Improvements in Gene Therapy

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.     

Muscular gene transfer using nonviral vectors

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.     

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.     

非病毒型纳米载体在基因治疗中的研究现状及展望

近 10年来 ,新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体 ,具有无毒性及免疫原性的优势 ,已作为高效阳离子载体用于基因转移。体外基因转移实验表明 ,纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体 ,如多聚氮丙啶及聚赖氨酸。本文对纳米载体的结构特点、性能、基因转移机制进行综述 ,并将其在体内外基因转移效率与其它非病毒载体作以比较     

Gene transfer into hematopoietic stem cells using lentiviral vectors

Gene transfer into hematopoietic cells is currently being used to modulate immune responses, to protect hematopoietic cells against cytotoxic drugs or viral genes, and to restore gene deficiencies due to either inborn genetic defects or acquired loss of regular gene function. In particular, gene addition strategies for inherited severe combined immunodeficiencies (SCID) due to adenosine deaminase (ADA) deficiency or defects of the interleukin-2 receptor gamma-chain represent potentially curative strategies based on gene transfer into hematopoietic cells using recombinant retroviral vectors. Since long-term correction of genetic defects in hematopoietic cells often requires transduction of hematopoietic stem cells, an effective gene transfer into stem cells with efficient long-term and multi-lineage transgene expression is the desired goal for these therapeutic strategies. However, gene transfer strategies with retroviral vectors unable to integrate into non-cycling cells are limited by the quiescent state of the stem cells that have to be stimulated by cytokines to induce cell cycle progression. To circumvent these barriers, lentiviral vector systems based on HIV-1 have recently been developed which are able to deliver and express genes in non-dividing cells both in vitro and in vivo. This review outlines the development and improvement of lentivirus-based gene transfer protocols and discusses the use of lentiviral vectors in preclinical gene therapy studies.     

Strategies for cancer gene therapy using adenoviral vectors

Modification of tumor cells using gene transfer either to enhance host immunity or to act directly on tumor cells is being intensively studied in animal models. Remarkable results have yielded to approved clinical protocols in the treatment of cancer patients using this approach. Several methods of gene delivery have been developed. This article is particularly devoted to the interest of the use of adenoviral vectors in the different strategies of cancer gene therapy.Abbreviations CSF Colony-stimulating factor - IL Interleukin - pfu plaque forming units     

Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases

Nonviral gene transfer is markedly enhanced by the application of in vivo electroporation (also denoted electro-gene transfer or electrokinetic enhancement). This approach is safe and can be used to deliver nucleic acid fragments, oligonucleotides, siRNA, and plasmids to a wide variety of tissues, such as skeletal muscle, skin and liver. In this review, we address the principles of electroporation and demonstrate its effectiveness in disease models. Electroporation has been shown to be equally applicable to small and large animals (rodents, dogs, pigs, other farm animals and primates), and this addresses one of the major problems in gene therapy, that of scalability to humans. Gene transfer can be optimized and tissue injury minimized by the selection of appropriate electrical parameters. We and others have applied this approach in preclinical autoimmune and/or inflammatory diseases to deliver either cytokines, anti-inflammatory agents or immunoregulatory molecules. Electroporation is also effective for the intratumoral delivery of therapeutic vectors. It strongly boost DNA vaccination against infectious agents (e.g., hepatitis B virus, human immunodeficiency virus-1) or tumor antigens (e.g., HER-2/neu, carcinoembryonic antigen). In addition, we found that electroporation-enhanced DNA vaccination against islet-cell antigens ameliorated autoimmune diabetes. One of the most likely future applications, however, may be in intramuscular gene transfer for systemic delivery of either endocrine hormones (e.g., growth hormone releasing hormone and leptin), hematopoietic factors (e.g., erythropoietin, GM-CSF), antibodies, enzymes, or numerous other protein drugs. In vivo electroporation has been performed in humans, and it seems likely it could be applied clinically for nonviral gene therapy.     

From leading role to the backstage: mesenchymal stem cells as packaging cell lines for in situ production of viral vectors

Gene therapy is based on the genetic manipulation of target cells. The genetic information required to genetically engineer these cells can be delivered through non-viral or viral vectors that present different biologic properties. The production of viral vectors for gene therapy depends on the nature of the cells transfected with plasmids containing the genetic information for recombinant viral assemblage. These so-called packaging cell lines (PCL) can be injected into the target organ, for the in situ transduction of target cells. There have been recent reports about the capacity of mesenchymal stem cells (MSCs) to target tumor cells. Different research groups, including our own, have isolated these MSCs, but they have not yet been studied as potential PCL to produce viral vectors. We propose here that a MSC packaging cell line could be employed for in situ gene therapy of solid tumors. The tropism of MSCs for tumor cells may render this PCL more efficient in that microenvironment, producing viral vectors for longer periods of time, shifting MSCs from target cell to the backstage level of viral gene therapy.     

Progress and outlook of inorganic nanoparticles for delivery of nucleic acid sequences related to orthopedic pathologies: a review

The anticipated growth in the aging population will drastically increase medical needs of society; of which, one of the largest components will undoubtedly be from orthopedic-related pathologies. There are several proposed solutions being investigated to cost-effectively prepare for the future--pharmaceuticals, implant devices, cell and gene therapies, or some combination thereof. Gene therapy is one of the more promising possibilities because it seeks to correct the root of the problem, thereby minimizing treatment duration and cost. Currently, viral vectors have shown the highest efficacies, but immunological concerns remain. Nonviral methods show reduced immune responses but are regarded as less efficient. The nonviral paradigms consist of mechanical and chemical approaches. While organic-based materials have been used more frequently in particle-based methods, inorganic materials capable of delivery have distinct advantages, especially advantageous in orthopedic applications. The inorganic gene therapy field is highly interdisciplinary in nature, and requires assimilation of knowledge across the broad fields of cell biology, biochemistry, molecular genetics, materials science, and clinical medicine. This review provides an overview of the role each area plays in orthopedic gene therapy as well as possible future directions for the field.     

The use of adenoviral vectors for genetic manipulation and analysis of primitive hematopoietic cells

Gene transfer into stem cells has long been studied as a means by which primitive hematopoietic cells could be characterized and manipulated. While a variety of strategies have been attempted, it still remains relatively difficult to perform direct stem cell analysis. In this review, we examine recent studies using adenovirus-based vectors as a means to achieve high-level gene transfer into primitive hematopoietic cell types.     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Cell type specific and inducible promoters for vectors in gene therapy as an approach for cell targeting

Gene therapy is used to correct genetic defects or to deliver new therapeutic functions to the target cells. Viral vectors are employed mainly as a gene delivery system. A great variety of viral expression systems have been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenoviral mediated gene transfer have been extensively studied and improved. Preclinical and clinical studies covering a large range of genetic disorders are currently underway to solve basic issues dealing with gene transfer efficiencies, regulation of gene expression, and potential risks of the use of viral vectors. The majority of clinical gene therapy trials that employ viral vectors perform ex vivo gene transfer into target cells. The main issue in potential clinical application of gene therapy is the need for increased gene transfer efficiency and target specificity associated with regulated gene expression at therapeutically relevant levels in vivo. Gene regulatory elements, such as promoters and enhancers, possess cell type specific activities and can be activated by certain induction factors (e.g., hormones, growth factors, cytokines, cytostatics, irradiation, heat shock) via responsive elements. A controlled and restricted expression of these genes can be achieved using such regulatory elements as internal promoters to drive the expression of therapeutic genes in viral vector constructs. In addition to high level and efficient gene expression, minimizing or excluding inappropriate gene expression in surrounding nontarget cells is of great importance for numerous gene therapeutic approaches. This contribution furnishes insight into the field of cell type specific promoter and enhancer systems which have been used for targeted and inducible expression of therapeutic genes in certain genetic disorders, viral infections, and malignancies. We also discuss promoters that represent attractive candidates for the construction of viral vectors.Abbreviations ADA Adenosine deaminase - AFP -Fetoprotein - AIDS Acquired immunodeficiency syndrome - CAT Chloramphenicol acetyltransferase - CD Cytosine deaminase - CEA Carcinoembryonic antigen - DMD Duchenne muscular dystrophy - 5-FC 5-Fluorocytosine - HIV Human immunodeficiency virus - LAD Leukocyte adherence deficiency - LCR Locus control region - LTR Long terminal repeats - MCK Muscle creatinine kinase - MLV Moloney murine leukemia virus - MMTV Mouse mammary tumor virus - PEPCK Phosphoenolpyruvate carboxykinase - PSA Prostate-specific antigen - SCLC Small cell lung cancer cells - SLPI Secretory leukoprotease inhibitor - SPA/B/C Human surfactant protein A/B/C - TAR Trans-activation-responsive - TNF Tumor necrosis factor-     

Integrase-defective lentiviral vectors--a stage for nonviral integration machineries

Gene vehicles derived from lentiviruses have become highly esteemed tools for gene transfer and genomic insertion in a wealth of cell types both in vivo and ex vivo. However, accumulating evidence of preferred insertion into actively transcribed genes, driven by biological properties of the parental human immunodeficiency virus type 1, has questioned the safety of this vector technology. As a consequence, integrase-defective lentiviral vectors [IDLVs], carrying an inactive integrase protein, have been developed and used with success for persistent in vivo gene transfer to quiescent or slowly dividing cells. We and others have shown that episomal DNA delivered by IDLVs may serve as a substrate for heterologous integration machineries, including recombinases and transposases, and homologous recombination triggered by nuclease-induced DNA damage. New vector systems that combine the best of lentiviral gene delivery and nonviral integration systems are under development. The first prototypes of such hybrid lentiviral vectors facilitate efficient gene transfer and show profiles of insertion that are not dictated by the biological constraints of the normal integration pathway and are, therefore, significantly different from the profile of conventional lentiviral vectors. The stage is set for further exploration of these vectors. In this review, we summarize the background and short history of hybrid IDLV-based vector systems and discuss their applicability in gene therapy and treatment of genetic disease.     

Thyroid hormone (T3)-modification of polyethyleneglycol (PEG)-polyethyleneimine (PEI) graft copolymers for improved gene delivery to hepatocytes

Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.     

载体与基因转染的研究进展

基因载体是指将基因或其它核酸物质运载到细胞中的工具.其化学本质可以是蛋白质或多肽、核酸、脂类、糖类、其它有机分子或它们的复合物.基因传递系统是基因治疗的重要组成部分,也是目前基因治疗的瓶颈.现有的基因载体包括两类.即病毒载体和非病毒载体.病毒载体转染效率高,但由于其转染具有免疫原性和致突变性限制了它的应用;非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.就各种载体的最新研究进展作一综述.     

Viral vectors in cancer immunotherapy: which vector for which strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.     

Gene Therapy for Metabolic Diseases of the Liver

Significant advances have been made in the field of liver-directed gene therapy. Many diseases are potential targets for gene therapy, including diseases that have exclusive liver involvement and those with systemic manifestations as a result of defective protein synthesis from the liver. Examples are Crigler-Najjar syndrome type 1, alpha(1)-antitrypsin deficiency and haemophilia A and B. Strategies for gene delivery include the use of viral and nonviral vectors. In addition to previously developed viral vectors, such as retroviruses, adenoviruses and adeno-associated viruses, new viral vectors such as lentiviruses are being investigated extensively. Nonviral vectors for gene delivery include liposomes and receptor-mediated gene therapy. A strategy to correct gene defects has been developed using chimaeric RNA/DNA oligonucleotides, and methods to inhibit aberrant or deleterious gene expression using ribozymes, antisense oligonucleotides and dominant-negative gene products are being developed. However, more research focusing on more efficient gene expression and safety will be required before gene therapy can be routinely applicable.     

Gene therapy using adeno-associated virus vectors

SUMMARY: The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAV's utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.     

Progress with retroviral gene vectors

Retroviral vectors have become a standard tool for gene transfer technology. Compared with other gene transfer systems, retroviral vectors have several advantages, including their ability to transduce a variety of cell types, to integrate efficiently into the genomic DNA of the recipient cells and to express the transduced gene at high levels. The relatively well understood biology of retroviruses has made possible the development of packaging cell lines which provide in trans all the viral proteins required for viral particle formation. The design of different types of packaging cells has evolved to reduce the possibility of helper virus production. The host range of retroviruses has been expanded by pseudotyping the vectors with heterologous viral glycoproteins and receptor-specific ligands. The development of lentivirus vectors has allowed efficient gene transfer to quiescent cells. This review describes different strategies adopted for developing vectors to be used in gene therapy applications.