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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
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
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
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
Amy H Lin, Yanzheng Liu, Cynthia Burrascano, Kathrina Cunanan, Christopher R Logg, Joan M Robbins, Noriyuki Kasahara, Harry Gruber, Carlos Ibañez, Douglas J Jolly
We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro...
April 2016: Human Gene Therapy Methods
Heather S Loring, Mai K ElMallah, Terence R Flotte
The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover...
April 2016: Human Gene Therapy Methods
Matthew J Benskey, Ivette M Sandoval, Fredric P Manfredsson
The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates...
February 2016: Human Gene Therapy Methods
Lluis Samaranch, John Bringas, Philip Pivirotto, Waldy San Sebastian, John Forsayeth, Krystof Bankiewicz
Accessing cerebrospinal fluid (CSF) from the craniocervical junction through the posterior atlanto-occipital membrane via cerebellomedullary injection (also known as cisternal puncture or cisterna magna injection) has become a standard procedure in preclinical studies. Such delivery provides broader coverage to the central and peripheral nervous system unlike local parenchymal delivery alone. As a clinical application, this approach offers a more reliable method for neurological gene replacement delivery in infants, where skull-mounted devices are not indicated...
February 2016: Human Gene Therapy Methods
Kyle Chamberlain, Jalish Mahmud Riyad, Thomas Weber
Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb...
February 2016: Human Gene Therapy Methods
Chris G Twitty, Oscar R Diago, Daniel J Hogan, Cindy Burrascano, Carlos E Ibanez, Douglas J Jolly, Derek Ostertag
Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity...
February 2016: Human Gene Therapy Methods
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