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PubMed1997

In situ molecular association of dystrophin with actin revealed by sensitized emission immuno-resonance energy transfer.

A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG2b antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80-90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.

Actins↗
PubMed1999

Nuclear transfer in the rhesus monkey: practical and basic implications.

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

Animals↗
PubMed1998

Somatic gene transfer to salivary glands.

Recent developments in gene transfer technology have expanded the range of in vivo experimentation and provided new insights that might be applicable to the treatment of human diseases. Somatic gene transfer may complement conventional transgenic animal experiments by allowing for more restricted gene expression. Salivary glands of rats are readily transduced in vivo by adenovirus vectors. This model has been used to demonstrate the effects of transferring a water channel (aquaporin) gene to glands that have been damaged by radiation. Submandibular glands that receive the aquaporin vector increase the stimulated salivary flow close to normal levels. The possible role of E2F1 in promoting cell regeneration in vivo was also explored. A vector expressing E2F1 was capable of increasing DNA synthesis in rat salivary glands, though complete mitosis was not observed. Future generations of vectors must overcome current limitations of efficiency, immunogenicity, and transient expression.

Animals↗
PubMed1997

Enhanced host defense after gene transfer in the murine p47phox-deficient model of chronic granulomatous disease.

The p47phox-/- mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus-based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox-/- marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox-/- mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% +/- 0.9% of phorbol myristate acetate-stimulated peripheral blood neutrophils at 4 weeks and 2.6% +/- 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox-/- mice were significantly lower than untreated p47phox-/- mice (0.89 +/- 0.30 colonies v 237.7 +/- 83.6 colonies at 24 hours, P < .02; 4.0 +/- 2.0 colonies v 110.2 +/- 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox-/- mice (P < .001). Thus, stem-cell-directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.

Animals↗
PubMed1997

Manipulation of immune responses via particle-mediated polynucleotide vaccines.

Polynucleotide vaccines are a new approach to immunization that promises qualitative advances in vaccine technology. These vaccines mimic infection in that they result in expression of pathogen gene products in situ, which can elicit both cell-mediated immune responses and humoral responses. This approach has been applied primarily to vaccines against viral diseases, but may be significant for vaccines directed toward bacterial pathogens. Auragen has developed a generally applicable gene transfer technology and, for vaccine applications, has focused on particle-mediated gene transfer to epidermis. Results demonstrate that Accell polynucleotide vaccines induce immune responses toward human immunodefficiency virus (HIV) antigens, influenza A virus antigens, and hepatitis B virus (HBV) antigens in rodent,s swine and primates. Cellular immune responses toward these antigens have been demonstrated in rodents. In a swine influenza a challenge model Accell vaccination provides protection equivalent to that of a commercial killed-whole-virus vaccine. Vaccination of mice by this method toward a Chlamydia pneumoniae major outer-membrane protein elicits a species-specific antibody response.

AIDS Vaccines↗
PubMedDate not supplied

Cancer immunogene therapy: a review.

Although immunotherapy has long held out promise as a specific, potent approach to cancer therapy, clinical applications have been unrewarding to date. However, advances in gene transfer technology and basic immunology have opened new avenues to stimulate antitumor immune responses including immunogene therapy. Many different approaches to immunogene therapy have been identified. These include transferring genes encoding proinflammatory proteins to tumor cells, suppressing immunosuppressive gene expression, and transferring proinflammatory genes and/or tumor antigen genes to professional antigen-presenting cells. In some cases, genes are transferred to tumor or antigen-presenting cells in situ. In others, gene transfer is performed ex vivo as part of preparing an anticancer vaccine. We discuss the underlying approach, relative success, and clinical application of various cancer immunogene therapy strategies, paying particular attention to immunogene therapy vaccines. Large numbers of preclinical studies have been reported, but only scattered clinical trial results have appeared in the literature. Although very successful preclinically, the ideal cancer immunogene therapy approach remains to be determined and will likely vary with tumor type. Clinical impact may be improved in the future as treatment protocols are refined.

Genetic Therapy↗
PubMed2006

Chorioallantoic placenta defects in cloned mice.

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

Animals↗
PubMed2007

Gene-modified tissue-engineered skin: the next generation of skin substitutes.

Tissue engineering combines the principles of cell biology, engineering and materials science to develop three-dimensional tissues to replace or restore tissue function. Tissue engineered skin is one of most advanced tissue constructs, yet it lacks several important functions including those provided by hair follicles, sebaceous glands, sweat glands and dendritic cells. Although the complexity of skin may be difficult to recapitulate entirely, new or improved functions can be provided by genetic modification of the cells that make up the tissues. Gene therapy can also be used in wound healing to promote tissue regeneration or prevent healing abnormalities such as formation of scars and keloids. Finally, gene-enhanced skin substitutes have great potential as cell-based devices to deliver therapeutics locally or systemically. Although significant progress has been made in the development of gene transfer technologies, several challenges have to be met before clinical application of genetically modified skin tissue. Engineering challenges include methods for improved efficiency and targeted gene delivery; efficient gene transfer to the stem cells that constantly regenerate the dynamic epidermal tissue; and development of novel biomaterials for controlled gene delivery. In addition, advances in regulatable vectors to achieve spatially and temporally controlled gene expression by physiological or exogenous signals may facilitate pharmacological administration of therapeutics through genetically engineered skin. Gene modified skin substitutes are also employed as biological models to understand tissue development or disease progression in a realistic three-dimensional context. In summary, gene therapy has the potential to generate the next generation of skin substitutes with enhanced capacity for treatment of burns, chronic wounds and even systemic diseases.

Animals↗
PubMed1995

[Molecular therapeutic control and gene therapy approaches].

The past decade has seen the successful application of genetic techniques to the investigation of the most important phenotypes of cancer cells; for example, the identification of particular molecules on the surface of cancer cells makes it possible to target these antigens and destroy them selectively via immunotoxins. At present, gene therapy is not a new treatment modality in most instances, but rather a new technology facilitating further exploration of preexisting treatment strategies, such as immunotherapy or chemotherapy, owing to the indirect approaches of contemporary clinical application. It can be anticipated that gene transfer technology will have a considerable impact on the way certain urological anticancer strategies are pursued in the future.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Current status and potential of embryo transfer and reproductive technology in dairy cattle.

Significant use of embryo transfer in dairy cattle commenced with the introduction of nonsurgical embryo recovery in the mid-1970s and developed with the use of nonsurgical transfers in the late 1970s. Numbers of registered Holstein calves from embryo transfer doubled yearly through 1980, after which the rate of increase slowed; the total reached nearly 19,000 calves in 1990. However, the efficacy of superovulation procedures and commercial success rates of transferred fresh embryos have not improved the past 10 to 15 yr. Fertilization rates in superovulated donors remain low. Although embryo-splitting techniques were perfected in the early 1980s, they are not used widely. A practical, commercial embryo-sexing procedure remains unavailable. Recent significant improvement is apparent in the technology of ultrasound-guided oocyte collection and in vitro oocyte maturation, fertilization, and embryo culture. In the future, this technology may be used in conjunction with sperm separated by sex with a flow cytometer. Modest numbers of embryo clones have been produced in several commercial programs via nuclear transfer techniques. However, the efficiency of gene transfer experiments involving ova of cattle and other domestic species has been low. Recently, DNA probe technology has begun to provide genotype information for cattle and will ultimately be applied to embryos.

Animals↗
PubMed2004

Gene transfer in tissue repair: status, challenges and future directions.

Wound repair involves a complex interaction of various cell types, extracellular matrix molecules and soluble mediators. Details on signals controlling wound cell activities are beginning to emerge. In recent years this knowledge has been applied to a number of therapeutic strategies in soft tissue repair. Key challenges include re-adjusting the adult repair process in order to augment diseased healing processes, and providing the basis for a regenerative rather than a reparative wound environment. In particular, the local delivery of pluripotent growth factor molecules to the injured tissue has been intensively investigated over the past decade. Limited success of clinical trials indicates that an important aspect of the growth factor wound-healing paradigm is the effective delivery of these polypeptides to the wound site. A molecular genetic approach in which genetically modified cells synthesise and deliver the desired growth factor in a time-regulated manner is a powerful means to overcome the limitations associated with the (topical) application of recombinant growth factor proteins. This article summarises repair mechanisms and their failure, and gives an overview of techniques and studies applied to gene transfer in tissue repair. It also provides perspectives on potential targets for gene transfer technology.

Adenoviridae↗
PubMed2004

[Recent developments in gene therapy].

Gene therapy is defined as the introduction of genetic material in a patient's cells with resulting therapeutic benefit. It is a promising new biomedical discipline that could potentially lead to new treatments for hereditary diseases, cardiovascular and neurologic disorders, cancer, diabetes and even infectious diseases. The introduction of genetic material into somatic cells requires gene delivery vectors. Since viruses have developed efficient means to introduce their own genetic material into cells they can be readily adapted as viral vectors for gene therapy. Preclinical studies in animal models have shown that therapeutic effects can be achieved after gene therapy for genetic, acquired and complex disorders. Furthermore, therapeutic effects have been obtained in several phase I/II gene therapy clinical trials for hemophilia, severe combined immune deficiency (SCID) and cancer. Gene transfer technology has improved significantly over the past few years and has led to the development of vectors which have fewer side-effects without compromising their efficacy, at least partly due the development of cell-type specific targetable vectors. Nevertheless, the success of gene therapy is still very much depending upon the continuous development of improved vector technologies which would hopefully and ultimately cure diseases which are refractory to current treatment paradigms.

Animals↗
PubMed2003

Total vascular exclusion of the liver enhances the efficacy of retroviral-mediated associated thymidine kinase and interleukin-2 genes transfer against multiple hepatic tumors in rats.

BACKGROUND: Recent advances in gene transfer technology render gene therapy an attractive treatment of disseminated liver metastases for which other treatments remain disappointing. In this setting, total vascular exclusion of the liver could improve gene transfer to cancer cells and prevent extrahepatic vector spreading during portal infusion of therapeutic genes. We evaluate the efficiency of combined herpes simplex virus type-1 thymidine kinase (HSV1-TK) and interleukin-2 retrovirus-mediated gene transfer through the portal vein, under total vascular exclusion of the liver, in a model of macroscopic multiple liver metastases in rats. METHODS: Multifocal liver metastases were established in BDIX rats with intraportal injection of DHDK12 colon cancer cells. On randomization, rats received either vector-producing cells or saline solution under total vascular exclusion of the liver. Vector-producing cells released retroviral vectors encoding Lac-Z in marking studies or HSV1-TK or interleukin-2 in therapeutic studies. Rats were either killed for pathologic studies, or followed for survival. RESULTS: Total vascular exclusion of the liver markedly improved gene transfer efficacy in marking studies. In therapeutic studies we observed a significant reduction in tumor volume of treated rats compared with untreated controls (2170 +/- 310 mm(3)). Although singular HSV1-TK or interleukin-2 gene transfer showed significant efficacy, the greatest tumor volume regression was observed in rats treated with combined HSV1-TK + interleukin-2 gene therapy (145 +/- 60 mm(3); P =.0001 vs control). This translated into an increased median survival rate compared with either control rats (P =.006) or rats treated with single gene therapy. CONCLUSION: In a rat model, a significant antitumoral effect against macroscopic multifocal liver metastases can be observed after retrovirus-mediated HSV1-TK and interleukin-2 gene transfer through the portal vein under total vascular exclusion of the liver, followed by ganciclovir administration. We believe that this well-tolerated and efficient therapeutic approach deserves clinical evaluation in patients with disseminated colorectal liver metastases.

Animals↗
PubMed2002

Basic requirements for the transfer of fermentation technologies to developing countries.

Traditional small-scale fermentation technologies offer considerable potential for stimulating development in the food industry of developing countries in light of their low cost, scalability, minimal energy and infrastructural requirements and the wide consumer acceptance of fermented products in these countries. Efficient transfer and adaptation of these technologies is, however, often limited by inadequate basic scientific knowledge of the processes involved and the lack of appropriate biological inoculants and process controls for these technologies. Basic infrastructures, such as suitably equipped laboratories with consistent working conditions, a constant supply of good quality water and reliable power supplies, are critical elements of a minimal technology base for transfer and adaptation of these technologies. Building the institutional capacity in developing countries to facilitate research and development geared toward a better understanding of the technologies applied in small-scale traditional fermentations is essential, as is the encouragement of governments to formulate supportive national policies, which promote small-scale agro-industrial development. Socioeconomic considerations play a critical role in the successful and sustainable transfer and adoption of technologies and their products in developing countries.

Developing Countries↗
PubMed2005

Gene transfer in endothelial dysfunction and hypertension.

Gene transfer represents a method for treatment of several cardiovascular disorders, including endothelial dysfunction and hypertension. For effective and safe gene therapy in vascular disease, a suitable therapeutic gene needs to be identified and delivered to the vasculature by appropriate delivery devices. In this chapter, we review the different vectors used, both viral and nonviral, suitable genes identified, and associated delivery devices. Several genes have been identified with a view to improve endothelial dysfunction, and we have elaborated the advantages and disadvantages of these approaches. Strategies to treat hypertension, both systemic and pulmonary, have also been described. The optimal vector has not yet been discovered although a wide variety of choices is available, each with properties that may render it suitable for specific applications. The individual characteristics of these vectors are described in relation to the proposed therapeutic paradigm. Although there are several unanswered questions in this arena, the future application of gene transfer technology to diseases of the vasculature holds significant promise.

Endothelium, Vascular↗
PubMedDate not supplied

Recent advances, prospects and problems in designing new strategies for oligonucleotide and gene delivery in therapy.

The field of gene therapy has been in rapid expansion since the first submissions of gene therapy trials in the early 1990s which provided encouraging results. Since then, many gene therapy protocols have been approved for phase I clinical trials for the treatment of inherited genetic diseases and cancer. The possibility of employing gene transfer technology to treat AIDS and neurologic diseases is currently under evaluation. Many gene delivery systems have been developed for in vivo studies and therapy. The efficiency of in vivo gene transfer, however, still needs to be optimized, even though significant advances have recently been achieved in improving gene delivery, gene regulation and avoidance of immune responses. This review provides a general outline focusing on the description of the most common gene delivery systems and on their current applications in therapeutics.

Adenoviridae↗
PubMed2004

Rapid detection and differentiation of Bartonella spp. by a single-run real-time PCR.

A Real-time PCR for the identification and differentiation of Bartonella spp. based on the detection of mutations in an internal region of the gltA gene by thermal analysis was developed. The assay included a simultaneous detection of the amplicons by direct hybridization of LCRed and fluorescein-labelled probes coupled with melting curve analysis by the use of fluorescence resonance energy transfer technology. The protocol allowed establishing genospecie identity in less than 1 h without a need for restriction enzyme digestion or gel electrophoresis. The method is simple, reproducible, rapid, specific and potentially transferable to clinical samples.

Bartonella↗
PubMed1998

Viral vectors, tools for gene transfer in the nervous system.

Viral vectors are becoming increasingly important tools to investigate the function of neural proteins and to explore the feasibility of gene therapy to treat diseases of the nervous system. This gene transfer technology is based on the use of a virus as a gene delivery vehicle. In contrast to functional analysis of gene products in transgenic mouse, viral vectors can be applied to transfer genes to somatic, post-mitotic cells of fully developed animals. To date, five viral vector systems are available for gene transfer in the nervous system. These include recombinant and defective herpes viral vectors, adenoviral vectors, adeno-associated viral vectors and lentiviral vectors. Of these vectors herpes and adenoviral vectors are the most common in use. To date, one of the main hurdles in applying these two vector systems is the focal immune response that occurs following intraparenchymal infusion. Despite this limitation, herpes and adenoviral vectors have been used successfully to modify the physiological response to injury in several rodent models of neurodegeneration. The first purpose of this review is to describe the principles of the generation of viral vectors and to discuss the advantages and disadvantages of the viral vector systems currently in use for gene transfer in the nervous system. Secondly, we give an overview of the performance of these vectors following direct infusion in the nervous system and review the results obtained with these vectors in animal models of neurodegeneration and regeneration. The results of these initial studies have provided a framework for future experiments based on gene transfer strategies with viral vectors to study normal physiology and pathology of the nervous system.

Adenoviridae↗