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Molecular Therapy. Methods & Clinical Development

Hsin-Yun Cheng, Yung-Song Wang, Po-Yuan Hsu, Chien-Yuan Chen, Yi-Chu Liao, Suh-Hang H Juo
Tissue plasminogen activator is the only U.S. FDA-approved therapy for ischemic stroke, while there is no specific medication for hemorrhagic stroke. Therefore, the treatment of acute stroke continues to be a major unmet clinical need. We explored the effects of miR-195 on neurovascular protection and its potential in treating acute stroke. Using both cellular and animal studies, we showed that miR-195's beneficial effects are mediated by four mechanisms: (1) anti-apoptosis for injured neural cells by directly suppressing Sema3A/Cdc42/JNK signaling, (2) neural regeneration by promoting neural stem cell proliferation and migration, (3) anti-inflammation by directly blocking the NF-kB pathway, and (4) improvement of endothelial functions...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Klaudia Kuranda, Sophie Caillat-Zucman, Sylvaine You, Roberto Mallone
Hematopoietic stem cell transplantation (HSCT) using unrelated cord blood (CB) donors is a suitable approach when an HLA-matched donor is not available. However, one important drawback is the risk of life-threatening viral infections prior to immune reconstitution, particularly from adenoviruses (AdVs). Although adoptive therapy with ex vivo expanded virus-reactive donor T cells has proven effective to treat these infections in HSCT recipients, the manufacturing process is complex and requires large numbers of cells, which is incompatible with CB donor units...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Shun-Hua Chen, Angel Chao, Chia-Lung Tsai, Shih-Che Sue, Chiao-Yun Lin, Yi-Zong Lee, Yi-Lin Hung, An-Shine Chao, Ann-Joy Cheng, Hsin-Shih Wang, Tzu-Hao Wang
The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Chris P Hughes, Neil M J O'Flynn, Maureen Gatherer, Michelle E McClements, Jennifer A Scott, Robert E MacLaren, Srinivas Goverdhan, Martin J Glennie, Andrew J Lotery
While anti-angiogenic therapies for wet age-related macular degeneration (AMD) are effective for many patients, they require multiple injections and are expensive and prone to complications. Gene therapy could be an elegant solution for this problem by providing a long-term source of anti-angiogenic proteins after a single administration. Another potential issue with current therapeutic proteins containing a fragment crystallizable (Fc) domain (such as whole antibodies like bevacizumab) is the induction of an unwanted immune response...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Hongman Song, Ronald A Bush, Yong Zeng, Haohua Qian, Zhijian Wu, Paul A Sieving
Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina. Unfortunately, most conventional, non-modified AAV vector serotypes penetrate the retina poorly from the vitreous; this limits efficient transduction and expression by target cells (retinal pigment epithelium and photoreceptors)...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Natalia Pacienza, Ryang Hwa Lee, Eun-Hye Bae, Dong-Ki Kim, Qisong Liu, Darwin J Prockop, Gustavo Yannarelli
Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Si Hyung Lee, Jin Young Yang, Sanjar Madrakhimov, Ha Yan Park, Keerang Park, Tae Kwann Park
Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Kousaku Ohno, Lluis Samaranch, Piotr Hadaczek, John R Bringas, Philip C Allen, Vivek Sudhakar, Diane E Stockinger, Christopher Snieckus, Michael V Campagna, Waldy San Sebastian, Jerusha Naidoo, Haifeng Chen, John Forsayeth, Ernesto A Salegio, Granger G C Hwa, Krystof S Bankiewicz
Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Ping Guo, Junping Zhang, Matthew Chrzanowski, Jianhe Huang, Helen Chew, Jenni A Firrman, Nianli Sang, Yong Diao, Weidong Xiao
Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Stosh Ozog, Craig X Chen, Elizabeth Simpson, Olivia Garijo, Nina D Timberlake, Petra Minder, Els Verhoeyen, Bruce E Torbett
Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Daniel Blessing, Gabriel Vachey, Catherine Pythoud, Maria Rey, Vivianne Padrun, Florian M Wurm, Bernard L Schneider, Nicole Déglon
Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo ...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Bence György, Elise J Meijer, Maryna V Ivanchenko, Kelly Tenneson, Frederick Emond, Killian S Hanlon, Artur A Indzhykulian, Adrienn Volak, K Domenica Karavitaki, Panos I Tamvakologos, Mark Vezina, Vladimir K Berezovskii, Richard T Born, Maureen O'Brien, Jean-François Lafond, Yvan Arsenijevic, Margaret A Kenna, Casey A Maguire, David P Corey
Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP...
June 14, 2019: Molecular Therapy. Methods & Clinical Development
Simone Merlin, Antonia Follenzi
Gene expression regulation is the result of complex interactions between transcriptional and post-transcriptional controls, resulting in cell-type-specific gene expression patterns that are determined by the developmental and differentiation stage of pathophysiological conditions. Understanding the complexity of gene expression regulatory networks is fundamental to gene therapy, an approach which has the potential to treat and cure inherited disorders by delivering the correct gene to patient specific cells or tissues by means of both viral and non-viral vectors...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Julia Fakhiri, Marc A Schneider, Jens Puschhof, Megan Stanifer, Verena Schildgen, Stefan Holderbach, Yannik Voss, Jihad El Andari, Oliver Schildgen, Steeve Boulant, Michael Meister, Hans Clevers, Ziying Yan, Jianming Qiu, Dirk Grimm
Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2-4 and GBoV (Gorilla BoV)...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Benjamin J Samelson-Jones, Valder R Arruda
Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Christopher T Lux, Sowmya Pattabhi, Mason Berger, Cynthia Nourigat, David A Flowers, Olivier Negre, Olivier Humbert, Julia G Yang, Calvin Lee, Kyle Jacoby, Irwin Bernstein, Hans-Peter Kiem, Andrew Scharenberg, David J Rawlings
Elements within the γ-hemoglobin promoters ( HBG1 and HBG2 ) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%)...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Fanny Collaud, Giulia Bortolussi, Laurence Guianvarc'h, Sem J Aronson, Thierry Bordet, Philippe Veron, Severine Charles, Patrice Vidal, Marcelo Simon Sola, Stephanie Rundwasser, Delphine G Dufour, Florence Lacoste, Cyril Luc, Laetitia V Wittenberghe, Samia Martin, Christine Le Bec, Piter J Bosma, Andres F Muro, Giuseppe Ronzitti, Matthias Hebben, Federico Mingozzi
Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Sonia Guedan, Hugo Calderon, Avery D Posey, Marcela V Maus
T cells engineered with chimeric antigen receptors (CARs) have emerged as a potent new class of therapeutics for cancer, based on their remarkable potency in blood cancers. Since the first clinical reports of their efficacy emerged 7 years ago, investigators have focused on the mechanisms and properties that make CARs effective or toxic, and their effects on T cell biology. Novel CAR designs coupled with improvements in gene transfer technology, incorporating advances in gene editing, have the potential to increase access to engineered cell therapies, as well as improve their potency in solid tumors...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Maria Castella, Anna Boronat, Raquel Martín-Ibáñez, Vanina Rodríguez, Guillermo Suñé, Miguel Caballero, Berta Marzal, Lorena Pérez-Amill, Beatriz Martín-Antonio, Julio Castaño, Clara Bueno, Olga Balagué, Europa Azucena González-Navarro, Carles Serra-Pages, Pablo Engel, Ramon Vilella, Daniel Benitez-Ribas, Valentín Ortiz-Maldonado, Joan Cid, Jaime Tabera, Josep M Canals, Miquel Lozano, Tycho Baumann, Anna Vilarrodona, Esteve Trias, Elías Campo, Pablo Menendez, Álvaro Urbano-Ispizua, Jordi Yagüe, Patricia Pérez-Galán, Susana Rives, Julio Delgado, Manel Juan
Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro , inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
Katja Stifter, Cornelia Schuster, Jana Krieger, Andreas Spyrantis, Bernhard Otto Boehm, Reinhold Schirmbeck
DNA vaccines against autoimmune type 1 diabetes (T1D) contain a nonpredictable risk to induce autoreactive T cell responses rather than a protective immunity. Little is known if (and how) antigen expression and processing requirements favor the induction of autoreactive or protective immune responses by DNA immunization. Here, we analyzed whether structural properties of preproinsulin (ppins) variants and/or subcellular targeting of ppins designer antigens influence the priming of effector CD8+ T cell responses by DNA immunization...
March 15, 2019: Molecular Therapy. Methods & Clinical Development
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