ABSTRACT
Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of major histocompatibility complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion, reduces handling, and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.
ABSTRACT
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.
Subject(s)
Fetal Hemoglobin , Receptors, Erythropoietin , Animals , Erythroid Cells , Fetal Hemoglobin/genetics , Macaca mulatta , Mice , Receptors, Erythropoietin/genetics , Repressor ProteinsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Inflammation/metabolism , Neoplasms/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, trkA/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Cell Line, Tumor , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/drug therapy , Disease Models, Animal , Gene Expression , Humans , Inflammation/drug therapy , Inflammation/genetics , Mice , NIH 3T3 Cells , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Rats , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Receptor, trkA/antagonists & inhibitors , Receptor, trkA/genetics , Signal Transduction/drug effects , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The goal of our study was to evaluate the in vitro and in vivo anti-angiogenic effects of ERß selective agonist, ß-LGND2, using human retinal microvascular endothelial cell (HRMVEC) cultures and a mouse model for oxygen-induced retinopathy (OIR). METHODS: The selectivity of ß-LGND2 was determined using binding and transactivation assays. The effects of ß-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: ß-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. ß-LGND2 inhibited in vitro hypoxia- or hyperoxia-induced cell death and the formation of endothelial tubular structures in an ERß-specific mechanism. However, ß-LGND2 did not inhibit significantly growth factor-induced HRMVEC proliferation and migration. Gene and protein studies revealed that OIR mice treated with ß-LGND2 had lower levels of pro-angiogenic factors, like VEGF and HIF1α. CONCLUSIONS: ß-LGND2 inhibited in vitro and in vivo pathologic neovascularization in the retina in an ERß-specific mechanism. These results show that ß-LGND2, a non-steroidal ERß selective agonist, could be a useful therapeutic for ocular diseases involving aberrant angiogenesis, like ROP, wet-AMD, and diabetic retinopathy.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Estrogen Receptor beta/agonists , Retinal Neovascularization/drug therapy , Animals , Cell Death/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Endothelial Cells/drug effects , Humans , Ligands , Mice , Mice, Inbred C57BLABSTRACT
PURPOSE: The goal of this study was to determine whether an estrogen receptor-ß (ERß)-selective agonist (GTx-822; GTx, Inc., Memphis, TN) could prevent hydrogen peroxide (H(2)O(2))-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection. METHODS: The selectivity of GTx-822 for ERß was determined by receptor-binding assay (RBA) and transactivation assay. Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide (t-BH) or hydrogen peroxide (H(2)O(2)) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell death ELISA. Mitochondrial membrane potential was measured with the JC-1 assay. Gene expression and protein expression and activation were quantitated with qRT-PCR and Western blot analysis. Phospho-protein arrays elucidated the activation of protein kinases. RESULTS: The RBA and transactivation assay revealed that GTx-822 is an ERß-selective agonist (K(i) = 0.53 nM). GTx-822 prevented oxidative stress in ARPE-19 cells. It preserved mitochondrial function and prevented cellular apoptosis. Pretreatment with GTx-822 increased ERß gene and protein expression during oxidative stress. Upregulation of the phase II antioxidant genes GPx-2 and HO-1 was also seen in an ERß-dependent mechanism. GTx-822 pretreatment induced phosphorylation of ERK1/2, PI3-K, and Bad. CONCLUSIONS: This is the first report to show that GTx-822, an ERß 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 ERß agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis.
Subject(s)
Estrogen Receptor beta/agonists , Mitogen-Activated Protein Kinases/metabolism , Oxidative Stress/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Quinolines/pharmacology , Retinal Pigment Epithelium/drug effects , Apoptosis , Blotting, Western , Cells, Cultured , Cytoprotection , Enzyme-Linked Immunosorbent Assay , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Gene Expression , Humans , Hydrogen Peroxide/toxicity , Membrane Potential, Mitochondrial/physiology , Phosphorylation , Radioligand Assay , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation , tert-Butylhydroperoxide/toxicityABSTRACT
PURPOSE: To elucidate the mechanism of 17-ß estradiol (17ß-E(2))-mediated protection of retinal pigment epithelium (RPE) from oxidative stress. METHODS: Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide or hydrogen peroxide in the presence or absence of 17ß-E(2). Reactive oxygen species (ROS) were measured using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell-death ELISA kit and Hoechst-3486 staining. Mitochondrial membrane potential was measured using the JC-1 assay. Cellular localization of estrogen receptor (ER) was evaluated by confocal microscopy. Gene expression and protein expression was quantified using qRT-PCR and western blotting. Superoxide dismutase and ATP levels were measured using commercial kits. RESULTS: ARPE-19 cells expressed significant amounts of ERα and ERß. Pretreatment with 17ß-E2 protected ARPE-19 cells from oxidative stress and apoptosis. 17ß-E(2) reduced the ROS levels and mitochondrial depolarization. The 17ß-E(2)-mediated cytoprotection was inhibited by ER antagonists ICI (ERα and ERß) and THC (ERß) but not by tamoxifen (ERα). Knockdown of ERß expression by siRNA abolished the protective effects of 17ß-E(2). Further, qRT-PCR analysis revealed that 17ß-E(2) pretreatment upregulated the expression of ERß and phase II cellular antioxidant genes. CONCLUSIONS: These results indicate that 17ß-E(2) protects ARPE-19 cells from oxidative stress through an ERß-dependent mechanism. 17ß-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.
Subject(s)
Estradiol/pharmacology , Estrogen Receptor beta/metabolism , Estrogens/pharmacology , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effects , Adenosine Triphosphate/metabolism , Apoptosis , Blotting, Western , Cells, Cultured , Cytoprotection , Enzyme-Linked Immunosorbent Assay , Estrogen Receptor beta/genetics , Humans , Hydrogen Peroxide/toxicity , Membrane Potential, Mitochondrial , Microscopy, Confocal , Mitochondria/drug effects , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/metabolism , tert-Butylhydroperoxide/toxicityABSTRACT
We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.