Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.
These results indicate that 17β-E(2) protects ARPE-19 cells from oxidative stress through an ERβ-dependent mechanism. 17β-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.
Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.
This is the first report to show that GTx-822, an ERβ agonist, can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. GTx-822 mediated cytoprotection was mediated through induction of both genomic and nongenomic pathways. The results of this study open new avenues for the use of a selective ERβ agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis.
PURPOSE. The goal of our study was to evaluate the in vitro and in vivo anti-angiogenic effects of ERb selective agonist, b-LGND2, using human retinal microvascular endothelial cell (HRMVEC) cultures and a mouse model for oxygen-induced retinopathy (OIR).METHODS. The selectivity of b-LGND2 was determined using binding and transactivation assays. The effects of b-LGND2 on pathologic neovascularization were evaluated in OIR mice by histology and retinal mounts stained with isolectin B4 to quantify aberrant angiogenesis. Gene expression and protein levels were evaluated using Q-PCR, angiogenesis protein array, and Western blotting. A cell death detection ELISA kit was used to evaluate HRMVECs following hypoxic and hyperoxic conditions. In vitro angiogenesis was evaluated by growth factor-induced proliferation, tube formation, and cell migration assays.RESULTS. b-LGND2-treated OIR mice had a reduced number of neovascular tufts compared to vehicle-treated animals and a significant amount of normal blood vessel maturation similar to normoxia controls. b-LGND2 inhibited in vitro hypoxia-or hyperoxia-induced cell death and the formation of endothelial tubular structures in an ERb-specific mechanism. However, b-LGND2 did not inhibit significantly growth factor-induced HRMVEC proliferation and migration. Gene and protein studies revealed that OIR mice treated with b-LGND2 had lower levels of pro-angiogenic factors, like VEGF and HIF1a.CONCLUSIONS. b-LGND2 inhibited in vitro and in vivo pathologic neovascularization in the retina in an ERb-specific mechanism. These results show that b-LGND2, a non-steroidal ERb selective agonist, could be a useful therapeutic for ocular diseases involving aberrant angiogenesis, like ROP, wet-AMD, and diabetic retinopathy. (Invest Ophthalmol Vis Sci. 2012; 53:5066-5075) DOI:10.1167/iovs.12-9627 O cular diseases, like wet age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and retinal vein occlusion, are characterized by pathologic neovascularization and vascular leakage.1 These diseases arise due to loss of retinal vasculature, which subsequently results in retinal ischemia. To compensate for a loss of oxygen in the retina, new blood vessels are formed aberrantly. Multiple signaling pathways are altered in the diseased retina that ultimately lead to activation of angiogenic factors (VEGF, TGFb, FGF, IGF1, and HIF, and so forth) as well as anti-angiogenic factors (pigment epithelium derived factor [PEDF], thrombospondins, endostatin, somatostatin, and so forth). A balance in the levels of pro-and anti-angiogenic proteins is critical in the formation and maintenance of normal retinal vasculature. In pathologic states associated with angiogenic factors, the balance is tipped in favor of abnormal blood vessel formation.1-5 Various therapeutic strategies, such as the use of antioxidants, anti-VEGF therapy, photodynamic therapy, laser photocoagulation, and corticosteroids, have been evaluated for the treatment of pathologic neovascularization. 6-9 ...
Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.
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