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PubMed2005

Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria.

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.

Animals↗
PubMed2004

Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers.

BACKGROUND: Human embryonic stem cells (hESCs) derived from blastocysts can propagate indefinitely in culture while maintaining pluripotency, including the ability to differentiate into cardiomyocytes (CMs); therefore, hESCs may provide an unlimited source of human CMs for cell-based therapies. Although CMs can be derived from hESCs ex vivo, it remains uncertain whether a functional syncytium can be formed between donor and recipient cells after engraftment. METHODS AND RESULTS: Using a combination of electrophysiological and imaging techniques, here we demonstrate that electrically active, donor CMs derived from hESCs that had been stably genetically engineered by a recombinant lentivirus can functionally integrate with otherwise-quiescent, recipient, ventricular CMs to induce rhythmic electrical and contractile activities in vitro. The integrated syncytium was responsive to the beta-adrenergic agonist isoproterenol as well as to other pharmacological agents such as lidocaine and ZD7288. Similarly, a functional hESC-derived pacemaker could be implanted in the left ventricle in vivo. Detailed optical mapping of the epicardial surface of guinea pig hearts transplanted with hESC-derived CMs confirmed the successful spread of membrane depolarization from the site of injection to the surrounding myocardium. CONCLUSIONS: We conclude that electrically active, hESC-derived CMs are capable of actively pacing quiescent, recipient, ventricular CMs in vitro and ventricular myocardium in vivo. Our results may lead to an alternative or a supplemental method for correcting defects in cardiac impulse generation, such as cell-based pacemakers.

Action Potentials↗
PubMed1992

A genetically engineered human IgG mutant with enhanced cytolytic activity.

A mutant chimeric anti-5-dimethylaminonaphthalene-1-sulfonyl human Ig gamma that exhibited augmented effector function was constructed. Utilizing directed mutagenesis, a serine residue near the carboxyl terminus of the human IgG1 H chain (Ser444) was replaced by a cysteine. Novel intermolecular disulfide bonds between Cys444 residues linked pairs of Ig "tail-to-tail" to form covalent dimers ((H2L2)2). These dimers were 200-fold more efficient, compared with monomeric human IgG1, at antibody-dependent complement-mediated cytolysis of hapten-bearing erythrocytes. The ability to enhance the cytolytic activity of an mAb by genetic engineering may be of value in immunotherapy.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1996

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

AIDS Vaccines↗
PubMed2002

Genetically engineered crops: their potential use for improvement of human nutrition.

Great success has been achieved in increasing agriculture productivity to fulfill human needs during the second half of the 20th century. However, there will be much greater challenges in the future. Based on the current population growth rate of 1.4% per year, the world's population is forecast to increase from the current level of approximately six billion to nine to twelve billion in 50 years. In addition to continuously increasing demand for agricultural production, there is an urgent need to improve the nutritional quality of human diets for this rapidly growing human population. Malnutrition is still a worldwide health issue. Macronutrient and micronutrient deficiencies are prevalent in developing countries, and over-consumption of certain nutrients in developed countries (e.g., saturated fatty acids) is associated with high incidence of certain chronic diseases. Furthermore, there will be declining natural resources such as arable land and water, and the challenges to humans must be met without further degrading the environment. Biotechnology offers a valuable tool to help achieve these goals. This review focuses on the most recent advances in biotechnology, which promise to improve human nutrition by enhancing the nutrient density of plant foods. Issues relating to the safety of food products from genetically engineered crops are also discussed.

Agriculture↗
PubMed2001

Utilizing genetically engineered bacteria to produce plant-specific glucosides.

Plant-derived glucosides have attracted much attention due to their widespread applications. This class of products is difficult to isolate or to synthesize in pure form because of the resulting low yields. Thus, simple approaches for the generation of such glucosides would be highly beneficial. We purified and characterized a novel glucosyltransferase from plant cell suspension cultures of Rauvolfia serpentina, which showed rather low substrate specificity. We obtained its cDNA and expressed the active recombinant protein in bacteria (Escherichia coli) with excellent plant-specific glucosylation efficiencies. Compared with the plant system, the bacteria delivered the new enzyme, which was in the form of a soluble or matrix-bound enzyme, approximately 1800 times more efficiently for the synthesis of a wide range of glucosides. More importantly, the engineered E. coli strain allowed for in vivo glucosylation and release of the product into the culture medium, as shown by the formation of arbutin, which is a potent inhibitor of human melanin biosynthesis with commercial value.

Arbutin↗
PubMed1999

Heterogeneity of multiorgan metastases of human lung cancer cells genetically engineered to produce cytokines and reversal using chimeric monoclonal antibodies in natural killer cell-depleted severe combined immunodeficient mice.

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Genetic engineering of pollen allergens for hayfever immunotherapy.

Type I allergy is an immunoglobulin E-mediated disease, representing a major health problem affecting more than 25% of the world's population. Grass, birch and ragweed pollen are a major source of environmental allergen. Specific immunotherapy is clinically proven to be an effective treatment for allergic conditions that involve the administration of crude extracts prepared from natural sources with potential life-threatening anaphylactic side effects. Recent developments in the molecular biology of pollen allergens have made it possible to design novel therapeutic approaches for improved and safer forms of specific immunotherapy. Hypoallergenic forms of major allergens with reduced immunoglobulin E epitopes have been produced using genetic engineering, whilst preserving other characteristics of the molecule that are able to induce a protective response. These modified forms are expected to make allergen-specific immunotherapy more widely used.

Administration, Oral↗
PubMed2000

Autologous, allogeneic tumor cells or genetically engineered cells as cancer vaccine against melanoma.

Melanoma is a prototype of immunogenic tumor to which various types of immunotherapy have been applied extensively over the past decades. Melanoma vaccines are designed for the purpose of immune modulation and subsequent anti-tumor effects in the process of an active specific immunotherapy. Previous attempts of these vaccines include immunization with whole tumor cells/cell lysates admixed with nonspecific adjuvants. While these vaccines generated enhanced anti-tumor immunity in a subset of patients, some of which showing prolonged survival compared to historical controls, no clinical benefit has so far been demonstrated in a properly controlled phase III study. New-generation melanoma vaccines, which are based on genetic modifications of tumor cells to express cytokines, generated long-lasting systemic anti-tumor immunity in animal models. Translation of these preclinical results primarily into melanoma patients with advanced diseases, shows the potential of these vaccines to induce systemic anti-tumor immune responses and in some instances tumor regression with acceptably low toxicity. Higher efficacy of this novel vaccine approach would be expected when used in a postsurgical adjuvant setting when the tumor load is small. Also other novel vaccine approaches such as dendritic cell-based therapy hold promise for the treatment of melanoma. But the clinical value of all these new approaches has to be analysed in prospectively randomized clinical studies.

Antigens, Neoplasm↗
PubMed2002

Kyolic and Pycnogenol increase human growth hormone secretion in genetically-engineered keratinocytes.

The amount of human growth hormone (HGH) decreases significantly after the age of 30. This decrease has been implicated as one of the major causes in the signs of aging, such as thinning of the skin and bones, a decrease in lean muscle mass and an increase in adipose tissue. Supplementing the body's dwindling supply with recombinant human growth hormone (rHGH) has been shown to reverse the signs and symptoms of aging. However, drawbacks in rHGH replacement therapy include prohibitively high cost, the need for repeated injection and side effects such as carpel tunnel syndrome, gynecomastia and insulin resistance. The purpose of this study was to establish an in vitro model using genetically-engineered keratinocytes to screen natural compounds for the ability to stimulate HGH secretion. We now report that a combination of equal amounts of L-arginine and L-lysine, aged garlic extract (Kyolic), S-allyl cysteine and Pycnogenol significantly increased secretion of HGH in this in vitro model. The data indicate that this in vitro model may be used to screen for other secretagogues.

Adjuvants, Immunologic↗
PubMed2002

Genetic engineering in insects of agricultural importance.

The past five years have witnessed the extension of genetic transformation techniques into 11 insect species covering four orders within the Insecta. While the robustness of these transformation systems can be improved, there is now a highly likely probability that transformation of a given insect species will ensue, provided transposable element-containing plasmid DNA can be effectively delivered to the embryo or some other life stage. These developments have shifted emphasis to concerns of transgene stability and the regulation of the rearing and release of these transgenic insects. They have also led to some elegant demonstrations of genetic sexing mechanisms in Drosophila melanogaster with the expectation that similar systems be extended into pest insect species. These developments and issues are discussed in this short review.

Animals↗
PubMed1991

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

Amino Acid Sequence↗
PubMed2001

Engraftment of genetically engineered amniotic epithelial cells corrects lysosomal storage in multiple areas of the brain in mucopolysaccharidosis type VII mice.

Cell-mediated gene therapy for visceral lesions of lysosomal storage diseases is promising; however, the treatment of central nervous system (CNS) lesions remains a challenge. In this study, we generated rat amniotic epithelial cells (AEC) that overexpress and secrete human beta-glucuronidase (GUSB) following transduction with an adenoviral vector encoding human GUSB. The AEC were used as donor cells for cell-mediated gene therapy of CNS lesions in mice with mucopolysaccharidosis type VII (MPSVII), a lysosomal storage disorder caused by an inherited deficiency of GUSB activity. After confirmation that the secreted GUSB was taken up mainly via mannose 6-phosphate receptors in primary cultured neurons, the AEC were transplanted into the brains of adult MPSVII mice. Histochemical analysis showed extensive GUSB activity throughout the ipsilateral hemisphere of the recipient brains, and pathological improvement of the lysosomal storage was observed even in regions far from the site of injection. These results suggest that intracerebral transplantation of genetically engineered AEC has therapeutic potential for the treatment of CNS lesions in lysosomal storage disorders.

Adenoviridae↗
PubMed2006

A genetically engineered anti-CD45 single-chain antibody-streptavidin fusion protein for pretargeted radioimmunotherapy of hematologic malignancies.

Acute myelogenous leukemia (AML) currently kills the majority of afflicted patients despite combination chemotherapy and hematopoietic cell transplantation (HCT). Our group has documented the promise of radiolabeled anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high, and cure rates are only 25% to 30% for relapsed AML. We now show the superiority of pretargeted radioimmunotherapy (PRIT) compared with conventional radioimmunotherapy using a recombinant tetravalent single-chain Ab-streptavidin (SA) fusion protein (scFv(4)SA) directed against human CD45, administered sequentially with a dendrimeric N-acetylgalactosamine-containing clearing agent and radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic (DOTA)-biotin. The scFv(4)SA construct was genetically engineered by fusing Fv fragments of the human CD45-specific BC8 Ab to a full-length genomic SA gene and was expressed as a soluble tetramer in the periplasmic space of Escherichia coli. The fusion protein was purified to >95% homogeneity at an overall yield of approximately 50% using iminobiotin affinity chromatography. The immunoreactivity and avidity of the fusion protein were comparable with those of the intact BC8 Ab, and the scFv(4)SA construct bound an average of 3.9 biotin molecules out of four theoretically possible. Mouse lymphoma xenograft experiments showed minimal toxicity, excellent tumor-specific targeting of the fusion protein and radiolabeled DOTA-biotin in vivo, marked inhibition of tumor growth, and cured 100% of mice bearing CD45-expressing tumors. These promising results have prompted large-scale cGMP production of the BC8 fusion protein for clinical trials to be conducted in patients with hematologic malignancies.

Amino Acid Sequence↗
PubMed1999

Survival of genetically engineered, adult-derived rat astrocytes grafted into the 6-hydroxydopamine lesioned adult rat striatum.

Astrocytes are potentially useful as vehicles for gene transfer into the CNS. As endogenous CNS cells, they possess secretory mechanisms and can be grown in vitro. We have developed an animal model of this system using autologous astrocyte grafts in Fischer 344 rats. Cultured cells were infected with an adenoviral vector containing the reporter gene lacZ in vitro and then grafted into the striatum of adult Fischer 344 rats previously lesioned with 6-OHDA. Survival of the cells and activity of the beta-galactosidase protein were followed for up to 21 days after injection. The grafted cells were shown to survive throughout the experimental period although the expression of transgene was reduced with time. If long-term expression of therapeutically active substances can be achieved, grafts of adult-derived astrocytes genetically engineered using recombinant adenoviral vectors could be employed in the treatment of Parkinson's disease and other neurological disorders.

Adenoviridae↗
PubMed2006

Genetic engineering of Nicotiana tabacum for reduced nornicotine content.

UNLABELLED: Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco. KEYWORDS: Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.

Cytochrome P-450 Enzyme System↗
PubMed1998

Ethical debates in genetic engineering: U.S. scientists' attitudes on patenting, germ-line research, food labeling, and agri-biotech issues.

A 1995 survey of 1,257 scientists working in the field of recombinant DNA research indicates wide areas of agreement as well as some noteworthy divisions when it comes to such thorny questions as patenting, germ-line research, food labeling, and biodiversity. In general, the scientists surveyed approve of patenting living organisms that result from rDNA research, but vary significantly on what should be patentable. They advocate human germ-line therapy, yet have reservations about using it for any but serious diseases. They oppose mandatory labeling of biologically engineered food products, but understand that the public has a right to know and advocate openness. Finally, they favor development of threats to biodiversity and maintain that publicly funded researchers should be legally obligated to consider the potential environmental effects of their research. Some clear differences arise between scientists working in industry and those in academia and between men and women.

Access to Information↗