Gene Therapy For Neurological Disorders Research Articles

Critical Issues in Gene Therapy for Neurologic Disease

Gene therapy for the nervous system is a newly emerging field with special issues related to modes of delivery, potential toxicity, and realistic expectations for treatment of this vital and highly complex tissue. This review focuses on the potential for gene delivery to the brain, as well as possible risks and benefits of these procedures. This includes discussion of appropriate vectors, such as adeno-associated virus, lentivirus, gutless adenovirus, and herpes simplex virus hybrid amplicons, and cell vehicles, such as neuroprogenitor cells. Routes of delivery for focal and global diseases are enumerated, including use of migratory cells, facilitation of vascular delivery across the blood-brain barrier, cerebrospinal fluid delivery, and convection injection. Attention is given to examples of diseases falling into different etiologic types: metabolic deficiency states, including Canavan disease and lysosomal storage disorders; and degenerative conditions, including Parkinson's disease and other neurodegenerative conditions.

Transduction of human neural progenitor cells using recombinant adeno-associated viral vectors.

Human neural progenitor cells (hNPCs) represent an attractive source for cell therapy of neurological disorders. Genetic modification of hNPCs may allow a controlled release of therapeutic proteins, suppress immune rejection, or produce essential neurotransmitters. In search of an effective gene delivery vehicle, we evaluated the efficiency of a recombinant adeno-associated viral (rAAV) vector expressing enhanced green fluorescent protein (CAGegfp). Our study demonstrated that CAGegfp efficiently transduced both proliferating and differentiated hNPCs in vitro. EGFP expression was detected as early as 1 day after exposure to CAGegfp and was detectable for up to 4 months. Following transduction, the growth rate of hNPCs slowed down, but they were still able to differentiate into neurons and glia. Furthermore, CAGegfp-modified hNPCs survived, differentiated and expressed EGFP after transplanting into spinal cord of adult rats. Our results indicated that rAAV vectors might be a useful tool in hNPC-based cell and gene therapy for neurological disorders.

6.脳疾患への遺伝子治療の応用(PS 3 脳神経外科における的確な遺伝子診断と治療)

6.脳疾患への遺伝子治療の応用(PS 3 脳神経外科における的確な遺伝子診断と治療)

Gene Therapy for Neurological Disorders

Gene therapy is an attractive strategy for neurological disorders, such as Parkinson’s disease and epilepsy, for several reasons. Introduction of genes with a therapeutic potential can be achieved locally, by...

Differentiation of transplanted bone marrow cells in the adult mouse brain.

Bone marrow transplantation is reportedly effective in preventing the progression of neurological deterioration in lysosomal storage disorders, although the mechanism underlying the therapeutic effects remains to be elucidated. Recent research on stem cell biology suggests that bone marrow cells contain nonhematopoietic stem cells, including brain precursor cells. To evaluate the contribution of bone marrow cells as carriers for cell and gene therapy of neurological disorders, we studied the fate of transplanted bone marrow cells in the adult mouse brain. Bone marrow cells were genetically marked with a retroviral vector containing the green fluorescence protein gene and then transplanted into irradiated mice by either systemic infusion or direct injection. To identify cell types, brain sections were stained with specific antibodies against neuronal cell markers-neuron specific enolase for neurons, glial fibrillary acidic protein (GFAP) for astrocytes, carbonic anhydrase II (CAII) for oligodendrocytes, and ionized calcium binding adaptor molecule 1 (Iba1) for microglia-and then examined under a confocal microscope. Twenty-four weeks after systemic infusion, transplanted cells expressed Iba1 but none of the other brain cell markers. Conversely, 12 weeks after direct injection, transplanted cells were stained with antibodies against GFAP, CAII, and Iba1. Bone marrow contains cells capable of differentiating into oligodendrocytes, astrocytes, and microglia when exposed to the brain microenvironment. Autologous bone marrow cells may be useful as carriers for ex vivo gene therapy for lysosomal disorders with neurological symptoms.

Regulatable and cell-type specific transgene expression in glial cells: prospects for gene therapy for neurological disorders

Regulatable and cell-type specific transgene expression in glial cells: prospects for gene therapy for neurological disorders

Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector.

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.

Gene Transfer into Rat Brain Using Adenoviral Vectors

Recombinant adenovirus vectors are attractive vehicles to deliver genes into the brain for the purposes of neurobiological research and for gene therapy of neurological diseases. This unit provides a comprehensive set of protocols for adenovirus vector-mediated gene transfer to the brain, including introduction of the vector into the brain by stereotaxic injection and preparation and processing of brain tissue for the evaluation of gene transfer. The potential side-effects of administering adenovirus vectors to the brain are discussed in detail. The unit also provides protocols for evaluating these side-effects (e.g., demyelination, inflammation, vector-mediated cytotoxicity, etc.). Finally, critical parameters for obtaining optimal gene transfer with minimum side-effects are presented.

Delivery of a Retroviral Vector Expressing Human β-Glucuronidase to the Liver and Spleen Decreases Lysosomal Storage in Mucopolysaccharidosis VII Mice

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Peripheral infection with adenovirus causes unexpected long-term brain inflammation in animals injected intracranially with first-generation, but not with high-capacity, adenovirus vectors: toward realistic long-term neurological gene therapy for chronic diseases.

Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8(+) cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.

HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part I. HSV-1 Structure, Replication and Pathogenesis

The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1) virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.

HSV-1-Based Vectors for Gene Therapy of Neurological Diseases and Brain Tumors: Part II. Vector Systems and Applications

Many properties of HSV-1 are especially suitable for using this virus as a vector to treat diseases affecting the central nervous system (CNS), such as Parkinson's disease or malignant gliomas. These advantageous properties include natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. Selective oncolysis in combination with modulation of the immune response mediated by replication-conditional HSV-1 vectors appears to be a highly promising approach in the battle against malignant glioma. Helper virus-free HSV/AAV hybrid amplicon vectors have great promise in mediating long-term gene expression in the PNS and CNS for the treatment of various neurodegenerative disorders or chronic pain. Current research focuses on the design of HSV-1-derived vectors which are targeted to certain cell types and support transcriptionally regulatable transgene expression. Here, we review the recent developments on HSV-1-based vector systems and their applications in experimental and clinical gene therapy protocols.

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer

N-methyl- d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca 2+ and sensitive to Mg 2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca 2+-impermeable and Mg 2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca 2+ permeability and Mg 2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg 2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.

Postsynaptic expression of Ca 2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer

The ability to artificially express a particular receptor protein in the postsynaptic sites of neurons in the central nervous system (CNS) would be useful for the study of synaptic function of cloned receptor genes as well as for gene therapy of neurological disorders caused by dysfunction of postsynaptic receptors. In this study, we aimed to express the cDNA of unedited GluR2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor that forms inwardly rectifying and Ca 2+-permeable channel in CNS neurons by using adenoviral-mediated gene transfer. For this purpose, we have constructed a recombinant adenovirus bearing an expression-switching unit, where the unedited GluR2 cDNA can be activated by the Cre recombinase-mediated excisional deletion of a stuffer DNA interposed between the promotor and the coding region. When PC12 cells were infected with this recombinant adenovirus together with an adenovirus expressing Cre recombinase, the inwardly rectifying and Ca 2+-permeable AMPA receptor channels were expressed in nearly 100% of infected cells. Two days after co-infection of cultured rat hippocampal neurons with these adenoviruses, fast excitatory neurotransmission in the glutamatergic synapse was mediated predominantly by the inwardly rectifying and Ca 2+-permeable AMPA receptor channels. This indicates that the native AMPA receptors in the postsynaptic sites of the glutamatergic synapse are replaced rapidly with recombinant receptors newly produced by the viral-mediated gene transfer.

Gene Therapy for Neurological Disorders and Brain Tumors

edited by E. Antonio Chiocca and Xandra O. Breakefield, 458 pp., ill., Totowa, NJ, Humana Press, 1998, $165.00 The methodologies employed in gene therapy are rapidly improving, leading to safer delivery vehicles that are more amenable for application to CNS disorders. Because the field is progressing so quickly, it is instructive to review periodically experimental data relating to gene delivery and to discuss the technological and clinical advances of gene therapeutic strategies, as this allows one to assess and devise new approaches effectively. Gene Therapy for Neurological Disorders and Brain Tumors seeks to accomplish this mission and its editors have assembled contributions from many investigators who work productively in gene therapy. An overview discusses cell types that comprise the CNS and viral vectors that are currently used, and outlines the issues regarding safety and efficacy of gene therapy (i.e., transgene expression duration, vector-mediated toxicity, and immune response). The volume has an unbalanced representation of the virus vectors currently used by investigators in the CNS gene delivery field. For example, there is a comprehensive discussion of retroviral vectors including life cycle, vector design, packaging cell lines, and packaging protocols. However, such murine-based vectors are poor choices for postmitotic neurons or quiescent glia. By contrast, adenoviral vectors, which have seen considerable use in the CNS, are covered in a rudimentary fashion that does not provide enough depth on these well-tested vectors. The chapters on HSV vectors are more …

Gene Therapy for Neurological Disorders and Brain Tumors

Gene Therapy for Neurological Disorders and Brain Tumors

Gene therapy for neurological disorders and brain tumors: E. A. Chiocca and X. O. Breakefield (Editors)

Gene therapy for neurological disorders and brain tumors: E. A. Chiocca and X. O. Breakefield (Editors)

Gene Therapy for Neurological Disorders and Brain Tumors

Gene Therapy for Neurological Disorders and Brain Tumors

Gene therapy of central nervous system tumors.

Recently, remarkable advances have been achieved in molecular and genetic researches of different kinds of general diseases, as well as in basic and clinical studies using gene therapy for central nervous system diseases. For brain tumors, clinical trials have been already started in more than 10 clinical protocols and more than 100 patients with malignant brain tumors. Nevertheless, there are still major issues that remain to be resolved for achieving better clinical results, such as delivery system of genetic material, regulatory methods of the intracellular expression of the transgene, antitumor efficacy, and tumor selectivity. In this paper, molecular genetic studies and the current state of gene therapy for neurological diseases, especially brain tumors, are described, and the future direction of this fascinating approach is discussed.

Potential use of herpes simplex virus (HSV) vectors for gene therapy of neurological disorders.

Advances in molecular biology and virus genetics have allowed the possibility of gene therapy using viral vectors for a variety of neurological diseases in which the genetic or biochemical basis is understood. A number of such vectors have now been constructed, including those derived from herpes simplex virus (HSV), adenovirus, retrovirus and adeno-associated virus, and used in preliminary in vitro experiments and in animal models. It is possible to package a foreign gene into such a vector which can then be targeted to specific regions of the nervous system. HSV is particularly appropriate for delivering genes to neurons in view of its ability to establish latent infection in these cells. Viral vectors have the potential to be used to treat such neurological conditions as malignant gliomas, Parkinson's disease, known single gene disorders and cerebral ischaemia. However, the technical problems which will need to be overcome are formidable and will not be easily solved. The problems include the efficient delivery of the vector to target cells, the maintenance and control of foreign gene expression, and the control of unwanted host immune responses.