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WO2006047299A2 - Cultures de tranches organotypiques et leurs applications - Google Patents

Cultures de tranches organotypiques et leurs applications Download PDF

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WO2006047299A2
WO2006047299A2 PCT/US2005/037924 US2005037924W WO2006047299A2 WO 2006047299 A2 WO2006047299 A2 WO 2006047299A2 US 2005037924 W US2005037924 W US 2005037924W WO 2006047299 A2 WO2006047299 A2 WO 2006047299A2
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cells
cell
culture
organotypic
brain
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WO2006047299A3 (fr
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Sean M. Kearns
Dennis Steindler
Bjorn Scheffler
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University of Florida
University of Florida Research Foundation Inc
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University of Florida Research Foundation Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
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    • C12N2503/00—Use of cells in diagnostics
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    • C12N2503/02—Drug screening
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the organotypic slice culture of the invention maintains the integrity of the cellular, cytoarchitecture, chemical and circuit functions of a pertinent section of a normal brain.
  • this system is used to study nigrostriatal pathway degeneration and methods for regeneration as well as a novel assay system for tumor growth and treatment.
  • Organotypic slice culture eliminates or minimizes these variables since the blood brain barrier and other tissues are not present. Metabolism of compound is easily assessed by sampling media. Dose at the target organ is easily controlled. [0004] Thus there is a need in the art to develop systems for the study of neurodegenerative disorders, drug discovery, tumor therapies and diagnosis of disorders. SUMMARY
  • slice cultures comprise the sagittal sections of the brain, for the study of nigrostriatal pathway degeneration and methods for regeneration as well as a novel assay system for tumor growth and treatment.
  • Organotypic slice cultures maintain the tissue organization and cytoarchitecture similar to the in vivo brain cellular organization and function.
  • tissue culture allows longer recording time compared to acute slices studies. This enables to study long-term phenomena, like delayed neuronal death, neurotoxicity or neurodegenerative processes.
  • tissue culture With this system one can perform continuous and simultaneous stimulation and recording of neuronal activity during several days.
  • the electronic/biological interface normal functioning allows measurement of biochemical molecules from the extracellular medium or to deliver chemical molecules into the slice cultures.
  • grafting of stem cells and microenvironmental manipulation by for example, using growth hormone, or an analogue thereof, or another substance leading to increased concentrations of growth hormone or analogues thereof, it is possible to modulate the proliferation and/or differentiation of neural stem cells and progenitor cells from the adult CNS.
  • the present invention thus provides new possibilities to treat injuries to or diseases of the central nervous system that predominantly affect oligodendroglia, astroglia or neuronal cells by modification of proliferation cell genesis and/or differentiation of neuronal stem cells or progenitor cells in the central nervous system.
  • the slice culture allows for control of the in vitro propagation of stem cells, progenitor cells and other cells, especially cells derived from the central nervous system, with, the potential to generate neurons, astrocytes or oligodendrocytes. Such cells may e.g. be used for therapeutic purposes in patients.
  • the invention also relates to an organotypic slice culture for determining cellular cytochemistry and architecture of, for example, sagittal regions in the brain affected by neurotoxins and neurodegenerative disorders.
  • the organotypic slice culture allows for the identification of therapeutic candidate agents that reverse the neurodegeneration of the brain for treatment of neural disorders.
  • the organotypic slice cultures allow for the determination of potential toxicity of candidate agents, the efficacy and dose determination of each compound to restore normal brain functioning.
  • the organotypic slice culture allows for the monitoring of the environment for detection of neurotoxins, especially in the event of a bioterrorist attack.
  • the organotypic slice culture allows for the identification of tumor agents and candidate therapeutic compounds that are low in toxicity and high in efficacy.
  • tumor cells directly implanted into the animal can be tracked in slice cultures past the point of lethal tumor growth. These slices also allow for the easy application of therapeutic agents and the quick visualization of their efficacy.
  • Treatment with a novel polymer based gene delivery system to introduce therapeutic genes, in an effort to halt tumor progression, has demonstrated the potential for limiting gene transfer to the tumor cells and not the surrounding normal explant tissue.
  • the organotypic slice culture assay is combined with high through put robotic, computer, and combinatorial library screening assays.
  • the initial screen is carried out by the high throughput screening assay.
  • examples of such assays are known to one of skill in the art.
  • Selected candidate drugs are then used in the organotypic slice culture, as described in detail in the Examples which follow, to identify those compounds which are therapeutic candidates or pose serious threats to an animal, such as for example, toxins, poisons and the like, especially those that affect the neural system.
  • a high throughput screening system for identifying therapeutic agents for treatment of neurological disorders comprises identifying a library of candidate compounds by robotic, computer screening and/or combinatorial libraries; and, contacting a test compound with a test organotypic brain slice culture and measuring (1) viability and (2) amount of dopamine production of the test organotypic brain slice culture; and, measuring (1) the viability and (2) the amount of dopamine production of a control organotypic brain slice culture; and, identifying a cellular dopamine inducing agent which induces production of dopamine in neural cells in the test culture as compared to the control culture, but does not reduce viability of the test culture in comparison with the control culture; wherein, the test organotypic brain slice culture and control brain slice culture contain viable cells and are derived from a mammal.
  • the candidate drugs are identified by laser desorption/ionization mass spectrometry, HPLC, ELISA, MALDI, SELEX, biochips or immunochemical assays.
  • a system for identifying therapeutic agents for treatment of neurological disorders comprises (a) contacting a test compound with a test organotypic brain slice culture and measuring (1) viability and (2) amount of dopamine production of the test organotypic brain slice culture; and, (b) measuring (1) the viability and (2) the amount of dopamine production of a control organotypic brain slice culture; and, (c) identifying a cellular dopamine inducing agent which induces production of dopamine in neural cells in the test culture as compared to the control culture, but does not reduce " viability of the test culture in comparison with the control culture; wherein, the test organotypic brain slice culture and control brain slice culture contain viable cells and are derived from a. mammal.
  • the mammal is selected from the group consisting of rats, rabbits, guinea pigs and mice.
  • the organotypic brain slice culture is an explant obtained from a brain region selected from the group consisting of hippocampus and cortex.
  • the organotypic brain slice culture is a section of tissue from about 100 ⁇ rn to about 500 ⁇ m thick. Dopamine levels are determined by extracellular secretions and loss of neuronal cyto architecture and neural circuits is a function of cell death. Viability of Che organotypic brain slice cultured is at least about six weeks in culture in the absence of a neurologically toxic agent.
  • the viability of the organotypic brain slice culture is determined by loss of tyrosine hydroxylase reactivity, visual inspection under a microscope; staining using vital dyes stains and immunohistochemical reagents specific for cell types or moieties present in normal and injured brain; reaction with antibodies to neurofilaments, glial fibrillary acidic protein, SlOO, microtubule associated protein, normal or phosphorylated tau, and synaptic proteins; biochemical assessment of metabolic activity; measurement of total or specific protein content; assessment of cellular function; and assessment of neural activity. Viability and neural circuitry functions of the organotypic brain slice culture is determined by measuring neurotransmitter secretion.
  • neurotransmitter secretion is stimulated by a method selected from the group consisting of electrical stimulation, ionic depolarization and application of neurotransmitter substance and the presence of a neurotransmitter selected from the group consisting of acetylcholine, ⁇ -amino butyric acid (GABA), glutamate a catecholamines, and neuropeptides.
  • a neurotransmitter selected from the group consisting of acetylcholine, ⁇ -amino butyric acid (GABA), glutamate a catecholamines, and neuropeptides.
  • Neurotransmitter secretion in a trans-well culture medium is determined by a method selected from the group consisting of immunoprecipitation, ELISA, gel electrophoresis, RIA and Western blotting.
  • a vector is targeted to cellular components of a slice culture, said vector expressing reporter genes is detectable in viable cells. Detection of reporter genes is indicative of a functioning neural circuit.
  • isolated stem cells are administered to trie organotypic brain slice culture, and these stem cells differentiate and integrate into the tr ⁇ ree dimensional cell/tissue environment of the organotypic brain slice culture.
  • Figure 1 is an image of an organotypic slice culture showing viability.
  • Figure 2 is an image of an organotypic slice culture in which lesions have been induced.
  • Figure 3 is an image showing embryonic stem cells derived from neural precursors engrafted in the SNc of an organotypic slice culture.
  • Figure 4 is an image showing whole bone marrow and hematopoietic stem cells transplanted in an organotypic slice culture.
  • Figure 5 is an organotypic slice culture of a brain tumor.
  • Figure 6 is an illustrative method of the invention showing the viability and integrity of the slice culture preparations and is a schematic representation of the slice culture procedure.
  • Figures 7 A to 7B are images showing slice culture viability and nigrostriatal circuit maintenance.
  • Figure 7A is a montage showing DiI (red) application in the striatum, and evidence of labeled cells in the SNc.
  • Hoechst labeling of the hippocampus shows an intact cyto architecture in the dentate gyrus (DG) as well as CAl.
  • the montage in Figure 7B shows TH (red) staining in the slice culture. There is robust staining in the SNc, the medial forebrain bundle (MFB), and in the striatum.
  • Figures 8A to 8C show 6-OHDA lesion of nigrostriatal slice cultures.
  • Figxire 8A shows TH staining of a slice culture that was exposed to 20 mM 6-OHDA.
  • Inset in Figure 8 A shows high magnification views of TH (red) staining in the control (left) and lesioned (right) regions of the substantia nigra (top) and the striatum (bottom).
  • Nigral TH staining after exposure to 6-OHDA is characterized by a loss of discrete TH labeling.
  • the histogram in Figure 8B shows the percent reduction in SNc cell bodies P ⁇ 0.05) and Figure 8C shows the reduction in optical density of TH staining in the striatum (P ⁇ 0.05).
  • Figures 9A to 9F show the applications for nigrostriatal slice cultures.
  • Figure 9A shows GFP positive ESNP's 2 weeks after engraftment. Addition of laminin enhanced the incorporation of ESNP's and increased their migratory potential after 2 weeks in cultiire.
  • GFP + ESNP's matured into Map2 expressing neurons (Figure 9C inset, green-GFP, red Map2) and exhibited extensive process outgrowth (Figure 9C, green-GFP, red-TH, blue- Hoechst).
  • ESNP's pretreated with, ventralizing agents generated TH neurons (red) that survived and matured in the striatum ( Figure 9D) and within the cortex (Figure 9E). Electrophysiological recording from ESNP-derived neurons engrafted into the striatum, and showed action potentials and postsynaptic currents, suggesting a neuronal maturation and integration into the slice culture neuronal architecture.
  • Organotypic slice culture systems are a novel technique that bridges the gap between in vitro and in vivo systems. These culture systems have been used for a variety of investigations into development of neuronal architecture and circuits. In addition these cultures allow for the direct observation of pathological conditions, such as demyelination, epilepsy, and on brain circuits and cell populations.
  • pathological conditions such as demyelination, epilepsy, and on brain circuits and cell populations.
  • the compositions and systems disclosed herein, are useful for diagnosis of neurological disorders, drug discovery, imaging and the like.
  • organotypic brain slice culture refers to sections or explants of brain tissue which are maintained in culture (Seil (1979) Review in Neiiroscience 4:105-177; Gahwiler (1981) J Neurosci Meth 4:329-342; Gahwiler (1984) Neuroscience 11 :751-760, Gahwiler (1988) Trends Neurosci 11 :484-490; Stoppini et al. (1991) J Neurosci Methods 37:173-182).
  • the slice culture comprises any synaptically connected two or more structures in the brain.
  • Examples include, nigrostriatal circuit, hippocampal and cortical structure, Telencephalon: cerebral cortex, corpora striata (caudate nucleus, internal capsule, putamen), and rhinencephalon (e.g., olfactory bulb, hippocampus, amygdala, septal region, and cingulate cortex); Diencephalon: thalamus, hypothalamus, pineal gland, and posterior lobe of the pituitary (a migrated portion of the hypothalamus); Midbrain, Mesencephalon: corpora quadrigemia (tectum— inferior and superior colliculi), tegmentum; Hindbrain; Metencephalon: cerebellum, pons; Myelencephalon: medulla oblongata; Brain Stem: pons, medulla, spinal cord.
  • the slice can be from any position, for example: Rostral/ Anterior; Caudal/ Posterior ; Dorsal; Ven
  • dopaminergic neuronal cells refers to those cells generally found in the region of the ventral midbrain (VM) known as the substantia nigra pars compacta that project to the striatum. The precursor cells are typically found near the midbrain/hindbrain junction of an intact brain.
  • Dopaminergic neurons can be characterized by their secretion of dopamine as a neurotransmitter and high levels of expression of tyrosine hydroxylase (TH), an enzyme that catalyzes the rate limiting step in the biosynthesis of dopamine.
  • TH tyrosine hydroxylase
  • normal cells refer to cells that are isolated from any animal tissue, organ, adult cells, precursor cells, bone marrow cells, and the like. These include, neural cells, epithelial cells, liver cells, kidney cells and the like.
  • Neuronal cells as defined herein, are cells that reside in the brain, central and peripheral nerve systems, including, but not limited to, nerve cells, glial cell, oligodendrocyte, microglia cells or neural stem cells.
  • Abnormal cells refer to cells isolated from cancers or have been damaged in any way, such as physical, chemical, mechanical damage. Examples of disease states, neural disorders, etc, from which such, abnormal cells can be obtained are given below.
  • Spynaptically connected refers to the specialized junctions through which cells of the nervous system signal to one another and to non-neuronal cells such as muscles or glands.
  • a synapse between a motor neuron and a muscle cell is called a neuromuscular junction. Synapses allow the neurons of the central nervous system to form interconnected neural circuits. They are thus crucial to the biological computations that underlie perception and thought. They also provide the means through which the nervous system connects to and controls the other systems of the body.
  • cells may be transfected with a nucleic acid of interest which encodes a neurologically relevant polypeptide.
  • the term "neurologically relevant peptide” generally refers to a peptide or protein which catalyzes a reaction within the tissues of the central nervous system. Such peptides may be naturally occurring neural peptides, proteins or enzymes, or may be peptide or protein fragments which have therapeutic activity within the central nervous system.
  • Examples of neurologically relevant peptides include neural growth factors, neurotransmitters and enzymes used to catalyze the production of important neurochemicals or their intermediates.
  • the peptide encoded by the nucleic acid may exogenous to the host or endogenous.
  • an endogenous gene that supplements or replaces deficient production of a peptide by the tissue of the host wherein such deficiency is a cause of the symptoms of a particular disorder.
  • the cell lines act as an artificial source of the peptide.
  • the peptide may be an enzyme which catalyzes the production of a therapeutic or neurologically relevant compound.
  • neurologically relevant compounds include tyrosine hydroxylase, nerve growth factor (NGF), brain derived neurotrophic factor (BDGF), basic fibroblast growth factor ( ⁇ FGF) and glial cell line derived growth factor (GDGF).
  • NGF nerve growth factor
  • BDGF brain derived neurotrophic factor
  • ⁇ FGF basic fibroblast growth factor
  • GDGF glial cell line derived growth factor
  • traumatic brain injury is art recognized and is intended to include the condition in which, a traumatic blow to the head causes damage to the brain, often without penetrating the skull.
  • the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure (ICP), and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow (CBF).
  • ICP intracranial pressure
  • CBF cerebral blood flow
  • CHI Closed head injury
  • Pre-injury administration of the muscarinic antagonist scopolamine facilitates recovery from brain injury, suggesting that rapid suppression of tine early immediate intense stimulation mediated by acetylcholine released cholinergic hyperexcitation, during the first few post-injury minutes post-injury is therapeutically advantageous.
  • other methods are needed that intervene at biologically significant steps so that recovery is assured and long-term risk factors for neurodegenerative diseases are avoided.
  • Neurodegenerative disorders Parkinson's; Alzheimer's) or autoimmune disorders (multiple sclerosis) of the central nervous system; memory loss; long term and short term memory disorders; learning disorders; autism, depression, benign forgetfulness, childhood learning disorders, close head injury, and attention deficit disorder; autoimmune disorders of the brain, neuronal reaction to viral infection; brain damage; depression; psychiatric disorders such as bi-polarism, schizophrenia and the like; narcolepsy/sleep disorders (including circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage; severance of the cerebrospinal nerve cord (CNS) and any damage to brain or nerve cells; neurological deficits associated with AIDS; tics (e.g.
  • Giles de Ia Tourette's syndrome Huntington's chorea, schizophrenia, traumatic brain injury, tinnitus, neuralgia, especially trigeminal neuralgia, neuropathic pain, inappropriate neuronal activity resulting in neurodysthesias in diseases such as diabetes, MS and motor neuron disease, ataxias, muscular rigidity (spasticity) and temporomandibular joint dysfunction; Reward Deficiency Syndrome (RDS) behaviors in a subject; neurotoxicity caused by alcohol or substance abuse (e.g. ecstacy, methamphetamine, etc.).
  • RDS Reward Deficiency Syndrome
  • Parkinson's disease independent of a specific etiology, is a chronic, progressive central nervous system disorder which usually appears insidiously in the latter decades of life. The disease produces a slowly increasing disability in purposeful movement. It is characterized by four major clinical features of tremor, bradykinesia, rigidity and a disturbance of posture. Often patients have an accompanying dementia. In idiopathic Parkinsonism, there is usually a loss of cells in the substantia nigra, locus ceruleus, and other pigmented neurons of the brain, and a decrease of dopamine content in nerve axon terminals of cells projecting from the substantia nigra.
  • RDS behaviors are those behaviors that manifests as one; or more behavioral disorders related to an individual's feeling of well-being with anxiety, anger or a craving for a substance.
  • RDS behaviors include, alcoholism, SUD, smoking, BMI or obesity, pathological gambling, carbohydrate bingeing, axis 11 diagnosis, SAB, ADD/ADHD, CD, TS, family history of SUD, and Obesity. All these behaviors, and others described herein as associated with RDS behaviors or genes involved in the neurological pathways related to RDS, are included as RDS behaviors as part of this invention. Additionally, many of the clinical terms used herein for many specific disorders that are RDS disorders are found in the Quick Reference to the Diagnostic Criteria From DSM-IVTM, The American Psychiatric Association, Washington, D. C, 1994.
  • Affective disorders including major depression, and the bipolar, manic- depressive illness, are characterized by changes in mood as the primary clinical manifestation.
  • Major depression is the most common of the significant mental illnesses, and it must be distinguished clinically from periods of normal grief, sadness and disappointment, and the related dysphoria or demoralization frequently associated with medical illness.
  • Depression is characterized by feelings of intense sadness, and despair, mental slowing and loss of concentration, pessimistic worry, agitation, and self-deprecation.
  • Physical changes can also occur, including insomnia, anorexia, and weight loss, decreased energy and libido, and disruption of hormonal circadian rhythms.
  • Mania is characterized by changes in mood as the primary symptom. Either of these two extremes of mood may be accompanied by psychosis with disordered thought and delusional perceptions. Psychosis may have, as a secondary symptom, a change in mood, and it is this overlap with depression that causes much confusion in diagnosis. Severe mood changes without psychosis frequently occur in depression and are often accompanied by anxiety.
  • schizophrenia refers to a psychiatric disorder ttiat includes at least two of the following: delusions, hallucinations, disorganized speech, grossly disorganized or catatonic behavior, or negative symptoms. Patients can be diagnosed as schizophrenic using the DSM-IV criteria (APA, 1994, Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition), Washington, D. C).
  • Alzheimer's Disease refers to a progressive mental deterioration manifested by memory loss, confusion and disorientation beginning in late middle life and typically resulting in death in five to ten years.
  • Pathologically, Alzheimer's Disease can be characterized by thickening, conglutination, and distortion of the intracellular neurofibrils, neurofibrillary tangles and senile plaques composed of granular or filamentous argentophilic masses with an amyloid core. Methods for diagnosing Alzheimer's Disease are known in the art.
  • NINCDS-ADRDA Alzheimer's Disease and Related Disorders Association
  • NINCDS-ADRDA National Institute of Neurological and Communicative Disorders and Stroke- Alzheimer's Disease and the Alzheimer's Disease and Related Disorders Association
  • autism refers to a state of mental introversion characterized by morbid self-absorption, social failure, language delay, and stereotyped behavior. Patients can be diagnosed as suffering from autism by using the DSM-IV criteria.
  • depression refers to a clinical syndrome that includes a persistent sad mood or loss of interest in activities, which lasts for at least two weeks in the absence of treatment. The DSM-IV criteria can be used to diagnose patients as suffering from depression.
  • Benign forgetfulness refers to a mild tendency to be unable to retrieve or recall information that was once registered, learned, and stored in memory (e.g., an inability to remember where one placed one's keys or parked one's car). Benign forgetfulness typically affects individuals after 40 years of age and can be recognized by standard assessment instruments such as the Wechsler Memory Scale (Russell, 1975, J. Consult Clin. Psychol 43:800-809).
  • childhood learning disorders refers to an impaired ability to learn, as experienced by certain children. Such learning disorders can be diagnosed by using the DSM-IV criteria.
  • close head injury refers to a clinical condition after head injury or trauma which condition can be characterized by cognitive and memory impairment. Such a condition can be diagnosed as "amnestic disorder due to a general medical condition" according to DSM-IV.
  • ADHD Attention-deficit disorder
  • ADD Attention-deficit disorder
  • Symptoms and signs include hyperactivity (e.g., ADDH and AD /HD, DSM-IV), impulsivity, emotional lability, motor incoordination and some perceptual difficulties.
  • Treatment has included psychostimulants, which while effective are controversial, and may cause troubling side effects such as dysphoria, headache and growth retardation.
  • Other drugs including the tricyclic antidepressants, appear to improve attention, but may be less effective than the psychostimulants.
  • subcellular localization refers to defined subcellular structures within a single nerve cell. These subcellularly defined structures are matched with unique neural proteins derived from, for example, dendritic, axonal, myelin sheath, presynaptic terminal and postsynaptic locations. Furthermore, mature neurons are differentiated into dedicated subtype fusing a primary neural transmitter such as cholinergic (nicotinic and mucarinic), glutamatergic, gabaergic, serotonergic, dopaminergic.
  • cholinergic neurotinic and mucarinic
  • glutamatergic glutamatergic
  • gabaergic gabaergic
  • serotonergic dopaminergic
  • a "pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • patient or “individual” are used interchangeably herein, and is meant a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • amelioration or treatment refers to a symptom which is approaches a normalized value, e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a nonxialized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • amelioration or treatment of depression includes, for example, relief from the symptoms of depression which include, but are not limited to changes in mood, feelings of intense sadness and despair, mental slowing, loss of concentration, pessimistic worry, agitation, and self- deprecation.
  • “treating Parkinson's disease” or “ ameliorating * ' as used herein means relief from the symptoms of Parkinson's disease which include, but are not limited to tremor, bradykinesia, rigidity, and a disturbance of posture.
  • “cancer” refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.
  • cancers are cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • leukemia refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease- acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • the present invention includes a method of treating leukemia, and, preferably, a method of treating acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythernic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leuk
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • sarcomas include, but not limited to a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma,
  • 'melanoma is taken to mean a tumor arising from the melanocyte system of the skin and other organs.
  • Melanomas include but not limited to, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
  • Carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • Carcinomas include but not limited to, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelial e adenoides, exophytic carcinoma
  • Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
  • Producer cells/packaging cells can be of any suitable cell type.
  • Producer cells are generally mammalian cells but can be, for example, insect cells.
  • the term, "producer cell” or “vector producing cell” refers to a cell which contains all the elements necessary for production of retroviral vector particles.
  • the producer cell is obtainable from a stable producer cell line.
  • the term "derived producer cell line” is a transduced producer cell line which has been screened and selected for high expression of a marker gene. Such cell lines support high level expression from the retroviral genome.
  • the term “derived producer cell line” is used interchangeably with the term "derived stable producer cell line” and the term “stable producer cell line.
  • the derived producer cell line includes but is not limited to a retroviral and/or a lentiviral producer cell, such as an HIV or EIAvV producer cell line.
  • envelope protein sequences, and nucleocapsid sequences are all stably integrated in the producer and/or packaging cell.
  • one or more of these sequences could also exist in episomal form and gene expression could occur from the episome.
  • packaging cell refers to a cell which contains those elements necessary for production of " infectious recombinant virus which are lacking in the RNA genome.
  • packaging cells typically contain one or more producer plasmids which express viral structural proteins (such as codon optimized gag-pol and env) but they do not contain a packaging signal.
  • packaging signal which is referred to interchangeably as “packaging sequence” or “psi” is used in reference to the non-coding, cis-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation.
  • this sequence has been mapped to loci extending from upstream of the major splice donor site (SD) to at least the gag start codon.
  • Simple packaging cell lines, comprising a pro virus in which the packaging signal has been deleted, have been found to lead to the rapid production of undesirable replication competent viruses through recombination. In order to improve safety, second generation cell lines have been produced wherein the 3'LTR of the provirus is deleted.
  • a further improvement involves the introduction of the gag-pol genes and the env gene on separate constructs so-called third generation packaging cell lines. These constructs are introduced sequentially to prevent recombination during transfection.
  • the packaging cell lines can be second generation packaging cell lines, third generation packaging cell lines etc In these split-construct, third generation cell lines, a further reduction in recombination may ⁇ be achieved by changing the codons.
  • This technique based on the redundancy of the genetic code, aims to reduce homology between the separate constructs, for example between the regions of overlap in the gag-pol and env open reading frames.
  • the packaging cell lines are useful for providing the gene products necessary to encapsidate and provide a membrane protein for a high titer vector particle production.
  • the packaging cell may be a cell cultured in vitro such as a tissue culture cell line. Suitable cell lines include but are not limited to mammalian cells such as murine fibroblast derived cell lines or human cell lines.
  • the packaging cell line is a primate or human cell line, such as for example: HEK293, 293 -T, TE671. HT1080.
  • test compound or “candidate drug” are used interchangeably and refer to any chemical entity, pharmaceutical, drug, and the like contemplated to be useful in. the treatment and/or prevention of a disease, illness, sickness, or disorder of bodily function., or otherwise alter the physiological or cellular status of a sample.
  • Test compounds comprise both known and potential therapeutic compounds and also toxins, siRNA and the like.
  • a test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • Organotypic slice culture systems represent a novel culture system that seeks to combine the advantages of both in vivo and in vitro experimentation.
  • the organotypic slice culture described allows for long term survival coupled with the choice of explants or sections of whole organ such as the brain, or, a discrete anatomical brain structure such as hippocampus or cerebellum.
  • a surprising feature of the organotypic culture system is the striking preservation of organotypic tissxie architecture: cellular anatomy resembles that in the intact brain, that is., synaptic inputs and function mimic that of the normal situation, and development continues in neonatal brain slices.
  • the invention provides a long term survival slice culture system of brain and neural sections.
  • the sections are derived from the brain and cell viability is at least about 3 weeks, preferably about 4 weeks, preferably, cell viability is up to six weeks.
  • the viability of the cells comprising the slice culture medium is detennined by individual cell viability and an intact neural circuitry.
  • An example of cell viability is discussed in detail in the examples which follow.
  • Standard viability assays can be used.
  • standard viability assay refers to the following protocols which measure the relative survival rates of cells in the slice culture such as cells exposed to a candidate therapeutic agent and control cells not so exposed.
  • Sue Ii standard viability assays include, without limitation: a) Neutral red uptake, wherein the living cells take up the dye, rather than excluding the dye and the percentage of cells containing dye is then measured in each sample and compared with results obtained in other samples, which may include control samples; b) Dye exclusion, which comprises contacting treated cells with a dye (Trypan Blue) which is excluded by live cells, wherein dead cells which take up the dye are counted and percent viability thereby determined for comparison with values observed in other samples, which may include control samples; c) Direct visualization (microscope); and d) Use of a modified MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide [Thiazolyl Blue]) Cytotoxicity Assay (Sigma). Additional known methods can be employed.
  • the slice culture is used for determining the nigrostriatal pathway degeneration du.e to disease, methods for cellular and neural circuitry regeneration as well as a novel assay system for tumor growth and treatment.
  • substantial TH immunoreactivity is observed in the substantia nigra (SN) and striatum.
  • Intact cell bodies of the SN and axons of the nigrostriatal circuit are observed in these cultures for 3.5 weeks in culture.
  • the nigrostriatal system in these long term explant cultures is being used to determine neurotoxic agents and their effects on dopamine neurons.
  • One of the important uses of the invention is analysis of neurotoxins in the environment, such as a bioterrorist attack on a population.
  • the slice cultures can be used to provide a continuous monitoring of, for example, air and water and the neurological effects on a human population.
  • this system is being used to screen replacement cell therapies using ES derived and adult stem cells.
  • the slice culture system provides an assay system for determining tumor cell growth and the effects of test compounds on inhibiting tumor cell growth.
  • an assay system for determining tumor cell growth and the effects of test compounds on inhibiting tumor cell growth.
  • An example of a tumor assay system is determining RG2 tumor cell growth and assessment of potential therapeutic strategies more efficiently.
  • tumor cells are directly implanted into the animal are tracked in slice cultures past the point of lethal tumor growth. These slices also allow for the easy application of therapeutic agents and the quick visualization of their efficacy. Treatment with a novel polymer based gene delivery system to introduce therapeutic genes, in an effort to halt tumor progression, has demonstrated the potential for limiting gene transfer to the tumor cells and not the surrounding normal explant tissue.
  • the organotypic slice culture systems are used to determine the development of neuronal architecture and circuits and also the effects of neurological disorders both on the development of the neural system and in a pathological state.
  • pathologies include, but not limited to demyelination, epilepsy on brain circuits and cell populations.
  • the interface model of slice culture generates mid-sagittal slices containing an intact nigrostriatal circuit, including the substantia nigra pars compacta (SNc), medial forebrain bundle, and striatum, in both mice and rat cultures.
  • organotypic slice cultures were generated from both mice and rats. All slices generated from rats, were from animals postnatal day 20 to postnatal day 23, and from mice postnatal day 15 to early adult. Animals were euthanized and quickly decapitated. The brains were cut into two sagittal halves and immersed in a preparation media (DMEM, L-ascorbic acid, L-Glutamate, and Penicillin/Streptomycin). The halves were then super-glued to the vibratome stage, medial surface down, and covered with cool molten 2% agar.
  • DMEM L-ascorbic acid
  • L-Glutamate L-Glutamate
  • Penicillin/Streptomycin Penicillin/Streptomycin
  • the stage was then placed in the vibratome chamber and filled with preparation media. Slices were cut between 300-400 ⁇ M, placed in cold preparation media and. scanned using a dissection microscope to select slices from the level of the media forebrain bundle. Slices, collected after selection, were immediately transferred to a transwell (Falcon) placed in a 6 well plate and incubated at 35 0 C and 5% CO 2 . Each transwell was suspended in 1.8 mL of "A" media, a proprietary culture media containing serum. The media was changed the next day and feeding was done every other day.
  • "A" media a proprietary culture media containing serum
  • organotypic brain slice culture employ sections of whole brain tissue or explants obtained from specific regions of the brain. Any region can be used to generate an organotypic brain slice culture.
  • the preferred source of the organotypic brain slice culture is explants obtained from specific regions of the brain, preferably the substantia nigra region.
  • Any mammal can be used as a tissue source for the explant that is used to generate the organotypic slice culture used in the present method.
  • the animal also serves as a tissue source and the organotypic slice culture can be established and maintained for a period sufficient to conduct the present methods.
  • Such mammals include, but are not limited to rats, mice, guinea pigs, monkeys, rabbits, embryos and humans.
  • the mammal used as a tissue source can be of any age.
  • the mammalian tissue source will be a neonatal mammal.
  • tissue for culturing from live animals the animal is quickly killed and decapitated, this generally being performed simultaneously.
  • the brain is then rapidly removed to a dissection media buffered to physiological pH.
  • a dissection media is a minimal essential media (MEM) buffered with 10 mM Tris, pH 7.2, and supplemented with antibiotic.
  • the brain or desired brain region is then isolated under a dissecting microscope under aseptic conditions. Entire brain tissue can be used to establish an organotypic brain slice culture. Alternatively, a specific area or region of the brain can be used as an explant source. The preferred regions for the source of the organotypic brain slice culture for assessing degeneration of dopamine releasing cells is the substantia nigra. [00086] Next small regions are separated from the tissue as slices or explants such that the surface to volume ratio allows exchange between the center of the tissue and the media. A variety of procedures can be employed to section or divide the brain tissues. For example., sectioning devices can be employed.
  • the size/thickness of the tissue section will be based primarily on the tissue source and the method used for sectioning/division. For example, preferred segments are from about 400 to about 500 ⁇ m in diameter and are made using a tissue chopper, razor blade, or other appropriate sectioning/microtome blade.
  • sections are separated and damaged tissue removed.
  • the sections of brain tissue are preferably manipulated in drops of dissecting media and placed on culture plate inserts in culture media. Excess media is drawn off, for example by using a tissue, and the culture is placed in an incubator.
  • the choice of culture media and culture conditions depends on the intended use, the source of tissue, and the length of time before the section is used in the present method. Examples of culture media include, but is not limited to 25% horse serum, 50% minimum essential media, 25% Hank's media, supplemented with antibiotic and L-glutamine. Examples of culture condition include, but are not limited to, 37 0 C, 5% CO 2 .
  • Organotypic b rain slice cultures are preferably used after they have stabilized following the trauma of transfer to culture, but before onset of decline. In general, it is preferable to use the slice cultures from about 1 week to about 4 weeks after they have been generated.
  • organotypic brain slice culture After the organotypic brain slice culture is obtain, it is tested for viability prior to the application of a test compound.
  • the viability/integrity of the organotypic slice culture is typically assessed at the initiation of each experiment in order to demonstrate the health of the preparation as well as to provide a measure of the amount of viable tissue present in the pretreated culture.
  • any method known in the art for determining viability can be used, such as discussed supra.
  • methods include, but are not limited to: visual inspection under a microscope; staining of sister cultures with vital dyes such as trypan blue; stains and immunohistochemical reagents specific for cell types or moieties present in normal and injured brain, such as silver stains, and antibodies to neurofilament, glial fibrillary acidic protein, SlOO, microtubule associated protein, normal or phosphorylated tau, and synaptic proteins; biochemical assessment of metabolic activity, such as with an MTT assay or of cellular leakiness, such as by a lactate dehydrogenase (LDH) assay; measurement of total or specific protein content; or assessment of cellular function, such as synaptic activity.
  • vital dyes such as trypan blue
  • stains and immunohistochemical reagents specific for cell types or moieties present in normal and injured brain such as silver stains, and antibodies to neurofilament, glial fibrillary acidic protein,
  • Neural activity is apprised by measuring secretions such as soluble ⁇ -amyloid precursor protein secretion under basal conditions or neurotransmitter secretion upon stimulation. Stimulation can be accomplished by electrical stimulation, ionic depolarization (typically with high potassium), or application of neurotransmitter substance. Secreted substances typically measured are neurotransmitters present in the neurons such as acetylcholine, ⁇ -amino butyric acid (GABA), glutamate, catecholamines, and neuropeptides. A skilled artisan can readily adapt any of the presently known viability test methods for use in the present invention.
  • an organotypic slice culture is typically transferred to a culture dish with media.
  • the culture media can either have a test compound present prior to the introduction of the tissue section or a test compound can be added to the media after the tissue section has been place in the culture dish.
  • a test substance will be first dissolved in appropriate vehicle, such as, but not limited to,
  • DMSO dimethyl sulfoxide
  • water a stock solution
  • physiological saline a stock solution
  • media a stock solution
  • a vehicle control test will be included when the present invention is used.
  • a range of doses is tested.
  • the range tested initially may be informed by prior knowledge of the effects of the substance or closely related substances on purified enzymes, dopamine production by cells in culture, or toxicity in other test systems.
  • the dose range is preferably from about 1 rJVI to about 100 ⁇ M.
  • a skilled artisan can readily develop a testing range for any particular compound or series of compounds.
  • the compound is typically applied to the tissue section for about 4 hours to about 21 days, preferably from about 1 day to about 7 days.
  • fresh media containing compound can be applied periodically; more frequently if rapid loss of compound due to chemical conversion or to metabolism is suspected.
  • Compounds that are assayed in the above method can be randomly selected or rationally selected or designed.
  • a compound is said to be randomly selected when the compound is chosen randomly without considering the structure of other identified active compounds.
  • An example of randomly selected compounds is the use a chemical library, a peptide combinatorial library, a growth broth of an organism, or a plant extract.
  • a "compound” is said to be rationally selected or designed when the compound is chosen on a nonrandom basis. Rational selection can be based on the target of action or the structure of previously identified active compounds.
  • compounds can be rationally selected or rationally designed by utilizing the structure of compounds that are presently being investigated for use in treating neural disorders, for example, Parkinson's,
  • the compounds of the present invention can be, as examples, peptides, small molecules, and vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the compounds of the present invention.
  • the period of time in which the test compound is contacted with the slice culture, the viability of cells in the slice culture and the level/degree of dopamine production by the treated and control cultures are assessed.
  • a variety of art known methods can be employed to determine the amount of dopamine present. Such methods include, but are not limited to: determining the amount of .dopamine secretion into the culture media using immunoprecipitation (Zhong et al (1994) J Biol. Ch em. 16:1217912184; Higaki et al. (1995) Neuron 14:651-659); radioimmunoassay (Naidu et al. (1995) J Biol. Chem.
  • dopamine levels can be assessed biochemically.
  • Biochemically insoluble material obtained from pellets of centrifugation of brain homogenates can be assessed as just described for brain homogenates.
  • Dopamine deposits can be visualized by tyrosine hydroxylase (TH) staining, standard histochemical stains such as silver, thioflavin S, and Congo red. It is well with the skill of the art to adapt dopamine detection methods for use in the present invention.
  • TH tyrosine hydroxylase
  • standard histochemical stains such as silver, thioflavin S, and Congo red. It is well with the skill of the art to adapt dopamine detection methods for use in the present invention.
  • a measurement is made of trie amount of viable tissue in the slices producing dopamine. This measurement is used to normalize the values for dopamine in the media and as a means for determining the toxicity of a test agent.
  • the viability assay is the one employed at the initiation of the experiment.
  • an assay that does not damage the slice is used both at the initiation and conclusion of the test period.
  • Such a use provides the highest accuracy and allows efficient assessment of toxicity of the compound; therapeutic compounds that result in the replacement of circuitry components; protection of circuitry components and the like. If this is not possible, either a survival assay is used at tine initiation of the experiment or an assay that destroys the slices and/or mimics a neurological disorder is performed on sister a culture.
  • the effects of test compounds on tumor cells is determined.
  • the effects of therapeutic compounds on the treatment of neoplastic disease or neoplastic cells is determined in many ways such as: (1) inhibition, to some extent, of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) redtiction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion and/or (8) relief, to some extent, of the severity or number of one or more symptoms associated with the disorder.
  • therapeutic compounds ameliorate tumor growth and/or proliferation as determined by the changes in tumor described supra.
  • tumors are ameliorated by about 50%, more preferably by about 75%, most preferably about 90%, 95%, 98%, 99%, 99.9% and 100%.
  • the present invention relates to a method for treating a neurodegenerative disease and/or symptoms thereof and/or preventing neurodegenerative disease and/or symptoms thereof, in a mammal, comprising, administering a DNA. molecule, such as a lentiviral vector, to a target cell in the brain or nervous system of the mammal.
  • a DNA. molecule such as a lentiviral vector
  • the DNA molecule may comprise a 3' flanking region which will stabilize the transcript made by the molecule and terminate transcription coming from the molecule, located 3' to the gene to be expressed. See for example, Moreira, A. et al, EMBO J., 14, 3809 (1995).
  • the 3' flanking region contains a transcription terminator and stabilizing elements such as a polyA region.
  • the 3' flanking region will be located where the transcript will terminate.
  • the preferred 3' flanking sequences include the 3' flanking regions from the genes encoding ⁇ -galactosidase, SV40, ⁇ -globin, ⁇ -globin, and human growth hormone.
  • the most preferred 3' flanking sequence is a 3' flanking sequence from a human growth hormone gene.
  • the preferred promoter elements include promoters from the genes encoding: myosin heavy chain ⁇ , myosin heavy chain ⁇ , insulin, somatostatin, glucagon, growth associated protein 43 IcDa, superior cervical ganglion clone 10, neurofilament-L, neurofilament-M, neurofilarnent-H, glial bifilary protein, PO, myelin associated glycoprotein, myelin basic protein, calcitonin-gene related peptide, and a neuron specific enolase.
  • the most preferred promoter element is a neuron specific enolase promoter.
  • Preferred recombinase sites include FRT and Lo xP sites.
  • Preferred terminators include transcription terminators for gastrin, C2 complement, and ⁇ -globin.
  • genes to be controlled will include genes expressing regulatory factors, signal transducers, and developmental factors.
  • the preferred genes whose expression is to be controlled includes genes expressing hormones, hormone receptors, neurotransmitters, neurotrophic factors, neurotrophic factor receptors, neuronal peptides, cell signaling molecules, and receptors for any of these peptides.
  • the most preferred genes whose expression is to be controlled includes genes expressing neuronal growth factors.
  • Cells transduced with a vector expressing the peptide of choice can b>e analyzed by any method known in tlie art. All of these principles may be applied independently, in combination, or in combination with other known methods of sequence identification.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl Acad. ScL U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al, FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,
  • preferred genes includes genes expressing hormones, hormone receptors, neurotransmitters, neurotrophic factors, neurotrophic factor receptors, neuronal peptides, cell signaling molecules, and receptors for any of these peptides.
  • Preferred genes whose expression is to be controlled includes genes expressing neuronal growth factors.
  • Useful vectors include viral and plasmid vectors. Generally, this involves inserting the selected DNA molecule into an expression system to which that DNA. molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein- coding sequences.
  • Recombinant genes may also be introduced into viruses, such as lenti virus, vaccinia virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, lambda vector systems gtl 1, gt WES, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYCL 77, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS + ⁇ /- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F.
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.
  • host-vector systems may be utilized to express the protein-encoding sequence(s).
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with, bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with vims (e.g., lentivirus, vaccinia vims, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a numb er of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA.) translation).
  • a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promoted mRNA synthesis.
  • the DNA sequences of eukaryotic promoters differ from those of prol ⁇ aryotic promoters.
  • eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
  • the SD sequences are complementary to the 3 '-end of the 16S rRISfA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosomes.
  • 16S rRISfA ribosomal RNA
  • the present invention also includes any host cells carrying the recombinatorial substrate.
  • Host cells include bacterial or animal cells, which may be used to maintain or propagate the recombinatorial substrate.
  • Host cells also encompass mammalian cells which have been transformed with the recombinatorial substrate.
  • a preferred approach is to introduce a nucleic acid coding for the recombinase through the use of viral vectors.
  • Viral vectors have the potential of achieving regional gene expression in organotypic slice cultures and in vivo.
  • the preferred vectors include Adenovirus ("Ad") (AkIi S. et al., Nat. Genet. 3, 224 (1993); Bajocchi, G. et al, Nat. Genet., 3, 229 (1993); Davidson, B., et al., Nat. Genet., 3, 219, (1993); Le Gal La Salle, G. et al, Science, 259, 988 (1993 ), adeno-associated virus (“AAV”) (Kaplitt, M.
  • Ad Adenovirus
  • HSV Herpes Simplex Virus
  • vectors should produce regional infections of a slice culture and expressed in a predictable time course. Bath application of virus results in widespread gene transfer and expression predominantly in glia on the edge of slice cultures and little expression in neurons (Casaccia- Bonnefil, P., et al., J. Neiirosci. Methods, 50, 341 (1993). Using a micropipette to deliver nanoliter quantities of virus directly to regions of the slice culture produced regional infections. Analysis of gene product expression in such cultures showed that it was limited to the microapplication site.
  • the term "retrovirus” includes: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (HEIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A.- MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and all other retroviridiae including lentiviruses.
  • MLV murine leukemia virus
  • HAIAV human immunodeficiency virus
  • HEIAV equine infectious anemia virus
  • MMTV mouse mammary tumor virus
  • RSV Rous sarcoma virus
  • retroviruses A detailed list of retroviruses may b e found in Coffin et al ("Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763). Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J. 3053- 3058).
  • the lentivirus group can be split into “primate” and "non-primate”.
  • primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SrV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis vims (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Details on the genomic structure of some lentiviruses may be found in the art.
  • a retrovirus initially attaches to a specific cell surface receptor.
  • the retroviral RNA genome is then copied to DNA by the virally encoded reverse transcriptase which is carried inside the parent virus.
  • This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome.
  • the provirus is typically referred to as the provirus.
  • the provirus is stable in the host chromosome during cell division and is transcribed like other cellular genes.
  • the provirus encodes the proteins and other factors required to make more virus, which can leave the cell by a process sometimes called "budding".
  • Each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, trie LTRs can control the expression of the viral genes.
  • Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R. and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA
  • U5 is derived from the sequence unique to the 5 'end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.
  • the site of transcription initiation is at the boundary between U3 and R in the left hand side LTR and the site of poly (A) addition (termination) is at the boundary between R and XJ5 in the right hand side LTR.
  • U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • Some retroviruses have any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tat, rev, tax and rex;.
  • gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • the env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to infection by fusion of the viral membrane with the cell membrane.
  • Retroviruses may also contain "additional" genes which code for proteins other than gag, pol and env. Examples of additional genes include in HIV, one or more of vif, vpr, vpx, vpu, tat, rev and nef. EIAV has, for example, the additional genes S2 and dUTPase.
  • Proteins encoded by additional genes serve various functions, some of " which may be duplicative of a function provided by a cellular protein.
  • EIAV for example, tat acts as a transcriptional activator of the viral LTR. It binds to a stable, stem-loop RZNA secondary structure referred to as TAR. Rev regulates and co-ordinates the expression of viral genes through rev-response elements (RRE).
  • RRE rev-response elements
  • the mechanisms of action of these two proteins are thought to be broadly similar to the analogous mechanisms in the primate viruses.
  • the function of S2 is unknown, hi addition, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.
  • retroviral vector systems are used as a delivery system, inter alia, for the transfer of a nucleic acid sequence to one or more sites of interest.
  • the transfer can occur in vitro, ex vivo, in vivo, or combinations thereof.
  • Retroviral vector systems have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).
  • a recombinant retroviral vector particle is capable of transducing a recipient cell with a nucleic acid sequence of interest. Once within the cell the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the DNA of the recipient cell.
  • vector genome refers to both to the RNA construct present in the retroviral vector particle and the integrated DNA construct. The term also embraces a separate or isolated DNA construct capable of encoding such an RNA genome.
  • a retroviral or lentiviral genome should comprise at least one component part derivable from a retrovirus or a lentivirus.
  • the term "derivable” is used in its normal sense as meaning a nucleotide sequence or a part thereof which need not necessarily be obtained from a virus such, as a lentivirus but instead could be derived therefrom.
  • the sequence may be prepared synthetically or by use of recombinant DNA techniques.
  • the genome comprises a psi region (or an analogous component which is capable of causing encapsidation).
  • the viral vector genome is preferably "replication defective" by which the genome does not comprise sufficient genetic information alone to enable independent replication to produce infectious viral particles within the recipient cell.
  • the genome lacks a functional eiiv, gag or pol gene.
  • the viral vector genome may comprise some or all of the long terminal repeats (LTRs).
  • LTRs long terminal repeats
  • the genome comprises at least part of the LTRs or an analogous sequence which is capable of mediating proviral integration, and transcription.
  • the sequence may also comprise or act as an enhancer-promoter sequence.
  • the sequences encoding retroviral gag, pol and env proteins are introduced into the cell and stably integrated into the cell genome; a stable cell line is produced which is referred to as the packaging cell line.
  • the packaging cell line produces the proteins required for packaging retroviral RNA but it cannot bring about encapsidation due to the lack of a psi region.
  • the helper proteins can package the psi-positive recombinant vector RNA to produce the recombinant virus stock. This can be used to transduce the nucleic acid sequence of " interest into recipient cells.
  • the present invention also provides a packaging cell line comprising a viral vector genome of the first aspect of the invention.
  • the packaging cell line may be transduced with a viral vector system comprising the genome or transfected with, a plasmid carrying a DNA construct capable of encoding the RNA genome.
  • the present invention also provides a retroviral (or lentiviral) vector particle produced by such a cell.
  • the second approach is to introduce the three different DNA sequences that are required to produce a retroviral vector particle i.e. trie env coding sequences, the gag-pol coding sequence and the defective retroviral genome containing one or more nucleic acid sequences of interest into the cell at the same time by transient transfection, is referred to as transient triple transfection.
  • Further components of the viral system which complement the vector genome may be present on one or more "producer plasmids" for transfecting into cells.
  • An example of a disease associated with a neural disorder is Parkinson's disease.
  • Parkinson's disease is a neurodegenerative disorder characterized by the loss of the nigrostriatal pathway; a progressive disorder resulting from degeneration of dopaminergic neurons within the substantia nigra. Although the cause of Parkinson's disease is not known, it is associated with the progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) mesencephalic neurons, inducing motor impairment. The characteristic symptoms of Parkinson's disease appear when up to 70% of TH-positive nigrostriatal neurons have degenerated.
  • TH dopaminergic
  • L-DOPA dihydroxyphenylalanine
  • dopamine supplied from cells implanted into the striatum would be able to substitute for lost nigrostriatal cells.
  • Clinical trials have suggested that mesencephalic TH positive neurons obtained from human embryo cadavers (aborted fetuses) can survive and function in the brains of patients with Parkinson's disease.
  • functional recovery has only been partial, and the efficacy and reproducibility of the procedure is limited.
  • the large amounts of tissue required to produce a therapeutic effect would be prohibitive.
  • xenotransplantation requires immunosuppressive treatment and is also controversial due to, for example, trie possible risk of cross-species transfer of infectious agents.
  • Another disadvantage is that, in current grafting protocols, no more than 5-20% of the expected numbers of grafted THI positive neurons survive. In order to develop a practicable and effective transplantation protocol, an alternative source of TH positive neurons is required.
  • the main advantage of the invention is that the organotypic cell slice cultures are used for grafting stem cells onto the slice culture allowing for growth and maturation and/or transduction with a suitable vector for use in treatment of patients with neurodegenerative diseases.
  • cells are transduced with a vector system comprising a viral genome and a heterologous sequence encoding a protein/peptide of interest.
  • Transduction with the vector system of the present invention may confer or increase the ability of the cell to produce catecholamines, confer or increase the ability of the cell to convert tyrosine to L-dopa and/or L-dopa to dopamine. Release of catecholamines can be measured by techniques known in the art, for example by using an electrochemical detector connected to an analytical cell.
  • the cell may be any cell which is susceptible to transduction. If the vector system is capable of transducing non-dividing cells (for example if it is a lentiviral system) then the cell may be a non-dividing cell such as a neuron. [000131] In a preferred embodiment the transduced cell forms part of a genetically modified neuronal cell line. Such a cell line may, for example, be transplanted into trie brain for the treatment of Parkinson's disease.
  • the cell is a cell in the striatum of a subject, such as a neuron or glial cell.
  • Direct gene transfer in vivo to such a cell may, for example, convert it into a dopamine-producer cell.
  • a further alternative strategy for therapy is to replace dopamine in the affected striatum by introducing the enzymes responsible for L-DOPA or dopamine synthesis (for example, tyrosine hydroxylase); or introduce potential neuroprotective molecules that may either prevent the TH-positive neurons from dying or stimulate regeneration and functional recovery in the damaged nigrostriatal system (Dunnet S. B. and Bjorklund A. (1999) Nature 399 A32-A39).
  • the enzymes responsible for L-DOPA or dopamine synthesis for example, tyrosine hydroxylase
  • tyrosine hydroxylase TH
  • AADC aromatic amino acid DOPA-decarboxylase
  • Parkinson's disease has been shown to be responsive to treatments that facilitate dopaminergic transmission in caudate-putamen.
  • genetically modified cells that express tyrosine hydroxylase, and thereby synthesize L-DOPA induce behavioral recovery in rodent models of PD (Wolff et al. (1989) PNAS (USA) 86:9011-14; Freed et al (1990) Arch. Neurol. 47:505-12; Jiao et al. (1993) Nature 262:4505).
  • the functional activity of tyrosine hydroxylase depends on the availability of its cofactor tetrahydrobiopterin (BH 4 ).
  • the level of cofactor may be insufficient in the denervated striatum, and so it is thought that GTP cyclohydrolase I, the enzyme that catalyses the rate limiting step on the pathway of BH 4 -synthesis, may also need, to be transduced to obtain sufficient levels of L-DOPA production in vivo.
  • the present invention provides a system for growth of cells, cell grafting, testing of therapeutic candidate compounds, vectors expressing therapeutic peptides for the treatment of neurodegenerative disease in a mammal, e.g., Parkinson's disease.
  • the present invention provides a method for treating a neurodegenerative disease and/or symptoms thereof and/or preventing neurodegenerative disease and/or symptoms thereof, in a mammal, comprising, administering a vector to a target cell in the brain or nervous system of the mammal, the vector comprising a nucleic acid sequence comprising a sequence encoding a growth factor, advantageously in operable linkage with or operably linked to a promoter sequence, wherein said growth factor is expressed in the target cell, thereby treating said neurodegenerative disease.
  • the invention envisions polypeptides wherein amino acids are substituted on the basis of charge and/or structural similarities. That is, in determining suitable analogs, homologs, derivatives or variants of, for example, human GDNF, the skilled artisan, without undue experimentation, can consider replacing amino acids in therein with amino acids of similar charge and/or structure so as to obtain a variant, homolog, derivative or variant; and, from making such changes, the skilled artisan can derive a suitable nucleic acid molecule coding sequence for the variant, homolog, derivative, or variant of GDNT, without any undue experimentation. Thus, the skilled artisan can consider charge and/or structure of human GDNF sequences or portions thereof, in constructing homologs, variants, analogs and derivatives and nucleic acid molecules coding therefor, without undue experimentation.
  • PCR or hybridization primers or probes and optimal lengths therefor reference is also made to Kajimura et al., GATA 7(4):71-79 (1990).
  • the vector system transduces a target site, wherein the vector system travels to the site by retrograde transport.
  • the cell body is where a neuron synthesizes new cell products.
  • Two types of transport systems carry materials from trie cell body to the axon terminals and back.
  • the slower system which moves materials 1-5 rnm per day is called slow axonal transport. It conveys axoplasm in one direction only (from the cell body toward the axon terminals (anterograde transport)).
  • Vector systems comprising rabies G protein are capable of retrograde transport (i.e. traveling towards the cell body).
  • the precise mechanism of retrograde transport is unknown, however. It is thought to involve transport of the whole viral particle, possibly in association with an internalized receptor.
  • the fact that vector systems comprising rabies G can be specifically be transported in this manner suggests that the env protein may be involved.
  • HSV, adenovirus and hybrid HSV/adeno-associated virus vectors have all been shown to be transported in a retrograde manner in the brain (Horellou and Mallet (1997) MoI Neurobiol 15(2) 241-256; Ridoux et al (1994) Brain Res.
  • adenoviral vector system expressing glial cell line derived neurotrophic factor (GDNF) into rat striatum allows expression in both dopaminergic axon terminals and cell bodies via retrograde transport (Horellou and Mallet (1997) as above; Bilang-Bleuel et al (1997) Proc. Natl. Acad. ScL USA 94:8818-8823).
  • GDNF glial cell line derived neurotrophic factor
  • Retrograde transport can be detected by a number of mechanisms known, in the art.
  • a vector system expressing a heterologous gene is injected into the striatum, and expression of the gene is detected in the substantia nigra. It is clear tliat retrograde transport along the neurons which extend from the substantia nigra to the basal ganglia is responsible for this phenomenon. It is also known to monitor labeled proteins or viruses and directly monitor their retrograde movement using real time confocal microscopy (Hirokawa (1997).
  • the present invention thus also provides the use of a vector system where the vector system is or comprises at least part of nerve growth factors to transduce a target site, which comprises the step of administration of the vector system to an administration site which is distant from the target site.
  • the target site may be any site of interest which is anatomically connected to the administration site.
  • the target site should be capable of receiving vector from the administration site by axonal transport, for example anterograde or (more preferably) retrograde transport.
  • axonal transport for example anterograde or (more preferably) retrograde transport.
  • retrograde transport for a given administration site, a number of potential target sites may exist which can be identified using retrograde tracers by methods known in the art (R ⁇ doux et al (1994).
  • intrastriatal injection of HSV/AAV amplicon vectors causes transgene expression in the substantia nigra, cortex, several thalamic nuclei (posterior, paraventricular, parafasicular, reticular), prerubral field, deep mesencephalic nuclei, mesencephalic grey nucleus, and intrastitial nucleus of the medial as well as dorsal longitudinal fasiculus (Constantini et al (1999).
  • a target site is considered to be "distant from the administration” if it is (or is mainly) located in a different region from the administration site.
  • the two sites may be distinguished by their spatial location, morphology and/or function.
  • the basal ganglia consist of several pairs of nuclei, the two members of each pair being located in opposite cerebral hemispheres.
  • the largest nucleus is the corpus striatum which consists of the caudate nucleus and the lentiform nucleus.
  • Each lentiform nucleus is, in turn, subdivided into a lateral part called the putamen and a medial part called the globus pallidus.
  • the substantia nigra and red nuclei of the midbrain and the subthalamic nuclei of the diencephalon are functionally linked to the basal ganglia. Axons from the substantia nigra terminate in the caudate nucleus or the putamen. The subthalamic nuclei connect with the globus pallidus. For conductivity in basal ganglia of the rat see Oorschot (1996) J. Comp. Neurol. 366:580-599.
  • the administration site is the striatum of the brain, in particular the caudate putamen.
  • Injection into the putamen can label target sites located in various distant regions of the brain, for example, the globus pallidus, amygdala, subthalamic nucleus or the substantia nigra. Transduction of cells in the pallidus commonly causes retrograde labeling of cells in tlie thalamus.
  • the (or one of the) target site(s) is the substantia nigra.
  • the vector system is injected directly into the spinal cord.
  • This administration site accesses distal connections in the brain stem and cortex.
  • the vector system may transduce a target cell.
  • the target cell may be a cell found in nervous tissue, such as a neuron, astrocyte, oligodendrocyte, microglia or ependymal cell.
  • the target cell is a glial cell, in particular a TH positive cell.
  • the vector system is preferably administered by direct injection.
  • Methods for injection into the brain are well known in the art (Bilang-Eleuel et al (1997) Pi-oc. Acad. Nat. Sci. USA 94:8818-8823; Choi-Lundberg et al (1998) Exp. Neurol. 154:261-275; Choi-Lundberg et al (1997) Science 275:838-841; and Mandel et al (1997) Proc. Acad. Natl. Sci. USA 94:14083-14088). Stereotaxic injections may be given.
  • the viral preparation is concentrated by ultracentrifugation.
  • the resulting preparation should have at least 10 s t.u./ml, preferably from 10 8 to 10 10 t.u./rnl, more preferably at least 10 9 t.u./ml.
  • the titer is expressed in transducing units per ml (t.u./ml) as titered on a standard D 17 cell line). It has been found that improved dispersion of transgene expression can be obtained by increasing the number of injection sites and decreasing the rate of injection (Horellou and Mallet (1997) as above).
  • injection sites usually between 1 and 10 injection sites are used, more commonly between 2 and 6.
  • the rate of injection is commonly between 0.1 and 10 ⁇ l/min, usually about 1 ⁇ l/min.
  • the vector system is administered to a peripheral administration site.
  • the vector may be administered to any part of the body from which it can travel to the target site by retrograde transport. In other words the vector may be administered to any part of the body to which a neuron within the target site projects.
  • the "periphery" can be considered to be all part of the body other than the CNS (brain and spinal cord).
  • peripheral sites are those which are distant to the ClSfS.
  • Sensory neurons may be accessed by administration to any tissue which is innervated by the neuron. In particular this includes the skin, muscles and the sciatic nerve.
  • the vector system is administered intramuscularly.
  • the system can access a distant target site via the neurons which innervate the inoculated muscle.
  • the vector system may thus be used to access the CNS (in particular the spinal cord and brain), obviating the need for direct injection into this tissue.
  • CNS in particular the spinal cord and brain
  • a non-invasive method for transducing a cell within the CNS Muscular administration also enables multiple doses to be administered over a prolonged period.
  • Another advantage with this system is that it is possible to target particular groups of cells (e.g. substantia nigra), or a particular neural tract by choosing a particular administration site.
  • the vector preferably encodes at least one or more molecules such as for example, tyrosine hydroxylase, GTP-cyclohydrolase I, aromatic amino acid dopa decarboxylase and vesicular monoamine transporter 2 (VMAT2) to be employed in the practice of the invention or for coding sequences for tyrosine hydroxylase, GTP- cyclohydrolase I, aromatic amino acid dopa decarboxylase and vesicular monoamine transporter 2 (VMAT2) to be expressed with the growth factor, e.g., GDNF by the lentiviral vector.
  • VMAT2 vesicular monoamine transporter 2
  • high-titer vims preparations it is highly desirable to use high-titer vims preparations in both experimental and practical applications.
  • Techniques for increasing viral titer include using a psi plus packaging signal as discussed above and concentration of viral stocks.
  • high titer means an effective amount of a retroviral vector or particle which is capable of transducing a target site such as a cell.
  • the term, "effective amount” means an amount of a regulated retroviral or lentiviral vector or vector particle which is sufficient to induce expression of the nucleic acid sequences of interest at a target site.
  • a high-titer viral preparation for a producer/packaging cell is usually of the order of 10 5 to 10 7 retrovirus particles per ml.
  • the viral preparation is concentrated by ultracentrifugation.
  • the resulting preparation should have at least 10 8 t.u./ml, preferably from 10 8 to 10 9 t.u./ml, more preferably at least 10 9 t.u./ml.
  • the titer is expressed in transducing units per ml (t.u./ml) as titered on a standard D17 cell line).
  • Other methods of concentration such as ultrafiltration or binding to and elution from a matrix may be used.
  • the expression products encoded by the nucleic acid sequences of interest may be proteins which are secreted from the cell. Alternatively the nucleic acid sequence expression products are not secreted and are active within the cell. For some applications, it is preferred for the nucleic acid sequence expression product to demonstrate a bystander effect or a distant bystander effect; that is the production of the expression product in one cell leading to the modulation of additional, related cells, either neighboring or distant (e.g. metastatic), which possess a common phenotype.
  • the presence of a sequence termed th.e central polypurine tract (cPPT) may improve the efficiency of gene delivery to non-dividing cells.
  • This cis-acting element is located, for example, in the EIAV polymerase coding region element.
  • the genome of the present invention comprises a cPPT sequence.
  • a method for producing a vector particle which comprises introducing a viral genome into a producer cell; a viral particle produced by such a system or method; a pharmaceutical composition comprising such a genome, system or particle; the use of such a genome, system or particle in the manufacture of a pharmaceutical composition to treat and/or prevent a disease; a cell wliich has been transduced with such a system; a method of treating and/or preventing a disease " by using such a genome, system, viral particle or cell.
  • the invention provides pharmaceutical compositions comprising the vector or one type of vector and other vector(s), as well as kits for preparing such compositions (e.g., the vector or the vector and the other vector(s) in one or more containers and pharmaceutically acceptable excipient, carrier, diluent, adjuvant, and the like in one or more additional containers, wherein said containers can be provided in one or more packages, for instance, packaged together or separately, and optionally including instructions for admixture and/or administration).
  • kits for preparing such compositions e.g., the vector or the vector and the other vector(s) in one or more containers and pharmaceutically acceptable excipient, carrier, diluent, adjuvant, and the like in one or more additional containers, wherein said containers can be provided in one or more packages, for instance, packaged together or separately, and optionally including instructions for admixture and/or administration).
  • the vector is pseudotyped.
  • the vector is pseudotyped.
  • One manner in which to achieve this is by engineering the virus envelope protein to alter its specificity.
  • Another approach is to introduce a heterologous envelope protein into the vector particle to replace or add to the native envelope protein of the virus.
  • pseudotyping means incorporating in at least a part of, or substituting apart of, or replacing all of, an env gene of a viral genome with a heterologous env gene, for example an env gene from another virus.
  • the vector system is pseudotyped -with a gene encoding at least part of the rabies G protein. In a further preferred embodiment of the present invention the vector system is pseudotyped with a gene encoding at least part of the VSV-G protein.
  • Parkinson's disease is characterized by the progressive loss in function of dopaminergic neurons.
  • the progressive loss of dopaminergic function interferes with the normal working of the neuronal circuitry necessary for motor control so that patients with.
  • PD show characteristic motor disturbances such as akinesia, rigidity and rest tremor.
  • Other symptoms include pain, impaired olfaction, alterations of personality and depression. Q ⁇ inn et al., (1997) Clin. Neurol. 6:1-13.
  • dopaminergic neuronal cells are generated using the methods described above.
  • the dopaminergic cells are then administered to the brain of the patient in need thereof to produce dopamine and restore behavioral deficits in the patient.
  • the cells are administered to the basal ganglia of the patient.
  • Alzheimer's disease involves a deficit in cholinergic cells in the nucleus basalis.
  • a subject having Alzheimer's disease may be treated by administering cells produced according to the method of the invention that are capable of producing acetylcholine.
  • Huntington's disease involves a gross wasting of the head of the caudate nucleus and putamen, usually accompanied by moderate disease of the gyrus.
  • a subject suffering from Huntington's disease can be treated by implanting cells produced according to the method of the invention that are capable of producing the neurotransmitters gamma amino butyric acid (GABA), acetylcholine, or a mixture thereof.
  • GABA gamma amino butyric acid
  • acetylcholine or a mixture thereof.
  • the vector expresses nucleic acid sequences of interest.
  • the nucleic acid sequence of interest is a neural growth factor, such as GDNF, e.g., human GDNF or analogs, variants, derivatives, or homologs thereof.
  • Nucleic acid sequences include any suitable nucleotide sequence, which need not necessarily be a complete naturally occurring DNA or RNA sequence.
  • the nucleic acid sequence can be, for example, a synthetic RNA/DNA sequence, a codon optimized RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof.
  • the sequence need not be a coding region. If it is a coding region, it need not be an entire coding region.
  • the RNA/DNA. sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation.
  • the sequence is, comprises, or is transcribed from cDNA.
  • nucleic acid sequences of interest also referred to as “heterologous sequence(s)", “heterologous gene(s)” or “transgene(s)" may be any one or more of, for example, a selection gene(s), marker gene(s) and therapeutic gene(s).
  • the nucleic acid sequence of interest may be a candidate gene which is of potential significance in a diseas e process.
  • the vector system of the present invention may, for example, be used for target validation purposes.
  • the nucleic acid sequences preferably have a therapeutic and/or diagnostic application.
  • Suitable nucleic acid sequences include, but are not limited to: sequences encoding enzymes, cytokines, chernokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, imrrxune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdomina ⁇ t negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumor suppressor protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives thereof (such as with an associated reporter group).
  • the nucleic acid sequences may also encode pro-drug activating enzymes.
  • the nucleic acid sequence is useful in the treatment of a neurodegenerative disorder, such as, Parkinson's disease.
  • a neurodegenerative disorder such as, Parkinson's disease.
  • growth factors such as GDNF, e.g., human GDNF or an analog, homolog, derivative or variant thereof, or an enzyme involved in dopamine synthesis or storage.
  • the enzyme may be one of the following: tyrosine hydroxylase, GTP-cyclohydrolase I and/or aromatic amino acid dopa decarboxylase.
  • the sequences of all three genes are available: Accession Nos. X05290, Ul 9523 and M76180 respectively.
  • the nucleic acid sequence may encode the vesicular monoamine transporter 2 (VMAT2, Accession number L23205.1).
  • the viral genome comprises a nucleic acid sequence encoding a neural growth factor alone or in combination with aromatic amino acid dopa decarboxylase, VMAT 2 and/or combinations thereo f.
  • a neural growth factor alone or in combination with aromatic amino acid dopa decarboxylase, VMAT 2 and/or combinations thereo f.
  • Such a genome may be used in the treatment of Parkinson's disease, in particular in conjunction with peripheral administration of L-DOPAL.
  • the nucleic acid sequence encodes a growth factor that blocks or inhibits degeneration in the nigrostriatal system.
  • An example of such a growth factor is a neurotrophic factor.
  • the nucleic acid sequence encodes glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), persephin growth factor, artemin growth factor, or neurturin growth factor, cilliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), pantropic neurotrophin, and other related or unrelated neurotrophic factors, (see for example, WO99/14235; WO00/18799; U.S. Pat. No. 6,090,778; U.S. Pat. No. 5,834,914; WO97/08196; U.S. Pat. No. 6,090,778; U.S. Pat. No.
  • a vector comprises one or more of these nucleic acid sequences encoding neurotrophic factors.
  • the nucleic acid sequence encode a neuroprotective factor.
  • the nucleic acid sequences encode molecules which prevent TH-positive neurons from dying or which stimulate regeneration and functional recovery in the damaged nigrostriatal system.
  • the nucleic acid sequence may encode all or part of the protein of interest, or a mutant, homologue or variant thereof.
  • the nucleic acid sequence may encode a fragment of the desired protein which functions in vivo in an analogous manner to the wild-type protein.
  • one of the nucleic acid sequences comprise a truncated form of the TH gene, lacking the regulatory domain.
  • the encoded product avoids feed-back inhibition by dopamine which may limit expression of the full- length enzyme.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99.9% identical to the subject sequence.
  • the homologues comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology b etween two or more sequences. Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • Gap penalties are assigned to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible— reflecting higher relatedness between the two compared sequences— will achieve a higher score than one with many gaps.
  • "Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. Por example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7- 58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (set FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol L ett 1999 177(1): 187-8).
  • the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a. matrix commonly used is the BLOSLJM62 matrix—the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the vector can also include one or more internal ribosome entry site(s) (IRES).
  • the viral vector can comprise two or more heterologous nucleic acid sequences of interest (e.g., GDNF and another nucleic acid sequence expressing a protein of interest useful in treating or preventing a neurodegenerative condition such as Parkinson's disease) .
  • heterologous nucleic acid sequences of interest e.g., GDNF and another nucleic acid sequence expressing a protein of interest useful in treating or preventing a neurodegenerative condition such as Parkinson's disease
  • there may be two or more transcription units within the vector genome one for each heterologous sequence, operably linked to an internal ribosome entry site (IRES) to initiate translation of the second (and subsequent) coding sequence(s) in a poly-cistronic message (Adam et al 1991 J. Virol. 65, 4985).
  • IRES elements inserted into retroviral vectors is compatible with the retroviral replication cycle and allows expression of multiple coding regions from a single promoter.
  • IRES elements When located between open reading frames in an RNA, IRES elements allow efficient translation of the downstream open reading frame by promoting entry of the ribosome at the IRES element followed by downstream initiation of translation.
  • the IRES(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) or cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or E1F4 IRES).
  • the IRES In order for the IRES to be capable of initiating translation of each nucleic acid sequence of interest, it should be located between or prior to such heterologous sequences in the vector genome.
  • the methods of the invention are used for the repopulation of destroyed cells in an organ in need of repair.
  • an organ in need of repair For example, brain, CNS, P]NS, and the like.
  • the stem cells can be are grafted onto the organotypic slice culture. Any cell can be used in the methods of the invention, including but not limited to, stem cells, thymocytes, precursor cells and the like.
  • a precursor cell population includes cells of a mesodermal derived cellular lineage, more particularly of hematopoietic lineage, endothelial lineage, muscle cell lineage, epithelial cell lineage and neural cell lineage.
  • a "precursor cell” can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell.
  • the term “precursor cell population” refers to a group of cells capable of developing into a more mature cell.
  • a precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of neuronal lineage).
  • stem cell lineage restricted i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of neuronal lineage.
  • totipotent cell refers to a cell capable of developing into all lineages of cells.
  • the term “totipotent population of cells” refers to a composition of cells capable of developing into all lineages of " cells.
  • the term “pluripotent cell” refers to a cell capable of developing into a variety (albeit not all) lineages and are at least able to develop into all neural cell lineages.
  • a pluripotent cell can differ from a totipotent cell by having the ability to develop into all cell lineages except endothelial cells.
  • a “pluripotent population of cells” refers to a composition of cells capable of developing into less than all lineages of cells but at least into all hematopoietic lineages.
  • a totipotent cell or composition of cells is less developed than a pluripotent cell or compositions of cells.
  • the terms “develop”, “differentiate” and “mature” all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized cell. Such terms can be used interchangeably for the purposes of the present application.
  • the invention is of application without limitation to ES cell type, and may suitably be applied to vertebrate cells, in particular mammalian cells, primate cells, rodent cells, and human cells.
  • ES cells it is intended to encompass embryonic stem cells, embryonic carcinoma cells, embryonic gonadal cells, embryo-derived pluripotential stem cells and germline-derived stem cells.
  • the term “population” refers to cells having the same or different identifying characteristics.
  • the term “lineage” refers to all of the stages of the development of a cell type, from the earliest precursor cell to a completely mature cell (i.e. a specialized cell).
  • a stem cell population of the present invention is capable of developing into cells of mesodermal cell lineage, of ectodermal cell lineage or of endodermal cell lineage.
  • mesodermal cells include cells of connective tissue, bone, cartilage, muscle, blood and blood vessel, lymphatic and lymphoid organ, notochord, pleura, pericardium, peritoneum, kidney and gonad.
  • Ectodermal cells include epidermal tissue cells, such as those of nail, hair, glands of the skin, the nervous system, the external sense organs (e.g., eyes and ears) and mucous membranes (such as those of the mouth and anus).
  • Endodermal cells include cells of the epithelium such as those of the pharynx, respiratory tract (except the nose), digestive tract, bladder and urethra cells.
  • Preferred cells within a stem cell population of the present invention include cells of at least one of the following cellular lineages: hematopoietic cell lineage, endothelial cell lineage, epithelial cell lineage, muscle cell lineage and neural cell lineage.
  • Other preferred cells within a stem cell population of the present invention include cells of erythroid lineage, endothelial lineage, leukocyte lineage, thrombocyte lineage, erythroid lineage (including primitive and definitive erythroid lineages), macrophage lineage, neutrophil lineage, mast cell lineage, megakaryocyte lineage, natural killer cell lineage, eosinophil lineage, T cell lineage, endothelial cell lineage and B cell lineage,
  • Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation. If desired, a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude" separation. For example, magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed.
  • Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the stem cells may be transformed with DNA which codes for different growth, factors and/or cytokines which will aid in the differentiation of the stem cells if the organ of interest is damaged to the extent that the microenvironment is not supportive of cell differentiation.
  • stem cells can be induced to differentiate into a desired phenotype by administering growth factors, hormones, cytokines and the like.
  • growth factors hormones, cytokines and the like.
  • the organotypic slice culture comprising isolated stem cells can be used to can be used to screen for candidate therapeutic agents (such as solvents, small molecule dri ⁇ gs, peptides, polynucleotides, and the like) or detection of undesirable environmental conditions (such as toxins, chemical and biological agents that can be used in a potential terrorist attack, and the like) that affect the characteristics of differentiated cells.
  • candidate therapeutic agents such as solvents, small molecule dri ⁇ gs, peptides, polynucleotides, and the like
  • undesirable environmental conditions such as toxins, chemical and biological agents that can be used in a potential terrorist attack, and the like
  • the organotypic slice culture system comprising isolated stem cells are used to screen factors that promote maturation, or promote proliferation and maintenance of such cells in long-term culture.
  • candidate maturation factors or growth factors are tested by adding them to system, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.
  • Particular screening applications of this invention relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook “In vitro Methods in Pharmaceutical Research", Academic Press, 1997, and U.S. Pat. No. 5,030,015).
  • Assessment of the activity of candidate pharmaceutical compounds generally involves administering a candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells and function of the slice culture (for example, the nigrostriatal circuit as described in the examples which follow) that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.
  • the screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug— drug interaction effects.
  • Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [ 3 H]thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (PP 375-410 in “In vitro Methods in Pharmaceutical Research,” Academic Press, 1997) for further elaboration.
  • the organotypic slice culture is used to screen for candidate drugs or compounds.
  • the candidate drugs or compounds used in the present method are classified by protection against 6-hydroxy dopamine mediated lesions, reversal of 6-hydroxy dopamine mediated lesions, and the degree of toxicity displayed.
  • the most preferred compounds identified using the present method will be non-toxic, showing no reduction in viability between treated and non-treated cultures. However, low toxicity levels may be tolerable for certain uses (e.g., in initial compound testing and design).
  • the preferred compounds reduce 6-hydroxy dopamine mediated lesions by about 50%.
  • 6- hydroxy dopamine mediated lesions are reduced by about 75%, more preferably, 6-hydroxy dopamine mediated lesions are reduced by about 80%, more preferably, 6-hydroxy dopamine mediated lesions are reduced by about 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 91°y 0 , 98%, 99% and 100%.
  • the compounds or therapeutic compositions can function to regenerate nerve cells, promote neurite outgrowth, and protect nerves from otherwise damaging treatments or conditions.
  • the compounds and compositions of this invention are useful in the diagnosis, cure, mitigation, treatment, or prevention of neurological conditions in animals, including humans, and in animals (including humans) exposed to neurodegenerative agents or having damaged nervous system cells.
  • Such conditions and disorders can be neurodegenerative disorders, neuropathic disorders, neurovascular disorders, traumatic injury of the brain, spinal cord, or peripheral nervous system, demyelinating disease of the central or peripheral nervous system,- metabolic or hereditary metabolic disorder of the central or peripheral nervous system, or toxin-induced- or nutritionally related disorder of the central or peripheral nervous system.
  • a neurodegenerative disorder can be, for example, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, cerebellar ataxia, or multisystem atrophy including, for example, olivopontocerebellar degeneration, striatonigral degeneration, progressive supranuclear palsy, Shy-Drager syndrome, spinocerebellar degeneration and corticobasal degeneration.
  • a demyelinating disease can be, for example, multiple sclerosis, Guillain-Barre syndrome, or chronic inflammatory demyelinating polyradiculoneuropathy.
  • a neurovascular disorder can be global cerebral ischemia, spinal cord ischemia, ischemic stroke, cardiogenic cerebral embolism, hemorrhagic stroke, lacunar infarction, multiple infarct syn dromes including multiple infarct dementia, or any disorder resulting in ischemia or ischemia/reperfusion injury of the central nervous system.
  • Traumatic injury of the central or peripheral nervous system can be, for example, concussion, contusion, diffuse axonal injury, edema, and hematoma associated with craniocerebral or spinal trauma, or axonal or nerve sheath damage associated with laceration, compression, stretch, or avulsion of peripheral nerves or plexi, and further includes nerve damage caused during surgery, such as prostate surgery.
  • a neuropathic disorder can be, for example, diafcetic neuropathy, uremic neuropathy, neuropathy related to therapy with drugs such as phenytoin, suramin, taxol, thalidomide, vincristine or vinblastine; or neuropathy/encephalopathy associated with infectious disease, such as, for example, encephalopathy related to HIV, rubella virus, Epstein-Barr vims, herpes simplex virus, toxoplasmosis, prion infection.
  • a metabolic disorder of the central nervous system can be, for example, status epilepticus, hypoglycemic coma, or Wilson's disease.
  • disease states can be induced in the organotypic slice culture by various means such as toxins, neural proteins, drugs, chemicals, siRNA, and then test candidate compounds, morphogenetic molecules, or cells, which will ameliorate the disease.
  • the compounds of the invention have utility in pharmacological compositions for the treatment and prevention of various neurological, ischemic, and inflammatory disorders.
  • the compounds also have utility in the treatment of traumatic injury to nervous tissue, or conditions associated with retinal and optic nerve damage.
  • the compounds of the invention may be prepared as a salt or derivative, as described above.
  • a compound of the invention can be administered to an animal or human- patient by itself or in pharmaceutical compositions where it is mixed with suitable carriers or excipients, at doses to treat or ameliorate various conditions.
  • the compounds according to the present invention preferably have sufficient stability, potency, selectivity, solubility and availability to be safe and effective in treating diseases, injuries and other abnormal conditions or insults to the central nervous system including the brain, the peripheral nerves, and other organs.
  • a therapeutically effective dose refers to that amount of the compound sufficient to effect an activity in a nerve or neuronal cell, to produce a detectable change in a cell or organism, or to treat a disorder in a human or other mammal.
  • treat in its various grammatical forms as used in relation to the present invention refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing, ameliorating or halting the deleterious effects of a disease state, disease progression, injury, wound, ischemia, disease causative agent (e.g., bacteria, protozoans, parasites, fungi, viruses, viroids and/or prions), surgical procedure or other abnormal or detrimental condition (all of which are collectively referred to as "disorders," as will be appreciated by the person of skill in Che art).
  • a “therapeutically effective amount" of a compound according to the invention is an amount that can achieve effective treatment, and such amounts can be determined in accordance with the present teachings by one skilled in the art.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, buccal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and optionally in a depot or sustained release formulation.
  • parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and optionally in a depot or sustained release formulation.
  • a targeted drug delivery system for example in a liposome coated with an antibody. The liposomes will be targeted to and taken up selectively by cells expressing the appropriate antigen.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can thus be used pharmaceutically.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers, well known to those in the art.
  • Such earners enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, quick-dissolving preparations, gels, syrups, shinies, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use of the compounds of this invention can be obtained by employing a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • trie pharmaceutical compositions also may comprise suitable solid or gel phase earners or excipients.
  • suitable solid or gel phase earners or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • disintegrating agents may be added, such, as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate or a number of others disintegrants [see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990)].
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, pressurized air, or other suitable gas or mixture.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, pressurized air, or other suitable gas or mixture.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, pressurized air, or other suitable gas or mixture.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils sucli as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for trie preparation of highly concentrated solutions.
  • the active ingredient maybe in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • the compounds may also be formulated in rectal compositions such a.s suppositories, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • rectal compositions such as a.s suppositories, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compounds of the invention may further be formulated in pharmaceutical or cosmetic compositions for topical application to the skin in the form of an aqueous, alcoholic, aqueous/alcoholic or oily solution, or of a dispersion of the lotion or serum type, of an emulsion having a liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice versa (W/O), or of a suspension or of an emulsion with a soft consistency of the aqueous or anhydrous gel, foam or cream type, or, alternatively, of microcapsules or microp articles, or of a vesicular dispersion of ionic and/or nonionic type, or may further be administered in the form of an aerosol composition comprising a pressurized propellent agent.
  • Liposomes and emulsions are well known examples of delivery vehicles or earners for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed. Additionally, the compounds may be delivered using a sustained-release system, such as semi permeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for stabilization may be employed.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose, to effect a therapeutic benefit, or to effect a detectable change in the function of a cell, tissue, or organ. More specifically, a therapeutically effective amount means an amount effective to prevent the development of or to alleviate the existing symptoms of the subject being treated. Determining the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • Toxicity and therapeutic efficacy of the compounds or compositions can be determined by standard pharmaceutical, pharmacological, and toxicological procedures in cell cultures or experimental animals. For example, numerous methods for determining the LD 5 o (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of trie population) exist. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio between LD 50 and ED 50 . Compounds and compositions exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays or animal studies can be used in formulating a range of dosages for use in humans. [See, for example, Fingl et al., in The Pharmacological Basis of Therapeutics, Ch. 1 p . 1 (1975)].
  • the compounds of the present invention may be administered by a single dose, multiple discrete doses or continuous infusion.
  • Some of the compounds preferably are non- peptidic, easily diffusible and relatively stable, they can be well-suited to continuous infusion.
  • Dose levels on the order of about 0.1 mg to about 10,000 mg of the active ingredient are useful in the treatment of the above conditions, with preferred levels being about 0.1 mg to about 1,000 mg.
  • the specific dose level, and thus the therapeutically- effective amount, for any particular patient will vary depending upon a variety of factors, including the activity of the specific compound employed and its bioavailability at the site of drug action; the age, body weight, general health, sex and diet of the patient; trie time of administration; the rate of excretion; drug combination; the severity of the particular disease being treated; and the form of administration.
  • in vitro dosage-effect results provide useful guidance on the proper doses for patient administration. Studies in animal models also are helpful. The considerations for determining the proper dose levels are available to the skilled person.
  • compounds can administered in lyophilized form.
  • 1 to 1000 mg of a compound of the present invention may be lyophilized in individual vials, together with a carrier and a buffer, such as mannitol and sodium phosphate.
  • the compound may be reconstituted in the vials with bacteriostatic water before administration.
  • the compounds of the present invention are preferably administered orally, rectally, parenterally or topically at least 1 to 6 times daily, and may follow an initial bolus dose of higher concentration..
  • any administration regiinen regulating the timing and sequence of drug delivery caji be used and repeated as necessary to effect treatment.
  • Such regimen may include pretreatrnent and/or co ⁇ administration with additional therapeutic agents.
  • the organotypic slice culture assay is combined with high through put robotic, computer, and combinatorial library screening assays.
  • the initial screen is carried out by the high throughput screening assay. Examples of such assays are known to one of skill in the art.
  • Selected candidate drugs are then used in the organotypic slice culture, as described in detail in the Examples which follow, to identify those compounds which are therapeutic candidates or pose serious threats to an animal, such as for example, toxins, poisons and the like, especially those that affect the neural system.
  • the methods of screening of the invention comprise using screening assays to identify, from a library of diverse molecules, one or more compounds having a desired activity.
  • a “screening assay” is a selective assay designed to identify, isolate, and/or determine the structure of, compounds within a collection that have a preselected activity. By “identifying” it is meant that a compound having a desirable activity is isolated, its chemical structure is determined (including without limitation determining the nucleotide and amino acid sequences of nucleic acids and polypeptides, respectively) the structure of and, additionally or alternatively, purifying compounds having the screened activity).
  • Biochemical and biological assays are designed to test for activity in a broad range of systems ranging from protein-protein interactions, enzyme catalysis, small molecule-protein binding, to cellular functions. Such assays include automated, semi-automated assays and HTS (high throughput screening) assays.
  • HTS HTS methods
  • many discrete compounds are preferably tested in parallel by robotic, automatic or semi-automatic methods so that large numbers of test compounds are screened for a desired activity simultaneously or nearly simultaneously. It is possible to assay and screen up to about 6,000 to 20,000, and even up to about 100,000 to 1,000,000 different compounds a day using the integrated systems of the invention.
  • target molecules are administered or cultured with the organotypic slice, including the appropriate controls.
  • screening comprises contacting each organotypic slice culture with a diverse library of member compounds, some of which are ligands of the target, under conditions where complexes between the target and ligands can form, and identifying which members of the libraries are present in such complexes.
  • screening comprises contacting a target enzyme with a diverse library of member compounds, some of which are inhibitors (or activators) of the target, under conditions where a product or a reactant of the reaction catalyzed by the enzyme produce a detectable signal.
  • inhibitors of target enzyme decrease the signal from a detectable product or increase a signal from a detectable reactant (or vice-versa for activators).
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks,” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library
  • a linear combinatorial chemical library is formed by combining a set of chemical building blocks (amino acids) in a large number of combinations, and potentially in every possible way, for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • a "library” may comprise from 2 to 50,000,000 diverse member compounds.
  • a library comprises at least 48 diverse compounds, preferably 96 or more diverse compounds, more preferably 384 or more diverse compounds, more preferably., 10,000 or more diverse compounds, preferably more than 100,000 diverse members and most preferably more than 1,000,000 diverse member compounds.
  • “diverse” it is meant that greater than 50% of the compounds in a library have chemical structures that are not identical to any other member of the library.
  • greater than 75% of the compounds in a library have chemical structures that are not identical to any other member of trie collection, more preferably greater than 90% and most preferably greater than about 99%.
  • the preparation of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton, et al, Nature, 354:84-88 (1991)).
  • Other chemistries for generating chemical diversity libraries can also be used.
  • Such chemistries include, but are not limited to, peptoids (PCT Publication No. WO 91/19735); encoded peptides (PCT Publication WO 93/20242); random bio-oligomers (PCT Publication No.
  • WO 92/00091 benzodiazepines (U.S. Pat. No. 5,288,514); diversomers, sxich as hydantoins, benzodiazepines and dipeptides (Hobbs, et al, Proc. Nat. Acad. Sci. USA, 90:6909-6913 (1993)); vinylogous polypeptides (Hagihara, et al, J. Amer. Chem. Soc. 114:6568 (1992)); nonpeptidal peptidomimetics with .beta.-D-glucose scaffolding (Hirschmann, et al, J. Amer. Chem.
  • High throughput screening can be used to measure the effects of drugs on complex molecular events such as signal transduction pathways, as well as cell functions including, but not limited to, cell function, apoptosis, cell division, cell adhesion, locomotion, exocytosis, and cell-cell communication.
  • Multicolor fluorescence permits multiple targets and cell processes to be assayed in a single screen. Cross-correlation of cellular responses will yield a wealth of information required for target validation and lead optimization.
  • the organotypic slice culture is run in tandem with a screening system
  • a screening system comprising a high magnification fluorescence optical system having a microscope objective, an XY stage adapted for holding a plate with an array of locations for holding the slice culture and having a means for moving the plate to align the locations with the microscope objective and a means for moving the plate in the direction to effect focusing; a digital camera; a light source having optical means for directing excitation light to cells in the array of locations and a means for directing fluorescent light emitted from the cells to the digital camera; and a computer means for receiving and processing digital data from the digital camera wherein the computer means includes: a digital frame grabber for receiving the images from the camera, a display for user interaction and display of assay results, digital storage media for data storage and archiving, and means for control, acquisition, processing and display of results.
  • the standard optical configurations use microscope optics to directly produce an enlarged image of the specimen on the camera sensor in order to capture a tiigh resolution image of the specimen.
  • This optical system is commonly referred to as "wide field" microscopy.
  • a high resolution image of the specimen can be created by a variety of other optical systems, including, but not limited to, standard scanning confocal detection of a focused point or line of illumination scanned over the specimen , and multi-photon scanning confocal microscopy, " both of which can form images on a CCD detector or by synchronous digitization of the analog output of a photomultiplier tube.
  • the output of a standard confocal imaging attachment for a microscope is a digital image that can be converted to the same format as the images produced by the other cell screening system embodiments described above, and can therefore be processed in exactly the same way as those images.
  • the overall control, acquisition and analysis in this embodiment is essentially the same.
  • the optical configuration of the confocal microscope system is essentially the same as that described above, except for the illuminator and detectors. Illumination and detection systems required for confocal microscopy have been designed as accessories to be attached to standard microscope optical systems such as that of the present invention (Zeiss, Germany). These alternative optical systems therefore can be easily integrated into the system as described above.
  • the present invention provides a method for analyzing cells comprising providing an array of locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecule in the cells; converting the fluorescent signals into digital data; and utilizing the digital data to determine the distribution, environment or activity of the fluorescent reporter molecule within the cells.
  • a major component of the new drug discovery paradigm is a continually growing family of fluorescent and luminescent reagents that are used to measure the temporal and spatial distribution, content, and activity of intracellular ions, metabolites, macromolecules, and organelles. Classes of these reagents include labeling reagents that measure the distribution and amount of molecules in living and fixed cells, environmental indicators to report signal transduction events in time and space, and fluorescent protein biosensors to measure target molecular activities within living cells.
  • a multiparameter approach that combines several reagents in a single cell is a powerful new tool for drug discovery.
  • This method relies on the high affinity of fluorescent or luminescent molecules for specific cellular components.
  • the affinity for specific components is governed by physical forces such as ionic interactions, covalent bonding (which includes chimeric fusion with protein-based chromophores, fluorophores, and lumiphores), as well as hydrophobic interactions, electrical potential, and, in some cases, simple entrapment within a cellular component.
  • the luminescent probes can be small molecules, labeled macromo lecules, or genetically engineered proteins, including, but not limited to green fluorescent protein chimeras.
  • fluorescent reporter molecules that can be used in the present invention, including, but not limited to, fluorescently labeled bio molecules such as proteins, phospholipids and DNA hybridizing probes.
  • fluorescent reagents specifically synthesized with particular chemical properties of binding or association have been used as fluorescent reporter molecules (Barak et ciL, (1997), J. Biol. Ch em. 272:27497-27500; Southwick et al., (1990), Cytometry 11:418- 430; Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp. 127-156).
  • Fluorescently labeled antibodies are particularly useful reporter molecules due to their high degree of specificity for attaching to a single molecular target in a mixture of molecules as complex as a cell or tissue.
  • the luminescent probes can be synthesized within the living cell or can be transported into the cell via several non-mechanical modes including diffusion, facilitated or active transport, signal-sequence-mediated transport, and endocytotic or pinocytotic uptake.
  • Mechanical bulk loading methods which are well known in the art, can also be used to load luminescent probes into living cells (Barber et al. (1996), Neuroscience Letters 207:17-20; Bright et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), pp. 153-173).
  • cells can be genetically engineered to express reporter molecules, such as GFP, coupled to a protein of interest as previously described (Chalfie and Prasher U.S. Pat. No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science 20 :448-455).
  • the luminescent probes accumulate at their target domain as a result of specific and high affinity interactions with the target domain or other modes of molecular targeting such as signal-sequence-mediated transport.
  • Fluoresce ⁇ tly labeled reporter molecules are useful for determining the location, amount and chemical environment of the reporter. For example, whether the reporter is in a lipophilic membrane environment or in a more aqmeous environment can be determined (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomolecidar Structure 24:405-434; Giuliano and Taylor (1995), Methods in Neuroscience 27.1-16).
  • the pH environment of the reporter can be determined (Bright et al. (1989), J.
  • certain cell types within an organism may contain components that can be specifically labeled that may not occur in other cell types.
  • neural cells often contain polarized membrane components. That is, these cells asymmetrically distribute macromolecules along their plasma membrane.
  • Connective or supporting tissue cells often contain granules in which are trapped molecules specific to that cell type (e.g., heparin, histamine, serotonin, etc.).
  • Most muscular tissue cells contain a sarcoplasmic reticulum, a specialized organelle whose function is to regulate the concentration of calcium ions within the cell cytoplasm.
  • Many nervous tissue cells contain secretory granules and vesicles in which are trapped neurohormones or neurotransmitters. Therefore, fluorescent molecules can be designed to label not only specific components within specific cells, but also specific cells within a population of mixed cell types.
  • fluorescent reporter molecules exhibit a change in excitation or emission spectra, some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a loss (quenching) or appearance of fluorescence, while some report rotational movements (Giuliano et al. (1995), Ann. Rev. of Biophysics andBiomol. Structure 24:405-434; Giuliano et al. (1995), Ivfethods in Neuroscience 27:1-16).
  • sampling of sample materials may be accomplished with a plurality of steps, which include withdrawing a sample from a sample container and delivering at least a portion of the withdrawn sample to organotypic slice culture. Sampling may also include additional steps, particularly and preferably, sample preparation steps.
  • sample preparation steps particularly and preferably, sample preparation steps.
  • only one sample is withdrawn into the auto-sampler probe at a time and only one sample resides in the probe at one time.
  • multiple samples may be drawn into the auto-sampler probe separated by solvents.
  • multiple probes may be used in parallel for auto sampling.
  • sampling can be effected manually, in a semi-automatic manner or in an automatic manner.
  • a sample can be withdrawn from a sample container manually, for example, with a pipette or with a syringe-type manual probe, and then manually delivered to a loading port or an injection port of a characterization system.
  • some aspect of the protocol is effected automatically C e -g- > delivery), but some other aspect requires manual intervention (e.g., withdrawal of samples from a process control line).
  • the sample(s) are withdrawn from a sample container and delivered to the characterization system, e.g.
  • auto-sampling may be done using a micropro cessor controlling an automated system (e.g., a robot ami).
  • the microprocessor is user- programmable to accommodate libraries of samples having varying arrangements of samples (e.g., square arrays with, "n-rows” by “n-columns", rectangular arrays with “n-rows” by “m- columns”, round arrays, triangular arrays with “r-” by “r-” by “r-” equilateral sides, triangular arrays with “r-base” by “s-” by “s-” isosceles sides, etc., where n, m, r, and s are integers).
  • Automated sampling of sample materials optionally may be effected with an auto-sampler having a heated injection probe (tip).
  • one or more systems, methods or both are used to identify a plurality of sample materials. Though manual or semi-automated systems and methods are possible, preferably an automated system or method is employed. A variety of robotic or automatic systems are available for automatically or programmably providing predeteimined motions for handling, contacting, dispensing, or otherwise manipulating materials in solid, fluid liquid or gas form according to a predetermined protocol. Such systems may be adapted or augmented to include a variety of hardware, software or both to assist the systems in determining mechanical properties of materials.
  • Hardware and software for augmenting the robotic systems may include, but are not limited to, sensors, transducers, data acquisition and manipulation hardware, data acquisition and manipulation software and the like.
  • Exemplary robotic systems are commercially available from CAVRO Scientific Instruments (e.g., Model NO. RSP9652) or BioDot (Microdrop Model 300O).
  • the automated system includes a suitable protocol design and execution software that can be programmed with information such as synthesis, composition, location information or other information related to a library of materials positioned with respect to a substrate.
  • the protocol design and execution software is typically in communication with robot control software for controlling a robot or other automated apparatus or system.
  • the protocol design and execution software is also in communication with data acquisition hardware/software for collecting data from response measuring hardware. Once the data is collected in the database, analytical software may be used to analyze the data, and more specifically, to determine properties of the candidate drugs, or the data may be analyzed manually.
  • a sample can be pre- fractionated according to size of proteins in a sample using size exclusion chromatography.
  • a size selection spin column is used for a biological sample wherein the amount of sample available is small.
  • the first fraction that is eluted from the column (“fraction 1") has the highest percentage of high molecular weight proteins; fraction 2 has a lower percentage of high molecular weight proteins; fraction 3 has even a lower percentage of high molecular weight proteins; fraction 4 has the lowest amount of large proteins; and so on.
  • a sample can be pre- fractionated by anion exchange chromatography.
  • Anion exchange chromatography allows pre-fractionation of the proteins in a sample roughly according to their charge characteristics.
  • a Q anion-exchange resin can be used (e.g., Q HyperD F, Biosepra), and a sample can be sequentially eluted with eluants having different pH's.
  • Anion exchange chromatography allows separation of compounds in a sample that are more negatively charged from other types of compounds.
  • a sample can be pre-fractionated by heparin chromatography. Heparin chromatography allows pre-fractionation of the compounds in a sample also on the basis of affinity interaction with heparin and charge characteristics.
  • Heparin a sulfated mucopolysaccharide, will bind compounds with positively charged moieties and a sample can be sequentially eluted with eluants having different pH's or salt concentrations. Samples eluted with an eluant having a low pH are more likely to be weakly positively charged. Samples eluted with an eluant having a high pH are more likely to be strongly positively charged. Thus, heparin chromatography also reduces the complexity of a sample and separates samples according to their binding characteristics. [000247] In yet another embodiment, a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated.
  • a CSF sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Glycosylated proteins will bind to the lectin column and non- glycosylated proteins will pass through the flow through. Glycosylated proteins are then eluted from the lectin column with an eluant containing a sugar, e.g., N-acetyl-glucosamine and are available for further analysis.
  • a sugar e.g., N-acetyl-glucosamine
  • a sample can be fractionated using a sequential extraction protocol.
  • sequential extraction a sample is exposed to a series of adsorbents to extract different types of molecules from a sample. For example, a sample is applied to a first adsorbent to extract certain proteins, and an eluant containing non-adsorbent proteins (i.e., proteins that did not bind to the first adsorbent) is collected. Then, trie fraction is exposed to a second adsorbent. This further extracts various proteins from the fraction. This second fraction is then exposed to a third adsorbent, and so on.
  • Any suitable materials and methods can be used to perform sequential extraction of a sample.
  • a series of spin columns comprising different adsorbents can be used.
  • a multi-well comprising different adsorbents at its bottom can be used.
  • sequential extraction can be performed on a probe adapted for use in a gas phase ion spectrometer, wherein the probe surface comprises adsorbents for binding samples.
  • the sample is applied to a first adsorbent on the probe, which is subsequently washed with an eluant. Samples that do not bind to the first adsorbent are removed with an eluant.
  • the compounds that are in the fraction can be applied to a second adsorbent on the probe, and so forth.
  • the advantage of performing sequential extraction on a gas phase ion spectrometer probe is that compounds that bind to various adsorbents at every stage of the sequential extraction protocol can be analyzed directly using a gas phase ion spectrometer.
  • protein samples can be separated by high-resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis.
  • a fraction containing a sample can be isolated and further analyzed by gas phase ion spectrometry.
  • two- dimensional gel electrophoresis is used to generate two-dimensional array of spots of samples. See, e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1 997).
  • the two-dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Academic Press ed., Methods In Enzymology vol. 1 82.
  • protein samples are separated by, e.g., isoelectric focusing, during which a sample is separated in a pH gradient until they reach a spot where their net charge is zero (i.e., isoelectric point).
  • This first separation step results in one-dimensional array of samples.
  • the samples in one dimensional array is further separated using a technique generally distinct from that used in the first separation step.
  • samples separated by isoelectric focusing are further separated using a polyacrylamide gel, such as polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).
  • SDS-PAGE gel allows further separation based on molecular mass of compounds.
  • two-dimensional gel electrophoresis can separate chemically different compounds in the molecular mass range from 1000-200,000 Da within complex mixtures.
  • Protein samples in the two-dimensional array can be detected using any suitable methods known in the art. For example, samples in a gel can be labeled or stained (e.g., Coomassie Blue or silver staining). The spot can be further analyzed by densitometric analysis or gas phase ion spectrometry. For example, spots can be excised from the gel and analyzed by gas phase ion spectrometry. Alternatively, the gel containing samples can be transferred to an inert membrane by applying an electric field.
  • a spot on the membrane that approximately corresponds to the molecular weight of a sample can be analyzed by gas phase ion spectrometry.
  • the spots can be analyzed using any suitable techniques, such as MALDI or SELDI.
  • HPLC high performance liquid chromatography
  • High throughput screening of drugs includes the isolation and purification and assaying of these samples in the organotypic slice culture.
  • HPLC can be xised to separate a mixture of sample based on their different physical properties, such as polarity, charge and size.
  • HPLC instruments typically consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Samples are separated by injecting an aliquot of the sample onto the column. Different samples in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. A fraction that corresponds to the molecul ar weight and/or physical properties of one or more molecules can be collected. The fraction can then be analyzed by gas phase ion spectrometry to detect potential drugs.
  • biochips are described in the art. These include, for example, protein biochips produced by Packard Bio Science Company (Meriden CT), Zyomyx (Hayward, CA) and Phylos (Lexington, MA).
  • a sample is placed on the active surface of a biochip for a sufficient time to allow binding. Then, unbound molecules are washed from the surface using a suitable eluant.
  • Analytes captured on the surface of a biochip can be detected by any method known in the art. This includes, for example, mass spectrometry, fluorescence, surface plasmon resonance, ellipsometry and atomic force microscopy. Mass spectrometry, and particularly SELDI mass spectrometry, is a particularly useful method for detection of the compounds of this invention.
  • a laser desorption time-of- flight mass spectrometer is used in embodiments of the invention, hi laser desorption mass spectrometry, a substrate or a probe comprising samples is introduced into an inlet system.
  • the samples are desorbed and ionized into the gas phase by laser from the ionization source.
  • the ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of-flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of candidate drugs of specific mass to charge ratio.
  • MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-MS is a method of mass spectrometry that involves the use of an energy absorbing molecule, frequently called a matrix, for desorbing proteins intact from a probe surface.
  • MALDI is described, for example, in U.S. patent 5,118,937 (Hillenkamp et al.) and U.S. patent 5,045,694 (Beavis and Chait).
  • the sample is typically mixed with a matrix material and placed on the surface of an inert probe.
  • Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid (“SPA”), cyano hydroxy cinnamic acid (“CHCA”) and dihydroxybenzoic acid.
  • SPA sinapinic acid
  • CHCA cyano hydroxy cinnamic acid
  • Other suitable energy absorbing molecules are known to those skilled in this art.
  • the matrix dries, forming crystals that encapsulate the analyte molecules. Then the analyte molecules are detected by laser desorption/ionization mass spectrometry.
  • MALDI-MS is useful for detecting the molecules if the complexity of a sample has been substantially reduced using the preparation methods described above.
  • SELDI-MS Surface-enhanced laser desorption/ionization mass spectrometry, or SELDI-MS represents an improvement over MALDI for the fractionation and detection of biomolecules, such as proteins, in complex mixtures.
  • SELDI is a method of mass spectrometry in which biomolecules, such as proteins, are captured on the surface of a protein biochip using capture reagents that are bound there. Typically, non-bound molecules are washed from the probe surface before interrogation.
  • SELDI is described, for example, in: United States Patent 5,719,060 ("Method and Apparatus for Desorption and Ionization of Analytes," Hutchens and Yip, February 1 7, 1998,) United States Patent 6,225,047 ("Use of Retentate Chromatography to Generate Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et al., "Time-of- flight mass spectrometry," in Encyclopedia of Analytical Chemistry, R. A. Meyers, ed., pp 11915-1 1918 John Wiley & Sons Chichesher, 2000.
  • Molecules on the substrate surface can be desorbed and ionized using gas phase ion spectrometry.
  • Any suitable gas phase ion spectrometers can be used as long as it allows compounds on the substrate to be resolved.
  • gas phase ion spectrometers allow quantitation of compounds.
  • a gas phase ion spectrometer is a mass spectrometer.
  • a substrate or a probe comprising compounds on its surface is introduced into an inlet system of the mass spectrometer.
  • the molecules are then desorbed by a desorption source such as a laser, fast atom bombardment, high energy plasma, electrospray ionization, thermospray ionization, liquid secondary ion MS , field desorption, etc.
  • the generated desorbed, volatilized species consist. of preformed ions or neutrals which are ionized as a direct consequence of the desoiption event.
  • Generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions.
  • the ions exiting the mass analyzer are detected by a detector.
  • the detector tl ⁇ en translates information of the detected ions into mass-to-charge ratios. Detection of the presence other substances will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of compounds bound to the substrate.
  • Any of the components of a mass spectrometer e.g., a desorption source, a mass analyzer, a detector, etc.
  • an immunoassay can be used to detect and analyze samples in a sample. This method comprises: (a) providing an antibody that specifically binds to a molecule of interest; (b) contacting a sample with the antibody; and (c) detecting the presence o f a complex of the antibody bound to the molecule of interest in the sample. [000264] Data generated by desorption and detection of compounds can be analyzed using any suitable means. In one embodiment, data is analyzed with the use of a programmable digital computer. The computer program generally contains a readable medium that stores codes.
  • Certain code can be devoted to memory that includes the location of each feature on a probe, the identity of the adsorbent at that feature and the elution conditions used to wash the adsorbent.
  • the computer also contains code that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location on the probe. This data can indicate the number of compounds detected, including the strength of the signal generated by each compound.
  • Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a compound detected and removing "outliers" (data deviating from a predetermined- statistical distribution). The observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated.
  • a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale. Then the signal strength detected for each sample or other biomolecules can be displayed in the form of relative intensities in the scale desired Ce.g., 100).
  • a standard e.g., a CSF protein
  • the computer can transform the resulting data into various formats for displaying.
  • spectrum view or retentate map a standard spectral view can be displayed, wherein the view depicts the quantity of compound reaching the detector at each particular molecular weight.
  • peak map a standard spectral view
  • mass map only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling compounds with nearly identical molecular weights to be more easily seen.
  • gel view each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels.
  • 3-D overlays several spectra can be overlaid to study subtle changes in relative peak heights.
  • two or more spectra can be compared, conveniently highlighting unique compounds. Profiles (spectra) from any two samples may be compared visually.
  • Spotfire Scatter Plot can be used, wherein molecules that are detected are plotted as a dot in a plot, wherein one axis of the plot represents the apparent molecular mass of the compounds detected and another axis represents the signal intensity of compounds detected. For each sample, compounds that are detected and the amount of compounds present in the sample can be saved in a computer readable medium.
  • Tau EGFP knock-in ES cells were used to permit visualization of ESNs in vital slice cultures. These cells are derived from the Jl ES cell line and carry the cDNA for EGFP targeted in-frame into exon 1 of the tau gene. This results in a fusion protein consisting of the first 31 amino acids of tau and EGFP. After /// vitro differentiation of this cell line, EGFP fluorescence has been found to be restricted to neuronal progeny. The generation of ES cell- derived neural precursors from tau EGFP knock-in ES cells was performed as described previously.
  • ES cells were aggregated to embryoid bodies, which, were subsequently plated and prop agated in ITSFn medium (DMEM-F12 supplemented with 5 ⁇ g/ml insulin, 50 ⁇ g/ml transferrin, 30 nM sodium selenite, and 5 ⁇ g/ml fibronectin) for 5—7 d. Cells were then trypsinized, tritxirated to a single-cell suspension, and replated in polyornithine-coated dishes.
  • ITSFn medium DMEM-F12 supplemented with 5 ⁇ g/ml insulin, 50 ⁇ g/ml transferrin, 30 nM sodium selenite, and 5 ⁇ g/ml fibronectin
  • DMEM-F12 DMEM-F12 supplemented with 25 ⁇ g/ml insulin, 50 ⁇ g/ml transferrin, 30 nM sodium selenite, 20 nM progesterone, 100 nM putrescine, 1 ⁇ g/ml laminin, and 10 ng/ml fibroblast growth factor 2.
  • Media, supplements, and growth factors were obtained from Invitrogen (Karlsruhe, Germany), R & D Systems (Wiesbaden, Germany), and Sigma (Taufkirchen, Germany).
  • donor cells were trypsinized and triturated through flame-polished Pasteur pipettes. They were then washed in calcium- and magnesium-free HBSS and concentrated to 5-8 x 10 4 cells/ ⁇ l.
  • ES cell-derived neural precursors were performed at day 10 ⁇ 1 in culture. This permitted monitoring of the maturation of ESNs for up to 21 d after application, while exposing donor cells to the most advanced stage of tissue differentiation.
  • the cells were suspended in a total volume of 0.2 ⁇ l and gently deposited on the surface of the slice centrally within the hilus of the dentate gyrus.
  • recipient cultures were washed thoroughly with medium to remove donor cells that had not migrated into the tissue. Migration and differentiation of the donor cells was studied by eplfluorescence microscopy in 2 d intervals. In some preparations (n-12), the perforant path was labeled with a rhodamine-conjugated anterograde tracer (Microraby; Molecular Probes, Leiden, The Netherlands).
  • Mouse ES cells (line Jl ) were aggregated to embryoid bodies and subsequently plated in ITSFn medium. After 5 days, cells were trypsinized and propagated for 5 days in polyornithine-coated dishes in a DMEM/F12-based medium supplemented with 10 ng ml "1 FGF2. They were then harvested and replated in medium supplemented ⁇ vith FGF2 and EGF (20 ng ml "1 ). In some experiments in vitro, the cells were propagated through an additional passage in medium supplemented with FGF2 and PDGF-AA (10 ng ml ""1 ). In vitro differentiation into astrocytes and oligodendrocytes was induced by growth- factor withdrawal.
  • ESGPs proliferating in the presence of FGF2 and EGF were transfected with a CMV- GFP/neo expression construct (pEGFP-Nl; Clontech, Palo Alto, CA) using FuGENETM 6 according to the manufacturers' instructions.
  • pEGFP-Nl CMV- GFP/neo expression construct
  • FuGENETM 6 FuGENETM 6 according to the manufacturers' instructions.
  • cells selected in G418- containing medium were washed in Ca 2+ /Mg 2+ -free Hanks' buffered salt solution and concentrated to 80,000 cells ml "1 .
  • donor cells 2 days after application. Fluorescence microscopy was used to monitor and document the distribution and morphology of the grafted cells at 2-day intervals. Proliferation of engrafted ESGPs was studied at days 2 and 10 after deposition. Slices were incubated with 10 ⁇ M BrdU (Sigma) for 48 hours, followed by thorough washing. BrdU- treated slices were excluded from functional studies and processed subsequently for immunofluorescence and confocal analysis (six slices at each time point).
  • the bath solution contained (in mM): 125 NaCl, 3 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2.0 CaCl 2 , 1.0 MgCl 2 , and 20 glucose, pH 7.3, NaOH.
  • glycine 5 ⁇ M was added to the extracellular solution.
  • MgCl 2 was omitted from the extracellular solution in some experiments.
  • pipettes contained (in mM): 110 cesium methanesulfonate, 2 MgCl 2 , 10 l,2-bis(2-amino-5-bromophenoxy)ethane-7V;iV;7V',iV-tetra-acetic acid, 2ATP, 1 OHEPES, 20 tetraethylammonium chloride, and 5 lidocaine N-ethyl bromide, pH 7.4, NaOH. Liquid junction potentials were not compensated. All of the chemicals were purchased from Sigma.
  • the standard pipette solution contained (in mM): 130 KCl, 2 MgCl 2 , 0.5 CaCl 2 , 5 BAPTA, 10 HEPES, 3 ⁇ a2ATP, and 0.1% Lucifer yellow (LY; Sigma), pH 7.25. Current signals were filtered at 3 or 10 kHz, and sampled at 5 or 30 IcHz. Capacitance and series resistance compensation (lip to 60%) were used to improve voltage-clamp control. Cells engrafted into slice cultures were identified by virtue of their green fluorescent protein (GFP) expression and chosen at random for functional analysis. During recording, donor cells were filled with LY by dialyzing the cytoplasm with the patch pipette solution.
  • GFP green fluorescent protein
  • mice IgG monoclonal antibodies to BrdU, (BD Biosciences, Heidelberg, Germany; 1 : 100) and GABA A receptors ( ⁇ -chain; clone BD17; Chemicon, Hofheim, Germany; 1:1000) as well as rabbit polyclonal antibodies to the AMPA receptor subunit glutamate receptor 1 (GIuRl) (Sigma; 1 :300) and the NMDA receptor subunit NMDARl (Chemicon; 1 :1000). All of the antigens were visualized using corresponding Cy3- and Cy5 -conjugated goat secondary antibodies (Dianova, Hamburg, Germany). Images were documented and three-dimensional reconstructions were performed using confocal microscopy and appropriate software (Leica, Pulheim, Germany). Data are expressed as means ⁇ SDs.
  • Organotypic slice culture systems are a novel technique that bridges the gap between in vitro and in vivo systems. These culture systems have been used for a variety of investigations into development of neuronal architecture and circuits. In addition these cultures allow for the direct observation of pathological conditions, such, as demyelination, epilepsy, and on brain circuits and cell populations.
  • the brain slice culture is an interface model of slice culture to generate mid-sagittal slices containing an intact nigrostriatal circuit, including the substantia nigra pars compacta (SNc), medial forebrain bundle, and striatum, in both mice and rat cultures.
  • SNc substantia nigra pars compacta
  • Organotypic slice cultures were generated from both mice and rats. All slices were generated from rats were from animals postnatal day 20 to postnatal day 23, and from mice postnatal day 15 to early adult. Animals were euthanized and quickly decapitated. The brains were cut into two sagittal halves and immersed in a preparation media (DMEM, L- ascorbic acid, L-Glutamate, and Penicillin/Streptomycin). The halves were then super-glued to the vibratome stage, medial surface down, and covered with cool molten 2% agar. The stage was then placed in the vibratome chamber and filled with preparation media.
  • DMEM L- ascorbic acid
  • L-Glutamate L-Glutamate
  • Penicillin/Streptomycin Penicillin/Streptomycin
  • Slices were cut between 300-400 ⁇ M, placed in cold preparation media and scanned using a dissection microscope to select slices from the level of the media forebrain bundle. Slices, collected after selection, were immediately transferred to a transwell (Falcon) placed in a 6 well plate and incubated at 35 0 C and 5% CO 2 . Each transwell was susp ended in 1.8 mL of "A" media, a proprietary culture media containing serum. The media was changed the next day and feeding was done every other day.
  • "A" media a proprietary culture media containing serum
  • Fig IA shows a brightfield image of an intact slice in the transwell chamber.
  • Figure IB and 1C show nissel and dapi staining of the hippocampus in a long term culture. This region is one of the most susceptible to hypoxia and oxidative stress, two difficult conditions encountered in slice culture. As can be seen the cyto architecture appears intact and the cell layers do not show any significant degeneration. This indicates that the slice, and the cells within, is viable.
  • This system represents a true bridge between in vivo and in vitro experimentation and allows direct manipulation of potential therapies and the neuronal environment that they are being placed in.
  • the slice culture system allows for a more complete observation of how intact and lesioned brain structures, such as myelinated fiber bundles, and glial scars, influence the effectiveness of potential therapies.
  • the experiments have also demonstrated that this culture assay is amenable to cell replacement transplants.
  • astrocyte-induced neurogenesis from adult neural stem cells glial cells should play an active rather than passive role in maintaining the neuronal population.
  • astrocytes are connected via gap junctions and form extensive networks. Incorporation of transplanted ES cell-derived astrocytes into such a network structure could provide new perspectives for both cell-mediated delivery of small molecules and modulation of neuronal function.
  • ES cell-derived neural precursors have been shown to develop into morphologically mature neurons and glia when grafted into brain and spinal cord.
  • ESNs ES cell-derived neurons
  • the ES cells were engineered to express enhanced green fluorescent protein (EGFP) only in neuronal progeny to study the functional properties of ESNs during integration into long-term hippocampal slice cultures.
  • EGFP enhanced green fluorescent protein
  • EGFP + donor neurons After incorporation into the dentate gyrus, EGFP + donor neurons display a gradual maturation of their intrinsic discharge behavior and a concomitant increase in the density of voltage-gated Na + and K + channels.
  • Integrated ESNs express AMPA and GABA A receptor subunits.
  • neurons derived, from ES cells receive functional glutamatergic and GAJBAergic synapses from host neurons. Specifically, it was demonstrated that host perforant path axons form synapses onto integrated ESNs. These synapses between host and ES cell-derived neurons display pronounced paired-pulse facilitation indicative of intact presynaptic short-term plasticity.
  • ES cell-derived neural precursors generate functionally active neurons capable of integrating into the brain circuitry.
  • ES cell-derived neural precursors incorporate widely throughout the CNS and differentiate into neurons, astrocytes, and oligodendrocytes. So far, functional studies on ES cell-derived neurons (ESNs) have been primarily restricted to monolayer cultures. In contrast, little is known about the functional maturation of individual ES-cell derived neurons after transplantation into CNS tissue.
  • Example 3 Incorporation of ES cell-derived neurons into hippocampal slice cultures [000300]
  • the ES cells used in this study express EGFP only in neuronal progeny and thus permit a reliable identification of donor derived neurons within the host tissue.
  • Donor cells were applied as immature neural precursors. Expression of EGFP indicative of neuronal differentiation became first visible at day 3 after implantation. Although this delayed expression of EGFP in donor-derived cells does not permit a well controlled investigation of the migration of these cells within the host tissue, the distribution of the engrafted EGFP + donor cells clearly indicated that they had invaded the slice preparation after deposition.
  • ESNs had migrated up to several hundred micrometers away from the engraftment site in a horizontal plane by 2 weeks after deposition. At this time, ESNs were found throughout the dentate gyrus hilar region. Most of the EGFP + cells were localized within or close to the DG granule cell layer, with only a few cells in the CA3 and CAl regions.
  • ESNs analyzed were functionally located within the granule cell layer or at its hilar border. The remainder of the neurons selected for patch-clamp analysis was located within the hilar region. During the first week, most EGFP + cells had round to oval cell bodies extending one or two small processes with few ramifications. At later stages, more complex neuronal phenotypes became visible.
  • ESNs express ionotropic AJVLPA and GABA receptors using antibodies against the GIuRl AMPA receptor subunit and the GABA A receptor ⁇ -chain, respectively. Both subunits were clearly detectable in the membrane of most EGFP + neurons. [000305] Then examined was whether the expression of these neurotransmitter receptor subunits reflects the presence of functional glutamatergic and GABAergic synapses on ESlSTs.
  • mEPSCs miniature EPSCs
  • AP 5 -sensitive EPSCs could be elicited by synaptic stimulation with a monopolar stimulation electrode and showed a characteristic nonlinear I- V relationship in the presence of ImM extracellular Mg 2+ .
  • Example 6 Development of synaptic input onto ESNs with time in culture [000308]
  • the distinct decay time course of AMPA and GABA A receptor mediated synaptic PSC as well as the scarcity of NMDA-mediated EPSCs in ESNs allowed a clear discrimination of spontaneous GABA ⁇ - and AMPA-mediated events in recordings in which blockers of neurotransmitter receptors were omitted.
  • a dramatic increase in the frequency of both AMPA- and GABA A -mediated spontaneous PSCs was observed after incorporation of ESNs into the host tissue.
  • Example 7 Incorporated ESNs receive input from host axonal projections [000310]
  • the hippocampal slice preparation used as recipient tissue for ESNs contains both the hippocampus and the entorhinal cortex.
  • Entorhinal cortex neurons give rise to the perforant path, the main afferent projection, to the hippocampus.
  • Preservation of the perforant path was visualized by depositing a small amount of rhodamine-conjugated dextran onto the entorhinal cortex.
  • ESGPs spread horizontally inside the slice preparation.
  • Donor cells migrated up to several hundred micrometers in the first week after engraftment. Migration inside the tissue occurred preferentially along endogenous fiber tracts. This was particularly prominent after deposition onto the EC (n ⁇ 25), from where the transplanted cells distributed along the perforant path and the Schaffer collaterals.
  • Transient outward K + currents [I K1A) ] were separated by subtracting the outward currents evoked after the -40 mV p re-pulses from those activated after a —110 mV pre-pulse.
  • I K(A > was present in 3/9 cells.
  • GFP + cells 4/31) expressed voltage-activated currents but lacked I 1C i 1 - and I ⁇ (p), which resembled ESGPs before transplantation.
  • a third population (20/31, >60%) of the recorded donor-derived astrocytes had prominent I ⁇ ( p ) typically found in 'passive' glial cells in mature hippocamp ⁇ s. There was no obvious association between these functional phenotypes and specific anatomical structures.
  • ES cell-derived astrocytes establish gap junctions with recipient glia [000318] A hallmark of astrocyte development in vivo is the formation of gap junctions. Individual astrocytes are coupled to dozens of neighboring cells to form an extensive syncytial network of interconnected glia.
  • Promoter driven marker genes e.g. GFP, YFP, AIk phos, and the like
  • GFP GFP
  • YFP YFP
  • AIk phos phos, and the like
  • Promoter driven marker genes are used to label specific pathways and circuits in the normal brain, or at-risk pathways and circuits within slices for detection of the circuitry and neural pathways. This allows for further analysis and manipulation (e.g. Using drugs, gene therapy) etc (and under real-time, live cell/slice culture conditions) to assay efficacy for neurological repair.
  • Example 12 A Method for a More Complete In Vitro Parkinson's Model: Slice Culture Bioassay for Modeling Maintenance and Repair of the Nigrostriatal Circuit [000323]
  • the air media interface model of slice culture was modified (Stoppini et al. , 1991, J Neiirosci Methods 37:173- 182), to generate a long term slice culture model that contains the nigrostriatal circuit. Once in the dish this circuit can be selectively targeted for degeneration using a variety of methods, including chemical lesions with 6-OHDA.
  • These slices can be cultured for up to 4 weeks in the dish and are amenable to patch recording and real time fluorescence microscopy to directly observe the neuronal circuitry.
  • embryonic stem cell derived neuronal precursors can be transplanted into the slices and their development tracked in real time.
  • ESNP's have been implanted into hippoca ⁇ rpal slices and show robust survival and engraftment.
  • Slice culture Preparation Slice cultures were generated from 10 mice po stnatal day 7 to 25, with a total of 48 slices cultured. Briefly, animals were euthanized under halothane, and their brains were cut at the midline into two saggital halves. These halves were then superglued, medial surface down to the vibratome stage, covered in cool, molten 2% agar, and immersed in cold preparation media (DMEM F 12, 100 ⁇ g/mL L-ascorbic acid, 2 mM L-glutamate, and antibiotic/antimycotic (Invitrogen, USA)).
  • DMEM F 12 100 ⁇ g/mL L-ascorbic acid, 2 mM L-glutamate, and antibiotic/antimycotic (Invitrogen, USA)
  • Slices were cut between 300-400 ⁇ M, and removed, using a large bore glass pipette, into a Petri dish with cold preparation media, and scanned using a dissection microscope to select slices. Typically 4-6 slices per animal from the appropriate levels were obtained, and then immediately transferred to a transwell (Falcon, USA), placed in a 6 well plate, and incubated at 35°C and 5% CO 2 . Each transwell was suspended in 1 mL of U A" media, a culture media containing 25 % horse serum (Kluge et al., 1998. Hippocampus 8:57-68).
  • ESNP transplants were derived as described by Okabe et al (Olcabe et al., 1996, Mech Dev 59:89-102) and TH expression was induced in cells using bFGF (10 ng/ml), FGF8 (100 ng/ml), SHH (500 ng/ml), PTN (100 ng/ml) (Sigma, USA) which were added to the culture media every day.
  • 50-100,000 cells were transplanted using a 5 ⁇ X Hamilton syringe to deliver 2 ⁇ L to the region in and around the substantia nigra or directly into the striatum.
  • 50 ⁇ g of laminin was added to a mixture of cells, 2% methylcellulose, and B media for transplant.
  • Immiinocytochemistiy and Quantification For morphological and phenotype analysis, slices were slices were fixed with 4% Immunocytochemistry and quantification: For morphological and phenotype analysis, slices were fixed with 4% paraformaldehyde for at least 24 h at 4 0 C. Sections were then either processed as whole mounts or embedded in paraffin and sections cut at I O ⁇ m.
  • aCSF aCSF containing, in mM: 125 NaCl 3 3 KCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 20 glucose, 1 MgCl 2 , and 2 CaCl 2 and maintained at 35° C during experiments.
  • Intracellular pipette solution comprised of, in mM: 145 K-gluconate, 10 HEPES, 10 EGTA, and 5 MgATP (pH 7.2, osmolarity 290).
  • 145 K- gluconate was replaced with 125 KCl and 20 K-gluconate.
  • FIG. 8 A shows the TH staining of a slice exposed to 6-OHDA, witli an almost complete lack of staining in the striatum, and a significant reduction in the medial forebrain bundle and SNC.
  • High magnification views of the nigral region show that there are fewer cells bodies, a loss o f intranigral fibers, and a loss of discreet TH labeling with an increase in diffuse TH staining.
  • High magnification views of the striatum illustrate the significant reduction in TH immunoreactivity post lesion (Figure 8A inset).
  • ESNP 's implanted into the SNc and striatum of slice cultures survive, incorporate and differentiate for at least 21 days post transplantation. These observations are consistent with those seen with both ES and fetal ventral mesencephalon, transplants into the striatum in vivo (Takagi et al., 2005). Prior to transplantation, some ESNP 's were treated for 7 days with the ventral ⁇ zing agents SHH, FGF8, and pleiotrophin, which increased the percentage of TH+ cells in vitro.
  • slice cultures can be obtained, thus scaling up the number of experiments per animal as well as the number of experiments that can be run simultaneously under similar growth conditions with the easily controlled screening of cellular reagents, drug libraries, or potentially toxic compounds.
  • recent developments in genetic models of neurodegeneration should allow for slice cultures of mutant brains without the use of neurotoxins. These cultures also should be extremely valuable in analyzing effects of mutations independent of confounding in vivo complications, hi addition, slice cultures allow for cells to be transplanted in a variety of locations in the brain; with the ability to directly manipulate the environment and the cells while in culture, there exist a variety of potential approaches to encourage circuitry reconstruction and axon regeneration that would be difficult to test using either dispersed cell cultures or in vivo models.

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Abstract

L'invention concerne un système de culture en tranches permettant une survie à long terme de section sagittales de souris et de rat. Ces sections peuvent être maintenues vivantes pendant des périodes de culture de plus de 6 mois. Ces systèmes de modèles de cultures en tranches offrent une occasion unique pour contrôler et modifier des circuits du cerveau dans un récipient et pour surveiller des cellules en temps réel. Ce système permet également d'observer, d'une part, la différenciation cellulaire dans ces tranches et, d'autre part, les effets de protéines morphogénétiques, telles que des molécules de la matrice extracellulaire, des médicaments ou des petites molécules. Ce système de culture unique offre un moyen innovant et utile pour la mise au point de thérapies cellulaires et moléculaires destinées au traitement de maladies neurodégénératives.
PCT/US2005/037924 2004-10-22 2005-10-21 Cultures de tranches organotypiques et leurs applications Ceased WO2006047299A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2103933A1 (fr) 2008-02-25 2009-09-23 KeyNeurotek Pharmaceuticals AG Dispositif et procédé de mesure d'impédance dans des tissus organotypiques
WO2010146211A1 (fr) * 2009-06-16 2010-12-23 Universidad Del País Vasco Modèle organotypique de maladies neurodégénératives, procédé d'obtention et utilisation de ce dernier
US8198083B1 (en) 2007-10-31 2012-06-12 William Gunter Loudon Organotypic slices of the central nervous system
EP2708889A1 (fr) 2012-09-12 2014-03-19 Universität Leipzig Procédé et dispositif permettant de prélever un échantillon tissulaire et de caractériser l'échantillon tissulaire par la détermination d'au moins une propriété électrique
WO2016172693A1 (fr) * 2015-04-23 2016-10-27 Rowan University Nouvelles cultures sur tranches et procédés pour le diagnostic de maladies de dégénérescence neuronale

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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EP0528978B1 (fr) * 1990-05-16 2002-10-16 Southern Research Institute Mikrocapsules pour liberation regulee et son utilisation pour stimuler la croissance de fibres nerveuses
WO2001055110A1 (fr) * 2000-01-24 2001-08-02 Neurosearch A/S Derives de l'isatine a activite neurotrophique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8198083B1 (en) 2007-10-31 2012-06-12 William Gunter Loudon Organotypic slices of the central nervous system
EP2103933A1 (fr) 2008-02-25 2009-09-23 KeyNeurotek Pharmaceuticals AG Dispositif et procédé de mesure d'impédance dans des tissus organotypiques
US10670576B2 (en) 2008-02-25 2020-06-02 Universität Leipzig Device and method for measuring impedance in organotypic tissues
WO2010146211A1 (fr) * 2009-06-16 2010-12-23 Universidad Del País Vasco Modèle organotypique de maladies neurodégénératives, procédé d'obtention et utilisation de ce dernier
ES2368054A1 (es) * 2009-06-16 2011-11-14 Universidad Del Pais Vasco Modelo organotipico de enfermedades neuro-degenerativas, proceso de obtencion y usos del mismo.
EP2708889A1 (fr) 2012-09-12 2014-03-19 Universität Leipzig Procédé et dispositif permettant de prélever un échantillon tissulaire et de caractériser l'échantillon tissulaire par la détermination d'au moins une propriété électrique
WO2016172693A1 (fr) * 2015-04-23 2016-10-27 Rowan University Nouvelles cultures sur tranches et procédés pour le diagnostic de maladies de dégénérescence neuronale

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