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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...
April 17, 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...
February 13, 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
Mario Mietzsch, Henrik Hering, Eva-Maria Hammer, Mavis Agbandje-McKenna, Sergei Zolotukhin, Regine Heilbronn
Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates...
February 2017: Human Gene Therapy Methods
Laura Adamson-Small, Mark Potter, Barry J Byrne, Nathalie Clément
The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium...
February 2017: Human Gene Therapy Methods
Peter Bell, Lili Wang, Shu-Jen Chen, Hongwei Yu, Yanqing Zhu, Mohamad Nayal, Zhenning He, John White, Deborah Lebel-Hagan, James M Wilson
Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence...
December 2016: Human Gene Therapy Methods
Adam E Snook, Trevor R Baybutt, Terry Hyslop, Scott A Waldman
There is an unmet need for improved therapeutics for colorectal cancer, the second leading cause of cancer mortality worldwide. Adjuvant chemotherapy only marginally improves survival in some patients and has no benefit in others, underscoring the clinical opportunity for novel immunotherapeutic approaches to improve survival in colorectal cancer. In that context, guanylate cyclase C (GUCY2C) is an established biomarker and therapeutic target for metastatic colorectal cancer with immunological characteristics that promote durable antitumor efficacy without autoimmunity...
December 2016: Human Gene Therapy Methods
Tangying Lily Lu, Omar Pugach, Robert Somerville, Steven A Rosenberg, James N Kochendefer, Marc Better, Steven A Feldman
The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials.(1-6) The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose.(7) This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production...
December 2016: Human Gene Therapy Methods
Priyanka Sharma, Sunishka M Wimalawansa, Gregory C Gould, R Michael Johnson, Katherine J D A Excoffon
Adipose-derived stem cells (ASCs) have shown potential in the treatment of a myriad of diseases; however, infusion of cells alone is unlikely to provide the full range of potential therapeutic applications. Transient genetic manipulation of ASCs could increase their repair and regeneration characteristics in a disease-specific context, essentially transforming them into drug-eluting depots. The goal of this study was to determine the optimal parameters necessary to transduce ASCs with recombinant adeno-associated virus (rAAV), an approved gene therapy vector that has never been associated with disease...
December 2016: Human Gene Therapy Methods
Huan-Ting Lin, Takashi Okumura, Yukinori Yatsuda, Satoru Ito, Hiromitsu Nakauchi, Makoto Otsu
Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT...
October 2016: Human Gene Therapy Methods
Larissa H Haut, Amanda L Gill, Raj K Kurupati, Ang Bian, Yan Li, Wynetta Giles-Davis, Zhiquan Xiang, Xiang Yang Zhou, Hildegund C J Ertl
Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis...
October 2016: Human Gene Therapy Methods
Mojgan Reza, Steve H Laval, Andreas Roos, Stephanie Carr, Hanns Lochmüller
Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo...
October 2016: Human Gene Therapy Methods
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