Victor Solodushko

Minimal piggyBac vectors for chromatin integration

We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co-delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full-length terminal domains required for successful plasmid-to-chromatin transgene delivery can be removed from the transgene delivery cassette to other parts of the plasmid without significantly impairing transposition efficiency. This is achieved by including in the same plasmid, an additional helper piggyBac sequence that contains both long terminal domains, but is modified to prevent its transposition into the host genome. This design decreases the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1500 to just 98 base pairs. By removing these sequences from the delivered gene cassette, they are no longer incorporated into the host genome which may reduce the risk of target cell transformation.

Dexamethasone and mifepristone increase retroviral infectivity through different mechanisms

Using adapted retroviruses for gene delivery is a modern and powerful tool in biological research as well as a promising approach for gene therapy. An important limitation for the extensive use of retroviral vectors is the low infection rate in target cells such as pulmonary vascular endothelial cells due to the insufficient infectivity of standard retrovirus supernatants that can only be overcome by complicated methods of virus concentration. This paper describes two easy methods to augment target cell infectivity, first by increasing the retroviral titer in the medium collected from packaging cells by stimulation of viral propagation with dexamethasone, and second, by increasing target cell sensitivity to retroviral infection by the glucocorticoid receptor antagonist, mifepristone. Using this method, we increased the infectivity of pulmonary microvascular endothelial cells from 16% to 85%. We demonstrate that mifepristone increased the susceptibility of target cells to retroviruses without increasing the viral titer of the supernatant. Dexamethasone, but not mifepristone, increased expression of delivered proteins such as GFP that are important for early identification of infected cells. Each, or both step(s), may be included in a standard protocol for retrovirus propagation and infection of target cells.

Hypoxia‐induced increases in glucose uptake do not cause oxidative injury or advanced glycation end‐product (AGE) formation in vascular endothelial cells

An increase in glucose uptake by endothelial cells exposed to hyperglycemia is the presumed initiating event that causes systemic vascular disease in individuals with diabetes. Diabetics do not develop clinically significant pulmonary vascular disease, however, despite the pulmonary circulations exposure to the same level of glucose. We hypothesized that pulmonary artery endothelial cells are protected from the detrimental effects of hyperglycemia because they take up less glucose than endothelial cells in the systemic circulation, either because of intrinsic differences between the two cell types or because the lower oxygen tension in the pulmonary arterial blood depresses glucose uptake. To test this hypothesis, we exposed normoglycemic and hyperglycemic bovine pulmonary artery (PAECs) and aortic endothelial cells (AECs) from the same animal to progressively lower oxygen tensions and determined glucose uptake. In contrast with our initial hypothesis, we detected no significant difference in glucose uptake between the two cell types. Furthermore, glucose uptake in both PAECs and AECs increased, not decreased, as the oxygen tension dropped; this oxygen‐dependent increase in glucose uptake in endothelial cells predominated over the hyperglycemia‐mediated decrease in glucose uptake that has been reported by others. Despite the increase in glucose uptake at lower oxygen tensions, we detected no corresponding increase in protein carbonylation or advanced glycation endproducts. These results demonstrate that small physiologically relevant changes in oxygen tension can have an important impact on glucose uptake in endothelial cells. These results also demonstrate that an increase in glucose uptake, by itself, is not sufficient to generate ROS‐mediated protein carbonylation or increase intracellular advanced glycation endproducts in vascular endothelial cells.

Mifepristone increases gamma-retroviral infection efficiency by enhancing the integration of virus into the genome of infected cells.

Gamma-retroviruses are commonly used to deliver genes to cells. Previously, we demonstrated that the synthetic anti-glucocorticoid and anti-progestin agent, mifepristone, increased gamma-retroviral infection efficiency in different target cells, independent of viral titer. In this study, we examine how this occurs. We studied the effect of mifepristone on different steps of viral infection (viral entry, viral survival, viral DNA synthesis and retrovirus integration into the host genome) in three distinct retroviral backbones using different virus recognition receptors. We also tested the potential role of glucocorticoid and progesterone receptors in mediating mifepristones ability to increase gamma-retroviral infectivity. We show that mifepristone increases gamma-retroviral infection efficiency by facilitating viral integration into the host genome and that this effect seems to be due to mifepristones anti-glucocorticoid, but not its anti-progestin, activity. These results suggest that inhibition of the glucocorticoid receptor enhances retroviral integration into the host genome and indicates that cells may have a natural protection again retroviral infection that may be reduced by glucocorticoid receptor antagonists.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

Serum can overcome contact inhibition in confluent human pulmonary artery smooth muscle cells.

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum’s stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.

The Glucocorticoid Receptor in Retroviral Infection

The hope that delivering genes to ailing tissues and organs can treat disease more effectively than drugs or surgery, has fueled the intense interest in gene therapy. Potential uses of gene therapy include replacing mutated genes with healthy ones, inactivating improperly functioning mutated genes, or introducing new genes into the body to help fight a specific disease. While the concept of gene therapy is easy to understand, technical difficulties have limited its practical use. Delivered genes that function admirably in cell culture may not function correctly in vivo, may have unexpected consequences on intracellular signaling pathways, or may transform cells raising the risk of iatrogenic malignancy. Retroviruses play a central role in gene delivery applications because they have a high infection efficiency and are able to induce stable mutagenesis in eukaryotic cells (Yi et al., 2005; Somia & Verma, 2000; Thomas et al., 2003). Stable incorporation of retroviral DNA into the host genome is advantageous, since long-term expression of the transgene, usually a requirement for prolonged therapeutic efficacy, is possible. While they are currently being used for in vitro and (animal) in vivo studies, the clinical use of retroviral vectors to deliver genes is still in its infancy. Among the reasons for the slow progress in adapting retroviruses to deliver genes are the concerns over potential adverse events when introduced into humans and a limited understanding of the mechanisms that affect retroviral function and expression in infected (target) cells. Both wild type and genetically modified retroviruses rely on the host cell to assist during its life cycle. Retroviral infection of cells, followed by integration of its genome into the host genome, is not always a certain process, however, and cellular and extracellular processes can influence these events. Therefore, while retroviral gene delivery is generally successful, the impact of viral infections on target cells remains less predictable and can be considered, basically uncontrollable. Despite the care with which viral vectors are generated, researchers ultimately rely on random events that can yield both positive and negative outcomes. In most eukaryotic cells, steroid hormones regulate a wide variety of physiological functions ranging from inflammation to pregnancy. There are five major classes of steroid hormones: glucocorticoids, mineralocorticoids, estrogens, androgens, and progestins. Steroid hormones from each class can complex with their specific receptors, and often with other transcription factors, to recognize DNA sequences called response elements. This mechanism of gene regulation by steroids is so potent and universal throughout the biosphere that it is not surprising that retroviruses have exploited the host nuclear steroid receptor regulatory system to expand their own genomes and improve their overall functionality.

Heterogeneous activation of p19Arf in pulmonary artery smooth muscle cells.

p19(ARF) is a tumor suppressor that leads to cell cycle arrest or apoptosis by stabilizing p53. p19(ARF) is not critical for cell cycle regulation under normal conditions, but loss of p19(ARF) is seen in many human cancers, and a murine p19(Arf) knockout model leads to malignant proliferation and tumor formation; its role in controlling nonmalignant proliferation is less defined. To examine this question, pulmonary artery smooth muscle cells (PASMC) were expanded in culture from a transgenic mouse in which the coding sequence of the p19(Arf) gene was replaced with a cDNA encoding green fluorescent protein (GFP), leaving the promoter intact. During the first 10 days in culture, wild-type, heterozygous, and knockout PASMC grew similarly, but, by day 14, p19(Arf)-deficient PASMC proliferated faster than p19(Arf) heterozygous or wild-type cells; reexpression of p19(Arf) prevented the increased proliferation. This time course correlated with activation of the p19(Arf) promoter, as indicated by the appearance of GFP positivity in p19(Arf)-deficient PASMC. By day 42, ∼80% of p19(Arf)-deficient cells were GFP-positive. When GFP-positive, p19(Arf)-deficient cells were sorted and subcultured separately, they remained GFP-positive, indicating that once cells had activated the p19(Arf) promoter, the promoter remained active in those and all subsequent daughter cells. In contrast, GFP-negative p19(Arf)-deficient cells gave rise to a combination of GFP-positive and -negative daughter cells over time. These results suggest that a subpopulation of PASMC are resistant to the signals that activate the p19(Arf) promoter, an event that would normally target these cells for arrest or cell death.