Use Of Gene Transfer Technology Research Articles

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Biosafety of Recombinant Adeno-associated Virus Vectors

It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

Use of gene transfer technology for functional studies in grapevine

The understanding of the genetic determinism of plant phenotypes requires the functional annotation of genes governing specific traits including the characterisation of their regulatory networks. A striking feature of the grapevine genome and proteome lies in the existence of large families related to wine attributes that have a higher gene copy number than in other sequenced plants. During speciation, the appearance of new adaptive functions is often based on the evolution of orthologous genes eventually associated with duplication (paralogous sequences) leading to new proteins and expression profiles. The presence of original features in grapevine, including perennial status, vegetative architecture, inflorescence/tendril, flower organisation (corolla), and fleshy fruit of considerable acidity with various flavonoid compounds, makes functional genomics an essential approach to link a gene to a trait. For grapevine, the current lack of high throughput genetic techniques (e.g. induced mutant collections) and the difficulties associated with genetic mapping (allele diversity, chimerism, generation time) highlights the critical role of transgenic technology for characterising gene function. Different techniques are available to obtain information about gene functioning, but the choice of a particular approach depends on the process investigated (e.g. metabolism, developmental, pathogen response) and the experimental purpose (e.g. induction of ectopic functions, promoter studies, subcellular localisation). After a brief overview of the development of grapevine biotechnology, this paper reviews the state-of-the-art gene transfer technology for grapevine and detailed examples of where transgenic technology has proven useful for studying gene function.

Differential gene expression of cotton cultivar CCRI24 during somatic embryogenesis

Regeneration is an essential step for recovery of transgenic plants following gene transfer. However, most cotton cultivars fail to respond to the current regeneration protocols for cotton. This hinders the use of gene transfer technology to improve this crop. Identification of the genes involved in cotton somatic embryogenesis (SE) may provide information that will help to improve regeneration protocols. To investigate the genes expressed during cotton SE, we constructed a suppression subtractive hybridization (SSH) library using cDNA from nonembryogenic callus as driver and those from embryogenic callus as tester. From this library, 368 cDNA clones that hybridized conspicuously to the forward-subtracted and unsubtracted tester probes, but not to the reverse-subtracted or unsubtracted driver probes, were obtained and sequenced. Among the 241 putative unigenes, the functions of 152 genes (63%) could be assigned using existing databases. In addition to many previously reported SE-related genes, some new genes, such as members of ethylene pathway and auxin pathway, were discovered in our library. The expression of eight genes, including an Aux/IAA responsive gene, was analyzed by reverse-transcription-polymerase chain reaction and found to be upregulated during the SE. This is in agreement with previous studies showing that embryogenesis involves auxin signaling.

Laboratory Forum: The Use of Gene Transfer Technology to Study the Pathophysiology of Erectile Dysfunction

ABSTRACT Introduction The past 25 years of basic science research on erectile physiology has been devoted to investigating the pathogenesis of erectile dysfunction. Research has led to a better understanding of the biochemical factors and intracellular mechanisms responsible for corporal smooth muscle contraction and relaxation, as well as the influence of endothelial-derived relaxing factors. Aim In this essay, we propose the use of gene transfer technology to study mechanisms of disease involved in penile vascular dysfunction. Methods The development of methods to deliver therapeutic genes to the penis has kindled a keen interest in treating ED with gene- and cell-based therapies. Results Gene therapy has delineated putative mechanisms of disease in animal models of erectile dysfunction. Conclusion Investigation of animal models using gene therapy may ultimately lead to mechanism-based therapies for the treatment of erectile dysfunction.

An overview on gene therapy programs

The 11th Annual Meeting of the American Society of Gene Therapy focused on clinical trials for the treatment of various pathological conditions, preclinical studies, use of gene transfer technology for genetic immunization purposes and problems related to the improvement of vector design. In this respect, a major emphasis was placed on safety issues, such as insertional mutagenesis and host immune responses to gene delivery systems.

Developing strategies for detection of gene doping

It is feared that the use of gene transfer technology to enhance athletic performance, the practice that has received the term 'gene doping', may soon become a real threat to the world of sport. As recognised by the anti-doping community, gene doping, like doping in any form, undermines principles of fair play in sport and most importantly, involves major health risks to athletes who partake in gene doping. One attraction of gene doping for such athletes and their entourage lies in the apparent difficulty of detecting its use. Since the realisation of the threat of gene doping to sport in 2001, the anti-doping community and scientists from different disciplines concerned with potential misuse of gene therapy technologies for performance enhancement have focused extensive efforts on developing robust methods for gene doping detection which could be used by the World Anti-Doping Agency to monitor athletes and would meet the requirements of a legally defensible test. Here we review the approaches and technologies which are being evaluated for the detection of gene doping, as well as for monitoring the efficacy of legitimate gene therapy, in relation to the detection target, the type of sample required for analysis and detection methods. We examine the accumulated knowledge on responses of the body, at both cellular and systemic levels, to gene transfer and evaluate strategies for gene doping detection based on current knowledge of gene technology, immunology, transcriptomics, proteomics, biochemistry and physiology.

Gene expression imaging with radiolabeled peptides.

An approach to image radiolabeled peptide localization at tumor sites by inducing tumor cells to synthesize membrane expressed human somatostatin receptor subtype 2 (hSSTr2) with a high affinity for radiolabeled somatostatin analogues is described. The use of gene transfer technology to induce expression of high affinity membrane hSSTr2 can enhance the specificity and degree of radiolabeled peptide localization in tumors. Employing this strategy, induction of high levels of hSSTr2 expression with selective tumor uptake of radiolabeled peptides was achieved in both subcutaneous non-small cell lung cancer and intraperitoneal ovarian cancer mouse human tumor xenograft models. The features of this genetic transduction imaging approach are: (1) constitutive expression of a tumor-associated receptor is not required; (2) tumor cells are altered to express a new target receptor or increased quantities of a constitutive receptor at levels which may significantly increase tumor targeting of radiolabeled peptides compared to uptake in normal tissues; (3) gene transfer can be accomplished by local or regional injection of adenoviral vectors; (4) it is feasible to target adenovirus vectors to tumor cells by modifying adenoviral tropism (binding) or by the use of tumor-specific promoters such that the hSSTr2 will be specifically expressed in the desired tumor; and (5) this technique can be used to image expression of a second therapeutic gene.

Imaging and therapy of tumors induced to express somatostatin receptor by gene transfer using radiolabeled peptides and single chain antibody constructs

The fields of radioimmunodetection and radioimmunotherapy began with an initial paradigm that a targeting molecule (eg, antibody) carrying a radioisotope had the potential of selectively imaging and delivering a therapeutic dose of radiation to tumor sites. A second paradigm was developed in which injection of the targeting molecule was separated from injection of a short-lived radioisotope-labeled ligand (so-called “pretargeting strategy”). This strategy has improved radioisotope delivery to tumors in animal models, enhanced radioimmune imaging in man, and therapeutic trials are in an early phase. We proposed a third paradigm to achieve radioisotopic localization at tumor sites by inducing tumor cells to synthesize a membrane expressed receptor with a high affinity for infused radiolabeled ligands. The use of gene transfer technology to induce expression of high affinity membrane receptors can enhance the specificity of radioligand localization, while the use of radioisotopes with the ability to deliver radiation damage across several cell diameters will compensate for less than perfect transduction efficiency. This approach was termed “Genetic Radioisotope Targeting Strategy.” Using this strategy, induction of high levels of gastrin releasing peptide receptor or human somatostatin receptor subtype 2 expression and selective tumor uptake of radiolabeled peptides was achieved. The advantages of the genetic transduction approach are (1) constitutive expression of a tumor-associated antigen/receptor is not required; (2) tumor cells are altered to express a new target receptor or increased quantities of an existing receptor at levels that may significantly improve tumor targeting of radiolabeled ligands compared with normal tissues; (3) gene transfer can be achieved by intratumoral or regional injection of gene vectors; (4) it is feasible to target adenovirus vectors to receptors overexpressed on tumor cells by modifying adenoviral tropism (binding) so that the virus will be targeted specifically to the desired tumor; and (5) it is possible to coexpress the receptor gene and a therapeutic gene, such as cytosine deaminase, for molecular prodrug therapy to produce an enhanced therapeutic effect.

Utilizing endocrine secretory pathways in salivary glands for systemic gene therapeutics.

Mammalian salivary glands are commonly used models of exocrine secretion. However, there is substantial experimental evidence showing the physiological existence of endocrine secretory pathways in these tissues. The use of gene transfer technology in vivo has allowed the unambiguous demonstration of these endocrine pathways. We and others have exploited such findings and evaluated salivary glands as possible target tissues for systemic applications of gene therapeutics. Salivary glands present numerous advantages for this purpose, including being well encapsulated, which limits extra-glandular vector dissemination, and having the luminal membranes of almost all parenchymal cells accessible via intraoral delivery of vectors through the main excretory ducts. Existing studies suggest that clinical benefits will result from salivary gland targeted systemic gene therapeutics.

As you sow, so shall you reap: the European gene vector production network.

In one of the best-known but most widely misinterpreted parables of the Bible, the Kingdom of God is compared to a farmer who throws seed randomly about, hoping that some of it may fall on good ground. In 1997, the Association Francaise contre les Myopathies (AFM) provided funding for the Gene Vector Production Network (GVPN) under the direction of Genethon III in Evry, France. By making various gene transfer vectors and cell lines freely available to researchers in France, throughout Europe, and beyond, AFM hoped to spark increased interest in gene transfer that could lead, ultimately, to successful clinical applications for neuromuscular and other diseases. Four years later, the first full GVPN meeting was held in Evry on October 4 and 5 to determine how well these first seeds were sown and to plan future plantings. Several hundred users and associates of the GVPN presented research using GVPN-supplied vectors and discussed ways to further enhance this unique tool. It is clear to me that the GVPN has correctly interpreted the ancient parable for modern times. The meeting presentations, poster and oral, covered a wide range of vectors (viral and nonviral) and investigations. Non-research presentations by the directors of the different GVPN centers showcased the physical facilities and specific abilities of the centers (currently in Evry, Nantes, and Marseilles, with a site in Barcelona possibly being added in the near future). The facilities described are all state-of-the-art and have varying capacities and specialties. Most of the research presentations focused on specific disease models (for example, protoporphyrias and immunodeficiencies, in addition to neuromuscular disease). However, a sizable proportion also described the use of gene transfer technology to dissect out specific mechanisms of newly discovered genes and gene products. The common theme throughout was the use of GVPN-supplied vectors. In his opening remarks, GVPN coordinator Mauro Mezzina pointed out that requests for GVPN vectors rose from 120 in 1997 to 631 so far in 2001. Although this may not seem like a large number, the lack of formal publicity for this service underlines the remarkable growth of this unique undertaking. In addition, requests are beginning to come in from around the world. However, as Mezzina noted, the value of the activity cannot be measured in numbers but rather must be evaluated in terms of the progress of science. To that end, it

Genetic engineering within the adult brain: Implications for molecular approaches to behavioral neuroscience

Currently, the most popular technology used to modify the molecular makeup of the nervous system is through germline modifications of early embryos. This allows to construct gene ‘knock-ins’ (gene overexpression) or ‘knock-outs’ (gene deletions). This technology leads to gene additions or deletions from the earliest developmental stages. This can potentially lead to compensatory genetic changes. The technology to achieve inducible and cell-type-specific changes in gene expression in transgenic animals has been established. However, it is not yet possible, to reliably turn a particular gene ‘on’ or ‘off’ exclusively in adult animals. Alternatively, the use of gene transfer technology in fully mature animals could overcome many of these shortcomings. Gene therapy is the use of nucleic acids as drugs, and uses gene transfer technology to genetically engineer adult animals. Viral and nonviral vectors have been modified to serve as vectors for nucleic acid sequences of interest. Thus, over the last two decades, methods have been developed to deliver particular nucleic acids directly to target tissues. Further technological advances allow delivery of transgenes or antisense mRNAs directly to predetermined cell types, as well as their delivery under the control of inducible promoter elements. Combined transgenic (i.e., germline modifications) and viral vector technology will also be very powerful in allowing the genetic modification of selected neuronal populations in adult animals. In this review, we discuss the potential of gene delivery to the brain to analyze the effect of genetic engineering of particular neuronal groups on behavior, as well as recent developments and applications of newly engineered vector systems to allow transgenesis within nervous structures of adult animals.

Hydroxychloroquine Enhances the Endocrine Secretion of Adenovirus-Directed Growth Hormone from Rat Submandibular GlandsIn Vivo

Use of gene transfer technology for treating single protein deficiency disorders requires delivery of therapeutic levels of the transgene product. We have suggested that salivary glands may provide a potentially valuable target site for certain systemic applications of gene therapeutics (He et al., Gene Ther. 1998;5:537-541). However, the ability of salivary glands to deliver therapeutic proteins to either the upper gastrointestinal tract via saliva or to the bloodstream, as required, must be carefully evaluated. In the anterior pituitary gland, human growth hormone (hGH) is secreted into the bloodstream via the regulated secretory pathway. However, when expressed from an adenoviral vector delivered to salivary glands, most hGH follows the regulated, tissue-specific, exocrine secretory pathway into saliva, where it is not therapeutically useful. We tested the hypothesis that the commonly used, FDA-approved drug hydroxychloroquine (HCQ) can divert adenovirus-directed hGH from this regulated secretory pathway in rat submandibular glands and enhance delivery into the bloodstream. In untreated rats, there was approximately 20-fold more vector-directed hGH in saliva than in serum. Administration of HCQ led to a shift of hGH secretion into the bloodstream. When delivered at doses of 1 or 10 mg/kg body weight, via intraperitoneal injection plus intraductal infusion, the saliva:serum hGH ratio was approximately 2:1. Such HCQ delivery did not significantly alter the total amount of hGH measured, but increased the serum level of hGH 5- to 6-fold. Also, HCQ had no significant effects on serum chemistries or hematological parameters. We conclude that HCQ is able to significantly enhance hGH secretion from salivary glands into the bloodstream and may be useful to facilitate clinical applications of gene therapeutics via salivary glands.

Gene therapy approaches for treating rheumatoid arthritis.

Current gene therapy approaches for treating rheumatoid arthritis have made use of gene transfer technology as an improved delivery system for emerging proteins and other biologicals whose activities may have therapeutic value. Preclinical research has focused on two primary directions, evaluation of methods of gene delivery and identification of gene products with antiarthritic potential. Although there are reports involving systemic gene delivery, the bulk of effort has focused on local, intraarticular administration using ex vivo and in vivo methods. Viral-based vectors, including adenovirus, adeno-associated virus and herpes simplex virus have the greatest efficiency of gene delivery after intraarticular injection and are capable of generating relevant levels of gene products in several animal models of disease. However, there are limitations to existing generations of these systems that currently preclude their clinical application. Those gene products found to be efficacious in animal models of rheumatoid arthritis include proteins that specifically block the activity of the primary inflammatory cytokines, and include interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factor and interleukin-1. Delivery and expression of genes encoding certain cytokines such as interleukins -4, -10, and -13 and viral interleukin-10, that block synthesis of inflammatory mediators and downregulate aspects of cellular and humoral immune pathways have been found beneficial. Although significant progress has been made, leading to Phase I clinical trials, there remain several hurdles to the routine practice of gene therapy for treatment of rheumatoid arthritis.

Gene Therapy for Restenosis

The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.

Toward a gene therapy for duchenne muscular dystrophy

AbstractDuchenne muscular dystrophy (DMD) is a wasting disease of muscle caused by mutations in a single gene on the X chromosome. Because no effective treatment is available, the disease inevitably is fatal by the third decade of life. Hence, the use of gene transfer technology to correct the biochemical defect (dystrophin‐deficiency) in muscle fibers of DMD patients may provide a promising therapeutic strategy. This review outlines recent progress toward understanding the molecular pathology of DMD, including identification of novel products of the DMD gene expressed in the nervous system and their involvement in neurological defects in DMD patients. We summarize germ‐line (transgenic) and somatic gene transfer studies performed on the mdx mouse model of DMD, using recombinant cDNA molecules. In light of these results, we discuss areas for future study, such as refinement of viral‐mediated gene transfer to muscle, and the prospects for gene therapy for human DMD patients. © 1995 Wiley‐Liss, Inc.

Agrobacterium as a tool for gene transfer in woody species: The state of the art and practical perspectives. A review

Abstract The Agrobacterium-mediated ability to transfer genes into organisms without sexual crossing provides breeders with new opportunities to improve the efficiency of plant production. Gene transfer offers advantages over classical genetic manipulation since plants are improved without disruption of the integrity of their genomes. Several useful genes isolated from microrganisms and affecting pest resistance, rooting ability, hormonal metabolism etc., are now available. These genes can be easily cloned into suitable Ti and Ri derived plasmid vectors and transferred into woody species. The scarce ability of most fruit trees to regenerate the whole plant from in vitro-cultured cells remains the main obstacle to a wider use of gene transfer technology.