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Adeno-Associated Virus-Based Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
The airway epithelium, while easily accessible to the gene therapist via aerosol or bronchoscopic lavage, is at a practical disadvantage with respect to its cell biology. Not only is the target epithelial layer composed of multiple cell types, but the submucosal glands, which are particularly rich in normal and mutant CFTR epithelial expression, are buried within the submucosa and theoretically less accessible. The airway epithelial cells in the mature adult human are extremely long-lived with proliferation rates as low as 1% to 5% and T1/2 estimated on the order of months (175). The wild-type human CFTR cDNA sequence is roughly 4.5 kb, leaving little room for promoter sequence in the AAV genome. Fortunately the 145 bp itr has been demonstrated to provide transcription promoter activity probably related to a consensus initiator sequence (60). This AAVCFTR vector functionally complements CF cell lines in vitro in both immortalized and primary CF nasal polyp cells (134,176).
Regulation of the α2-Macroglobulin Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Friedemann Horn, Ursula M. Wegenka, Peter C. Heinrich
Whereas α2-M concentrations in rat serum are dramatically elevated during an acute phase reaction, α2-M serum levels in man are constitutively high. This difference is also found in cultured cells. Rat hepatocytes in primary culture synthesize α2-M only marginally, but produce large amounts after IL-6 stimulation. In contrast, constitutive high α2-M protein and mRNA levels are found in primary human lung fibroblasts and hepatocytes, and in Hep G2 cells. To understand this different regulation, we compared the IL-6 responsiveness of the human and rat α2-M promoters by transient transfection studies in Hep G2 cells. Unexpectedly, the human α2-M promoter responded about as well as the rat promoter to IL-6. The acute phase response element for the human promoter was localized and shown to be organized similar to the rat α2-M APRE: it contains two binding sites for APRF arranged in a spacing similar to that in the rat APRE, and the APRF binds to the two sites in a cooperative manner. Therefore, we have no evidence from our data that the different regulation of human and rat α2-M synthesis can be accounted for by differently regulated promoters. We cannot exclude, however, the possibility that additional regulatory elements enhancing the basal promoter activity are located either upstream of − 4.8 kb or downstream of the transcriptional start site.
Lipoprotein(a) and Fibrinolysis
Published in Pia Glas-Greenwalt, Fibrinolysis in Disease Molecular and Hemovascular Aspects of Fibrinolysis, 2019
Lindsey A. Miles, Edward F. Plow
All members of the plasminogen/apo(a) gene families share a high degree of homology in their coding regions as well as in the 5′ flanking sequence proximal to the ATG translation initiation codon. However, comparison of the 5′ sequences of the apo(a)-related genes and the plasminogen-related genes shows divergence to less than 50% homology at the extreme 5′ flanking region.30 This predicts that regulation of these gene products may be distinct and this has been borne out in studies demonstrating that strength of the basal promoter activity of these gene products is different.30
Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies – a spotlight on hemophilia
Published in Expert Opinion on Biological Therapy, 2022
Oscar G Segurado, Ruhong Jiang, Steven W Pipe
Pre-clinical studies in HemA have shown that injection of dual AAV vectors appears efficacious in treating HemA, as two AAV vectors containing Staphylococcus pyogenes Cas 9 (SpCas9) and guide RNA with human B-domain deleted FVIII were used to integrate the human FVIII into the albumin locus, resulting in production of FVIII by the liver in mice (Figure 2). The amelioration of the HemA phenotype persisted for at least 7 months with no obvious off-target effects or signs of liver toxicity, leading the authors to hypothesize that permanent FVIII replacement may be possible using this method [58]. This study provides the basis to initiate clinical development programs incorporating two vectors: The first vector incorporates the gRNA and CRISPR/Cas 9 machinery; the second vector incorporates the FVIII transgene precisely located in a so-called ‘safe harbor’ designed to leverage the promoter activity of the albumin locus [25]. For the treatment of genetic liver disorders, the albumin locus provides the best promoter for targeted integration of donor genes. A ‘safe harbor’ means that the location of genome integration will not pose a risk to the host and will perform predictably [59].
Association of Elongation Factor Tu GTP-binding Domain-containing 2 Gene (EFTUD2) Polymorphism with the Risk of Hepatitis B Virus Infection
Published in Immunological Investigations, 2022
Anran Tian, Yuwen Li, Haozhi Fan, Pingping Hu, Ruirui Xu, Hui Yuan, Jinyuan Cai, Wen Zhang, Ming Yue, Jun Li, Chen Dong, Chuanlong Zhu
A dual-luciferase reporter assay was used to assess the promoter activity. HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C in humidified CO2 (5%). Subsequently, 5 × 105 cells were seeded per well in 24-well plates. On reaching approximately 80% confluence, cells were transfected with the prepared pGL3-rs3809756-A, pGL3-rs3809756-C, or pGL3-basic (negative control) using Lip8000 (Beyotime, China). The pRL-CMV-expressing Renilla luciferase gene vector was co-transfected into HepG2 cells as an internal control. Transfected cells were harvested and analyzed 24 h after transfection. Three independent transfection experiments were performed, and each transfection was performed in triplicate. The ratio of firefly luciferase activity to Renilla luciferase activity was used to determine the relative luciferase activity (RLA).
Approaching complexity: systems biology and ms-based techniques to address immune signaling
Published in Expert Review of Proteomics, 2020
Joseph Gillen, Caleb Bridgwater, Aleksandra Nita-Lazar
Reporter assays constitute the in vitro stimulation of a promoter and reporter gene pair to measure a response [2]. These assays can be extremely useful and are a robust method for high throughput screens of different drugs or compounds by the direct measurement of expressed gene products or the indirect changes in signal molecules regulated by genes. This is accomplished by transfecting a desired cell line to express a transcription factor activated promoter and a common reporter gene. The cells can then be stimulated to drive promoter activity. Ligand or agonist induction of the transcription factors leading to expression of the reporter gene. In the case of a luciferase reporter gene, luciferin, a luciferase substrate, can be added and the resulting luminescence measured as a corollary of stimulation. The unattractive variables of reporter assays tend to revolve around the limits in experimental design: reporter assays may take vast resources and long time periods to design and to target assays to transcription factors. Common reporter assays exploit the activity of reporter genes producing β-galactosidase, green fluorescent protein (GFP), and luciferase [2].