Primer on medical genomics. Part X: Gene therapy

Gene therapy is defined as any therapeutic procedure in which genes are intentionally introduced into human somatic cells. Both preclinical and clinical gene therapy research have been progressing rapidly during the past 15 years; gene therapy is now a highly promising new modality for the treatment of numerous human disorders. Since the first clinical test of gene therapy in 1989, more than 600 gene therapy protocols have been approved, and more than 3000 patients have received gene therapy. However, at the time of writing this article, no gene therapy products have been approved for clinical use. This article explains the potential clinical scope of gene therapy and the underlying pharmacological principles, describes some of the major gene transfer systems (or vectors) that are used to deliver genes to their target sites, and discusses the various strategies for controlling expression of therapeutic transgenes. Safety issues regarding clinical use of gene therapy are explored, and the most important technical challenges facing this field of research are highlighted. This review should serve as an introduction to the subject of gene therapy for clinician investigators, physicians and medical scientists in training, practicing clinicians, and other students of medicine.     

Herpes simplex type-1 virus-based vectors for gene therapy

Recent advances in recombinant nucleic acid technologies and the understanding of the molecular biology of diseases provide the basis for gene therapy's ability to impact on the clinical practice of medicine. Herpes simplex virus type 1 (HSV-1) has features that make it suitable as a vector for gene therapy. These properties include: 1) wide host range and high efficiency of gene transfer; 2) large transgene capacity which is provided by deletion of genes unnecessary for viral replication; 3) unique ability of entering a state of latency in neurons; 4) specific oncolytic effect of some mutants by deletion of certain early genes. Three types of vectors designed for gene therapy have been developed from this virus, termed replication-defective vectors, replication-conditional vectors and amplicon vectors. In this review, we describe the recent advances in HSV-1-based vector systems and their applications for gene therapy.     

Prostate cancer gene therapy

With the advance in genetic engineering, tumor biology and immunology, gene therapy has been recognized as a promising new treatment option for cancer including prostate cancer. Several clinical trials of prostate cancer gene therapy are currently underway, using therapeutic genes which include suicide genes, immunomodulatory genes, tumor suppressor genes and anti-oncogenes. Although the gene therapy for prostate cancer as a clinical alternative is still early stage which requires several technological breakthrough, some information obtained from clinical trial indicates full potential of prostate cancer gene therapy. Concordant progress both in the basic research and gene therapy technology will make prostate cancer gene therapy ready for wide-scale of practice in the future. In this report, general concept and current progress in prostate cancer gene therapy are summarized.     

Prostate cancer gene therapy

With the advance in genetic engineering, tumor biology and immunology, gene therapy has been recognized as a promising new treatment option for cancer including prostate cancer. Several clinical trials of prostate cancer gene therapy are currently underway, using therapeutic genes which include suicide genes, immunomodulatory genes, tumor suppressor genes and anti-oncogenes. Although the gene therapy for prostate cancer as a clinical alternative is still early stage which requires several technological breakthrough, some information obtained from clinical trial indicates full potential of prostate cancer gene therapy. Concordant progress both in the basic research and gene therapy technology will make prostate cancer gene therapy ready for wide scale of practice in the future. In this report, general concept and current progress in prostate cancer gene therapy are summarized.     

Gene therapy for peripheral arterial disease

Introduction: Gene therapy has emerged as a novel therapy to promote angiogenesis in patients with critical limb ischemia (CLI) caused by peripheral artery disease. Researchers working in this area have focused on pro-angiogenic factors, such as VEGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). Based on the elaborate studies and favorable results of basic research using naked plasmid DNA (pDNA) encoding these growth factors, some clinical Phase I and Phase II trials have been performed. The results of these studies demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI patients. However, the Phase III clinical trials have so far been limited to HGF gene therapy. Because one pitfall of the Phase III trials has been the limited transgene expression achieved using naked pDNA alone, the development of more efficient gene transfer systems, such as ultrasound microbubbles and the needleless injector, as well as the addition of other genes will make these novel therapies more effective and ease the symptoms of CLI.

Areas covered: This study reviews the previously published basic research and clinical trials that have studied VEGF, FGF and HGF gene therapies for the treatment of CLI. Adjunctive therapies, such as the addition of prostacyclin synthase genes and the development of more efficient gene transfer techniques for pDNA, are also reviewed.

Expert opinion: To date, clinical studies have demonstrated the safety of gene therapy in limb ischemia but the effectiveness of this treatment has not been determined. Larger clinical studies, as well as the development of more effective gene therapy, are needed to achieve and confirm beneficial effects.     

Nitric oxide synthase gene therapy: progress and prospects

NOS gene therapy has been the focus of extensive research as dysfunction of this enzyme has been implicated in several cardiovascular diseases. Research has concentrated on comparing the effect of gene delivery of NOS isoforms (eNOS, iNOS and nNOS) in healthy and diseased animal models on intimal hyperplasia, restenosis, vascular tone and ischemia-reperfusion injury. Most results demonstrate therapeutic benefits following vascular gene delivery of all NOS in pre-clinical models of cardiovascular disease. eNOS has been shown to have particular promise as it promotes re-endothelialisation and inhibits intimal hyperplasia in injured blood vessels. The ultimate goal is to translate the benefit of NOS gene therapy in animal models into clinical practise. To develop NOS gene therapy for clinical use further work needs to be undertaken to improve delivery systems and vectors to minimise detrimental side-effects and enhance positive treatment outcomes. This review focuses on current research on NOS gene therapy in cardiovascular disease and identifies the next steps that would be necessary to lead to clinical trials.     

肝癌基因治疗的基础研究与临床应用

肝癌基因治疗发展至今已有30余年,基因治疗的策略、载体和治疗基因随着科学的发展不断更新。近年来由于基础研究的重要突破,肝癌基因治疗逐步由基础研究走向临床治疗,并在临床实践过程中不断积累经验,不断成熟。本文将结合自身工作就肝癌基因治疗的基础研究与临床应用作一简短综述。     

New Generation of Plasmid Backbones Devoid of Antibiotic Resistance Marker for Gene Therapy Trials

Since it has been established that the injection of plasmid DNA can lead to an efficient expression of a specific protein in vivo, nonviral gene therapy approaches have been considerably improved, allowing clinical trials. However, the use of antibiotic resistance genes as selection markers for plasmid production raises safety concerns which are often pointed out by the regulatory authorities. Indeed, a horizontal gene transfer to patient''s bacteria cannot be excluded, and residual antibiotic in the final product could provoke allergic reactions in sensitive individuals. A new generation of plasmid backbones devoid of antibiotic resistance marker has emerged to increase the safety profile of nonviral gene therapy trials. This article reviews the existing strategies for plasmid maintenance and, in particular, those that do not require the use of antibiotic resistance genes. They are based either on the complementation of auxotrophic strain, toxin–antitoxin systems, operator–repressor titration, RNA markers, or on the overexpression of a growth essential gene. Minicircles that allow removing of the antibiotic resistance gene from the initial vector will also be discussed. Furthermore, reported use of antibiotic-free plasmids in preclinical or clinical studies will be listed to provide a comprehensive view of these innovative technologies.     

Gene therapy in orthopaedics

Genes are composed of deoxyribonucleic acid (DNA), the hereditary material of all nucleated cells. One way in which genes function is to direct the synthesis of specific proteins. When a gene is transferred to and expressed within a cell, the recipient cell produces the protein encoded by the transferred gene. This process forms the basis for gene therapy, which can be defined as the transfer of genes to patients for therapeutic purposes. Both genetic and acquired disorders may be treated, or even cured, by gene therapy. Potential orthopaedic applications include the treatment of arthritis, tumors, osteoporosis, and genetic diseases such as osteogenesis imperfecta, as well as the enhancement of tissue repair and regeneration. Impressive preclinical progress has been made in several of these areas. A phase I clinical trial of gene therapy for rheumatoid arthritis has just been completed. Orthopaedic gene therapy should become a clinical reality during the next decade.     

Gene therapy for Parkinson's disease: from non-human primates to humans

Gene therapy strategies in non-human primate models of Parkinson's disease (PD) are beginning to produce results consistently, and have been successfully translated to clinical trials. Although not all of the therapeutic efforts based on gene therapy have demonstrated clinical efficacy, the stereotactic techniques and at least three different beneficial genes that have been delivered to patients have been proven to be safe. The adeno-associated virus has been used as an effective and safe delivery vehicle for the first three, single therapeutic transgenes (ie, glutamic acid decarboxylase, aromatic l-amino acid decarboxylase, and neurturin) to be tested in trials. In addition, the larger lentivirus, which has been used for the codelivery of up to three therapeutic genes in parkinsonian non-human primates, has also being used in a trial in humans. Additional preclinical and clinical research is required to advance the understanding of PD and its potential treatments. Gene therapy, however, has the potential to be a safe and effective therapeutic option for an increasing number of patients with PD in the near future. In this review, the pertinent scientific research related to the use of gene therapy for the treatment of PD is summarized, with a particular focus on the accomplishments and challenges during the past 2 years.     

Cell-mediated and direct gene therapy for bone regeneration

INTRODUCTION: Bone regeneration is required for the treatment of fracture non/delayed-unions and bone defects. However, most current treatment modalities have limited efficacy, and newer therapeutic strategies, such as gene therapy, have substantial benefit for bone repair and regeneration. AREAS COVERED: This review discusses experimental and clinical applications of cell-mediated and direct gene therapy for bone regeneration. The review covers literature on this subject from 2000 to February 2012. EXPERT OPINION: Direct gene therapy using various viral and non-viral vectors of cell-mediated genes has been demonstrated to induce bone regeneration, although use of such vectors has shown some risk in human application. Osteoinductive capability of a number of progenitor cells isolated from bone marrow, fat, muscle and skin tissues, has been demonstrated by genetic modification with osteogenic genes. Cell-mediated gene therapy using such osteogenic gene-expressing progenitor cells has shown promising results in promoting bone regeneration in extensive animal work in recent years.     

Gene transfer to treat rheumatoid arthritis

Intraarticular transfer of exogenous genes could be useful for treatment of rheumatoid arthritis. The joints are not easy for drug accession while gene therapy can deliver therapeutic genes directly into the joints. Also, long-term expression of the transferred genes is expected. Anti-inflammatory genes, including interleukin-1 receptor antagonist gene, have been used in experimental arthritis models and in a clinical trial for treatment of rheumatoid arthritis. However, during gene therapy of other diseases, tragic accidents associated with viral vectors occurred. Since then, they have hampered clinical application of the gene therapy. Genes for treatment should be selected carefully and gene delivery methods should be optimized to resume clinical trials.     

Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy.

A long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) is showing promise for targeted gene delivery and systemic gene therapy. Lipoplexes directed by ligands such as folate, transferrin or anti-transferrin receptor scFv, showed tumor-targeted gene delivery and expression in human breast, prostate, head and neck cancers. The two elements, ligand/receptor and liposome composition, work together to realize the goal of functional tumor targeting of gene therapeutics. The tumor suppressor gene, p53, has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation. The systemically delivered ligand-liposome-p53 gene therapeutics resulted in efficient expression of functional wild-type p53, sensitizing the tumors to chemotherapy and radiotherapy. This is a novel strategy combining current molecular medicine with conventional chemotherapy and radiotherapy for the treatment of cancer. The systemic delivery of normal tumor suppressor gene p53 by a non-viral, tumor-targeted delivery system as a new therapeutic intervention has the potential to critically impact the clinical management of cancer.     

Gene therapy for Parkinson's disease using recombinant adeno-associated viral vectors

Existing strategies for gene therapy in the treatment of Parkinson's disease include the delivery of genes encoding dopamine (DA)-synthesising enzymes, leading to localised production of DA in the striatum; genes encoding factors that protect nigral neurons against ongoing degeneration, such as glial cell line-derived neurotrophic factor; and genes encoding proteins that produce the inhibitory transmitter gamma-aminobutylic acid (GABA) in the subthalamic nucleus (STN), thus suppressing the hyperactive STN. Recombinant adeno-associated viral (rAAV) vectors, which are derived from non-pathogenic viruses, have been shown to be suitable for clinical trials. These rAAVs have been found to transduce substantial numbers of neurons efficiently and to express transgenes in mammalian brains for long periods of time, with minimum inflammatory and immunological responses. In vivo imaging using positron emission tomography is useful for monitoring transgene expression and for assessing the functional effects of gene delivery. Vector systems that regulate transgene expression are necessary to increase safety in clinical applications, and the development of such systems is in progress.     

Gene therapy for Parkinson’s disease

Significant progress has been made in the field of gene therapy for Parkinson’s disease (PD). Successful vehicles for gene transfer into the central nervous system have been developed and clinical efficacy and safety have both been shown in various animal models of PD. Further optimisation of dosing, timing and location of gene therapy delivery as well as the ability to regulate and prolong gene expression will be important for the commencement of human trials. Current gene therapy models for PD have focused on two treatment strategies. One is the replacement of biosynthetic enzymes for dopamine synthesis and the second strategy is the addition of neurotrophic factors for protection and restoration of dopaminergic neurones. Concepts of neuroprotection and restoration of the nigrostriatal pathway will become important themes for future genetic treatment strategies for PD and may include, in addition to neurotrophic factors, genes to prevent apoptosis or detoxify free radical species. This review will highlight the recent literature on gene therapy for PD and summarise general approaches to gene therapy.     

Gene therapy for Parkinson's disease

Significant progress has been made in the field of gene therapy for Parkinson's disease (PD). Successful vehicles for gene transfer into the central nervous system have been developed and clinical efficacy and safety have both been shown in various animal models of PD. Further optimisation of dosing, timing and location of gene therapy delivery as well as the ability to regulate and prolong gene expression will be important for the commencement of human trials. Current gene therapy models for PD have focused on two treatment strategies. One is the replacement of biosynthetic enzymes for dopamine synthesis and the second strategy is the addition of neurotrophic factors for protection and restoration of dopaminergic neurones. Concepts of neuroprotection and restoration of the nigrostriatal pathway will become important themes for future genetic treatment strategies for PD and may include, in addition to neurotrophic factors, genes to prevent apoptosis or detoxify free radical species. This review will highlight the recent literature on gene therapy for PD and summarise general approaches to gene therapy.     

Update on the use of nonhuman primate models for preclinical testing of gene therapy approaches targeting hematopoietic cells.

Transfer of genes into hematopoietic stem cells or primary lymphocytes has been a primary focus of the gene therapy field for more than a decade because of the wide variety of congenital and acquired diseases that potentially could be cured by successful gene transfer into these cell populations. However, despite success in murine models and in vitro, progress has been slow, and early clinical trials were disappointing due to inefficient gene transfer into long-term repopulating cells. The unique predictive value of nonhuman primate or other large animal models has become more apparent, and major advances in gene transfer efficiency have been made by utilizing these powerful but expensive and complex systems. This review summarizes more recent findings from nonhuman primate investigations focusing on hematopoietic stem cells or lymphocytes as target populations, and highlights specific preclinical issues, including safety. Results from studies using standard retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors are discussed. Judicious application of these models should continue to be a priority, and advances should now be tested in proof-of-concept clinical trials.     

Kunjin virus replicons: an RNA-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications

The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the Australian flavivirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphavirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8+ T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced 'danger signals'. The identification of KUN replicon mutants that induce increased levels of IFN-alpha/beta has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications.     

Gene therapy for relapsed breast cancer

Gene therapy for advanced breast cancer is anticipated to be a useful therapeutic approach. Strategies in ongoing clinical protocols can be divided into four groups: (1) suppression of oncogenes or transfer of tumor-suppressor genes: (2) enhancement of immunological response: (3) transfer of suicide genes: (4) protection of bone marrow using drug resistance genes. We have started a clinical study of multidrug resistance (MDR1) gene therapy. Patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation (PBSCT) with MDR1-transduced hematopoietic cells, and then were treated with docetaxel. Three patients have been treated so far, and in vivo enrichment of MDR1-transduced cells with docetaxel treatment has been seen. There has been no apparent adverse effect from the MDR1 gene transfer.     

Electrotransfer of therapeutic molecules into tissues

Electroporation is a physical method for the delivery of various molecules into cells by application of controlled external electrical fields that transiently increase permeability of the cell membrane. This technique is now widely used as an alternative to viral gene delivery for transfection of therapeutic genes into different tissues. Gene electrotransfer holds great potential for clinical application due to the ease of preparation of large quantities of endotoxin-free plasmid DNA, the control and reproducibility of this method, and the development of electric pulse generators approved for clinical use. Electroporation has been utilized mainly for DNA vaccination against infectious diseases and cancer. It has also been used for the delivery of other therapeutic genes, mainly cytokines, used in the treatment of various diseases, including cancer, arthritis, multiple sclerosis and inflammation, following organ transplantation. Electroporation as a delivery system for chemotherapeutic drugs, termed antitumor electrochemotherapy, is already at the clinical stage and is being used routinely in several oncology centers in Europe. In addition, the first clinical trials for electrogene therapy of cancer are ongoing. Therefore, it can be presumed that electrotransfer of therapeutic genes into tissues will soon form a validated alternative to viral delivery systems in a clinical setting.