PEGylated helper-dependent adenoviral vector expressing human Apo A-I for gene therapy in LDLR-deficient mice.

Helper-dependent adenoviral (HD-Ad) vectors have great potential for gene therapy applications; however, their administration induces acute toxicity that impairs safe clinical applications. We previously observed that PEGylation of HD-Ad vectors strongly reduces the acute response in murine and primate models. To evaluate whether PEGylated HD-Ad vectors combine reduced toxicity with the correction of pathological phenotypes, we administered an HD-Ad vector expressing the human apolipoprotein A-I (hApoA-I) to low-density lipoprotein (LDL)-receptor-deficient mice (a model for familial hypercholesterolemia) fed a high-cholesterol diet. Mice were treated with high doses of HD-Ad-expressing apo A-I or its PEGylated version. Twelve weeks later, LDL levels were lower and high-density lipoprotein (HDL) levels higher in mice treated with either of the vectors than in untreated mice. After terminal killing, the areas of atherosclerotic plaques were much smaller in the vector-treated mice than in the control animals. Moreover, the increase in pro-inflammatory cytokines was lower and consequently the toxicity profile better in mice treated with PEGylated vector than in mice treated with the unmodified vector. This finding indicates that the reduction in toxicity resulting from PEGylation of HD-Ad vectors does not impair the correction of pathological phenotypes. It also supports the clinical potential of these vectors for the correction of genetic diseases.

Drug-virus interaction: effect of administration of recombinant adenoviruses on the pharmacokinetics of docetaxel in a rat model.

Modern cancer therapy combines recombinant viruses with traditional chemotherapeutic agents that are metabolized by hepatic cytochrome P450 3A4 (CYP3A4). A single dose of recombinant adenovirus (Ad) expressing beta-galactosidase (AdlacZ) significantly alters CYP3A2, the correlate of CYP3A4, in rats for 14 days. Recombinant adenovirus expressing human p53 (Adp53) also suppresses CYP3A2. Plasma clearance of docetaxel (DTX) in animals given AdlacZ (3.38+/-0.22 l h(-1) kg(-1)) was significantly lower than that of those given DTX alone (7.35+/-1.22 l h(-1) kg(-1), P

PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile.

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.

Development of formulations that enhance physical stability of viral vectors for gene therapy.

This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites. We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions. Adenovirus and adeno-associated virus expressing E. coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants. X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations. Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at -20 and 4 degrees C. Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degrees C. Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degrees C. Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degrees C for 1 year after an initial loss of 0.5 log due to processing. Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product. Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization. In summary, lyophilized preparations that can be shipped and stored at 25 degrees C offer a solution to the current problem of distribution of viral vectors for clinical trials.

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells.

Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.

Development of novel formulations that enhance adenoviral-mediated gene expression in the lung in vitro and in vivo.

Despite remarkable progress in the development of both viral and non-viral gene delivery vectors for cystic fibrosis therapy, low efficiency of gene transfer to the airway epithelium is a major obstacle to clinical application. Here we develop formulations that enhance cellular absorption of adenoviral vectors. We selected excipients from a panel of pharmaceutically acceptable com-pounds known to enhance drug absorption. Transduction efficiency of the virus in the presence of each ingredient was assessed in vitro and in vivo. Mannitol and chitosan substantially enhanced transduction efficiency in vitro and augmented expression in vivo by 4 and 8 log units, respectively. The most successful formulation (a blend of sucrose, mannitol, and Pluronic F68) transduced 100% of an A549 cell population in vitro and produced areas of intense gene expression in both large and small airways in vivo with minimal toxicity. Dose response studies also indicate that when placed in this formulation, the viral dose can be lowered by 1/2 log while maintaining superior levels of transgene expression. This formulation also enhanced the physical stability of the virus. No significant loss in titer was detected from a lyophilized formulation after storage at 25 degrees C for 30 days.

Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages.

We investigated the cellular basis for secretion of inflammatory cytokines in mice following intravenous administration of adenoviral vectors (Ad). Serum inflammatory cytokines including interleukin-6 (IL-6), IL-12, and tumor necrosis factor-alpha (TNF-alpha) were detected as early as 6 h following intravenous injection of Ad-expressing Escherichia coli beta-galactosidase (Ad-lacZ). Ad-lacZ readily accumulated in the splenic marginal zone 1 h after intravenous infusion, where both dendritic cells (DCs) and macrophages were transduced and activated within 6 h. Flow cytometric analyses showed that the expression of Ia and CD86 antigens was markedly enhanced on splenic DCs indicating their activation in vivo by Ad-lacZ. Upon ex vivo culture, these early-activated splenic DCs spontaneously produced high levels of IL-6 and IL-12. By contrast, activated splenic macrophages spontaneously secreted only IL-6. Elimination of tissue macrophages and splenic DCs in vivo considerably reduced the early release of IL-12, IL-6, and TNF-alpha and significantly blocked the specific cellular immune response to Ad and the transgene product in vivo. Our findings indicate that preferential activation of DCs and macrophages may account for Ad-triggered acute inflammatory response in vivo in mice. Moreover, DCs and macrophages may play different roles in this process in terms of their abilities to produce distinct patterns of inflammatory cytokines.

"Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung.

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.

Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions.

PEGylation is the covalent attachment of activated monomethoxy poly(ethylene) glycols (MPEGs) to free lysine groups of therapeutic proteins. This technology has enhanced the physical stability of proteins and ablated humoral immune responses generated against them. In this study, adenoviral vectors were modified with MPEGs activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Under proper buffering conditions, reactions were complete within 2 hr. Transduction efficiency of PEGylated adenoviruses was not compromised by neutralizing antibodies to native adenovirus in vitro. These preparations retained titers that were significantly greater than those of the unconjugated virus after storage at 42, 25, 4, and -20 degrees C. Stability profiles of PEGylated preparations at -20 degrees C suggest that glycerol could be eliminated from formulations without significant loss of viral titer. PEGylated adenoviruses produced a two- to threefold increase in transduction in the lung when administered by intratracheal injection and a fivefold increase in transduction in the liver when administered intravenously.

Characterization of the lung Krüppel-like transcription factor gene and upstream regulatory elements.

We previously described the isolation and characterization of the cDNA for lung Krüppel-like factor (LKLF), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the -490/-72bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from -157/-72bp was found to be necessary for upregulating expression. In transfection assays, mutations of the -138/-111bp region resulted in approximately 70-80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues.

Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

In vitro and in vivo assessment of adenovirus 41 as a vector for gene delivery to the intestine.

In order to identify suitable adenoviral vectors for efficient delivery of transgenic proteins and peptides to the intestine, the ability of adenovirus types 5 and 41 (an enterotropic serotype) to bind to and enter undifferentiated and differentiated enterocytes was assessed. FACS analysis showed no significant difference between the virions in their ability to bind to undifferentiated Caco-2 cells as 81.6% of the cellular population bound adenovirus 5 (Ad 5) and 79.8% bound Ad 41. Both virions were also efficiently internalized in this cell type as 99.6% of the cells took up Ad 5, while 95.9% took up Ad 41. In studies with differentiated enterocytes, probable targets for oral gene delivery but rather resistant to adenovirus-mediated gene transfer, 28.4% of the population internalized the Ad 5 vector and less than 10% bound the virus. Adenovirus 41 was efficiently internalized in differentiated enterocytes as 89.6% of the cellular population took up the virus while 37.4% bound the virus. These results were consistent with those observed in vivo in rat jejunum. Thus, molecularly engineered Ad 41-based recombinants could be highly efficient vectors for delivery of transgenic proteins to differentiated enterocytes.

Ligand binding sites of Na,K-ATPase.

Our studies have concentrated on two aspects of the Na,K-ATPase, the first relates to the identification of amino acids involved in binding Na+ and K+ during the catalytic cycle and the second involves defining how cardiac glycosides inhibit the enzyme. To date, three amino acids, Ser775, Asp804 and Asp808, all located in transmembrane regions five and six, have been shown to play a major role in K+ binding. These findings are based on site directed mutagenesis and expression studies. In order to understand how cardiac glycosides interact with the Na,K-ATPase, studies again involving mutagenesis coupled with expression have been used. More specifically, amino acid residues have been substituted in an ouabain sensitive alpha subunit using random mutagenesis, and the ability of the resulting enzyme to confer resistance to ouabain sensitive cells was determined. Interestingly, the amino acids of the alpha subunit which alter ouabain sensitivity cluster in two major regions, one comprised of the first and second transmembrane spanning domains and the extracellular loop joining them, and the second formed by the extracellular halves of transmembrane regions four, five, six and seven. As noted above, transmembrane regions five and six also contain the three amino acid residues Ser775, Asp804 and Asp808 which play a key role in cation transport, possibly binding K+. Thus, it is reasonable to propose that cardiac glycosides bind to two sites, the N- terminal region and the central region which contains the cation binding sites. Cardiac glycoside binding to the center region may lock the cation transport region into a configuration such that the enzyme cannot go through the conformational change required for ion transport.

Beta cyclodextrins enhance adenoviral-mediated gene delivery to the intestine.

PURPOSE: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum. METHODS: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique. RESULTS: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression. CONCLUSIONS: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.

Development of a highly efficient purification process for recombinant adenoviral vectors for oral gene delivery.

Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.

Factors that influence stability of recombinant adenoviral preparations for human gene therapy.

This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.

Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium.

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.

The absence of accessible vitronectin receptors in differentiated tissue hinders adenoviral-mediated gene transfer to the intestinal epithelium in vitro.

PURPOSE: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. METHODS: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the beta-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. RESULTS: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. CONCLUSIONS: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding.

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.