1,721,044 research outputs found
Site-specific integration by the adeno-associated virus rep protein
No full textInserting genetic information at precise locations into the human genome has been the goal of gene transfertechnology for almost two decades. The spectacular progress of mammalian genetics in the last two decades has led to thedevelopment of technology for genome editing and homologous recombination in human somatic cells that is finally approachingefficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locationsby enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could verywell be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesi
Site-specific integration into the human genome: Ready for clinical application?
No full textInserting genetic information at precise locations into the human genome has been the goal of the gene therapy community for almost two decades. Despite their spectacular progress in many fields of mammalian genetics, genome editing and homologous recombination are still too inefficient to be applied to human primary cells and tissues, the targets of any medical application. Site-specific integration, or the insertion of genes at known locations by enzymes that target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology
Tracking gene-modified T cells in vivo.
No full textIdentification, monitoring, and analysis of genetically modified cells in the peripheral blood are an important component of the clinical follow-up of patients treated by hematopoietic cell gene therapy. Analysis of gene-marked peripheral blood cells provides crucial information on gene transfer efficiency as well as on the nature and characteristics of the genetically modified cells, and may provide early evidence of the occurrence of potentially detrimental side effects. T lymphocytes are a convenient target for this type of analysis, due to their abundance and their relatively long life span in vivo. Tracking of gene-marked T cells is based on relatively simple, FACS- and PCR-based techniques, which may be applied to monitoring genetically modified T cells as well as T cells derived from transplanted, genetically modified hematopoietic stem cells. This chapter provides a description of these techniques and clues to their rational use in a clinical setting
Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors
Full text linkUncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications
Acute onset of hypertensive encephalopathy in a dog with right adrenal pheochromocytoma and neoplastic invasion of the caudal vena cava: Case report and review of the literature
No full textBackground: Canine pheochromocytomas (PCCs) are rare tumors of the adrenal medulla. Clinical signs are often vague, resulting in intermittent catecholamine over secretion or neoplastic invasion of adjacent structures. Case Description: A 12-year-old Epagneul Breton dog with a 1-year history of chronic kidney disease, was examined for acute onset of severe neurological signs. Based on clinical and instrumental data, hypertensive encephalopathy was suspected. Cardiac and abdominal ultrasound were performed. Severe hypertensive cardiopathy and a right adrenal gland mass with invasion of the caudal vena cava were diagnosed. Computed tomography imaging confirmed the suspect of invasive malignant neoplasia. Emergency pharmacological therapy was started to reduce systemic pressure, improve clinical signs, and stabilize the dog in view of surgical resolution. After initial improvement, patient conditions abruptly worsened, and euthanasia was elected. Histology examination confirmed a right adrenal PCC, with caval invasion. Conclusion: To the authors' conclusions, acute hypertensive encephalopathy is a peculiar manifestation of PCCs. Ultrasound is a useful, and rapid test to suspect PCC as it can detect adrenal alterations, caval invasion, metastasis, and cardiac sequelae consistent with the condition. PCC can mimic multiple affections, and be misinterpreted, especially when a concurrent disease has already been diagnosed. Veterinarians need to be aware that comorbidities could mask clinical signs and delay diagnosis. Furthermore, this clinical case reminds us to include PCC also in the differential diagnosis of dogs with an acute onset of severe neurological signs
Comparison of Genetic and Reinforcement Learning Algorithms for Energy Cogeneration Optimization
Full text linkLarge process plants generally require energy in different forms: mechanical, electrical, or thermal (in the form of steam or hot water). A commonly used source of energy is cogeneration, also defined as Combined Heat and Power (CHP). Cogeneration can offer substantial economic as well as energy savings; however, its real-time operation scheduling is still a challenge today. Multiple algorithms have been proposed for the CHP control problem in the literature, such as genetic algorithms (GAs), particle swarm optimization algorithms, artificial neural networks, fuzzy decision making systems and, most recently, reinforcement learning (RL) algorithms.This paper presents the comparison of a RL approach and a GA for the control of a cogenerator, using as a case study a thermal power plant serving a factory during the year 2021. The two methods were compared based on an earnings before interest, taxes, depreciation, and amortization (EBITDA) metric. The EBITDA that could be obtained using the RL algorithm, exceeds both the EBITDA that could be generated using a per-week genetic algorithm and the one from the manual scheduling of the CHP. Thus, the RL algorithm proves to be the most cost-effective strategy for the control of a CHP
Specific knock-down of C-terminal dominant mutation in Rhodopsin gene by CRISPR/Cas9 system
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An efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector
Full text linkThe Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer
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