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Human Gene Therapy Methods

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https://www.readbyqxmd.com/read/28934862/protocol-for-efficient-generation-and-characterization-of-adeno-associated-viral-aav-vectors
#1
Andreas Jungmann, Barbara Leuchs, Hugo A Katus, Jean Rommelaere, Oliver J Müller
AAV vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high AAV titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure which results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.
September 21, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28870117/pseudotyped-lentiviral-vectors-one-vector-many-guises
#2
Alok Vishnu Joglekar, Salemiz Sandoval
Viruses have evolved specialized molecular mechanisms to transfer their genome efficiently into host cells. Viruses can be repurposed into viral vectors to achieve controlled gene transfer to desired cells. One of the most popular class of vectors, lentiviral vectors, transduce mammalian cells efficiently. Lentiviral vectors are pseudotyped with various heterologous viral envelopes to alter their tropism. While the most common example is the envelope glycoprotein from vesicular stomatitis virus (VSVG), many other viral proteins have also been used...
September 4, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28854814/function-and-safety-of-lentivirus-mediated-gene-transfer-for-csf2ra-deficiency
#3
Miriam Hetzel, Takuji Suzuki, Anna Rafiei Hashtchin, Paritha Arumugam, Brenna Carey, Marc Schwabbauer, Alexandra Kuhn, Johann Meyer, Axel Schambach, Johannes Van Der Loo, Thomas Moritz, Bruce C Trapnell, Nico Lachmann
Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder of pulmonary surfactant accumulation and hypoxemic respiratory failure caused by mutations in CSF2RA (encoding the granulocyte/macrophage-colony stimulating factor (GM-CSF) receptor α-chain (CD116)), which results in reduced GM-CSF-dependent pulmonary surfactant clearance by alveolar macrophages. While no pharmacologic therapy currently exists for hPAP, we recently demonstrated that endotracheal instillation of wild-type or gene-corrected mononuclear phagocytes (pulmonary macrophage transplantation, PMT) results in a significant and durable therapeutic efficacy in a validated murine model of hPAP...
August 30, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28826344/scalable-lentiviral-vector-production-using-stable-hek293sf-producer-cell-lines
#4
Aziza P Manceur, Howard Kim, Vanja Misic, Nadejda Andreev, July Dorion-Thibaudeau, Stéphane Lanthier, Alice Bernier, Sonia Tremblay, Anne-Marie Gélinas, Sophie Broussau, Rénald Gilbert, Sven Ansorge
Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field towards developing suspension processes that are conducive to large scale production. We describe here a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a Green Fluorescent Protein (GFP)-expressing lentiviral vector in the 10<sup>6</sup> Transduction Units (TU)/ml range without optimization...
August 21, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28817977/a-short-and-efficient-transduction-protocol-for-mouse-hematopoietic-stem-cells-with-lentiviral-vectors
#5
María Fernández-García, Cristina Mesa, Elena Almarza, Juan Bueren, Rosa Yañez
Transduction of hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors (LVs) constitutes a new therapeutic option for the treatment of various monogenic diseases affecting the lymphohematopoietic system. The development of detailed preclinical studies of gene therapy in animal disease models constitutes an essential step in expanding the application of gene therapy in a wide variety of inherited and acquired diseases. Here we describe an efficient protocol to transduce HSPCs from wild-type and Fanconi anemia mice with either gene-marking or therapeutic LVs...
August 17, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28806885/adenovirus-particle-quantification-in-cell-lysates-using-light-scattering
#6
Adrian Hohl, Anne Sophie Ramms, Christian Dohmen, Klaus Mantwill, Andrea Bielmeier, Andreas Kolk, Andreas Ruppert, Roman Nawroth, Per Sonne Holm
Adenoviral vector production for therapeutic applications is a well-established routine process. However, current methods for measurement of adenovirus particle titers as a quality characteristic require highly purified virus preparations. While purified virus is typically obtained in the last step of downstream purification, rapid and reliable methods for adenovirus particle quantification in intermediate products and crude lysates to allow for optimization and validation of cell cultures and intermediate downstream processing steps are currently not at hand...
August 15, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28750596/-rt-qpcr-for-detection-of-and-differentiation-between-rna-and-dna-of-hiv-1-based-lentiviral-vectors
#7
Melanie Pavlovic, Nina Koehler, Martina Anton, Anna Dinkelmeier, Maren Haase, Thorsten Stellberger, Ulrich Busch, Armin E Baiker
The purpose of the described method is the detection of and differentiation between RNA and DNA of HIV-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering operations methods for the detection of the HIV-1 based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here a qPCR method targeting the LTR U5 subunit and the start sequence of the packaging signal ψ is described...
July 27, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28750559/suppression-of-choroidal-neovascularization-in-mice-by-subretinal-delivery-of-multigenic-lentiviral-vectors-encoding-anti-angiogenic-micrornas
#8
Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, Lars Aagaard, Toke Bek, Jacob Giehm Mikkelsen, Thomas Juhl Corydon
Lentivirus-based vectors have been used for the development of potent gene therapies. Here, we present application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA, and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells...
July 27, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28741380/genetic-modification-of-t-cells-with-chimeric-antigen-receptors-cars-a-laboratory-manual
#9
Viktoria Golumba-Nagy, Johannes Kuehle, Hinrich Abken
Redirected T cells genetically modified with a chimeric antigen receptor (CAR) induced spectacular remissions of so far refractory leukemia/lymphoma in early phase trials attracting interest to use CAR T cells in a variety of other applications including solid cancer and non-malignant diseases. However, extensive pre-clinical explorations demand highly effective and robust procedures for the genetic modification of blood T cells; the same applies for engineering with a recombinant T cell receptor (TCR). We present laboratory procedures in a step-by-step protocol to engineer human and mouse T cells with a CAR by retro- or lentiviral transduction for further pre-clinical testing...
July 25, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28817345/reverse-transcription-quantitative-polymerase-chain-reaction-for-detection-of-and-differentiation-between-rna-and-dna-of-hiv-1-based-lentiviral-vectors
#10
Melanie Pavlovic, Nina Koehler, Martina Anton, Anna Dinkelmeier, Maren Haase, Thorsten Stellberger, Ulrich Busch, Armin E Baiker
The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28817344/a-guide-to-approaching-regulatory-considerations-for-lentiviral-mediated-gene-therapies
#11
Michael White, Roger Whittaker, Carolina Gándara, Elizabeth A Stoll
Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is nonpathogenic, and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28817343/suppression-of-choroidal-neovascularization-in-mice-by-subretinal-delivery-of-multigenic-lentiviral-vectors-encoding-anti-angiogenic-micrornas
#12
Anne Louise Askou, Josephine Natalia Esther Benckendorff, Andreas Holmgaard, Tina Storm, Lars Aagaard, Toke Bek, Jacob Giehm Mikkelsen, Thomas Juhl Corydon
Lentivirus-based vectors have been used for the development of potent gene therapies. Here, application of a multigenic lentiviral vector (LV) producing multiple anti-angiogenic microRNAs following subretinal delivery in a laser-induced choroidal neovascularization (CNV) mouse model is presented. This versatile LV, carrying back-to-back RNApolII-driven expression cassettes, enables combined expression of microRNAs targeting vascular endothelial growth factor A (Vegfa) mRNA and fluorescent reporters. In addition, by including a vitelliform macular dystrophy 2 (VMD2) promoter, expression of microRNAs is restricted to the retinal pigment epithelial (RPE) cells...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28747142/development-of-the-first-world-health-organization-lentiviral-vector-standard-toward-the-production-control-and-standardization-of-lentivirus-based-gene-therapy-products
#13
Yuan Zhao, Hannah Stepto, Christian K Schneider
Gene therapy is a rapidly evolving field. So far, there have been >2,400 gene therapy products in clinical trials and four products on the market. A prerequisite for producing gene therapy products is ensuring their quality and safety. This requires appropriately controlled and standardized production and testing procedures that result in consistent safety and efficacy. Assuring the quality and safety of lentivirus-based gene therapy products in particular presents a great challenge because they are cell-based multigene products that include viral and therapeutic proteins as well as modified cells...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28712309/improving-mirna-delivery-by-optimizing-mirna-expression-cassettes-in-diverse-virus-vectors
#14
Elena Herrera-Carrillo, Ying Poi Liu, Ben Berkhout
The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28683573/multimodal-lentiviral-vectors-for-pharmacologically-controlled-switching-between-constitutive-single-gene-expression-and-tetracycline-regulated-multiple-gene-collaboration
#15
Maike Stahlhut, Axel Schambach, Olga S Kustikova
Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied...
August 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28627251/direct-liquid-chromatography-mass-spectrometry-analysis-for-complete-characterization-of-recombinant-adeno-associated-virus-capsid-proteins
#16
Xiaoying Jin, Lin Liu, Shelley Nass, Catherine O'Riordan, Eric Pastor, X Kate Zhang
The requirement for robust analytical methods to characterize adeno-associated virus (AAV) vectors is immediate, as the field advances more AAV gene therapies into the clinic and onto commercialization. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency. Thus, complete characterization of the constituent viral capsid proteins of AAV vectors, including their sequences and post-translational modifications (PTMs), is highly recommended to ensure AAV product quality and consistency...
June 16, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28605938/a-guide-to-approaching-regulatory-considerations-for-lentiviral-mediated-gene-therapies
#17
Michael White, Roger Whittaker, Elizabeth Ann Stoll
Lentiviral vectors are increasingly the gene transfer tool of choice for gene or cell therapies, with multiple clinical investigations showing promise for this viral vector in terms of both safety and efficacy. The third-generation vector system is well-characterized, effectively delivers genetic material and maintains long-term stable expression in target cells, delivers larger amounts of genetic material than other methods, is non-pathogenic and does not cause an inflammatory response in the recipient. This report aims to help academic scientists and regulatory managers negotiate the governance framework to achieve successful translation of a lentiviral vector-based gene therapy...
June 12, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28504553/aavs1-targeted-plasmid-integration-in-aav-producer-cell-lines
#18
Yuxia Luo, Amy Frederick, John M Martin, Abraham Scaria, Seng H Cheng, Donna Armentano, Samuel C Wadsworth, Karen A Vincent
Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield...
June 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28488944/a-scalable-and-accurate-method-for-quantifying-vector-genomes-of-recombinant-adeno-associated-viruses-in-crude-lysate
#19
Jianzhong Ai, Raed Ibraheim, Phillip W L Tai, Guangping Gao
Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate...
June 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28463571/accurate-identification-and-quantification-of-dna-species-by-next-generation-sequencing-in-adeno-associated-viral-vectors-produced-in-insect-cells
#20
Magalie Penaud-Budloo, Emilie Lecomte, Aurélien Guy-Duché, Sylvie Saleun, Alain Roulet, Céline Lopez-Roques, Benoît Tournaire, Benjamin Cogné, Adrien Léger, Véronique Blouin, Pierre Lindenbaum, Philippe Moullier, Eduard Ayuso
Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS)...
June 2017: Human Gene Therapy Methods
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