs with AAV8 vectors carrying human factor VIII (hFVIII), along with the proteasome inhibitor bortezomib, we observed a phenotypic improvement in both dogs that persisted in one dog...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
María C Rosales Gerpe, Jacob P van Vloten, Lisa A Santry, Jondavid de Jong, Robert C Mould, Adrian Pelin, John C Bell, Byram W Bridle, Sarah K Wootton
Organotypic slice cultures recapitulate many features of an intact organ, including cellular architecture, microenvironment, and polarity, making them an ideal tool for the ex vivo study of viruses and viral vectors. Here, we describe a procedure for generating precision-cut ovine and murine tissue slices from agarose-perfused normal and murine melanoma tumor-bearing lungs. Furthermore, we demonstrate that these precision-cut lung slices can be maintained up to 1 month and can be used for a range of applications, which include characterizing the tissue tropism of viruses that cannot be propagated in cell monolayers, evaluating the transducing properties of gene therapy vectors, and, finally, investigating the tumor specificity of oncolytic viruses...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Jenny A Greig, Jayme M L Nordin, Christine Draper, Deirdre McMenamin, Edward A Chroscinski, Peter Bell, John T Gray, Laura K Richman, James M Wilson
Liver metabolism disorders are attractive targets for gene therapy, because low vector doses can reverse the buildup of toxic metabolites in the blood. Crigler-Najjar syndrome is an inherited disorder of bilirubin metabolism that is caused by the absence of uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) activity. This syndrome is characterized by hyperbilirubinemia and jaundice. Unfortunately, current phototherapy treatment is not effective long term. We intravenously injected phototherapy-rescued adult UGT1 knockout mice with 2...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Achille François, Mohammed Bouzelha, Emilie Lecomte, Frédéric Broucque, Magalie Penaud-Budloo, Oumeya Adjali, Philippe Moullier, Véronique Blouin, Eduard Ayuso
Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro , based on detection of vector genome replication in trans -complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Eloise Hudry, Eva Andres-Mateos, Eli P Lerner, Adrienn Volak, Olivia Cohen, Bradley T Hyman, Casey A Maguire, Luk H Vandenberghe
Adeno-associated viral vectors (AAVs) have demonstrated potential in applications for neurologic disorders, and the discovery that some AAVs can cross the blood-brain barrier (BBB) after intravenous injection has further expanded these opportunities for non-invasive brain delivery. Anc80L65, a novel AAV capsid designed from in silico reconstruction of the viral evolutionary lineage, has previously demonstrated robust transduction capabilities after local delivery in various tissues such as liver, retina, or cochlea, compared with conventional AAVs...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Misty L Noble-Vranish, Shuxian Song, Kyle P Morrison, Dominic M Tran, Ryan R Sun, Keith R Loeb, George W Keilman, Carol H Miao
We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [fc ] = 1...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Ekramy E Sayedahmed, Ahmed O Hassan, Rashmi Kumari, Weiping Cao, Shivaprakash Gangappa, Ian York, Suryaprakash Sambhara, Suresh K Mittal
Several human and nonhuman adenovirus (AdV) vectors including bovine AdV type 3 (BAdV-3) were developed as gene delivery vectors to supplement and/or elude human AdV (HAdV)-specific neutralizing antibodies (vector immunity). Here we evaluated the vaccine immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA) of a H5N1 influenza virus in a dose escalation study in mice with the intranasal (IN) or intramuscular (IM) route of inoculation in comparison with the HAdV type C5 (HAdV-C5) vector (HAd-H5HA) expressing HA of a H5N1 influenza virus...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Mor Ngom, Suzan Imren, Tobias Maetzig, Jennifer E Adair, David J H F Knapp, Jalila Chagraoui, Iman Fares, Marie-Eve Bordeleau, Guy Sauvageau, Philippe Leboulch, Connie Eaves, Richard Keith Humphries
Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34+ and CD34+ CD45RA- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Nicole K Paulk, Katja Pekrun, Gregory W Charville, Katie Maguire-Nguyen, Michael N Wosczyna, Jianpeng Xu, Yue Zhang, Leszek Lisowski, Bryan Yoo, Jose G Vilches-Moure, Gordon K Lee, Joseph B Shrager, Thomas A Rando, Mark A Kay
Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Jessica Hartmann, Robert C Münch, Ruth-Therese Freiling, Irene C Schneider, Birgit Dreier, Washington Samukange, Joachim Koch, Markus A Seeger, Andreas Plückthun, Christian J Buchholz
Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 1012 variants...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
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