Showing papers in "Nature Biotechnology in 1997"

Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.

Abstract: Retroviral vectors derived from lentiviruses such as HIV-1 are promising tools for human gene therapy because they mediate the in vivo delivery and long-term expression of transgenes in nondividing tissues. We describe an HIV vector system in which the virulence genes env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. These data demonstrate the potential of lentiviral vectors in human gene therapy.

Yeast surface display for screening combinatorial polypeptide libraries

Abstract: Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

Genome-wide expression monitoring in Saccharomyces cerevisiae

Abstract: The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

Abstract: To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

αv Integrins as receptors for tumor targeting by circulating ligands

Abstract: Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.

Solid stress inhibits the growth of multicellular tumor spheroids

Abstract: In normal tissues, the processes of growth, remodeling, and morphogenesis are tightly regulated by the stress field; conversely, stress may be generated by these processes. We demonstrate that solid stress inhibits tumor growth in vitro, regardless of host species, tissue of origin, or differentiation state. The inhibiting stress for multicellular tumor spheroid growth in agarose matrices was 45 to 120 mm Hg. This stress, which greatly exceeds blood pressure in tumor vessels, is sufficient to induce the collapse of vascular or lymphatic vessels in tumors in vivo and can explain impaired blood flow, poor lymphatic drainage, and suboptimal drug delivery previously reported in solid tumors. The stress-induced growth inhibition of plateau-phase spheroids was accompanied, at the cellular level, by decreased apoptosis with no significant changes in proliferation. A concomitant increase in the cellular packing density was observed, which may prevent cells from undergoing apoptosis via a cell-volume or cell-shape transduction mechanism. These results suggest that solid stress controls tumor growth at both the macroscopic and cellular levels, and thus influences tumor progression and delivery of therapeutic agents.

Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain.

Abstract: Small protein domains, capable of specific binding to different target proteins have been selected using combinatorial approaches. These binding proteins, called affibodies, were designed by randomization of 13 solvent-accessible surface residues of a stable alpha-helical bacterial receptor domain Z, derived from staphylococcal protein A. Repertoires of mutant Z domain genes were assembled and inserted into a phagemid vector adapted for monovalent phage display. Two libraries, each comprising approximately 4 x 10(7) transformants, were constructed using either an NN(G/T) or an alternative (C/A/G)NN degeneracy. Biopanning against the target proteins Taq DNA polymerase, human insulin, and a human apolipoprotein A-1 variant, showed that in all cases significant enrichments were obtained by the selection procedures. Selected clones were subsequently expressed in Escherichia coli and analyzed by SDS-PAGE, circular dichroism spectroscopy, and binding studies to their respective targets by biospecific interaction analysis. The affibodies have a secondary structure similar to the native Z domain and have micromolar dissociation constants (KD) for their respective targets.

Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.

Abstract: Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAC containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAC, and germline transmission of the intact BAC has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.

Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines

Abstract: In recent years there has been considerable progress towards the development of expression systems for the display of heterologous polypeptides and, to a lesser extent, oligosaccharides on the surface of bacteria or yeast. The availability of protein display vectors has in turn provided the impetus for a range of exciting technologies. Polypepttde libraries can be displayed in bacteria and screened by cell sorting techniques, thus simplifying the isolation of proteins with high affinity for ligands. Expression of antigens on the surface of nonvirulent microorganisms is an attractive approach to the development of high-efficacy recombinant live vaccines. Finally, cells displaying protein receptors or antibodies are of use for analytical applications and bioseparations.

Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress.

Abstract: Transgenic tobacco seedlings that overexpress a cDNA encoding an enzyme with both glutathione S-transferase (GST) and glutathione peroxidase (GPX) activity had GST- and GPX-specific activities approximately twofold higher than wild-type seedlings. These GST/GPX overexpressing seedlings grew significantly faster than control seedlings when exposed to chilling or salt stress. During chilling stress, levels of oxidized glutathione (GSSG) were significantly higher in transgenic seedlings than in wild-types. Growth of wild-type seedlings was accelerated by treatment with GSSG, while treatment with reduced glutathione or other sulfhydryl-reducing agents inhibited growth. Therefore, overexpression of GST/GPX can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool.

A bioactive designer cytokine for human hematopoietic progenitor cell expansion

Abstract: Efficient expansion of hematopoietic progenitor cells requires, at least, the simultaneous stimulation of the receptors c-kit and gp130. While c-kit is activated by SCF; gp130, in cells which do not express sufficient amounts of IL-6R, can be activated by the complex of soluble IL-6R (sIL-6R) and IL-6. The therapeutic use of IL-6/sIL-6R, however, has been hampered by the high concentrations of the sIL-6R protein required. We have designed a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain, that was expressed to high levels. On gp130 expressing cells the fusion protein turned out to be fully active at 100 to 1,000-fold lower concentration than the combination of unlinked IL-6 and IL-6R. The fusion protein was used to effectively expand human hematopoietic progenitor cells ex vivo in a dose dependent fashion.

Metal-recognition by repeating polypeptides.

Abstract: Attachment of proteins to metal surfaces has the potential to improve our understanding of protein adhesion and has applications in sensor technology. Repeating polypeptides able to bind to metallic gold or chromium were selected from a population of approximately 5 million different polypeptides. Each polypeptide contained several direct repeats of identical peptide units 14 or 28 amino acids long. The metal-recognizing polypeptides were found to retain their binding properties when freed from the protein used to select them. One gold-binding polypeptide's avidity for gold was found to be dependent on the number of repeats and the presence of salt.

Stress tolerance: the key to effective strains of industrial baker's yeast

Abstract: Application of yeasts in traditional biotechnologies such as baking, brewing, distiller's fermentations, and wine making, involves them in exposure to numerous environmental stresses. These can be encountered in concert and sequentially. Yeast exhibit a complex array of stress responses when under conditions that are less than physiologically ideal. These responses involve aspects of cell sensing, signal transduction, transcriptional and posttranslational control, protein-targeting to organelles, accumulation of protectants, and activity of repair functions. The efficiency of these processes in a given yeast strain determines its robustness, and to a large extent, whether it is able to perform to necessary commercial standards in industrial processes. This article reviews aspects of stress and stress response in the context of baker's yeast manufacturing and applications, and discusses the potential for improving the general robustness of industrial baker's yeast strains, in relation to physiological and genetic manipulations.

Molecular evolution of an arsenate detoxification pathway by DNA shuffling.

Abstract: Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pI258 encoded by genes arsR, arsE, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12–fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.

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

Abstract: 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.

Increasing the serum persistence of an IgG fragment by random mutagenesis.

Abstract: The major histocompatibility complex (MHC) class l-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fcγ1 fragment, the affinity for binding to FcRn at pH 6,0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the home-ostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.

Applications of the green fluorescent protein in cell biology and biotechnology

Abstract: The recent emergence of an autofluorescent protein, the green fluorescent protein (GFP), has opened the door for the convenient use of intact living cells and organisms as experimental systems in fields ranging from cell biology to biomedicine. We present an overview of some of the major applications of GFP, namely its use in protein tagging and in monitoring gene expression as well as its potential in a variety of biological screens.

Selecting effective antisense reagents on combinatorial oligonucleotide arrays.

Abstract: An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

Abstract: The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

Design and production of novel tetravalent bispecific antibodies

Abstract: We have produced novel bispecific antibodies by fusing the DNA encoding a single chain antibody (ScFv) after the C terminus (CH3-ScFv) or after the hinge (Hinge-ScFv) with an antibody of a different specificity. The fusion protein is expressed by gene transfection in the context of a murine variable region. Transfectomas secrete a homogeneous population of the recombinant antibody with two different specificities, one at the N terminus (anti-dextran) and one at the C terminus (anti-dansyl). The CH3-ScFv antibody, which maintains the constant region of human IgG3, has some of the associated effector functions such as long half-life and Fc receptor binding. The Hinge-ScFv antibody which lacks the CH2 and CH3 domains has no known effector functions.

An encapsulation system for the immunoisolation of pancreatic islets

Abstract: Over a thousand combinations of polyanions and polycations were tested to search for new polymer candidates that would be suitable for encapsulation of living cells. The combination of sodium alginate, cellulose sulfate, poly (methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride was most promising. In parallel, a novel multiloop chamber reactor was developed to control the time of complex formation and to negate gravitational effects such as pancreatic islet sedimentation and droplet deformation during the encapsulation process. Encapsulated rat islets demonstrated glucose-stimulated insulin secretion in vitro, and reversed diabetes in mice. This new capsule formulation and encapsulation system allows independent adjustments of capsule size, wall thickness, mechanical strength, and permeability, which may offer distinct advantages for immunoisolating cells.

Transformation of wheat with high molecular weight subunit genes results in improved functional properties

Abstract: The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering

Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase

Abstract: Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane fractions was verified by immunoblotting with OmpA antisera. Inclusion of the organophosphorus hydrolase signal sequence was necessary for achieving enzymatic activity on the surface. More than 80% of the OPH activity was located on the cell surface as determined by protease accessibility experiments. Whole cells expressing OPH on the cell surface degraded parathion and paraoxon very effectively without any diffusional limitation, resulting in sevenfold higher rates of parathion degradation compared with whole cells with similar levels of intracellular OPH. Immobilization of these live biocatalysts onto solid supports could provide an attractive means for pesticide detoxification in place of immobilized enzymes, affording a reduced diffusional barrier.

A Dominant Mutant Receptor From Arabidopsis Confers Ethylene Insensitivity in Heterologous Plants

Abstract: Ethylene (C_2H_4) is a gaseous hormone that affects many aspects of plant growth and development. Ethylene perception requires specific receptors and a signal transduction pathway to coordinate downstream responses. The etr1-1 gene of Arabidopsis encodes a mutated receptor that confers dominant ethylene insensitivity. Evidence is presented here that etr1-1 also causes significant delays in fruit ripening, flower senescence, and flower abscission when expressed in tomato and petunia plants. The ability of etr1-1 to function in heterologous plants suggests that this pathway of hormone recognition and response is highly conserved and can be manipulated.

Transgenic plants: An emerging approach to pest control

Abstract: Insect pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt). A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

Drug metabolism by Escherichia coli expressing human cytochromes P450.

Abstract: The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.

Plant-derived vaccine protects target animals against a viral disease

Abstract: The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa

Abstract: Patients with retinitis pigmentosa (RP) typically develop night blindness early in life due to loss of rod photoreceptors. The remaining cone photoreceptors are the mainstay of their vision; however, over years or decades, these cones slowly degenerate, leading to blindness. We created transgenic pigs that express a mutated rhodopsin gene (Pro347Leu). Like RP patients with the same mutation, these pigs have early and severe rod loss; initially their cones are relatively spared, but these surviving cones slowly degenerate. By age 20 months, there is only a single layer of morphologically abnormal cones and the cone electroretinogram is markedly reduced. Given the strong similarities in phenotype to that of RP patients, these transgenic pigs will provide a large animal model for study of the protracted phase of cone degeneration found in RP and for preclinical treatment trials.

Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV

Abstract: Species-specific differences in the enzymatic inactivation of peptides is an important consideration in the evaluation of therapeutic efficacy. We demonstrate that glucagon-like peptide 2 (GLP-2), shown to be highly intestinotrophic in mice, promotes an increase in intestinal villus height but has no trophic effect on small bowel weight in rats. The reduced intestinotrophic activity of GLP-2 in rats is attributable to inactivation by the enzyme dipeptidyl peptidase IV (DPP-IV). GLP-2(1-33) was degraded to GLP-2(3-33) following incubation with human placental DPP-IV or rat serum but not by serum from DPP-IV-deficient rats. Administration of rat GLP-2 to DPP-IV-deficient rats was associated with markedly increased bioactivity of rat GLP-2 resulting in a significant increase in small bowel weight. A synthetic GLP-2 analog, r[Gly2]GLP-2, with an alanine to glycine substitution at position 2, was resistant to cleavage by both DPP-IV and rat serum in vitro. Treatment of wild-type rats with r[Gly2]GLP-2 produced a statistically significant increase in small bowel mass. DPP-IV-mediated inactivation of GLP-2 is a critical determinant of the growth factor-like properties of GLP-2.