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Human Gene Therapy Methods

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
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
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
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...
September 7, 2016: Human Gene Therapy Methods
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
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
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
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
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
Alicia D Powers, Bryan A Piras, Robert K Clark, Timothy D Lockey, Michael M Meagher
Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required...
June 2016: Human Gene Therapy Methods
Kizito-Tshitoko Tshilenge, Baptiste Ameline, Michel Weber, Alexandra Mendes-Madeira, Steven Nedellec, Marine Biget, Nathalie Provost, Lyse Libeau, Véronique Blouin, Jack-Yves Deschamps, Guylène Le Meur, Marie-Anne Colle, Philippe Moullier, Virginie Pichard, Fabienne Rolling
Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents...
June 2016: Human Gene Therapy Methods
Alexander Taschauer, Antonia Geyer, Sebastian Gehrig, Julia Maier, Haider Sami, Manfred Ogris
Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery...
June 2016: Human Gene Therapy Methods
Nathan VanderVeen, Nicholas Raja, Elizabeth Yi, Henry Appelman, Philip Ng, Donna Palmer, Daniel Zamler, Marta Dzaman, Pedro R Lowenstein, Maria G Castro
Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity "gutless" adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible "Tet-On" activation system that induces anti-GBM immunity...
June 2016: Human Gene Therapy Methods
Philipp Schlegel, Regina Huditz, Eric Meinhardt, Kleopatra Rapti, Nicolas Geis, Patrick Most, Hugo A Katus, Oliver J Müller, Raffi Bekeredjian, Philip W Raake
Cardiac gene therapy is a promising approach for treating heart diseases. Although clinical studies are ongoing, effective and targeted transgene delivery is still a major obstacle. We sought to improve and direct transgene expression in myocardium by ultrasound-targeted microbubble destruction (UTMD). In pigs, adeno-associated virus-derived (AAV) vectors harboring the luciferase reporter gene were delivered via retroinfusion into the anterior interventricular coronary vein (AIV). AAV vectors were either loaded to lipid microbubbles before injection or injected unmodified...
April 2016: Human Gene Therapy Methods
Balaji Balakrishnan, Giridhara R Jayandharan
The present study was designed to visualize the cellular trafficking of adeno-associated virus (AAV) vectors in general and AAV5 serotype vectors in particular. We fluorescently labeled AAV5 wild-type and a mutant (S652A) virus and studied their infection process by live cell imaging and confocal microscopy. Our data demonstrate considerable difference in the ability of these vectors to reach the nuclear compartment within the first 6 hr after infection.
April 2016: Human Gene Therapy Methods
Sofia M Calado, Francisco Diaz-Corrales, Gabriela A Silva
Diabetic retinopathy (DR) is one of the major complications of diabetes mellitus. It is characterized by retinal microvascular changes caused by chronic exposure to hyperglycemia, leading to low tissue oxygenation and ultimately to neovascularization. Laser photocoagulation and vitrectomy are the most efficient treatments for DR, but display severe side effects such as the destruction of the healthy retina. Another clinical approach uses antiangiogenic agents to prevent and delay progression of neovascularization, but these require recurrent local administrations that increase the possibility of retinal detachment, vitreous hemorrhage, and cataract formation...
April 2016: Human Gene Therapy Methods
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