Gene therapy for diseases of the nervous system.

Advances in molecular biology and recombinant DNA technologies have contributed to our understanding of the molecular basis of many diseases. Now the possibility of gene transfer into normal cells to produce a gene product of therapeutic potential, or into diseased cells to correct the pathologic alteration, promises to revolutionize medical practice. In contemporary medicine, many therapeutic strategies focus on the link between a biochemical deficiency and the ensuing disorder. The treatment of noninfectious disease is often based on replacement therapy; medication is given to compensate for biochemical defects and to prevent or reverse the progression of disease. Although conventional therapies seldom alter the fundamental cause of a disease, gene therapy potentially could correct, at a molecular level, the genetic abnormalities contributing to its pathogenesis. Treatment directed at specific molecular alterations associated with the development of neurologic disease provides expectations of more effective and less toxic therapy. The development of gene therapy for nervous system tumors has progressed rapidly and may be prototypical in the development of therapies for inherited and acquired disorders of the nervous system. We describe possible strategies for using gene therapy to treat nervous system disorders, and we review recent advances in gene therapy for nervous system tumors.     

The application of DNA repair vectors to gene therapy

The nature of DNA, the sequence of the human genome and our increased understanding of the genetic basis of many inherited and acquired disorders have made the possibility of curing diseases a reality. The modulation of a host's genome is now the ultimate goal in the treatment of genetic diseases. Historically, gene therapy recognized two very different approaches: gene replacement or augmentation and gene repair. Gene repair precisely targets and corrects the chromosomal mutation responsible for a genetic and/or acquired disorder. Many recent advances have been made in this area of research.     

Genetics of colon cancer.

Strikingly rapid advances in the identification of genetic events that are important in colonic carcinogenesis have been made in the past several years. Specific inherited (adenomatous polyposis coli gene) and acquired (ras gene point mutations; c-myc gene amplification; allelic deletion at specific sites on chromosomes 5, 17, and 18) genetic abnormalities appear to be capable of mediating steps in the progression from normal to malignant colonic mucosa. Understanding these genetic factors and how they influence cellular function will have a profound effect on medical practice. High-risk populations will be (and are being) identified by genetic markers, thus allowing prevention and screening to be more precisely targeted to the population at risk; intervention strategies will be designed on the basis of the known cellular defects of neoplastic colonic mucosa; and new molecular preventive and therapeutic approaches can be developed.     

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep–wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.     

From genotype to phenotype: genetics and medical practice in the new millennium

The completion of the human genome project will provide a vast amount of information about human genetic diversity. One of the major challenges for the medical sciences will be to relate genotype to phenotype. Over recent years considerable progress has been made in relating the molecular pathology of monogenic diseases to the associated clinical phenotypes. Studies of the inherited disorders of haemoglobin, notably the thalassaemias, have shown how even in these, the simplest of monogenic diseases, there is remarkable complexity with respect to their phenotypic expression. Although studies of other monogenic diseases are less far advanced, it is clear that the same level of complexity will exist. This information provides some indication of the difficulties that will be met when trying to define the genes that are involved in common multigenic disorders and, in particular, in trying to relate disease phenotypes to the complex interactions between many genes and multiple environmental factors.     

The genetics of idiopathic generalized epilepsy: implications for the understanding of its aetiology

Epilepsy is one of the most common neurological disorders. Both inherited and acquired factors contribute to its multifactorial pathogenesis. A genetic predisposition plays a major role in the aetiology of the common idiopathic generalized epilepsies. Susceptibility genes for two syndromes of idiopathic generalized epilepsies, the benign familial neonatal convulsions and juvenile myoclonic epilepsy, have been assigned to the chromosomal regions 20q13 (EBN1), 8q24 (EBN2) and 6p21 (EJM1). Positional cloning of the mutations causing these traits will help to elucidate the molecular pathways of epileptogenesis and will imply a classification on a neurobiological basis. Insights into the underlying impairment of neuronal excitability should provide new concepts for the development of rational treatment strategies.     

Toll-like receptors are key participants in innate immune responses

During an infection, one of the principal challenges for the host is to detect the pathogen and activate a rapid defensive response. The Toll-like family of receptors (TLRs), among other pattern recognition receptors (PRR), performs this detection process in vertebrate and invertebrate organisms. These type I transmembrane receptors identify microbial conserved structures or pathogen-associated molecular patterns (PAMPs). Recognition of microbial components by TLRs initiates signaling transduction pathways that induce gene expression. These gene products regulate innate immune responses and further develop an antigen-specific acquired immunity. TLR signaling pathways are regulated by intracellular adaptor molecules, such as MyD88, TIRAP/Mal, between others that provide specificity of individual TLR- mediated signaling pathways. TLR-mediated activation of innate immunity is involved not only in host defense against pathogens but also in immune disorders. The involvement of TLR-mediated pathways in auto-immune and inflammatory diseases is described in this review article.     

Genetic, cellular and immune approaches to disease therapy: past and future

Advances in immunology and molecular genetics have accelerated our understanding of the genetic and cellular basis of many diseases. At the same time, remarkable progress in recombinant DNA technology has enabled the development of molecular and cellular treatments for infectious diseases, inherited disorders and cancer. This Perspective is intended to give a sample of the progress over the past ten years in cellular, genetic and immune therapy of disease. During this time, monoclonal antibody technology and cellular transplantation have begun to come of age in biomedicine. Innovations in gene delivery have not only catalyzed the nascent field of human gene therapy, but may also ultimately impact human health by advancing recombinant vaccine technology.     

Molecular genetics of tyrosine 3-monooxygenase and inherited diseases

Tyrosine 3-monooxygenase (tyrosine hydroxylase, TH) catalyzes the initial and rate-limiting step in the catecholamine biosynthesis. Alteration in TH activity is involved in the pathogenesis of certain disorders derived from catecholaminergic dysfunction. In the present review, we focus on recent advances in molecular genetic study of TH function and inherited diseases. Knockout mice lacking TH gene show severe catecholamine depletion and perinatal lethality. Mice heterozygous for the TH mutation exhibit defects in some neuropsychological functions. Dopamine-deficient mice impair motor control and operant learning during postnatal development. In addition, some point mutations in the human TH gene underlie the inherited diseases, including the recessive form of L-DOPA-responsive dystonia, parkinsonism in infancy, or progressive encephalopathy. These mutations indeed appear to reduce TH activity or influence expression of TH protein. Advances in molecular genetic studies provide a deeper understanding of the relationship between the alteration in TH activity and the pathology of catecholaminergic systems.     

Introductory Lecture: Genetic Approaches to CNS Disorders with Particular

Abstract

The tools of molecular biology will bring the field of human genetics into a new era by permitting the analysis of the genetic contribution to disease. Most single gene disorders, inherited in a Mendelian fashion, will be molecularly diagnosed. In addition, the genetic susceptibility of common, complex diseases such a schizophrenia can be clarified, even though the conditions are not inherited as Mendelian characteristics. The mapping of the human genome will increase the rate at which new disease genes are identified and isolated. Finally, the development of genetically engineered animal models will help to dissect the steps involved in physiological and pathophysiological processes and thereby enhance our understanding of complex biological systems.     

Molecular Therapy and Prevention of Liver Diseases

Molecular analyses have become an integral part of biomedical research as well as clinical medicine. The definition of the genetic basis of many human diseases has led to a better understanding of their pathogenesis and has in addition offered new perspectives for their diagnosis, therapy and prevention. Genetically, human diseases can be classified as hereditary monogenic, acquired monogenic and polygenic diseases. Based on this classification, gene therapy is based on six concepts (1) gene repair, (2) gene substitution, (3) cell therapy, (4) block of gene expression or function, (5) DNA vaccination and (6) gene augmentation. While major advances have been made in all areas of gene therapy during the last years, various delivery, targeting and safety issues need to be addressed before these strategies will enter clinical practice. Nevertheless, gene therapy will eventually become part of the management of patients with various liver diseases, complementing or replacing existing therapeutic and preventive strategies.     

Molecular therapy and prevention of liver diseases

Molecular analyses have become an integral part of biomedical research as well as clinical medicine. The definition of the genetic basis of many human diseases has led to a better understanding of their pathogenesis and has in addition offered new perspectives for their diagnosis, therapy and prevention. Genetically, human diseases can be classified as hereditary monogenic, acquired monogenic and polygenic diseases. Based on this classification, gene therapy is based on six concepts: (1) gene repair, (2) gene substitution, (3) cell therapy, (4) block of gene expression or function, (5) DNA vaccination and (6) gene augmentation. While major advances have been made in all areas of gene therapy during the last years, various delivery, targeting and safety issues need to be addressed before these strategies will enter clinical practice. Nevertheless, gene therapy will eventually become part of the management of patients with various liver diseases, complementing or replacing existing therapeutic and preventive strategies.     

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

Genetic Variants in Diseases of the Extrapyramidal System

Knowledge on the genetics of movement disorders has advanced significantly in recent years. It is now recognized that disorders of the basal ganglia have genetic basis and it is suggested that molecular genetic data will provide clues to the pathophysiology of normal and abnormal motor control. Progress in molecular genetic studies, leading to the detection of genetic mutations and loci, has contributed to the understanding of mechanisms of neurodegeneration and has helped clarify the pathogenesis of some neurodegenerative diseases. Molecular studies have also found application in the diagnosis of neurodegenerative diseases, increasing the range of genetic counseling and enabling a more accurate diagno-sis. It seems that understanding pathogenic processes and the significant role of genetics has led to many experiments that may in the future will result in more effective treatment of such diseases as Parkinson’s or Huntington’s. Currently used molecular diagnostics based on DNA analysis can identify 9 neurodegenerative diseases, including spinal cerebellar ataxia inherited in an autosomal dominant manner, dentate-rubro-pallido-luysian atrophy, Friedreich’s disease, ataxia with ocu-lomotorapraxia, Huntington''s disease, dystonia type 1, Wilson’s disease, and some cases of Parkinson''s disease.     

Rigid nanoparticle-based delivery of anti-cancer siRNA: Challenges and opportunities

Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized.     

Molecular background of progressive myoclonus epilepsy

Research on human inherited diseases provides a powerful tool to identify an intrinsically important subset of genes vital to healthy functioning of the organism. Progressive myoclonus epilepsies (PMEs) are a group of rare inherited disorders characterized by the association of epilepsy, myoclonus and progressive neurological deterioration. Significant progress has been made in elucidating the molecular background of PMEs. Here, progress towards understanding the molecular pathogenesis of PMEs is reviewed using the most common single cause of PME, Unverricht-Lundborg disease, as an example. Mutations in the gene encoding cystatin B (CSTB), a cysteine protease inhibitor, are responsible for the primary defect in Unverricht-Lundborg disease. CSTB-deficient mice, produced by targeted disruption of the mouse Cstb gene, display a phenotype similar to the human disease, with progressive ataxia and myoclonic seizures. The mice show neuronal atrophy, apoptosis and gliosis as well as increased expression of apoptosis and glial activation genes. Although significant advances towards understanding the molecular basis of Unverricht-Lundborg disease have been achieved, the physiological function of CSTB and the molecular pathogenesis of the disease remain unknown.     

Gene therapy flexes muscle

This commentary highlights the promising results of recent studies in animal models of Duchenne muscular dystrophy and amyotrophic lateral sclerosis that have clearly demonstrated the potential of gene therapy for tackling these diseases. In the absence of effective drugs or other treatments, these advances in gene therapy technology represent the best hope for those patients and families that are blighted by these diseases. BACKGROUND: Diseases characterized by progressive muscle degeneration are often incurable and affect a relatively large number of individuals. The progressive deterioration of muscle function is like the sword of Damocles that constantly reminds patients suffering from these diseases of their tragic fate, since most of them will eventually die from cardiac or pulmonary dysfunction. Some of these disorders are due to mutations in genes that directly influence the integrity of muscle fibers, such as in Duchenne muscular dystrophy (DMD), a recessive X-linked genetic disease. Others result from a progressive neurodegeneration of the motoneurons that are essential for maintaining muscle function, such as in amyotrophic lateral sclerosis (ALS), also commonly known as Lou Gehrig's disease. The genetic basis of DMD is relatively well understood as it is due to mutations in the dystrophin gene that encodes the cognate sarcolemmal protein. In contrast, the cause of ALS is poorly defined, with the exception of some dominantly inherited familial cases of ALS that are due to gain-of-function mutations in the gene encoding superoxide dismutase (SODG93A). Gene therapy for these disorders has been hampered by the inability to achieve widespread gene transfer. Moreover, since familial ALS is due to a dominant gain-of-function mutation, inhibition of gene expression (rather than gene augmentation) would be required to correct the phenotype, which is particularly challenging.     

Perspectives and limitations of gene expression profiling in rheumatology: new molecular strategies

The deciphering of the sequence of the human genome has raised the expectation of unravelling the specific role of each gene in physiology and pathology. High-throughput technologies for gene expression profiling provide the first practical basis for applying this information. In rheumatology, with its many diseases of unknown pathogenesis and puzzling inflammatory aspects, these advances appear to promise a significant advance towards the identification of leading mechanisms of pathology. Expression patterns reflect the complexity of the molecular processes and are expected to provide the molecular basis for specific diagnosis, therapeutic stratification, long-term monitoring and prognostic evaluation. Identification of the molecular networks will help in the discovery of appropriate drug targets, and permit focusing on the most effective and least toxic compounds. Current limitations in screening technologies, experimental strategies and bioinformatic interpretation will shortly be overcome by the rapid development in this field. However, gene expression profiling, by its nature, will not provide biochemical information on functional activities of proteins and might only in part reflect underlying genetic dysfunction. Genomic and proteomic technologies will therefore be complementary in their scientific and clinical application.     

Dissecting the molecular mechanisms of cancer through bioinformatics-based experimental approaches

Cancer is a disease of aberrant gene expression characterized by inappropriate (temporal or quantitative) expression of positive mediators of cell proliferation in conjunction with diminished expression of negative mediators of cell growth. Alteration of the normal balance of these positive and negative mediators leads to the abnormal growth of cells and tissues that typify neoplastic disease. Development of a better understanding of the genetic and epigenetic mechanisms that induce neoplastic transformation and drive the cancer phenotype is essential for continued progress towards the design of practical molecular diagnostics and effective treatment strategies. Over the past decades, molecular techniques that facilitate the assessment of gene expression, identification of gene mutations, and characterization of chromosome abnormalities (numeric and structural) have been established and applied to cancer research. However, many of these techniques are slow and labor-intensive. More recently, high-throughput technologies have emerged that generate large volumes of data related to the genetics and epigenetics of cancer (or other disorders). These advances in molecular genetic technology required the development of sophisticated bioinformatic tools to manage the large datasets generated. The combination of high-throughput molecular assays and bioinformatic-based data mining strategies has significantly impacted our understanding of the molecular pathogenesis of cancer, classification of tumors, and now the management of cancer patients in the clinic. This article will review basic molecular techniques and bioinformatic-based experimental approaches used to dissect the molecular mechanisms of carcinogenesis.