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D S Strayer

Publications and source records attributed to D S Strayer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (</=5 kb). The rationale for developing SV40 as a gene therapy vector is reviewed. Our studies with rSV40 gene transfer have focused mostly on hematopoietic progenitor cells (CD34+) and their derivatives, and on gene delivery to the liver. In both settings, in vitro and in vivo, SV40 has proven to be very effective. It is thus a promising gene delivery vehicle that can complement others currently in use or under development.

Animals↗
PubMed1999

Comparative effects of virulent and avirulent poxviruses on cell cycle progression.

We studied the impact of tumorigenic poxviral infection on key regulators of cell cycle progression. Malignant fibroma virus (MV) is a virulent poxvirus that causes severe immunological impairment in vivo and in vitro. It also directs expression of important cellular regulatory proteins, such as p53. Its avirulent relative, Shope fibroma virus (SFV), has little effect on the immune system or p53. Accordingly we examined the effects of MV and SFV on the cell cycle in RK-13 rabbit kidney fibroblasts. MV caused an accumulation of cells in G2/M phase and decreased the percentage of cells in G0/G1. Prolongation of G2/M phase was associated with increased levels of cyclin B protein, decreases in cyclin A and cdc2 proteins, and diminished cdc2 activity. In contrast SFV did not affect cellular cycling detectably. SFV infection was accompanied by large increases in cyclin A and cdc2 proteins and increased cdc2 activity. Thus alterations in cell cycle transit during virus infection may reflect active direction in which virus induces changes in cell cycle regulators. Such changes may be important in the differences in virulence between MV and SFV.

Animals↗
PubMed1999

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line.

Alpha 1-antitrypsin (alpha 1AT) deficiency disease is one of the more common hereditary disorders that affects the liver and lung. The liver disease of alpha 1AT deficiency is generally thought to be caused by the accumulation of an abnormal alpha 1AT protein in hepatocytes, whereas the lung disease is thought to be due to a relative lack of the normal protein in the circulation. Therefore, one possible approach to prevent and treat alpha 1AT disease is to both inhibit the expression of the mutated alpha 1AT gene, and to provide a means of synthesizing the normal protein. To do this, we designed specific hammerhead ribozymes that were capable of cleaving the alpha 1AT mRNA at specific sites, and constructed a modified alpha 1AT cDNA not susceptible to ribozyme cleavage. Ribozymes were effective in inhibiting alpha 1AT expression in a human hepatoma cell line using a newly developed simian virus (SV40) vector system. In addition, the hepatoma cell line was stably transduced with a modified alpha 1AT cDNA that was capable of producing wildtype alpha 1AT protein, but was not cleaved by the ribozyme that decreased endogenous alpha 1AT expression. These results suggest that ribozymes can be employed for the specific inhibition for an abnormal alpha 1AT gene product, the first step in designing a gene therapy for the disease. The findings also suggest that the novel SV40-derived vector may represent a fundamental improvement in the gene therapeutic armarmentarium.

Blotting, Northern↗
PubMed1999

Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection.

Human immunodeficiency virus type 1 (HIV-1) encodes several proteins that are packaged into virus particles. Integrase (IN) is an essential retroviral enzyme, which has been a target for developing agents to inhibit virus replication. In previous studies, we showed that intracellular expression of single-chain variable antibody fragments (SFvs) that bind IN, delivered via retroviral expression vectors, provided resistance to productive HIV-1 infection in T-lymphocytic cells. In the current studies, we evaluated simian-virus 40 (SV40) as a delivery vehicle for anti-IN therapy of HIV-1 infection. Prior work suggested that delivery using SV40 might provide a high enough level of transduction that selection of transduced cells might be unnecessary. In these studies, an SV40 expression vector was developed to deliver SFv-IN (SV(Aw)). Expression of the SFv-IN was confirmed by Western blotting and immunofluorescence staining, which showed that > 90% of SupT1 T-lymphocytic cells treated with SV(Aw) expressed the SFv-IN protein without selection. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression and syncytium formation, was potently inhibited in cells expressing SV40-delivered SFv-IN. Levels of inhibition of HIV-1 infection achieved using this approach were comparable to those achieved using murine leukemia virus (MLV) as a transduction vector, the major difference being that transduction using SV40 did not require selection in culture whereas transduction with MLV did require selection. Therefore, the SV40 vector as gene delivery system represents a novel therapeutic strategy for gene therapy to target HIV-1 proteins and interfere with HIV-1 replication.

Blotting, Western↗
PubMed1999

Gene delivery to the liver using simian virus 40-derived vectors.

We describe here the development and testing of simian virus 40 (SV40)-derived vectors to deliver foreign genetic material to the liver. Based on current understanding of the biology of wild-type SV40, it should be possible to exploit several important attributes of this virus, including efficient replication and gene expression, almost universal infectivity, and low immunogenicity if large T-antigen is deleted, to deliver DNA to the liver effectively. Our studies in cultured hepatocytes and in vivo, using both reporter constructs and transgenes of therapeutic interest, provide strong experimental support for this prediction. These successes indicate that SV40 may play an important role in gene delivery to the liver.

Animals↗
PubMed1999

Infection with vaccinia virus alters regulation of cell cycle progression.

The effect of vaccinia virus (VV) on cell cycle progression and its regulators was studied. Infected cultures showed significantly increased transit through G1, decreasing the percentage of cells in G1 and increasing the percentage in S phase. The numbers of cells in G2/M were not affected. Because of the increased S-phase fraction at the expense of G1, expression of cyclins and cyclin-dependent kinases (Cdks) that regulate cell cycle checkpoints was examined. Transcripts for cyclins A and B, Cdk2, and Cdc2 were decreased in VV-infected cells as infection progressed. The amounts of p53 and p27 proteins decreased after 12 and 24 h of infection, respectively. The Cdc2 and Cdk2 protein levels were decreased with increasing time after infection. Taken together, these findings would be expected to lead to more cells in S phase and G2/M, as was observed. Therefore, VV actively modulates expression of cellular regulators of the cell cycle and alters cell cycle progression.

Animals↗
PubMed1998

Effects of overexpression of Ran/TC4 mammalian cells in vitro.

We investigated the effect of overexpression of Ran/TC4 on cell cycle progression. Ran/TC4 (ras-related nuclear protein) is a highly conserved 25-kDa GTP-binding protein that, in concert with its guanine-nucleotide-exchange factor RCC1, is involved in signal transduction. Ran and RCC1 act on nuclear transport of RNA and protein, cell cycle regulation at the G1/S interphase, chromatin decondensation after mitosis, and chromosome stability. These two proteins are essential for the coupling of DNA synthesis with the onset of mitosis. The cDNA for rabbit Ran/TC4 was identified in a cDNA library using degenerate oligonucleotide probes devised on the basis of deduced protein sequence data. This cDNA was cloned into pCDM8 expression vector to yield a plasmid, pTC4, in which Ran/TC4 expression is driven by the cytomegalovirus intermediate early promoter. Both a human tumor cell line, MCF7, and a normal rabbit fibroblast line, RK-13, were tested. Following transfection with pTC4 we observed an increase in Ran/TC4 transcript levels. Transfection with pTC4 prolonged the duration of S phase in both MCF7 and RK-13 cells and led to reduced cell proliferation and decreased total cell numbers. DNA fragmentation was seen in pTC4-transfected cultures but not in control cultures. These findings underscore the function of Ran/TC4 as a molecular switch that guides the cell to completion of DNA synthesis before it enters mitosis and suggest that its overexpression may greatly alter cell cycle kinetics and cell viability.

Amino Acid Sequence↗
PubMed1998

Lung function and bacterial proliferation in experimental neonatal pneumonia in ventilated rabbits exposed to monoclonal antibody to surfactant protein A.

Surfactant protein A (SP-A) increases the resistance of surfactant to inhibition by plasma and other proteins. In a previous study we found that a monoclonal anti-SP-A antibody (R 5) increased the sensitivity of surfactant to inhibition by fibrinogen in vivo and in vitro. SP-A has been shown to stimulate microbial phagocytosis and killing by alveolar macrophages. We hypothesized that using R 5 to inactivate SP-A in an animal model mimicking congenital group B streptococcal (GBS) pneumonia might result in increased bacterial proliferation and a deterioration in lung function. Newborn near term rabbits were delivered by Cesarean section, anesthetized, tracheotomized, and ventilated for 5 h in a plethysmograph system allowing measurement of dynamic lung-thorax compliance. Postnatally the animals received one intratracheal injection (5 ml/kg) of R 5, nonspecific IgG, or normal saline. At 30 min all animals received a standard dose of an encapsulated GBS strain by intratracheal injection. The number of bacteria (mean log10 CFU/g lung +/- S.D.; CFU = colony forming unit) was evaluated in lung homogenates. Histologic lung sections were judged by light microscopy. Bacterial proliferation was similar in rabbits treated with the monoclonal antibody (9.33 +/- 0.39; n = 14) and in control animals receiving saline (9.16 +/- 0.35; n = 14) or nonspecific IgG (9.26 +/- 0.31; n = 11). No significant differences were noted on the histologic analysis or in measurements of lung function. We conclude that intratracheal instillation of a monoclonal anti-SP-A antibody did not increase bacterial proliferation in GBS-infected newborn rabbits. These findings suggest that SP-A does not play an important role in protection against encapsulated GBS strains in the neonatal period.

Animals↗
PubMed1998

Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent.

We have described a novel gene transfer system, in which replication-incompetent, T antigen-deleted simian virus-40 (SV40) is used as the transduction vehicle. We report here successful immunization using such an SV40-derived viral vector. Hepatitis B surface antigen (HBsAg) cDNA was cloned downstream of two tandem SV40 early promoters to yield a T antigen-deficient SV40 derivative, SV(HBS). Cultured TC7 cells were exposed to SV(HBS), and expression of HBsAg was detected 24 h later by Northern blot and RT-PCR analysis. Immunochemistry and Western blot analysis were also performed 24 h after infection to detect expression of HBsAg. Once it was ascertained that we could express HBsAg in this way, we used SV(HBS) to elicit anti-HBs. SV(HBS) was injected intraperitoneally or subcutaneously into mice every 4 weeks. These mice were bled every 2 weeks and their sera assayed for antibody activity against HBsAg and SV40. Production of anti-HBs was measured by ELISA and confirmed by Western blot analysis, both of which demonstrated significant levels of anti-HBs after the second injection. We also tested production of anti-SV40 antibodies by the ability of sera to neutralize SV(HBS) infectivity. We found no evidence of neutralization of SV(HBS) infectivity even after eight inoculations. Thus, replication-incompetent SV40 is itself not a strong antigen. Our data suggest that SV40-based transduction systems may be a useful vehicle for immunization and for other gene transfer applications when a need for multiple inoculations is anticipated.

Animals↗
PubMed1998

Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells.

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of 125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of 125I-A2R and 125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of approximately 32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was approximately 210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Viral vectors for gene therapy: past, present and future.

Gene delivery has been attempted in both experimental and clinical settings. These studies have shown that therapeutic gene transfer is possible, but it has not yet arrived as a practicable therapeutic intervention. This is due in large part to the inability of the vectors used to convey genetic material to a desired location in sufficient quantity and for long enough time to be effective. Current research on viral vectors for gene therapy has focused on reengineering viruses currently being tested as delivery agents, modifying the host to facilitate viral gene transfer and developing new viruses for use in gene transfer. It is too early to know which of these approaches will be effective; however, these ongoing studies are likely to make available in the future an array of gene delivery vehicles with different strengths and weaknesses. It is reasonable to expect that several of the vectors now being studied will prove useful for some therapeutic applications.

Journal Article↗
PubMed1997

Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies.

The reactivity of two rat monoclonal antibodies was studied. These antibodies, A2R and A2C, bind a 32 kDa alveolar type II cell membrane receptor for surfactant protein A. A2R and A2C also bind apical cell membranes of ciliated and nonciliated cells of the conducting airways. Because this reactivity suggested possible utility in targeting those cells for therapeutic gene transfer, the binding activity of these two antibodies was examined in human tissues. In conducting airways, A2R and A2C bound apical epithelial cell membranes throughout the embryologic period studied: from 15 weeks of gestation, through maturity. Reactivity was more restricted to ciliated cells of the airways as maturation progressed. In the peripheral lung, A2C and A2R only bound most cells in the early developing lung, but mainly type II cells in mature lungs. Other normal tissues recognized by these antibodies included crypt lining cells of the adult and fetal stomach, large bile duct epithelium, and pancreatic acinar cells. All of these cells derive from embryonic foregut endoderm. Other normal tissues, both of endodermal and nonendodermal origin, were negative. Pulmonary carcinomas were studied. A2C and A2R recognized all non-small cell carcinomas of the lung tested. In contrast, none of the small cell carcinomas or carcinoid tumors of the lung were recognized by these antibodies. The function of p32 in these diverse cell types is not clear, but whatever its role in these tissues, antibodies versus p32 may potentially be used to target gene or drug therapy to the normal or malignant cells they recognize.

Adult↗
PubMed1997

Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells.

Stable, efficient gene transfer to normal human peripheral blood mononuclear cells (PBMC) is a prerequisite for therapy of a number of diseases, both hereditary and acquired, affecting these cells. Current approaches to gene transfer to PBMC entail ex vivo mitogenic stimulation and multiple transduction steps followed by selection, usually of progenitor populations. Thus, the ability to transfer gene expression to normal, resting PBMC could complement gene transfer strategies that target dividing precursor cells. We report successful short-term transduction of human PBMC using two different SV40-derived viral vectors SV40-derivative viruses were constructed by cloning cDNAs for firefly luciferase (luc), or hepatitis B surface antigen (HBSAg), into shuttle plasmids to create the SV40 derivative viruses SVluc and SV(HBS) respectively. Both genes were cloned downstream from SV40 early promoter. Normal, resting, human PBMC were exposed to these viruses, and unselected cultured cells were assayed 24 to 48 h later for expression of transduced genes by immunochemistry and Northern blot analysis. Expression of both luciferase and HBSAg was detected using both approaches. Levels of expression of luciferase were slightly higher in PBMC which were stimulated with concanavalin A (con A). Conversely, expression of HBSAg was less in con A-stimulation did not alter infectivity of PBMC by SV40-derivative virus. While longevity and stability of expression in vitro are as yet unknown, this demonstration of successful gene transfer to resting, normal human PBMC, assayed on unselected cells, suggests that SV40-based transduction systems may be potential candidates for use in transient gene transfer to mononuclear blood cells.

Blotting, Northern↗
PubMed1997

Surfactant protein-A receptor-mediated inhibition of calcium signaling in alveolar type II cells.

Receptor-mediated inhibition of cellular activating signals is not well understood. Type II alveolar cells secrete surfactant in response to such secretagogs as terbutaline, calcium (Ca) ionophores (e.g., ionomycin [Io]), and adenosine triphosphate (ATP). A cell membrane receptor for SP-A, one of the surfactant proteins, regulates secretion by negative feedback. We used quantitative fluorescence microscopy to study the effects of SP-A on alterations in cytosolic Ca2+ ([Ca2+]i) elicited by surfactant secretagogs. Freshly isolated type II cells were loaded with Fura-2, then treated with secretagog, in the presence or absence of SP-A. Io and ATP produced biphasic increases in cytosol [Ca2+]i, reflecting first Ca2+ release from intracellular stores, and then influx through the cell membrane. Thapsigargin (TG) and Io directly initiate Ca2+ release; ATP elicits Ca2+ release via receptor-mediated mechanisms. Ca2+ release causes cell membrane Ca channels to open by as yet poorly understood mechanisms. Io itself acts as an additional Ca2+ channel. SP-A blocks much of the Ca2+ release and some of the Ca2+ influx elicited by these secretagogs. Antibody against SP-A receptor restores secretagog-induced Ca2+ fluxes from inhibition by SP-A, confirming that the inhibitory activity of SP-A is mediated through its receptor. Type II cells incubated in Ca2+-free medium plus SP-A show diminished Ca2+ release responses to TG or ATP, suggesting that the action of SP-A to prevent secretagog initiated increases in [Ca2+]i may reflect its ability to block Ca2+ release from cytoplasmic Ca stores. The feedback inhibition of surfactant secretion by SP-A may, correspondingly, be a manifestation of this effect. Because recent work suggests that TGF-beta also inhibits Ca2+ fluxes, SP-A and TGF-beta could be representative of a group of physiologic regulators that act by modulating intracellular Ca signaling.

Adenosine Triphosphate↗
PubMed1996

SV40 as an effective gene transfer vector in vivo.

SV40 was used to transduce gene expression in vitro and in vivo. Using cloned SV40 genome, we replaced large T antigen gene (Tag) with a polylinker, and inserted firefly luciferase, controlled by SV40 early promoter. Transfection into Tag-expressing cells yielded Tag-deficient virus, SVluc. SVluc was Tag-deficient and therefore replication-deficient in cells that did not supply Tag. SVluc transduced functional luciferase expression in vitro. BALB/c mice were inoculated with SVluc, and their tissues were assayed 3-21 days post-inoculation (dpi) for luciferase protein production and enzyme activity. Luciferase protein was detected by immunohistochemistry throughout the experiment, from 3 to 21 dpi. There was no inflammatory reaction against SVluc-infected cells at any time, in any tissue studied. Luciferase activity was first detected by luminometry 14 dpi, and remained level through day 21. Thus, replication-deficient recombinant SV40 can mediate gene transfer in vitro and in vivo.

Animals↗