Biomedical subjects

R J Debs

Publications and source records attributed to R J Debs.

At least 19 recordsLinked to original sources

PubMed2006

Systemic co-administration of depsipeptide selectively targets transfection enhancement to specific tissues and cell types.

Depsipeptide, a histone deacetylase (HDAC) inhibitor, kills tumor cells much more effectively than normal cells, and can produce significant antitumor activity in human cancer patients. Depsipeptide also increases the expression of lipoplex-delivered genes in cultured tumor cells, as well as following direct intra-tumoral injection. We now show that co-intravenous (i.v.) injection of depsipeptide with polyethylenimine (PEI):DNA complexes significantly increases the expression of PEI-delivered genes in normal, as well as in tumor-bearing mice. At the tissue level, depsipeptide-mediated enhancement of gene expression was selectively targeted to the lung, liver and spleen. At the cellular level, depsipeptide significantly increased the expression of the i.v., PEI co-delivered wild-type human p53 gene in metastatic breast cancer cells, but not in adjacent normal cells. Thus, the ability of depsipeptide to enhance the expression of systemically delivered genes is selectively targeted at both the tissue and cellular levels, without requiring the use of ligand- or promoter-based approaches. Analyzing HDAC-based targeting of gene expression may identify host genes that control the expression of systemically delivered genes.

Animals↗

PubMed2001

Evaluation of the role of lipoprotein metabolism genes in systemic cationic liposome-mediated gene transfer in vivo.

Germ line gene disruption and gene insertion are often used to study the function of selected genes in vivo. We used selected knockout and transgenic mouse models to attempt to identify lipoprotein-related genes and gene products that regulate the process of intravenous cationic liposome-DNA complex (CLDC)-based gene delivery. Several observations suggested that proteins involved in lipoprotein metabolism might be important in influencing the delivery and/or expression of CLDC. First, in vitro transfection of either K562 or CHO cells by CLDCs was enhanced by the presence of a functional low-density lipoprotein receptor (LDLR). Second, pretreatment of mice with 4-aminopyrazolopyrimidine (4APP), an agent that alters lipoprotein profiles in mice, significantly decreased expression of luciferase (luc) after intravenous injection of CLDC-luc complexes in mice. Therefore, we tested mouse model systems either deficient for, or overexpressing, selected genes involved in lipoprotein metabolism, for their potential to regulate intravenous, CLDC-based gene delivery. Although homozygous knockout mutation in the apoE gene caused a significant decrease in gene expression in many tissues of apoE-deficient mice, mice with homozygous deletion of both the apoE and LDLR genes showed wild-type levels of gene transfer efficiency. Thus, a secondary event, produced by homozygous deletion of apoE, but compensated for by the concomitant deletion of LDLR, and/or effects resulting from strain-related, genetic background differences, appeared to play a significant role in mediating intravenous, CLDC-based gene delivery. Secondary alterations resulting from germ line knockouts, as well as epigenetic effects produced by strain differences, may limit the ability to assign specific, gene transfer-related functions to the deleted gene.

Animals↗

PubMed2001

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

Animals↗

PubMed2000

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo.

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.

Animals↗

PubMed1999

Gene expression along the cerebral-spinal axis after regional gene delivery.

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.

Animals↗

PubMed1999

Systemic gene delivery expands the repertoire of effective antiangiogenic agents.

Cationic liposome-DNA complex (CLDC)-based intravenous gene delivery targets gene expression to vascular endothelial cells, macrophages and tumor cells. We used systemic gene delivery to identify anti-angiogenic gene products effective against metastatic spread in tumor-bearing mice. Specifically, CLDC-based intravenous delivery of the p53 and GM-CSF genes were each as effective as the potent antiangiogenic gene, angiostatin, in reducing both tumor metastasis and tumor angiogenesis. Combined delivery of these genes did not increase anti-tumor activity, further suggesting that each gene appeared to produce its antimetastatic activity through a common antiangiogenic pathway. CLDC-based intravenous delivery of the human wild type p53 gene transfected up to 80% of tumor cells metastatic to lung. Furthermore, it specifically induced the expression of the potent antiangiogenic gene, thrombospondin-1, indicating that p53 gene delivery in vivo may inhibit angiogenesis by inducing endogenous thrombospondin-1 expression. CLDC-based delivery also identified a novel anti-tumor activity for the metastasis suppressor gene CC3. Thus, CLDC-based intravenous gene delivery can produce systemic antiangiogenic gene therapy using a variety of different genes and may be used to assess potential synergy of combined anti-tumor gene delivery and to identify novel activities for existing anti-tumor genes.

Angiostatins↗

PubMed1999

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

Animals↗

PubMed1999

Topical gene delivery to murine skin.

We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations > or =0.25 microg per microl were required to achieve maximal expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders.

Administration, Topical↗

PubMed1998

Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo.

The factors controlling cationic liposome-DNA complex (CLDC)-based gene transfer in cells and in animals are poorly understood. We found that cell surface heparin/heparan sulfate-bearing proteoglycans mediate CLDC-based gene transfer and expression both in cultured cells and following intravenous gene delivery into animals. CLDC did not transfect Raji cells, which lack proteoglycans, but did efficiently transfect Raji cells stably transfected with the proteoglycan, syndecan-1. Fucoidan, heparin, or dextran sulfate, all of which are highly anionic polysaccharides, each blocked CLDC-mediated transfection both in cultured cells and following intravenous injection into mice, but had no effect on transfection by either recombinant adenovirus infection or electroporation. Intravenous pretreatment of mice with heparinases, which specifically cleave heparan sulfate molecules from cell surface proteoglycans, blocked intravenous, CLDC-mediated transfection in mice, confirming that proteoglycans mediate CLDC gene delivery in vivo. Modulation of proteoglycan expression may prove useful in controlling the efficiency of, as well as targeting the sites of, CLDC-based gene transfer in animals.

Adenoviridae↗

PubMed1997

Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery.

We have characterized the relationships between the design of cationic liposomes as a gene transfer vehicle, their resulting biodistribution and processing in animals, and the level and sites of gene expression they produce. By redesigning conventional cationic liposomes, incorporating cholesterol (chol) as the neutral lipid and preparing them as multilamellar vesicles (MLV), we increased the efficiency of cationic liposome:DNA complex (CLDC)-mediated gene delivery. Expression of the luciferase gene increased up to 1,740-fold and of the human granulocyte-colony stimulating factor (hG-CSF) gene up to 569-fold due to prolonged circulation time of injected CLDC, and increased uptake and retention in tissues. The level of gene expression per microgram of DNA taken up per tissue was 1,000-fold higher in lung than in liver, indicating that in addition to issues of delivery and retention of injected DNA, tissue-specific host factors also play a central role in determining the efficiency of expression. Vascular endothelial cells, monocytes, and macrophages are the cell types most commonly transfected by intravenous injection of CLDC.

Animals↗

PubMed1996

Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes.

Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells. African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-1 and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA-containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes.

Animals↗

PubMed1995

A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery.

We have developed three catioinic amphiphiles based on the structure 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)imidazolinium chloride. Although these three compounds differ only in the structure of the hydrophobic acyl chains, they differ greatly in their ability to mediate in vivo and in vitro gene delivery. Moreover, in vitro efficiency is not predictive of in vivo efficiency. The myristoyl form is the most effective compound in vitro, and the oleoyl form is the most effective compound in vivo. The compounds readily form suspensions in aqueous media, both in the pure form and as mixtures with either cholesterol or dioleoylphosphatidylethanolamine. These suspensions can be sonicated to produce smaller particles. Particle size, electron microscopy, and the ability to capture glucose suggest that these lipids form liposomes on suspension in aqueous media. When mixed with plasmid DNA, the lipid particles appear to fuse and form larger particles. Fusion is maximal at the critical DNA:lipid ratio where extensive aggregation and precipitation are observed. Therefore, these compounds behave similarly to other cationic liposome-forming lipids upon interaction with DNA.

Animals↗

PubMed1994

The role of tumor necrosis factor-alpha in acute murine cytomegalovirus infection in BALB/c mice.

The role of tumor necrosis factor-alpha (TNF alpha) in acute lethal and sublethal murine cytomegalovirus (MCMV) infection in BALB/c mice was examined. During the course of acute infection, TNF alpha was not detectable in the serum or bronchoalveolar lavage (BAL) fluids, while TNF alpha was uniformly detected in both serum and BAL following intravenous administration of lipopolysaccharide (LPS). Administration of recombinant murine (rMu) TNF alpha did not consistently alter the virus content of tissues during acute infection. Passive transfer of purified polyclonal immunoglobulin containing neutralizing antibody to TNF alpha did not alter mortality or MCMV replication in tissues during acute infection but did block the TNF alpha response when LPS was administered to BALB/c mice. Thus, TNF alpha appears to play little role in the course and outcome of acute MCMV infection.

Acute Disease↗

PubMed1992

Cationic lipid-mediated transfection of liver cells in primary culture.

We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.

Animals↗

PubMed1991

Treatment of Mycobacterium avium-intracellulare complex infection in beige mice with free and liposome-encapsulated streptomycin: role of liposome type and duration of treatment.

Current treatments for Mycobacterium avium-intracellulare complex (MAC) infections are generally ineffective. Thus, the potential of free or liposome-encapsulated streptomycin to treat acute MAC infection was investigated in beige mice. Free streptomycin administered intramuscularly 5 days a week (150 mg/kg) was effective in the liver, spleen, and lungs. At 4 weeks, liposome-encapsulated streptomycin, administered intravenously in weekly doses (15 mg/kg), reduced the colony-forming units in the liver and spleen by about the same extent as a 50- to 100-fold higher dose of free drug. With 4 weekly injections of liposomes, the colony-forming units in the liver and spleen were lower by 2.4 and 2.9 log units, respectively, compared to untreated controls, even by the end of 12 weeks. The effects of unilamellar and multilamellar liposomes were similar. These observations suggest that liposome encapsulation not only targets streptomycin to infected cells but also increases the residual activity of the drug.

Animals↗

PubMed1991

Chemotherapeutic potential of free and liposome encapsulated streptomycin against experimental Mycobacterium avium complex infections in beige mice.

We investigated in two experiments the chemotherapeutic role of streptomycin on the progression of Mycobacterium avium complex (MAC) disease in beige mice. In the first experiment, streptomycin 100 mg/kg given intramuscularly (im) five days a week for four weeks caused a significant reduction of the colony forming unit (cfu) counts of MAC from spleen, lungs and liver. In the same experiment, streptomycin, given in an encapsulated form in multilamellar liposomes at 15 mg/kg in two intravenous (iv) injections caused greater reduction of cfu in the three tissues. In the second experiment, the effect of free streptomycin at 150 mg/kg given im five days a week for eight weeks was compared with 15 mg/kg of streptomycin encapsulated in unilamellar liposomes given iv in four injections (one day and at three weekly intervals) with no further treatment within the eight weeks. Similar results as in the first experiment were obtained. In both experiments, liposome encapsulation resulted in several-fold increase in the chemotherapeutic efficacy when the data was expressed as reduction of cfu counts per unit dose of the drug.

Animals↗

PubMed1991

Reduction in intensity of Pneumocystis carinii pneumonia in mice by aerosol administration of gamma interferon.

Pneumocystis carinii pneumonia in patients with AIDS may result from impaired local release of gamma interferon from lung lymphocytes and subsequent failure of macrophage activation. We tested this hypothesis with mice rendered immunodeficient by selective depletion of CD4+ lymphocytes and inoculated intratracheally with P. carinii. After aerosol administration of recombinant murine gamma interferon, the intensity of P. carinii infection in these mice was reduced in comparison with that in mice not treated with gamma interferon. Histologic scoring of lung tissue did not reveal additional pulmonary inflammation attributable to gamma interferon. Aerosol administration of gamma interferon may reduce the intensity of P. carinii infection by augmentation of host defense, despite persistent CD4+ lymphocyte depletion.

Aerosols↗

PubMed1990

Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor.

We describe a procedure for assessing the functional activity in vivo of a glucocorticoid receptor derivative, T7X556, a mammalian transcriptional regulator that has been overexpressed in Escherichia coli and purified to homogeneity. The protein was assessed with DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride) liposomes, which are internalized by cultured mammalian cells. T7X556 protein delivered in this manner localized rapidly to the nucleus and selectively enhanced expression from glucocorticoid response element-linked promoters, properties that are characteristic of this receptor derivative when it is synthesized endogenously in mammalian cells. Thus, in vivo activities of T7X556 were not disrupted by expression in bacteria or by biochemical purification. In general, liposome-mediated delivery may permit functional analyses of proteins that have been expressed in heterologous cells and manipulated in vitro.

Animals↗