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Human Gene Therapy Methods

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https://www.readbyqxmd.com/read/28193101/how-to-successfully-screen-random-aav-display-peptide-libraries-in-vivo
#1
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 14, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28042946/improvement-of-de-novo-cholesterol-biosynthesis-efficiently-promotes-the-production-of-human-immunodeficiency-virus-type-1-derived-lentiviral-vectors
#2
Nathalie Holic, Sophie Frin, Ababacar Khalil 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 amount of vectors. 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 in culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR)...
January 2, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28192678/assaying-the-stability-and-inactivation-of-aav-serotype-1-vectors
#3
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
https://www.readbyqxmd.com/read/28166648/characteristics-of-minimally-oversized-adeno-associated-virus-vectors-encoding-human-factor-viii-generated-using-producer-cell-lines-and-triple-transfection
#4
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
https://www.readbyqxmd.com/read/28140663/correction-to-hum-gene-ther-methods-2016-27-5-187-196
#5
(no author information available yet)
No abstract text is available yet for this article.
February 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28125909/efficient-gene-delivery-and-expression-in-pancreas-and-pancreatic-tumors-by-capsid-optimized-aav8-vectors
#6
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
https://www.readbyqxmd.com/read/28125901/onebac-2-0-sf9-cell-lines-for-production-of-aav1-aav2-and-aav8-vectors-with-minimal-encapsidation-of-foreign-dna
#7
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
https://www.readbyqxmd.com/read/28117600/sodium-chloride-enhances-recombinant-adeno-associated-virus-production-in-a-serum-free-suspension-manufacturing-platform-using-the-herpes-simplex-virus-system
#8
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
https://www.readbyqxmd.com/read/27903094/effects-of-self-complementarity-codon-optimization-transgene-and-dose-on-liver-transduction-with-aav8
#9
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
https://www.readbyqxmd.com/read/27903079/preclinical-evaluation-of-a-replication-deficient-recombinant-adenovirus-serotype-5-vaccine-expressing-guanylate-cyclase-c-and-the-padre-t-helper-epitope
#10
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
https://www.readbyqxmd.com/read/27897048/a-rapid-cell-expansion-process-for-production-of-engineered-autologous-car-t-cell-therapies
#11
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
https://www.readbyqxmd.com/read/27820963/adeno-associated-virus-5-transduces-adipose-derived-stem-cells-with-greater-efficacy-than-other-adeno-associated-viral-serotypes
#12
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
https://www.readbyqxmd.com/read/27763786/application-of-droplet-digital-pcr-for-estimating-vector-copy-number-states-in-stem-cell-gene-therapy
#13
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
https://www.readbyqxmd.com/read/27604324/a-partial-e3-deletion-in-replication-defective-adenoviral-vectors-allows-for-stable-expression-of-potentially-toxic-transgene-products
#14
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
https://www.readbyqxmd.com/read/27650213/adeno-associated-viral-vectors-raav-transduce-mature-human-adipocytes-in-three-dimensional-slice-cultures
#15
Sonja Kallendrusch, Nicolas Schopow, Sonja C Stadler, Hildegard Büning, Ulrich Hacker
Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity and the metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral vectors (rAAV) as a versatile tool to transduce human mature adipocytes in organotypic 3-dimensional (3-D) tissue cultures...
September 21, 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27529507/debate-on-germline-gene-editing
#16
Nathalie Cartier-Lacave, Robin Ali, Seppo Ylä-Herttuala, Kazuto Kato, Bernard Baetschi, Robin Lovell-Badge, Luigi Naldini, Adrian Thrasher
No abstract text is available yet for this article.
August 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27480111/evaluation-of-an-optimized-injection-system-for-retinal-gene-therapy-in-human-patients
#17
M Dominik Fischer, Doron G Hickey, Mandeep S Singh, Robert E MacLaren
Many retinal gene therapy clinical trials require subretinal injections of small volumes of adeno-associated viral (AAV) vector solutions in patients with retinal dystrophies, using equipment not specifically designed for this purpose. We therefore evaluated an optimized injection system in order to identify variables that might influence the rate of injection and final dose of vector delivered. An optimized injection system was assembled with a 41G polytetrafluoroethylene tip for retinal gene therapy. Flow rate was recorded at relevant infusion pressures (2-22 psi [14-152 kPa]), different target pressures (0...
August 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27440556/optimizing-a-method-for-the-quantification-by-quantitative-real-time-polymerase-chain-reaction-of-host-cell-dna-in-plasmid-vector-batches-used-in-human-gene-therapy
#18
Serge Ferro, Isabelle Fabre, Xavier Chenivesse
Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis...
August 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27431826/development-of-optimized-aav-serotype-vectors-for-high-efficiency-transduction-at-further-reduced-doses
#19
Chen Ling, Baozheng Li, Wenqin Ma, Arun Srivastava
We have described the development of capsid-modified next-generation AAV vectors for both AAV2 and AAV3 serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses. We have also described the development of genome-modified AAV vectors, in which the transcriptionally inactive, single-stranded AAV genome was modified to achieve improved transgene expression. Here, we describe that combination of capsid modifications and genome modifications leads to the generation of optimized AAV serotype vectors, which transduce cells and tissues more efficiently, both in vitro and in vivo, at ∼20-30-fold reduced doses...
August 2016: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/27477497/optimisation-of-internally-deleted-dystrophin-constructs
#20
Mojgan Reza, Steven Hector Laval, Stephanie Jan Carr, Hanns Lochmuller
Duchenne muscular dystrophy is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered as a potential strategy for treatment of DMD, aiming to restore the missing protein. One of the major challenges of this method is the large size of the dystrophin cDNA of ~14 kb, exceeding the packaging capacity of conventional viral vectors. 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 characterised in relation to their structure and function in vivo...
July 31, 2016: Human Gene Therapy Methods
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