Recent advances in gene therapy and future prospects

Gene therapy, which is treatment of diseases by introducing normal genes into the body, is becoming feasible as the result of advances in genetic engineering. The hematopoietic stem cells have been considered as the appropriate target for gene transfer in many genetic diseases for which allogeneic bone marrow transplantation has been employed successfully. However, there are still many problems to be solved. In particular, expression from retrovirally transduced genes in bone marrow cells has been transient and unstable. On the other hand, an alternative approach to somatic cell gene therapy using nonhematopoietic cells, including skin fibroblasts, endothelial cells, keratinocytes, and lymphocytes, has been shown to possess several advantages. This kind of approach is usually applied to supplementation therapy in not only hereditary disorders but also various acquired diseases, such as cancer or infectious diseases. Recently, clinical application of gene transfer into lymphocytes to treat cancer and immunodeficiency have been approved at NIH (USA). The trial could represent the start of a new era in molecular medicine.     

Regulatable systemic production of monoclonal antibodies by <Emphasis Type="Italic">in vivo</Emphasis> muscle electroporation

The clinical application of monoclonal antibodies (mAbs) potentially concerns a wide range of diseases including, among others, viral infections, cancer and autoimmune diseases. Although intravenous infusion appears to be the simplest and most obvious mode of administration, it is very often not applicable to long-term treatments because of the restrictive cost of mAbs certified for human use and the side effects associated with injection of massive doses of antibodies. Gene/cell therapies designed for sustained and, possibly, regulatable in vivo production and systemic delivery of mAbs might permit to advantageously replace it. We have already shown that several such approaches allow month- to year-long ectopic antibody production by non-B cells in living organisms. Those include grafting of ex vivo genetically modified cells of various types, in vivo adenoviral gene transfer and implantation of encapsulated antibody-producing cells. Because intramuscular electrotransfer of naked DNA has already been used for in vivo production of a variety of proteins, we have wanted to test whether it could be adapted to that of ectopic mAbs as well. We report here that this is actually the case since both long-term and regulatable production of an ectopic mAb could be obtained in the mouse taken as a model animal. Although serum antibody concentrations obtained were relatively low, these data are encouraging in the perspective of future therapeutical applications of this technology in mAb-based immunotherapies, especially in developing countries where cost-effective and easily implementable technologies would be required for large-scale applications in the context of severe chronic viral diseases such as HIV and HCV infections.     

Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases?

There are 50 ways to leave your lover (Simon 1987) but many more to kill your brain cells. Several neurodegenerative diseases in humans, like Alzheimer’s disease, have been intensely studied but the underlying cellular and molecular mechanisms are still unknown for most of them. For those syndromes where associated gene products have been identified their biochemistry and physiological as well as pathogenic function is often still under debate. This is in part due to the inherent limitations of genetic analyses in humans and other mammals and therefore experimentally accessible invertebrate in vivo models, such as Caenorhabditis elegans and Drosophila melanogaster, have recently been introduced to investigate neurodegenerative syndromes. Several laboratories have used transgenic approaches in Drosophila to study the human genes associated with neurodegenerative diseases. This has added substantially to our understanding of the mechanisms leading to neurodegenerative diseases in humans. The isolation and characterization of Drosophila mutants, which display a variety of neurodegenerative phenotypes, also provide valuable insights into genes, pathways, and mechanisms causing neurodegeneration. So far only about two dozen such mutants have been described but already their characterization reveals an involvement of various cellular functions in neurodegeneration, ranging from preventing oxidative stress to RNA editing. Some of the isolated genes can already be associated with human neurodegenerative diseases and hopefully the isolation and characterization of more of these mutants, together with an analysis of homologous genes in vertebrate models, will provide insights into the genetic and molecular basis of human neurodegenerative diseases.     

Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

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.     

Perspectives on methylmalonic acidemia resulting from molecular cloning of methylmalonyl CoA mutase

Methylmalonyl CoA mutase deficiency (methylmalonic acidemia) has been a paradigm for biochemical and somatic cell genetic approaches to human disease. Recently, genes encoding this enzyme have been cloned from several species. These studies have provided information about the primary structure and evolution of this enzyme, the mutations which underlie its deficiency state, and the structure-function determinants which are required for its activity. Gene transfer studies now permit restitution of this enzyme to genetically deficient cells and may enable somatic gene therapy to be undertaken. Molecular genetic studies not only provide more detailed information about this enzyme, but introduce new perspectives on the molecular mechanisms and dynamics of its function and raise new questions about the dyshomeostatic consequences of its deficiency.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

诱导性多潜能干细胞(iPS cells)——现状及前景展望

主要从 iPS细胞发展历程、获得 iPS细胞的几个关键步骤 (如基因导入方式、诱导 iPS细胞所需因子组合与小分子化合物运用和体细胞种类选择等)、病人或疾病特异性 iPS细胞、iPS细胞体内外诱导分化与其衍生物的临床应用和制备无遗传修饰的(genetic modification-free) iPS细胞的可行性与前景等方面对 iPS细胞最新研究进展做评述．日本和美国研究小组先后用4种基因将小鼠(2006年8月)和人(2007年11～12月)的体细胞在体外重编程为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells),此后在短短两年多时间内,iPS 细胞的研究和关注度呈爆炸式增长．体细胞重编程、去分化和多潜能干细胞来源等一系列热点问题再次成为干细胞和发育生物学等研究的热点和焦点．与胚胎干细胞(embryonic stem cells,ES cells)一样,iPS细胞在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官．迄今,在体外已由 iPS细胞定向诱导分化出功能性的多种成熟细胞．因此,iPS细胞研究不仅具有重要理论意义,而且在再生医学、组织工程和药物发现与评价等方面极具应用价值．     

Gene therapy for lipid disorders

Lipid disorders are associated with atherosclerotic vascular disease, and therapy is associated with a substantial reduction in cardiovascular events. Current approaches to the treatment of lipid disorders are ineffective in a substantial number of patients. New therapies for refractory hypercholesterolemia, severe hypertriglyceridemia, and low levels of high-density lipoprotein cholesterol are needed: somatic gene therapy is one viable approach. The molecular etiology and pathophysiology of most of the candidate diseases are well understood. Animal models exist for the diseases and in many cases preclinical proof-of-principle studies have already been performed. There has been progress in the development of vectors that provide long-term gene expression. New clinical gene therapy trials for lipid disorders are likely to be initiated within the next few years.     

Mapping Approaches to Gene Identification in Humans

Although a number of human genes that cause disease have been traced through the defective product, most genetic defects are recognized only by phenotype. When the biochemical defect is unknown, a gene can be located only through molecular approaches based on coinheritance (genetic linkage) of the disease phenotype with a particular allele of a polymorphic DNA marker that has already been mapped to a specific chromosomal region. Linkage studies in affected families have already localized genes for several important diseases, including cystic fibrosis. Finding a genetic linkage in families in which a disease segregates requires that the human genetic map have a large number of polymorphic markers; when the map is dense enough, any disease gene can be located by linkage to a known marker. Many DNA segments with a high degree of polymorphism are being found and mapped as markers in normal reference pedigrees. Genetic linkage mapping has implications even broader than its application to prenatal diagnosis or therapeutic strategy; analyzing mutations in important genes will illuminate basic mechanisms in molecular biology and the early events that lead to cancer and other disorders.     

Reporter genes in transgenic mice

Althoughin vivo models utilizing endogenous reporter genes have been exploited for many years, the use of reporter transgenes to dissect biological issues in transgenic animals has been a relatively recent development. These transgenes are often, but not always, of prokaryotic origin and encode products not normally associated with eukaryotic cells and tissues. Some encode enzymes whose activities are detected in cell and tissue homogenates, whereas others encode products that can be detectedin situ at the single cell level. Reporter genes have been used to identify regulatory elements that are important for tissue-specific gene expression or for development; they have been used to producein vivo models of cancer; they have been employed for the study ofin vivo mutagenesis; and they have been used as a tool in lineage analysis and for marking cells in transplanation experiments. The most commonly usedin situ reporter gene islacZ, which encodes a bacterial -galactosidase, a sensitive histochemical marker. Although it has been used with striking success in cultured cells and in transgenic mouse embryos, its postnatalin vivo expression has been unreliable and disappointing. Nevertheless, the ability to express reporter genes in transgenic mice has been an invaluable resource, providing insights intoin vivo biological mechanisms. The development of newin vivo models, such as those in which expression of transgenes can be activated or repressed, should produce transgenic animal systems that extend our capacity to address heretofore unresolved biological questions.     

Animal models of gene therapy

Somatic gene therapies are based on the introduction of genes in somatic cells in an attempt to correct a gene defect, to induce a resistance or to add a particular activity. In their principle, they are not very different from organ grafts and do not set specific ethic problems. Their application to human therapy has to be subjected to a critical evaluation of their harmlessness and efficiency. For this purpose, animal models of somatic gene therapy are essential. Such therapy have been tried in bone marrow and endothelial cells, in fibroblasts, keratinocytes hepatocytes, but also by direct transfer of genes in the organism. These different approaches are briefly reviewed and compared in this article.     

Fromin vitro toin vivo. Progress in the use of cultured cells for human therapy

The use of cultured cells with the ultimate goal of using the cells or their products for human therapy has experienced an exponential growth during the last decade. Stable cell cultures have been established and genetically modified to obtain high quality products for protein replacement therapy or vaccines. Cells have also been directly isolated from the human organism and, after their expansionin vitro, been retransferred as skin grafts for treatment of burns or for cancer therapy by activated lymphocytes. With the explosive development of molecular biology techniques, it is now possible to genetically modifyex vivo, cells derived from the human body. These modifications should allow targeted expression of therapeutic genes into specific cells which will, upon retransfer to the body, exert their therapeutic action in a diseased organism.Abbreviations ADA adenosine deaminase - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN interferon - IL interleukin - TIL tumor infiltrating lymphocytes     

Multimodality molecular imaging of disease progression in living subjects

The enormous advances in our understanding of the progression of diseases at the molecular level have been supplemented by the new field of ‘molecular imaging’, which provides for in vivo visualization of molecular events at the cellular level in living organisms. Molecular imaging is a noninvasive assessment of gene and protein function, protein–protein interaction and/or signal transduction pathways in animal models of human disease and in patients to provide insights into molecular pathogenesis. Five major imaging techniques are currently available to assess the structural and functional alterations in vivo in small animals. These are (i) optical bioluminescence and fluorescence imaging techniques, (ii) radionuclide-based positron emission tomography (PET) and single photon emitted computed tomography (SPECT), (iii) X-ray-based computed tomography (CT), (iv) magnetic resonance imaging (MRI) and (v) ultrasound imaging (US). Functional molecular imaging requires an imaging probe that is specific for a given molecular event. In preclinical imaging, involving small animal models, the imaging probe could be an element of a direct (‘direct imaging’) or an indirect (‘indirect imaging’) event. Reporter genes are essential for indirect imaging and provide a general integrated platform for many different applications. Applications of multimodality imaging using combinations of bioluminescent, fluorescent and PET reporter genes in unified fusion vectors developed by us for recording events from single live cells to whole animals with high sensitivity and accurate quantification are discussed. Such approaches have immense potential to track progression of metastasis, immune cell trafficking, stem cell therapy, transgenic animals and even molecular interactions in living subjects.     

酶类药物现状、问题及展望

随着生物制药的迅速发展,许多酶类药物应运而生,在治疗代谢疾病、心血管疾病、癌症等诸多疾病上发挥着越来越重要的作用。但是酶类药物也存在一些不足,如潜在的免疫原性、较短的体内半衰期,以及较差的组织靶向性,影响了酶类药物的疗效和应用。为克服这些缺点,人们已开发出多种技术,如通过糖基化、聚乙二醇修饰等分子工程技术提升酶蛋白药效,另一方面酶基因疗法也已成功用于多种酶缺陷疾病的治疗。基于酶类药物的迅速发展和广泛的应用前景,本文对酶类药物的现状进行较详细的阐述,并对酶类药物的优势、所存在的问题及未来发展趋势进行分析和评述。     

Primary immune deficiencies unravel the molecular basis of immune response

Primary immune deficiencies (PID) represent inborn errors of immunity. Over the years, detailed analysis of the clinical and laboratory features associated with these unique and rare disorders have shed light on the complex array of signals and processes that govern development and activation of the immune system. While the first examples of PID pertained to severe defects in lymphoid development, more recently a variety of gene defects have been identified in humans that do not compromize the ability to generate lymphocytes, but rather result in profound immune dysregulation. In many cases, identification of the molecular and cellular bases of PID has preceeded development of animal models by gene targeting. Finally, since the very first cases reported in humans, PID have also represented a unique tool to investigate the efficacy of novel therapeutic approaches (from molecular therapy to hematopoietic stem cell transplantation to somatic cells gene therapy), that have been applied or may apply to a variety of more common human diseases.     

The human genome project--some implications of extensive "reverse genetic" medicine

Impressive progress has been made during the past several decades in understanding the pathogenesis of human genetic disease. The tools of molecular biology have allowed the isolation of many disease-related genes by forward and a few by reverse genetics, and the imminent completion of a complete human genetic linkage map will accelerate the genetic characterization of many more genetic diseases. The major impacts of the molecular characterization of human genetic diseases will be 1. To increase markedly the number of human diseases that we recognize to have major genetic components. We already understand that genetic diseases are not rare medical curiosities with negligible societal impact, but rather constitute a wide spectrum of both rare and extremely common diseases responsible for an immense amount of suffering in all human societies. The characterization of the human genome will lead to the identification of genetic factors in many more human diseases, even those that now seem too multifactorial or polygenic for ready understanding. 2. To allow the development of powerful new approaches to diagnosis, detection, screening and even therapy of these disorders aimed directly at the mutant genes rather than at the gene products. This should eventually allow much more accurate and specific management of human genetic disease and the genetic factors in many human maladies. The preparation of a fine-structure physical map of the entire human genome together with an overlapping contiguous set of clones spanning entire chromosomes or large portions of chromosomes is rapidly becoming feasible, and the information that will flow from this effort promises eventually to affect the management of many important genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)