Simulated lung transfection by nebulization of liposome cDNA complexes using a cascade impactor seeded with 2-CFSME0-cells.

The aim of this study was to devise a simulation aerosol system for quasirealistic gene transfection that could eventually be used to study the characteristics of aerosol delivery, stability, delivery efficiency, and expression efficacy of gene products. It consisted of (1) a PARI aerosol generator and PARI LL jet; (2) an Andersen cascade impactor with a calibrated vacuum pump, fitted with a glass "throat," nebulizer in which stages were seeded with pulmonary cells of interest (e.g., 2-CFSME0-); and (3) a hot room set to 37 degrees C and approximately 70% relative humidity. Cell viability remained at 95% to 99%. A prostaglandin G/H synthase (PGH)- and a human alpha 1-antitrypsin (AAT)-expressing plasmid, respectively, driven by a cytomegalovirus promoter (pCMV4-PGH, pCMV4-AAT) and a heat-insensitive placental alkaline phosphatase (PAP)-expressing plasmid driven by a Rous sarcoma virus promoter (pRSV-PAP) were employed; cationic liposomes consisted of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol/DOPE (DC-Chol/ DOPE). The fluorescent dye Toto-1 was used to visualize aerosol distribution and to monitor cellular uptake. Alternatively, pCMV4-PGH deposited onto impactor stages covered with nitrocellulose membranes was hybridized with random primer-32P-radiolabeled pCMV4-PGH and autoradiographed. The mass median aerodynamic diameter (MMAD) of the plasmid, liposomes, and liposome-plasmid complexes and their effect on the mass output were monitored. A majority of gene product was delivered to stages 1 through 5, corresponding to an area ranging from the pharynx to the terminal bronchi, excluding the alveolar space. A corresponding, although very low, transfection of cells with pRSV-PAP was found, with the majority of transfected cells on stages 4 and 5. The MMAD was significantly affected by the presence of the DNA constructs alone or by DNA constructs complexed with cationic liposomes; the control phosphate buffered saline (PBS) MMAD of 2.3 microns increased to 3.5 microns for DC-Chol liposomes and 4.5 microns for the DC-Chol/PGH complex; DOTMA-based liposomes and liposome DNA complexes precipitated during aerosolization. Mass output was reduced for cationic liposomes from 0.61 g/min (PBS control) to 0.35 g/min. Large plasmid (pRSV-PAP, 10.1 kb) was more rapidly degraded by aerosolization than smaller plasmid (pCMV4-AAT, 6.2 kb) although complexation with cationic liposomes provided some protection in both cases.

Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.

The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.

No lung toxicity after repeated aerosol or intravenous delivery of plasmid-cationic liposome complexes.

The safety aspects of human gene therapy are of paramount importance in developing an ideal system for gene transfer. Lipofection using DNA in the form of a plasmid has been shown to successfully transfect the lungs when administered either intravenously or by aerosol. We have shown that repeated intravenous or aerosol administration of a plasmid containing the recombinant human alpha 1-antitrypsin gene and a cytomegalovirus promoter complexed to cationic liposomes results in no adverse effects on pulmonary histology, lung compliance, lung resistance, or alveolar-arterial oxygen gradient. Immunohistochemistry and Western blot analysis confirm successful gene transfer using this delivery system. We conclude that plasmids complexed to cationic liposomes may be a safe and efficacious delivery system for in vivo gene transfer to the lungs. Using this delivery system, in vivo gene therapy to the lungs can be achieved by either intravenous or aerosol administration of the transgene.

Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.

Gene therapy for inflammatory diseases.

The concept of gene therapy may be broadened to include transient gene therapy (gene therapeutics) as a potential intervention in prevention and treatment of diseases which are a consequence of triggering the inflammatory response. Functioning genes can be delivered in vivo by a variety of technologies. Liposome technology is particularly attractive for gene therapeutics because plasmid DNA constructs can be delivered using liposomes which express transiently and do not readily incorporate into the host genome. In the lungs, DNA may be targeted by either intravenous or airway delivery. Airway delivery may be achieved either by direct injection into the airways or by aerosolizing liposome-DNA constructs. Expression of transgenes might also be targeted to areas of inflammation by choosing DNA constructs which contain the appropriate regulatory regions. Several genes have been cloned which are directly relevant to manipulating the inflammatory response and this technology could, in theory, by using either sense or antisense constructs, provide an opportunity to increase or decrease proteins relevant to the inflammatory response. Because of rapid progress in the technology of molecular biological techniques, and rapid progression of human trials using gene transfer methodologies, it is likely that extension of gene therapy to acute diseases such as those which are characterized by inflammation will open a new vista for pharmacological approaches to these complex diseases.

Aerosol and intravenous transfection of human alpha 1-antitrypsin gene to lungs of rabbits.

In vivo gene transfer to the lungs is possible either by an intravenous or an airway route of administration. A plasmid containing the recombinant human alpha 1-antitrypsin (h alpha 1AT) gene and a cytomegalovirus promoter complexed to cationic liposomes was given either intravenously or by aerosol to New Zealand White rabbits. Both routes of administration resulted in successful transfection and expression of the h alpha 1AT gene. h alpha 1AT mRNA and protein were detected for at least 7 days. Immunohistochemical staining showed h alpha 1AT protein in the pulmonary endothelium following intravenous administration, in alveolar epithelial cells following aerosol administration, and in the airway epithelium by either route. After intravenous injection of radiolabeled plasmids, autoradiographs showed localization of plasmid in endothelial cells, especially at arterial bifurcations, and at the alveolar level. A plasmid-liposome delivery system for gene therapy to the lungs may permit targeting of the DNA to subsets of lung cells by selection of the route of delivery and may permit a broad application of gene therapy to acute as well as chronic diseases.

Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2).

The present studies demonstrate cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger which was isolated from a rat intestinal cDNA library. The cloned cDNA recognizes two transcripts in poly(A)+ RNA from the stomach, jejunum, ileum, liver, large intestine, and uterus. Based on deduced amino acid sequences, this clone shares sequence homology with the other known Na+/H+ exchanger isoforms (NHE-1, NHE-3, and NHE-4) except for its 5' end. Overall, the protein exhibits 47.8%, 41.2%, and 56.2% amino acid sequence identity to NHE-1, NHE-3, and NHE-4, respectively. The hydropathy profile of the predicted protein shows 10 transmembrane domains, suggesting a protein with transport characteristics. The tissue distribution differs from that of the other Na+/H+ exchanger isoforms. The cDNA hybridizes to two closely related transcripts in the mRNA of these tissues, which suggests that the predominant transcript of this clone is alternatively spliced. Transfection of this cDNA into Na+/H+ exchanger-deficient mutant fibroblasts (PS120 cells) results in functional Na+/H+ exchange activity. These data suggest that we have cloned a member of the Na+/H+ exchanger family with tissue-specific expression. We suggest the designation of NHE-2 for this Na+/H+ exchanger.

Expression of human growth hormone fusion genes in cultured lung endothelial cells and in the lungs of mice.

We sought to develop genetic therapy for acute lung diseases by introducing genes into lung cells in vivo that were only transiently expressed. To that end, we introduced a gene encoding a physiologically relevant secreted human protein into bovine lung endothelial cells in culture and into the lungs of mice using the technique of lipofection. We exposed cultured endothelial cells to a plasmid containing the coding region for human growth hormone (hGH) driven by a metallothionein (MT) promoter. In cells lipofected with the plasmid containing the MT promoter, expression of the hGH gene in medium was low (peak = 30 ng hGH/24 h/60-mm dish), but expression was markedly increased by addition of either dexamethasone (peak = 91) or cadmium (peak = 120). Lipofection with the same construct except a thymidine kinase promoter showed no cadmium response. We gave mice 5,000 ppm ZnSO4 in their drinking water and 24 h later injected intravenously plasmid containing the MT promoter complexed to liposomes. Mice were killed 1, 3, and 5 days after injection, and hGH production by minced lung, liver, and kidneys was determined in vitro. Neither kidneys nor liver produced detectable hGH. However, hGH was produced by the lungs, beginning on day 1, peaking on day 3 (approximately 1.0 ng hGH/24 h/g tissue), and declining by day 5. Lungs from mice injected either with DNA alone or with liposome alone did not produce hGH. mRNA specific for hGH was demonstrated in the lungs by polymerase chain reaction amplification of cDNA followed by agarose gel electrophoreses.(ABSTRACT TRUNCATED AT 250 WORDS)

Dexamethasone up-regulates mannose receptor activity by increasing mRNA levels.

The macrophage mannose receptor is highly susceptible to modulation by a variety of inflammatory and anti-inflammatory agents. Previous studies have demonstrated that mannose receptor activity is dramatically enhanced in rat bone marrow macrophages following treatment with dexamethasone. In the present study we have investigated potential mechanisms that might be involved in this up-regulation. Uptake of ligands by the mannose receptor was increased 2.5-fold in a dose- and time-dependent manner. Maximal stimulation was seen following treatment of macrophages with 0.1-1.0 microgram/ml of dexamethasone for 24-48 h. Dexamethasone treatment increased both the number of cell surface binding sites and total cellular binding activity to 250% of control levels. In addition, total receptor protein as measured by immunoprecipitation was increased 2.5-fold. Neither the maturation rate nor the turnover rate of the protein was altered by dexamethasone treatment. Using an oligonucleotide probe derived from sequence data from the cloned human receptor cDNA, we investigated the effect of dexamethasone on the expression of mannose receptor mRNA. Following incubation with dexamethasone for 12-24 h, the level of mRNA was significantly increased. These results demonstrate that dexamethasone treatment of rat bone marrow macrophages induces synthesis of new receptor protein through an increase in the level of mannose receptor mRNA.

In vivo molecular analysis of lymphokines involved in the murine immune response during Schistosoma mansoni infection. I. IL-4 mRNA, not IL-2 mRNA, is abundant in the granulomatous livers, mesenteric lymph nodes, and spleens of infected mice.

Using Northern Blot analysis, the endogenous levels of IL-4 and IL-2 mRNA in the spleens, mesenteric lymph nodes, and granulomatous livers of male CBA/J mice in the acute phase of infection with Schistosoma mansoni have been quantified. High levels of IL-4 mRNA were detected in all three tissues from infected mice, whereas none was detected in tissues from normal, uninfected, age-matched mice. Isolation of the granulomas from the livers of infected mice and subsequent extraction of total RNA from these lesions resulted in a 70-fold enrichment of IL-4 message compared with the whole, unseparated granulomatous liver tissue. Hence, the predominant source of the IL-4 mRNA detected in livers from infected mice appears to be the schistosome egg-induced granulomas within these livers. In contrast, IL-2 mRNA was never detected in any of these tissues from either infected or normal mice. Control experiments were performed that ruled out the possibility that this inability to detect IL-2 mRNA was due to a difference in the efficacy of the IL-4 and IL-2 probes or due to a selective lability of IL-2 message. These data imply that IL-4-producing, Th2 lymphocytes are active in and possibly integral to the granulomatous, delayed-type hypersensitivity response characteristic of this infection, and directly challenges the current hypothesis that delayed-type hypersensitivity responses are exclusively mediated by Th1 lymphocytes.

A reliable method for northern blot analysis using synthetic oligonucleotide probes.

We have developed a method for using short (30-42 base pair) synthetic oligonucleotide DNA probes in Northern blot assays. The method involves labeling the probes to high specific activity, very stringent hybridization and wash conditions, and the presence of several inhibitors of nonspecific binding in the hybridization buffer. We have tested this method with several probes obtained from local and commercial sources. The results with every probe used were high signal-to-noise ratios in an exposure time range of 30 min to 7 days.

Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains.

Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.

Synthesis and stability of steroid sulfatase in fibroblasts from multiple sulfatase deficiency.

Multiple sulfatase deficiency is a lysosomal storage disorder, which can be divided into group I with severe and group II with moderate deficiencies in sulfatases. Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis and stability of this enzyme in multiple sulfatase-deficiency fibroblasts. Fibroblasts from both groups synthesized steroid sulfatase of apparently normal size and stability, while the apparent rate of enzyme synthesis and catalytic properties of steroid sulfatase were affected to a variable extent. Cell lines were observed, that synthesized normal amounts of steroid-sulfatase polypeptides, which were catalytically inactive, as well as cell lines that synthesized diminished amounts of catalytically active steroid sulfatase.

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase.

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.

Heterogeneity in late-onset metachromatic leukodystrophy. Effect of inhibitors of cysteine proteinases.

The synthesis of arylsulfatase A polypeptides was followed in fibroblasts from 11 patients with late-onset forms of metachromatic leukodystrophy. In 10 cell lines, the apparent rate of synthesis was 20%-70% as measured by the amount of [35S]arylsulfatase A secreted in the presence of 10 mM NH4Cl. The specific activity of the secreted arylsulfatase A was normal. The residual activity of arylsulfatase A was below 10% except for one cell line in which it was 20%. The activity of arylsulfatase A and the degradation of sulfatides was partially restored in these fibroblast lines by treatment with irreversible (peptidyl diazomethyl ketones) or competitive (leupeptin) inhibitors of cysteine proteinases. Thus, the mutation(s) in these cell lines led to the synthesis of arylsulfatase. A polypeptides with increased susceptibility to cysteine proteinases. Multiple allelic mutations within this group of late-onset metachromatic leukodystrophy were suggested by the clinical heterogeneity, the variability of the residual activity, and in the response to inhibitors of cysteine proteinases. In fibroblasts from one patient, the apparent rate of synthesis of arylsulfatase A was less than 5%. Furthermore, inhibitors of cysteine proteinases were without effect, suggesting that the mutation in this patient is different from the others.

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts.

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.