Complementation cell lines for viral vectors to be used in gene therapy

Viral vectors provide a highly efficient method for the transfer of foreign genes into a variety of quiescent or dividing eukaryotic cells from many animal origins. While recombinant vectors derived from an increasing number of mammalian viruses (herpes simplex virus, autonomous and non-autonomous parvoviruses, poxviruses, retroviruses, adenoviruses available today, vectors based on murine retroviruses and human adenoviruses constitute preferential candidates for the delivery of marker or therapeutic genes into human somatic cells. The availability of such vectors has made possible the recent transition of human gene therapy from laboratory benches to clinical settings. Most current recombinant vectors have been generated by deleting essential viral genes in order to make space available for the introduction of passenger genes. Such vectors are therefore unable to replicate in the absence of these critical gene products and their production relies on the development of stable complementation cell lines providingin trans the missing viral functions. Although complementation (or packaging) cell lines are available for both adenovirus and retrovirus vectors, their respective drawbacks still limit their use to research applications and phase I clinical trials. The future success or failure of human gene therapy will therefore rely on the production of improved generations of packaging cell lines that can produce safer and more efficient vectors which are fully adapted to large scale production and clinical applications.     

Retroviral vectors

The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology.     

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.     

Prospects for virus-based gene therapy for cystic fibrosis

Gene therapy for cystic fibrosis (CF) could potentially be accomplished with one of several recombinant virus vectors, including a murine retrovirus (MMuLV), adenovirus, or adeno-associated virus (AAV). All these vectors take advantage of their respective viruses' mechanisms for delivery of viral DNA to cells, evasion of lyosomal degradation, and optimization of the levels and duration of expression of viral (or vector) DNA. Each has its own unique life cycle, however. The differences among these viruses result in certain advantages and disadvantages, such as the requirement of retroviruses for active cell division, and the potential pathogenic effects from expression of certain adenovirus genes present in adenovectors. While no single vector may be optimal for CF gene therapy in humans, new techniques, such as receptor-mediated gene transfer, seek to take advantage of the desirable properties of one or more of the virus-based systems while avoiding certain potential hazards.     

Adeno-associated viral vectors for gene transfer and gene therapy.

Adeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression in vivo without immune response or toxicity. Over the past few years, many applications of rAAVs as therapeutic agents have demonstrated the utility of this vector system for long-lasting genetic modification and gene therapy in preclinical models of human disease. New production methods have increased rAAV vector titers and eliminated contamination by adenovirus. In addition, vectors for regulatable gene expression and vectors retargeted to different cells have been engineered. These advancements are expected to accelerate and facilitate further animal model studies, providing validation for use of rAAVs in human clinical trials.     

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics

Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.     

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

Gene therapy: the first two decades and the current state-of-the-art

The concept of gene therapy was envisioned soon after the emergence of restriction endonucleases and subcloning of mammalian genes in phage and plasmids. Over the ensuing decades, vectors were developed, including nonviral methods, integrating virus vectors (gammaretrovirus and lentivirus), and non-integrating virus vectors (adenovirus, adeno-associated virus, and herpes simplex virus vectors). Preclinical data demonstrated potential efficacy in a broad range of animal models of human diseases, but clinical efficacy in humans remained elusive in most cases, even after decades of experience in over 1000 trials. Adverse effects from gene therapy have been observed in some cases, often because of viral vectors retaining some of the pathogenic potential of the viruses upon which they are based. Later generation vectors have been developed in which the safety and/or the efficiency of gene transfer has been improved. Most recently this work has involved alterations of vector envelope or capsid proteins either by insertion of ligands to target specific receptors or by directed evolution. The disease targets for gene therapy are multiple, but the most promising data have come from monogenic disorders. As the number of potential targets for gene therapy continues to increase, and a substantial number of trials continue with both the standard and the later generation vector systems, it is hoped that a therapeutic niche for gene therapy will emerge in the coming decades.     

Reverse genetics of negative-stranded RNA viruses: a global perspective

The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. The fundamental insights gleaned from the reverse genetics-based studies over the last several years have provided a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. The recombinant viruses have been exploited also as vectors for devising targeted therapies for non-viral diseases such as malignancies, and in gene therapy for inherited disorders. This review provides a brief summary of the stumbling blocks and the successes in the development of the technology for the negative-stranded RNA viruses. The many and varied applications of the recombinant vectors are also outlined.     

Artificial and engineered chromosomes: developments and prospects for gene therapy

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals.     

Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer

Gene therapy, the correction of dysfunctional or deleted genes by supplying the lacking component, has long been awaited as a means to permanently treat or reverse many genetic disorders. To achieve this, therapeutic DNA must be delivered to the nucleus of cells using a safe and efficient delivery vector. Although viral-based vectors have been utilized extensively due to their innate ability to deliver DNA to intact cells, safety considerations, such as pathogenicity, oncogenicity and the stimulation of an immunological response in the host, remain problematical. There has, however, been much progress in the development of safe non-viral gene-delivery vectors, although they remain less efficient than the viral counterparts. The major limitations of non-viral gene transfer reside in the fact that it must be tailored to overcome the intracellular barriers to DNA delivery that viruses already master, including the cellular and nuclear membranes. In particular, nuclear transport of the therapeutic DNA is known to be the rate-limiting step in the gene-delivery process. Despite this, much progress had been made in recent years in developing novel means to overcome these barriers and efficiently deliver DNA to the nuclei of intact cells. This review focuses on the nucleocytoplasmic delivery of DNA and mechanisms to enhance to non-viral-mediated gene transfer.     

Adeno-associated virus-mediated gene delivery approaches for the treatment of CNS disorders

Over the last few years, a large number of preclinical and clinical studies have demonstrated the potential of gene therapy applications using adeno-associated viral (AAV) vectors. Gene transfer via AAV vectors has been particularly successful for the treatment or adjunct therapy of several CNS disorders. The present review summarizes the progress on AAV gene delivery models for three different CNS disorders. In particular, we discuss advances in AAV-mediated gene transfer strategies in animal models of Parkinson's disease, Alzheimer's disease and spinal cord trauma and summarize the results from the first clinical studies using AAV systems.     

Recombinant gene expression in human umbilical vein endothelial cells transduced by retroviral vectors

Endothelial cells are attractive targets for gene transfer because of their immediate contact with the bloodstream, and, therefore, they might serve as vehicles for therapeutic drug delivery. Recently, we and others reported that endothelial cells of animal origin efficiently express both secretory and nonsecretory recombinant proteins. We now show that human endothelial cells are also capable of expressing a recombinant gene following transduction with retroviral vectors. Human umbilical vein endothelial cells were transduced with either the N2 or the SAX vector. Following selection with G418, cells transduced by both vectors were found to express neophosphotransferase activity, the product of the neomycin resistance gene. The fact that a recombinant gene can be readily inserted and efficiently expressed into human endothelial cells suggests that these cells may be able to serve a role in human gene therapy.     

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.     

Gene therapy of neoplastic liver diseases

Since advanced liver cancer lacks effective therapy in most cases, a considerable interest has been drawn towards gene therapy. Natural or chimerical genes can be transferred to the tumour itself, the non-tumoral liver, or even distant tissues using a variety of vectors administered by intratumoral or intravascular routes. The desired selectivity in gene expression can be achieved by increasing the specificity of gene delivery or by controlling gene expression with tumour-specific promoters, such as alpha-fetoprotein or carcinoembryonic antigen. There are two main approaches to gene therapy of liver cancer aiming at killing directly malignant cells or at improving the host's defensive systems, respectively. The former include replacing the lost function of tumour suppressor genes, inhibiting the action of activated oncogenes, sensitising tumour cells to prodrugs, or infecting the tumoral tissue with viruses that replicate selectively in cancer cells. Host defences can be improved by stimulating the antitumoral immune response, or by interfering with tumour vessel formation. Progress in gene therapy of liver cancer depends very much on information collected from well-designed clinical trials. This information includes knowledge of whether an efficient gene transfer has been achieved and what is the duration and magnitude of gene expression in the transduced tissues. Hopefully, magnetic resonance or positron emission tomography (PET) may turn out to be reliable procedures for tracing transgene expression in humans. Pre-clinical evidence and early clinical trials strongly suggest that there is a place for gene therapy of liver malignancies.     

Engineering chromosomes for delivery of therapeutic genes

The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.     

The emerging fields of suicide gene therapy and virotherapy

Gene therapy is defined as a technology aimed at modifying the genetic component of cells for therapeutic benefit. ‘Suicide genes’ can be introduced into cancer cells to make them more sensitive to chemotherapeutics or toxins. Chemotherapeutic suicide gene therapy approaches are known as gene-directed enzyme prodrug therapy or gene-prodrug activation therapy. Other approaches include replacement gene therapy, antisense strategies and induction of resistance to normal cells. All gene therapy strategies share a common component, which is the need for a selective delivery vehicle or vector with tumor-targeting capabilities. This need has led to the in-depth investigation of viruses as new vectors for gene therapy.     

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.     

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin Ⅰ,or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.     

Gene therapy using retroviral vectors

Gene therapy is a novel approach for treating various congenital and acquired genetic disorders, including cancer, heart disease, and acquired immune deficiency syndrome. Amongst possible gene delivery systems, retroviral vector mediated gene transfer has been the most extensively studied and has been approved for use in over 40 current Phase I/II clinical trials for the treatment of various disorders, primarily cancers. Recent technological improvements include the optimization of vector production by concentration and lyophilization, resulting in high titers of vectors, as well as the large-scale production of vector-produced cells for the treatment of brain cancer. Present clinical protocols require specialized care centers with expertise in molecular biology and cell transplantation. Considerable effort is under way to develop retroviral vectors that can be both injected directly into the body and targeted to specific cell types within the body. Such vectors could be administered to patients by physicians in their offices. Successful development of this new technology would greatly expand the clinical potential of gene therapy.