Assessing viral gene therapy in neuroendocrine models

A simple method of manipulating neuronal gene expression would greatly facilitate the design of experiments to increase our understanding of and ability to treat diseases of the CNS. However, until recently most transfection methods could only deliver DNA into dividing cells and it was only possible to manipulate neuronal gene expression through the production of transgenic animals. The development of powerful new viral-based gene transfer systems has generated a great deal of research interest in the field of therapeutic gene transfer during the last decade. One of the most powerful and versatile gene delivery systems currently available is the recombinant adenovirus (Ad) vector. These vectors can transfect postmitotic neurons in the CNS, but have not yet been fully evaluated as CNS gene therapy vectors. Brattleboro rats contain a point mutation in the arginine vasopressin (AVP) gene that results in a pathological phenotype characterized by a lack of circulating AVP. This decrease in AVP in turn causes the characteristics signs of diabetes insipidus, with the production of large volumes of dilute urine and a compensatory drinking of large volumes of water (equivalent to the body weight of the rat per day). We have shown that injection of an Ad encoding the arginine vasopressin cDNA into the supraoptic nuclei of the hypothalamus results in the long-term reversal of this pathological phenotype. This was demonstrated by reduced daily water intake and micturition, as well as increased urine osmolality lasting 4 months. The highly characterized Brattleboro rat model of hypothalamic diabetes insipidus, therefore, provides the means to examine noninvasively the efficacy of viral and nonviral gene therapy strategies in the CNS.     

Therapeutic gene transfer to the nervous system using viral vectors

The past few years have been marked by substantial progress in preclinical studies of therapeutic gene transfer for neurologic disease using viral-based vectors. In this article, the authors review the data regarding (1). treatment of focal neuronal degeneration, exemplified by Parkinson disease, ischemia, and trauma models; (2). treatment of global neurologic dysfunction, exemplified by the mucopolysaccharidoses and other storage diseases; (3). peripheral nervous system diseases including motor neuron disease and sensory neuropathies; and (4). the use of vectors expressing neurotransmitters to modulate functional neural activity in the treatment of pain. The results suggest that a number of different viral vectors may be appropriate for gene transfer to the central nervous system for specific disease processes, and that for the peripheral nervous system herpes simplex virus-based vectors appear to have special utility. The results of the first human gene therapy trials for neurologic disease, which are just now beginning, will be crucial in defining the next step in the development of this therapy.     

Retroviral transfection of the lacZ gene from Liz-9 packaging cells to glioma spheroids.

Despite the development of numerous vectors for gene transfection to gliomas, patient survival length remains unaffected in clinical trials. For glioma gene therapy to be successful, the extent of gene transfer to the solid tumor tissue has to be high. In the present work we review some of the vector types and strategies so far utilized in experimental and clinical glioma gene therapy. Since gene transfer efficacy into solid glioma tissue is unknown for many vectors, we studied the gene transfer efficacy into multicellular spheroids derived from a human glioma cell line GaMg as well as into spheroids derived from human glioma biopsies (glioblastoma multiforme, GBM). A replication deficient retroviral vector from the Liz 9 packaging cell line was used for transfer of the bacterial beta-galactosidase lacZ gene into the target tissue. Gene transfer was obtained by adding medium containing virus from the producer cells to the target tissue. The experiments were also conducted with EGF (epidermal growth factor) added to the medium. The data show that the transfection rate ranged from 0-4.5% where the transfection efficacy was higher in spheroids after the addition of EGF. Most of the transfected cells were found at the surface, but transfected cells could also be observed in the center of the spheroids. We conclude that using this vector system, the transfection efficacy was low, even if the number of replicating cells was increased by adding EGF. The findings are consistent, and may partly explain, the lack of effect using this vector system during in vivo studies.     

Loss of gene expression in lentivirus- and retrovirus-transduced neural progenitor cells is correlated to migration and differentiation in the adult spinal cord

Gene transfer into multipotent neural progenitor cells (NPC) and stem cells may provide for a cell replacement therapy and allow the delivery of therapeutic proteins into the degenerating or injured nervous system. Previously, murine leukemia virus-based retroviral vectors expressing GFP from an internal EF-1alpha promoter and lentiviral vectors expressing GFP from a hybrid CMV/beta-actin promoter have been described to be resistant to stem cell specific gene silencing. Therefore, we investigated whether these viral vectors allow stable in vivo gene expression in genetically modified NPC isolated from the adult rat spinal cord. In vitro, NPC genetically modified to express GFP using the described retroviral vector showed strong GFP expression in undifferentiated NPC. However, in vitro differentiation resulted in the loss of GFP expression in 50% of cells. Grafting of BrdU-prelabeled NPC to the spinal cord resulted in a loss of GFP expression in 70% and 95% of surviving NPC at 7 and 28 days post-grafting, respectively. The loss in gene expression was paralleled by the differentiation of NPC into a glial phenotype. Transgene downregulation although less profound was also observed in cells modified with lentiviral vectors, whereas in vivo lentiviral gene transfer resulted in stable transgene expression for up to 16 months. Thus, in vivo gene expression in genetically engineered neural progenitor cells is temporally limited and mostly restricted to undifferentiated NPC using the viral vectors tested.     

The therapeutic use of gene therapy in inflammatory demyelinating diseases of the central nervous system

PURPOSE OF REVIEW: Gene therapy protocols aimed to deliver therapeutic molecules into the central nervous system may represent an alternative therapeutic strategy in patients affected by inflammatory demyelinating diseases of the central nervous system where systemic therapies have shown limited therapeutic efficacy possibly owing to the blood-brain barrier, a major obstacle for the entry of therapeutic molecules into the central nervous system. RECENT FINDINGS: Among inflammatory demyelinating diseases of the central nervous system, gene therapy approaches have been so far developed almost exclusively for multiple sclerosis. However, the chronic/relapsing nature of the disease, the restriction to the central nervous system of the pathological process as well as the necessity to inhibit the ongoing inflammatory process but also to foster endogenous remyelinating pathways, have posed several questions which still need to be properly addressed for the development of a successful gene therapy strategy in multiple sclerosis patients. SUMMARY: The gene therapy approaches for multiple sclerosis have been so far developed and tested only in rodents and monkeys with experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The results of these studies clearly indicate that the delivery of therapeutic genes within the central nervous system is superior to the peripheral delivery. In particular, the intracerebral delivery of genes coding for anti-inflammatory and/or neurotrophic molecules, using gene vectors derived from non-replicative viruses, showed to inhibit not only the detrimental function of blood-borne mononuclear effector cells but also to foster proliferation and differentiation of surviving oligodendrocytes within demyelinated areas. Here, we summarize the most recent findings of this novel area of research.     

Viral vectors for gene delivery to the central nervous system

The potential benefits of gene therapy for neurological diseases such as Parkinson's, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer's are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features.     

Gene therapy in epilepsy

Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson's disease with an excellent safety profile.
Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronic models of epileptogenesis, as well as assessment of safety in toxicological studies.     

Targeted transgene expression in rat brain using lentiviral vectors

Direct gene transfer to the adult brain is dependent on vectors that transduce non-dividing cells, such as lentiviral vectors. Another aspect of the development of gene therapy to the brain is the need for cell-specific transgene expression. Expression from vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors has been reported to be mainly neuron specific in the brain. We constructed cell-specific lentiviral vectors using the neuron-specific enolase (rNSE) or the glial fibrillary acidic protein (hGFAP) promoters and compared them to the ubiquitous human cytomegalovirus promoter (hCMV), a hybrid CMV/beta-actin promoter (CAG) and the promoter for human elongation factor 1 alpha (EF1 alpha). Our results showed that the hGFAP promoter was expressed only in glial cells, whereas rNSE was purely neuron specific, showing that VSV-G is pantropic in the rat striatum. We conclude that the VSV-G allows transduction of both glial and neuronal cells and the promoter dictates in what cell type the transgene will be expressed. The expression of transgenes exclusively in astrocytes would allow for local delivery of secreted transgene products, such as glial cell line-derived neurotrophic factor (GDNF), circumventing the anterograde transport that may induce unwanted side effects.     

Progress in gene therapy for Duchenne muscular dystrophy

Gene transfer research for Duchenne muscular dystrophy (DMD) has brought the goal of successful treatment of this devastating, inherited disease closer to being a reality. Although gene therapeutic approaches for DMD patients are not yet in clinical use, recent advances using DMD animal models are encouraging. Progress in vector design, such as high-capacity adenoviral vectors, targeted adenoviral vectors, and heterodimerization of DNA delivered by adeno-associated virus (AAV) vectors have advanced the field considerably. The recent studies into the pharmacologic-induced read-through of stop codons, the increased study of utrophin and its upregulation, and the introduction of point mutation correction using chimeric oligonucleotides have expanded the field, providing new avenues of inquiry.     

Genetic therapy in gliomas: historical analysis and future perspectives

High-grade gliomas are relatively frequent in adults, and consist of the most malignant kind of primary brain tumor. Being resistant to standard treatment modalities such as surgery, radiation, and chemotherapy, it is fatal within 1 to 2 years of onset of symptoms. Although several gene therapy systems proved to be efficient in controlling or eradicating these tumors in animal models, the clinical studies performed so far were not equally successful. Most clinical studies showed that methodologies that increase tumor infection/transduction and, consequently confer more permanent activity against the tumor, will lead to enhanced therapeutic results. Due to the promising practical clinical benefits that can be expected for the near future, an exposition to the practicing neurosurgeon about the basic issues in genetic therapy of gliomas seems convenient. Among the main topics, we shall discuss anti-tumoral mechanisms of various genes that can be transfected, the advantages and drawbacks of the different vectors utilized, the possibilities of tumor targeting by modifications in the native tropism of virus vectors, as well as the different physical methods for vector delivery to the tumors. Along with the exposition we will also review of the history of the genetic therapy for gliomas, with special focus on the main problems found during the advancement of scientific discoveries in this area. A general analysis is also made of the present state of this promising therapeutic modality, with reference to the problems that still must be solved and the new paradigms for future research in this area.     

Pseudotype-dependent lentiviral transduction of astrocytes or neurons in the rat substantia nigra

Gene transfer to the central nervous system provides powerful methodology for the study of gene function and gene–environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. The aim of the present study was to determine patterns of tropism exhibited by pseudotyped lentiviral vectors in the rat substantia nigra, in order to evaluate their utility for gene transfer in experimental models of Parkinson's disease. Isogenic lentiviral vector particles encoding a GFP reporter were pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), or Moloney murine leukemia virus (MuLV). Adult male Lewis rats received unilateral stereotactic infusions of vector into the substantia nigra; three weeks later, patterns of viral transduction were determined by immunohistological detection of GFP. Different pseudotypes gave rise to transgene expression in restricted and distinct cellular populations. VSV and MV pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. In contrast, LCMV- and MuLV-pseudotyped lentivirus produced transgene expression exclusively in astrocytes; the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of LCMV or MuLV. These data suggest that pseudotyped lentiviral vectors will be useful for experimental gene transfer to the rat substantia nigra. In particular, the availability of neuronal and astrocytic-targeting vectors will allow dissociation of cell autonomous and cell non-autonomous functions of key gene products in vivo.     

Targeted gene therapy to antigen-presenting cells in the central nervous system using hematopoietic stem cells

BACKGROUND: Hematopoietic stem cells (HSC) have been previously used as vectors for gene therapy of systemic disease. The effectiveness of HSC-mediated gene therapy largely depends on efficient gene delivery into long-term repopulating progenitors and targeted transgene expression in an appropriate progeny of the transduced pluripotent HSCs. In the present study, we examined the feasibility of using HSC transduced with self-inactivating (SIN) lentiviral vectors for the delivery of gene therapy to the central nervous system (CNS). MATERIAL AND METHODS: We constructed two SIN lentiviral vectors, EF.GFP and DR.GFP, to express the green fluorescent protein (GFP) gene controlled solely by the promoter of either a housekeeping gene EF-1alpha or the human HLA-DRalpha gene, which is selectively expressed in antigen-presenting cells. RESULTS: We demonstrated that both vectors efficiently transduced human pluripotent CD34+ cells capable of engrafting NOD/SCID mice. Only the DR.GFP vector mediated transgene expression in the murine CNS containing human HLA-DR+ cells. These cells express surface markers characteristic of resident CNS microglia. Furthermore, human dendritic cells derived from transduced and engrafted human cells potently stimulated allogeneic T cell proliferation. CONCLUSIONS: The present study demonstrated successful targeting of transgene expression to CNS microglia after stable gene transduction of pluripotent HSC.     

中枢神经系统基因治疗的病毒载体

重组病毒载体因其自然感染途径而成为有效的基因转移手段。迄今为止，许多病毒载体，包括逆转录病毒、腺病毒、单纯疱疹病毒、腺相关病毒、慢病毒及多种杂交型病毒载体等，已应用于中枢神经系统基因治疗的基础和临床研究。本对其主要研究进展进行了综述。     

Translational paradigms in cerebrovascular gene transfer.

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.     

Immunology of neurological gene therapy: How T cells modulate viral vector-mediated therapeutic transgene expression through immunological synapses

Gene therapy has been shown to be a powerful new approach to the treatment of brain diseases. Brain neurodegenerations, brain tumors, inherited brain diseases, and autoimmune disorders are currently recognized as proper targets for gene therapeutics. Advances in the development of viral vectors (especially improvements in their immune profiles), the capacity to regulate transgene expression, and identification of appropriate therapeutic constructs have made the transition into clinical trials for gene therapy possible. One particular remaining challenge is the immune response that could be raised against either the viral vectors themselves or any regulatory or therapeutic transgenes. Because of the structure of brain immune responses, viral gene transfer into the brain can, under certain circumstances, be invisible to the systemic immune response and thus not generate a deleterious immune attack. If, however, the systemic immune system is primed against any vector antigen, the systemic immune response eliminates transgene expression and thus curtails the therapeutic efficacy of gene therapy. Mechanistic studies of brain immune responses indicate that the adaptive arm of the immune system may indeed be able to kill transduced cells. To move neurological gene therapy into the clinic in an effective and safe manner, these are the developments needed: novel viral vectors that either display a reduced capacity to stimulate an adaptive immune response or become invisible to the immune system after the delivery of the vector genome to the nucleus of transduced cells, and ways either to steer the immune response away from cytotoxic responses or to induce tolerance to gene therapy products.     

Recombinant micro-genes and dystrophin viral vectors

An effective gene therapy for Duchenne muscular dystrophy ideally relies on the ability to provide long-term expression to muscle tissue of the missing protein, dystrophin. Early work in the mdx mouse using a 6.3 kb mini-dystrophin cDNA, carried out in either adenoviral or retroviral vectors was generally successful, however, expression was only transient. In an attempt to remedy this problem, two approaches are being investigated. The first of these is a hybrid vector system that combines the efficacy of gene transfer into skeletal muscle of adenoviral vectors with the long-term stability of retroviral vectors. The second utilises the inherently efficient transducing properties and stability of the adeno-associated viral delivery system. Using highly truncated micro-dystrophin cDNAs we have shown that both vector systems were able to restore dystrophin and dystrophin-associated protein expression at the plasma membrane of mdx mice for prolonged periods of time. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. These studies suggest that hybrid adenoviral-retroviral and adeno-associated viral vectors are capable of ameliorating dystrophic pathology at the cellular level and as such are useful tools in the development of a gene therapy for Duchenne muscular dystrophy.     

Neuroprotective effect of a CNTF-expressing lentiviral vector in the quinolinic acid rat model of Huntington's disease.

Neurodegenerative diseases represent promising targets for gene therapy approaches provided effective transfer vectors. In the present study, we evaluated the effectiveness of LacZ-expressing lentiviral vectors with two different internal promoters, the mouse phosphoglycerate kinase 1 (PGK) and cytomegalovirus (CMV), to infect striatal cells. The intrastriatal injection of lenti-beta-Gal vectors lead to 207, 400 +/- 11,500 and 303,100 +/- 4,300 infected cells in adult rats, respectively. Importantly, the beta-galactosidase activity was higher in striatal extracts from PGK-LacZ-injected animals as compared to CMV-LacZ animals. The efficacy of the system was further examined with a potential therapeutic gene for the treatment of Huntington's disease, the human ciliary neurotrophic factor (CNTF). PGK-LacZ- or PGK-CNTF-expressing viruses were stereotaxically injected into the striatum of rats, 3 weeks later the animals were unilaterally lesioned with 180 nmol of quinolinic acid (QA). Control animals displayed 148 +/- 43 apomorphine-induced rotations ipsilateral to the lesion 5 days postlesion as compared to 26 +/- 22 turns/45 min in the CNTF-treated group. The extent of the striatal damage was significantly diminished in the CNTF-treated rats as indicated by the 52 +/- 9.7% decrease of the lesion volume and the sparing of DARPP-32, ChAT and NADPH-d neuronal populations. These results further establish that lentiviruses may represent an efficient gene delivery system for the screening of therapeutic molecules in Huntington's disease.     

Gene therapy for the central nervous system in the lysosomal storage disorders

Although great promise has been made in the field of gene therapy, a number of difficulties must be solved before successful human studies can be completed. These issues involve safety, immunological reactions to the vectors and their transgene products, persistent transgene expression, and ability to repeat administrations of the vector safely. A major hurdle that must be overcome is the ubiquitous delivery of the transgene throughout the nervous system. Significant gene delivery to the CNS of murine models of LSD has been accomplished, but we await the successful treatment of the nervous system in a larger mammalian model of LSD. As yet there is no perfect vector that can solve all of these problems. It is likely that vector technology will evolve into hybrid vectors also using synthetic components that will increase safety and efficacy of recombinant vectors. The treatment of the CNS remains complicated, but progress is being made in this area. Clinical trials already planned will give us increasing information as to the ideal gene therapy for the CNS.     

Regulated protein expression for in vivo gene therapy for neurological disorders: progress, strategies, and issues

The field of in vivo gene therapy has matured to the point where there are numerous clinical trials underway including late-stage clinical trials. Several viral vectors are especially efficient and support lifetime protein expression in the brain and a number of clinical trials are underway for various progressive or chronic neurological disorders including Parkinson's disease, Alzheimer's disease, and Batten's disease. To date, however, none of the vectors in clinical use have any direct way to reverse or control their transgene product in the event continued protein expression should become problematic. Several schemes that use elements within the vector design have been developed that allow an external drug or pro-drug to alter ongoing protein expression after in vivo gene transfer. The most promising and most studied regulated protein expression methods for in vivo gene transfer are reviewed. In addition, potential scientific and clinical advantages of transgene regulation for gene therapy are discussed.