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Human Gene Therapy Methods

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
Elena Herrera-Carrillo, Ying Poi Liu, Ben Berkhout
The RNA interference (RNAi) 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 (siRNAs) can trigger the selective knockdown of a target mRNA. For durable gene silencing, either artificial short hairpin RNA (shRNA) or microRNA (miRNA) encoding transgene constructs have been developed...
July 16, 2017: Human Gene Therapy Methods
Maike Stahlhut, Axel Schambach, Olga 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...
July 6, 2017: Human Gene Therapy Methods
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
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
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
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
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
Maria Schnödt, Hildegard Büning
Adeno-associated viral (AAV) vectors have emerged as one of the most popular gene transfer systems in both research and clinical gene therapy. As AAV vectors are derived from a stealth, nonpathogenic virus and lack active integrase activity, these vectors are frequently applied for in vivo gene therapy of liver, muscle, and other postmitotic tissues. Although long-term transgene expression from AAV vector episomes is reported from these tissues, the episomal nature of AAV-once regarded as disadvantage-has become an attractive feature for gene-editing approaches targeting proliferating cells...
June 2017: Human Gene Therapy Methods
Jakob Körbelin, Martin Trepel
Adeno-associated virus (AAV) has emerged as a very promising gene therapy vector. To enable tissue-directed gene expression, many artificially generated AAV variants have been established, often isolated from large pools of mutated capsids. Random peptide libraries displayed on AAV capsids have been used successfully to select vectors targeted to a given target cell or tissue in vitro and in vivo. However, the published methodology for screening of AAV libraries to isolate vectors with selective tissue tropism after intravenous administration in vivo has not been described in sufficient detail to address all critical steps...
June 2017: Human Gene Therapy Methods
Maria B Kostina, Alexader V Sass, Elena A Stukacheva, Igor V Korobko, Eugeny D Sverdlov
A set of vectors for Cre recombinase-dependent expression of hybrid suicidal FCU1 transgene was constructed which included two-plasmid system where FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants when a single plasmid bears both transgenes. To improve safety profile and specificity in cancer gene therapy applications, as well as to assure stable propagation of plasmids in bacterial cells, Cre/LoxP system components were optimized. Bicistronic vector with Cre expression cassette placed between LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared to double-plasmid system in enzyme-prodrug suicide cancer gene therapy scheme...
April 27, 2017: Human Gene Therapy Methods
(no author information available yet)
No abstract text is available yet for this article.
April 2017: Human Gene Therapy Methods
Olivia Wilkins, Allison M Keeler, Terence R Flotte
Lentivirus-mediated transduction of autologous T cells with a chimeric antigen receptor (CAR) to confer a desired epitope specificity as a targeted immunotherapy for cancer has been among the first human gene therapy techniques to demonstrate widespread therapeutic efficacy. Other approaches to using gene therapy to enhance antitumor immunity have been less specific and less effective. These have included amplification, marking, and cytokine transduction of tumor infiltrating lymphocytes, recombinant virus-based expression of tumor antigens as a tumor vaccine, and transduction of antigen-presenting cells with tumor antigens...
April 2017: Human Gene Therapy Methods
Vanessa S Bandeira, Hélio A Tomás, Evren Alici, Manuel J T Carrondo, Ana S Coroadinha
Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination)...
April 2017: Human Gene Therapy Methods
Naoya Uchida, Kareem N Washington, Brian Mozer, Charlotte Platner, Josiah Ballantine, Luke P Skala, Lydia Raines, Anna Shvygin, Matthew M Hsieh, Lloyd G Mitchell, John F Tisdale
Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34(+) cell-derived erythroid cells from healthy individuals...
April 2017: Human Gene Therapy Methods
Nathalie Holic, Sophie Frin, Ababacar K Seye, Anne Galy, David Fenard
The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs...
April 2017: Human Gene Therapy Methods
Douglas B Howard, Brandon K Harvey
Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze-thaw cycles, the resulting transduction efficiency became variable at 60-120% of a single thaw...
February 2017: Human Gene Therapy Methods
Bindu Nambiar, Cathleen Cornell Sookdeo, Patricia Berthelette, Robert Jackson, Susan Piraino, Brenda Burnham, Shelley Nass, David Souza, Catherine R O'Riordan, Karen A Vincent, Seng H Cheng, Donna Armentano, Sirkka Kyostio-Moore
Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated...
February 2017: Human Gene Therapy Methods
(no author information available yet)
No abstract text is available yet for this article.
February 2017: Human Gene Therapy Methods
Min Chen, Kyungah Maeng, Akbar Nawab, Rony A Francois, Julie K Bray, Mary K Reinhard, Sanford L Boye, William W Hauswirth, Frederic J Kaye, Georgiy Aslanidi, Arun Srivastava, Maria Zajac-Kaye
Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC...
February 2017: Human Gene Therapy Methods
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