MAMMALIAN NON-NEURONAL CELLS THAT ARE MODIFIED TO STORE AND RELEASE A NEUROTRANSMITTER

Abstract: Mammalian non-neuronal cells are modified to store and release a neurotransmitter. The cells can be modified by loading the cells, with a neurotransmitter in culture. The cells can be genetically modified to produce, store, and release the neurotransmitters. Also, the neurotransmitter is released selectively by the cells in a quantal fashion. Further disclosed are methods for making such cells and to methods for using such cells to treat neurodegenerative, psychiatric,, and neuromuscular disorders in mammals.

Description: MAMMALIAN NON-NEURONAL CELLS THAT ARE MODIFIED TO STORE AND RELEASE A NEUROTRANSMITTER CLA OF PRIORITY This application claims priority under 35 U.S.C. § 119(e) to United States provisional patent application 60/110,360, filed November 30, 1998. TECHNICAL FIELD OF THE INVENTION This invention relates to mammalian non-neuronal cells that are modified to store and release a neurotransmitter. BACKGROUND OF THE INVENTION The localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) genes within a single genomic locus, and the co-expression of both proteins throughout the cholinergic system, strongly suggests that synthesis and storage of acetylcholine (ACh) are tightly controlled. (Bέjanin et al, 269 J. Biol. Chem 21944-21947 (1994); Erickson et al, 2691 Biol. Chem. 21929-21935 (1994); Berrard et al, 65 J. Neurochem. 939-942 (1995); Misawa etal, 6 NeuroReport 965-968 (1995)) But what about release? A variety of cell lines that do not express the ChAT or VAChT genes nonetheless display a calcium-dependent quantal release mechanism for acetylcholine that was discovered after loading the cells in culture with acetylcholine (Dan & Poo, 359 Nature 733-736 (1992); Israel et al, 25 Neurochem. International 103-109 (1994); Falk-Vairant et al, 45 J. Neurosci. Res. 195-201 (1996); Falk-Vairant etal, 75 Neuroscience 353-360 (1996); Falk-Vairant et al, 93 Proc. Natl. Acad. Sci. USA 5203-5207 (1996)) or after their transfection with ChAT cDNA (Kirnura et al, 314 Fedn. Eur. Biochem. Socs. Lett. 409-4 12 (1992); Fisher et al, 61 J. Neurochem. 1323-1332 (1993)). Taken together, these data demonstrate that the proteins involved in release are expressed independently of ChAT or VAChT. This does not imply that the release mechanism is not specific for a given transmitter. The specificity of the release mechanism was studied in the crucial work by Docherty et al, 330 Nature 64-66 (1987), who found that glutamate (Gin) is co-released from cholinergic and GABAergic synaptosomes. This observation was confirmed for cholinergic electric organ synaptosomes (Vyas & Bradford, 82 Neurosci. Lett. 58-64 (1987)), which co-release glutamate under high calcium conditions (Israel et al, 22 Neurochem. International 53-58 (1993)). If cells that release acetylcholine (ACh) were loaded with γ-aminobutyric acid (GAB A) or glutamate, would they release these transmitters as well? SUMMARY OF THE INVENTION The present invention is directed to mammalian non-πeuronal cells that are modified to store and release a neurotransmitter. The cells can be modified by loading the cells, with a neurotransmitter in culture. Preferably, the cells are genetically modified to produce, store, and release the neurotransmitters. In another embodiment, the neurotransmitter is released selectively by the cells in a quantal fashion. The invention is further directed to processes for making such cells and to methods for using such cells to treat neurodegenerative, psychiatric, and neuromuscular disorders in mammals. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows release of acetylcholine (ACh), glutamate (GIu), or γ-aminobutyrate (GAB A) from loaded cells. Transmitter release was monitored using the three che iluminescent procedures. Methods for loading the cells with neurotransmitter and for monitoring their release are described in EXAMPLES 1 and 2. Release was evaluated in an aliquot of cell suspension corresponding to cellular transmitter contents of 0.5 nmol for acetylcholine loaded cells, 1 nmol for glutamate loaded cells, and 5 nmol for GAB A loaded cells. Cellular contents were measured beforehand as described in TABLE 1. Calcium-dependent release was elicited by adding ionophore A23187 (6 μ ) and then calcium 10 mM in the case of acetylcholine and glutamate release, and 2.5 mM for GAB A release. This lower calcium concentration avoids possible interference with the GAB A assay procedure. Some cells specifically released one transmitter, while others co-released two of them. Calibration was obtained by injecting a standard when release decreased. FIG. 2 is a set of bar graphs showing the release specificities of the different cell lines loaded with transmitter. The different cell lines displayed different release specificities for the transmitter they contain. The cell lines were loaded with either acetylcholine (ACh), glutamate (Glu), or GABA as described in the description of FIG. 1. The cerebral endothelial cell line RBE4 was specific for acetylcholine, while the neuroblastom N18TG-2 cell line released GABA. The co-release of acetylcholine and GABA, but not glutamate, was found with the glial cell line C6BU-1 and with the neuroblastoma-glioma cells NGl 08-15. The fibroblastic cell line L-M(TK " ) co-released acetylcholine and glutamate but not GABA. In all cases, release was elicited with ionophore A23187 and calcium, as indicated in the description of FIG. 1. Release was expressed by dividing the amplitude (pmol) by the time to peak, and normalized for lnmol of cellular transmitter content (n = number of tests). FIG. 3 shows the construction of the plasmid pcDNARSV puro. FIG. 4 is a set of bar graphs and graphs FIG. 4A is a set of bar graphs that show ChAT activity measured in standard conditions in some stable transfectants after co-transfection with a rat ChAT cDNA construct in a ratio of 10 (A) or 5 (B) with the selection vector. FIG. 4B is a set of graphs that illustrate time and protein dependencies. ChAT activity in this clone reaches about 65 pmol/min x μg protein with a K m for acetylCoA around 5 μM. FIG. 5 is a Western blot that shows an immunoblot analysis of ChAT protein performed on A24 clone (lane 2) and a negative clone (lane 1). FIG. 6 is a set of graphs. FIG. 6 A is a graph that shows results from clone A24. The light emission elicited by calcium addition was calibrated by addition of a known amount of acetylcholine. No light emission was observed in the absence of acetylcholinesterase. Addition of Triton after release allows the determination of the residual content. FIG. 6B is a graph that shows that, at equal acetylcholine amounts during the test shown in FIG. 6A, the release from control cells artificially loaded with external acetylcholine appears very similar to acetylcholine release from the stable ChAT transfectant A24. FIG. 7 shows ChAt activity in a selected clone (A24). - FIG. 8 is a photograph that shows that when a VAChT cDNA construct was introduced in the stably transfected cell line A24 expressing the rat ChAT, clones selected in zeocin containing media displayed a transient elongated morphology with long processes, seen at low density of cells. FIG. 9 is a graph and a Western blot. FIG. 9 illustrates the kinetics of vesamicol binding for the clone A24-56 B m ^ =2.9 pmol/mg protein; KD= 16 nM). FIG. 9B shows immunoblot analysis of two clones for the VAChT protein: lane 1 is A24-23; lane 2 is A24-56; lane 3 is parental A24. FIG. 10 is a plasmid map that shows the construction of plasmid TVAChT pZeoSV2. See EXAMPLE 3. FIG. 11 is a set of polyacrylamϊde gels which showe that RBE4 cells endogenously express vesicular proteins (synaptobrevin (or VAMP), synaptotagmin, and SV2) and other proteins of the SNARE complex (SNAP23) but at different levels. DETAILED DESCRIPTION OF THE INVENTION Non-neuronal cells may be genetically modified by transfecting the cells with a gene that encodes an enzyme that causes production of a neurotransmitter in the cells. The gene that encodes an enzyme is generally transfected in a suitable vector. The cells are preferably further modified by transfection with a gene that regulates storage, release of the neurotransmitter, or both. For example, the cells may be transfected with a gene that encodes a vesicular transporter for the neurotransmitter in the cells, such as VAChT. More preferably, the cells are also modified by transfection with a selection gene. Suitable selection genes include antibiotic resistance genes, such as a puromycin resistance gene. The puromycin resistance gene is conveniently transfected into cells in a suitable plasmid, such as in plasmid pcDNA RSV puro. Examples of enzymes that cause production of a neurotransmitter in cells include choline acetyltransferase, which causes production in cells of the neurotransmitter acetylcholine; tyrosine hydroxylase, which causes production in cells of the neurotransmitter L-Dopa/dopamine; glutamic acid decarboxylase, which causes production in cells of the neurotransmitter γ-aminobutyric acid (GABA); and tryptophan hydroxylase, which causes production in cells of the neurotransmitter serotonin. In this specification, the words "mammal" and "mammalian" refer to any mammal. Mammals, include, for example, laboratory animals such as rats, mice, etc; farm animals, such as horses, cattle, sheep, goats, etc; and pet animals, such as dogs, cats, etc; and primates, such as apes, monkeys, orangutans, and humans. The non-neuronal cells are preferably mammalian endothehal cells, mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells. More preferably, mammalian brain endothehal cells, mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells are human brain endothehal cells, human retinal cells, human fibroblast cells, or human glial cells. The endothehal cells are preferably brain endothehal cells, most preferably human brain endothehal cells. Examples of endothehal cells include the endothehal cells described in PCT International patent application WO 96/11278, incorporated by reference. In particular, the endothehal cells can be the RBE4 cells deposited under the identification no. I- 1142 with the Collection National de Micro-organismes (CNCM) held by the Institut Pasteur, 28 rue de Docteur Roux, 75724 PARIS CEDEX 15 FRANCE. The endothehal cells preferably are from a continuous, cloned cell line. Examples of mammalian retinal cells are those described in PCT International patent application WO 97/40139, incorporated by reference. In particular, the retinal cells can be primary retinal pigmentary epithelial cells or the immortalized retinal pigmentary epithelial IO/LD7/4, in the Collection Nationale de Cultures de Micro-organismes, under the identification no. 1-1694. Alternatively, the retinal cells can be primary retinal endothehal or the immortalized retinal endothehal IO/JG2/1 cells in the Collection Nationale de Cultures de Micro-organismes, under the identification no. 1-1695. Examples of neurotransmitters include acetylcholine, glutamate, γ-aminobutyric acid, glycine, dopa ine, norepinephrine, serotonin (5-hydroxvtryptamine), and histamine. The preferred neurotransmitter is acetylcholine. In another embodiment, the invention is a process for preparing the genetically modified (i.e., genetically modified) mammalian non-neuronal cells that produce, store, and release the neurotransmitters described above. The process comprises transfecting mammalian non-neuronal cells with a gene that encodes an enzyme that, in turn, causes production in the cells of a neurotransmitter. Preferably, the transfected cells are from a continuous, cloned cell line. The cells are preferably further modified by transfection with a gene that regulates storage, express the neurotransmitter, or both; or modified by transfection with a selection gene, or both a regulatory and a selection gene, as described above. In another embodiment, the transfected mammalian non-neuronal cells selectively release the enzyme, or selectively co-release the enzyme and a second enzyme that causes production of a second neurotransmitter. For example, certain brain endothehal cells, such as RBE4 cells, selectively release acetylcholine. Certain neuroblastoma cells, such as N18TG-2 cells, selectively release γ-aminobutyric acid. In yet another embodiment, the invention relates to a method of treating a neurodegenerative disorder in a mammal. The method comprises administering to a mammal in need of such treatment mammalian non-neuronal cells that are modified to store and release a neurotransmitter. Preferably, the mammalian non-neuronal cells administered are the genetically modified cells, described above, that produce, store, and release the neurotransmitter. Preferably, the mammal treated is a human, and the mammalian non-neuronal cells are human non-neuronal cells. Examples of mammals in need of treatment include mammals that suffer from Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy or stroke, or psychiatric disorders. More specifically, when the neurotransmitter is acetylcholine, the method is capable of treating mammals that suffer from cholinergic deficiency, such as, for example, metabolic encephalopathy with cholinergic deficiency. When the neurotransmitter is L-Dopa/dopamine, the method is capable of treating mammals that suffer from Parkinson's disease. When the neurotransmitter is γ-ammobutyric acid, the method is capable of treating mammals that suffer from Huntington's disease, epilepsy, or stroke. When the neurotransmitter is serotonin, the method is capable of treating mammals that suffer from psychiatric disorders. The cells may be administered by methods known in the art. For example, the cells may be administered by direct intracranial injection, systemic intravenous (IV.) injection, intra-arterial injection, or systemic intra-carotid injection. Alternatively, the cells are directed to a specific region of the brain by grafting, The cells may also be encapsulated and used to deliver neurotransmitters, according to known encapsulation technologies, including microencapsulatioπ (see, e.g., United States patents 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference), (b) macroencapsulation (see, e.g., United States patents 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and published PCT patent applications WO 92/19195, WO 95/05452, each incorporated herein by reference). If the cells are encapsulated, we prefer macroencapsulation, as described in United States patents 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and published PCT patent application WO 95/05452, each incorporated herein by reference. Cell number in the devices can be varied; preferably each device contains between 10 3 - 10 ? cells, most preferably 10 5 to 10 7 cells. A large number of macroencapsulation devices may be implanted in the subject, we prefer between one to 10 devices. The cells of the invention are also suitable for use in treating a mammal suffering from a neuromuscular disorder. The method comprises administering to a mammal in need of such treatment mammalian non-neuronal cells that are modified to store and release acetylcholine to the neuromuscular junction of the mammal. Preferably, the non-neuronal cells are the genetically modified cells, described above, that produce, store, and release the neurotransmitter. Preferably, the mammal treated is a human, and the mammalian non-neuronal cells are human non-neuronal cells. The neuromuscular disorder may be, for example, myasthenia gravis. The details of one or more embodiments of the invention are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, All patents and publications cited in this specification are incorporated by reference. Apart from the foregoing provisions, the invention also comprises other provisions, which will become apparent from the following description referring to EXAMPLES of how to carry out the process forming the subject of the present invention, and to the attached drawings. The following EXAMPLES are presented in order to more fully illustrate the preferred embodiments of the invention. It must be clearly understood, however, that these EXAMPLES are given solely in order to illustrate the subject of the invention, without in any, way implying a limitation. EXAMPLE 1 CELL CULTURE AND LOADING OF CELLS WITH ACh, Glu OR GABA The different cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamine 2 mM, sodium pyruvate 10 mM, and 10% fetal calf serum. Cultures were carried out at 37°C in a 5.5% CO 2 -94.5% air mixture. For the acetylcholine loading, the cells were maintained for 5 days in DMEM without choline and then 10 mM acetylcholine was added in the culture dish for 15 hr after inhibiting acetylcholinesterases with phospholine (5-10 "5 M) added 5 min before acetylcholine. Harvested cells were washed 5 times for 5 min at 800 g in choline-free DMEM. For glutamate loaded cells, glutamate 10 mM was added to the culture medium instead of glutamine for 15 hr, and the cells were washed several times in glutamate-free DMEM and finally in mammalian rebs. For GABA loaded cells, 10 mM GABA was added overnight and washed the harvested cells in normal DMEM. An aliquot (3 μl) of an adequately diluted cell suspension (10-20»10 6 cells/ml) was disrupted to measure ihe cellular transmitter contents, using the three different specific assay mixtures described below. For acetylcholine loaded cells, we used 0,4% Triton X-100, while a 10 times lower detergent concentration was used for glutamate, so as to avoid a loss of sensitivity of the reaction mixture. The GABA reaction mixture was also affected by the detergent, so the cells were in this case disrupted by heat (80°C 5 sec) and then assayed. TABLE 1 shows the transmitter contents of the different cell lines. EXAMPLE 2 REACTION MIXTURES FOR MONITORING RELEASE For acetylcholine, this mixture comprised 100 μl choline oxidase (WAKO) 250 U/ml, 50 μl horseradish peroxidase type II (Sigma) 2 mg/ l, 100 μl luminol (Merck) 1 mM in Tris buffer pH 8.6. The stock solutions are stored at -20°C. 15 μl of the reaction mixture was then added to 250 μl sucrose 120 mM-Tris 120 mM buffer pH 8.6 and 3μl of acetylcholinesterase (1000 U/ml) previously cleaned on Sephadex G50. For more details see Israel & Lesbats, 37 I. Neurochem. 1475-1483 (1981); Israel & Lesbats, 39 J. Neurochem. 248-250 (1982); Israel & Lesbats, 67 J. Neurochem. 2624-2627 (1996). The glutamate reaction mixture comprised 50 μl glutamate dehydrogenase (Sigma) 2600 U/ml 100 μl NAD(P)H-FMN oxidoreductase (Boehringer) 20 U/ml, 80 μl luciferase from Vibri flscheri (Sigma) 25 mg/ml, 250 μl NAD (β nicotinamide adenine dinucleotide) (Sigma) 6.6 mg/ml in Tris buffer 0.2 M pH 8, and of 25 μl FMN (flavin mononucleotide sodium salt) (Sigma) 0.12 mg ml. The reaction mixture was stored at -20°C. To 250 μl sucrose 120 mM in Tris buffer 120 mM pH 7.2, we added 50 μl of the reaction mixture and 5μl of n-decyl aldehyde (Sigma) 2 μl/ml, the aldehyde emulsion being shaken before each addition (Israel et al., 22 Neurochem. International 53-58 (1993)). The GABA reaction rnixture consisted of 100 μl GABAase (Boehringer) 50 U/ml, 100 μl NAD(P)H-FMN oxidoreductase (Boehringer) 40 U/ml, 200 μl Luciferase from Vibrio fischeri (Sigma) 50 mg/ml. This mixture was stored at -20°C. To 250 μl sucrose 120 mM Tris buffer 120 mM pH 8.6, we added 40 μl of the enzyme mixture plus 25 μl/assay of a substrate mixture consisting of 100 μl NAD 19.9 mg/ml in Tris buffer 0.2 M pH 8.6, 100 μl of FMN at 0.12 mg/ml, and 50 μl ketoglutaric acid (Sigma) 8.4 mg/ml in Tris buffer 0.2 M pH 8 (Israel & Lesbats, 67 . Neurochem. 2624-2627 (1996)). Results of EXAMPLES J and 2. We first demonstrated that various cell types could indeed be loaded with any of the three transmitters. TABLE 1 gives the transmitter content of the loaded cells for the three transmitters which were added to the culture medium from 12-15 hr at a concentration of 10 mM, The harvested cells were then washed 4 or 5 times by centrifugation to remove the extracellular transmitter and were resuspended at about 10-20«10 6 cells/ml. The intracellular transmitter content was determined by disrupting the cells (3 μl of suspension adequately diluted) in the specific reaction mixture for measuring acetylcholine, glutamate, or GABA. The three chemiluminescent procedures used have been previously described (Israel & Lesbats, 37 J. Neurochem. 1475-1483 (1981); Israel & Lesbats, 39 J. Neurochem. 248-250 (1982); Israel & Lesbats, 67 J. Neurochem. 2624-2627 (1996); Fosse et al, 47 J. Neurochem. 340-349 (1986). For cells having a 10 μm radius the volume should be about 4/3 πr 3 « 4« 10 3 μm 3 W4-10 "6 μl. With 10-20-10 6 cells/ml, the total cell volume would be in the range of 40-80 μl/ml. The data in TABLE 1 show an average transmitter concentration of 0.35 μmol ml, whichever transmitter or cell line was considered. The cellular transmitter concentration is thus in the range of 4.3-8.7 mM, approaching the extracellular. concentration of 10 mM. The different assay mixtures that contain high amounts of esterase, GAB Aase or glutamate dehydrogenase, reduce to negligible levels any trace of transmitter on the extracellular side of the membrane, only transmitters incorporated in the cell remain non degraded. The loaded cells retained the incorporated transmitter for more than 5 hr., which enabled the performance of numerous release determinations. FIG. 1 shows release of the three transmitters from loaded cells, as elicited by calcium after ionophore A23187 addition. In spite of the fact that release may be elicited as soon as calcium is added to the extracellular solution in the range of 0.2-2 mM in the presence of ionophore, we used higher calcium concentrations of 2-10 mM to compare the different cells in order to be sure that we had a maximum release rate. Release was monitored using three chemiluminescent procedures. The specificity of these procedures was shown in the particular case of acetylcholine, since release measured biochemically, paralleled the detection by eletrophysiological procedures using myoballs in contact with releasing cells as described by Dan & Poo, 359 Nature 733-736 (1992). In addition the co-release of acetylcholine and glutamate was initially found using radioactive tracers (Vyas & Bradford, 82 Neurosci. Lett. 58-64 (1987)) and was confirmed with the chemiluminescent procedure. For a given transmitter, the cell lines studied were directly comparable, being tested for release at the same ionophore and calcium concentration, and for the same cellular content of the loaded transmitter. Some cells are specific for a given transmitter, while others co-released two of them. Four different release phenotypes were characterized, as shown in FIG. 2. The cerebral endothelial ceil line RBE4 (Roux et al, 159 J. Cell Physiol. 101-113 (1994)) and neuroblastoma N18TG-2 cells were specific for acetylcholine and GAB A respectively, and did not release the other two transmitters. The glial cell line C6BU-1 released acetylcholine and GABA but not glutamate, while the fibroblastic cell line L-M(TK) released acetylcholine and glutamate but not GABA. Neuroblastoma-glioma NG108-15 cells displayed the release properties of the parent cell lines. In controls previously described we have shown that acetylcholine release from loaded cells was fully calcium dependent, could be triggered by electrical stimulation, and displayed quantal jumps like at natural synapses (Falk-Vairant etal, 45 I. Neurosci. Res. 195-201 (1996); Falk-Vairant et al, IS Neuroscience 353-360 (1996); Falk-Vairant et al, 93 Proc. Natl. Acad. Sci. USA 5203-5207 (1996)). Among the cells that did not release acetylcholine (such as Nl 8TG-2, NIEl 15, MMQ, myeloma), the N 18TG-2 cell line was particularly silent. We therefore used it for mediatophore transfection and were able to restore quantal release (Falk-Vairant et l, 45 I. Neurosci. Res. 195-201 (1996); Falk-Vairant et al, 75 Neuroscience 353-360 (1996); Falk-Vairant et al, 93 Proc. Natl. Acad. Sci. USA 5203-5207 (1996)). Discussion of results of EXAMPLES 1 and 2. The most widely accepted mechanism for release is that synaptic vesicles docked to plasma membranes proteins form a "flickering" pore that releases the vesicular content, the specificity depending on which transmitter is stored. The occasional full fusion event will give rise to a capacitance change. A less commonly held view is that the "flickering" pore may release the transmitter directly from the cytoplasmic compartment, particularly when the vesicle is not docked, Whichever mechanism is considered, knowledge of the specificity of the translocation step for the different transmitters is of value. We have established that cell lines may express a calcium-dependent quantal release mechanism for acetylcholine which is independent of their ability to synthesize or store the transmitter (Dan & Poo, 359 Nature 733-736 (1992); Falk-Vairant et al, 45 J. Neurosci. Res. 195-201 (1996); Falk-Vairant et al, 75 Neuroscience 353-360 (1996); Falk-Vairant et al, 93 Proc. Natl. Acad. Sci. USA 5203-5207 (1996)), the size of the quantal steps being comparable to sub-miniatures (Vautrain et al, 32 . Neurosci. Res. 245-254 (1992)). If a vesicle becomes docked to the releasing site, the higher intravesicular acetylcholine concentration should increase the size of the packet, as may be the case for secreting exocrine cells. Even in the absence of synthesis and storage, the translocation step of the release machinery is operational in certain cell types when they are loaded with acetylcholine. The specificity of this translocation step corresponds to several release phenotypes. Some are acetylcholine or GABA specific, while others co-release two of the three transmitters. Other release phenotypes will probably be discovered by loading cells with other transmitters. Are we dealing with isoforms of pore-forming structures, or with completely different proteins that are able to translocate the different transmitters? Transmitter loaded cells offer eventual possibilities for investigating the specificity of calcium-dependent release steps. Moreover, the recovery in homogeneous cell lines of different transmitter release phenotypes found in central or peripheral synapses should help the molecular and pharmacological characterization of proteins supporting the specificity of release. Of particular interest is the RBE4 cell line that may be grafted in brain and perhaps to correct an eventual deficit in neurotransmission. EXAMPLE 3 PLASMID CONSTRUCTS Material and methods. The following procedure was carried out with the ChAT construct in the Flip vector. The 2,3 kb EcoRl fragment with the whole rat protein-coding sequence (Brice etal, 23 I. Neurosci. Res. 266-273 (1989)) has been inserted into the Sail site of the Flip vector containing the SV40 promoter and the. The Torpedo marmorata coding sequence of VAChT cDNA (BamHl-SnaBl; 2.2 kb) (Varoqui et al, 342 FEBS Lett. 97-102 (1994)) was subcloned in the BamHl-EcoRV pZeoSV2 (Invitrogen), as indicated below. Orientation of the inserted cDNA was confirmed by restriction. EXAMPLE 4 CELL CULTURE, STABLE TRANSFECTION AND ANALYSIS OF GENE EXPRESSION RBE4 cells were plated on 100 mm Petri dishes previously coated with collagen (rat tail type I) and grown in αMEM - Ham's F10 (1/1) medium with glutamate (Life Technologies) supplemented with 10% fetal calf serum, basic fibroblast growth factor (1 ng/ ml, Boehringer) and G418 (300 μg/ml) as described by Roux et al, 159 J. Cell Physiol. 101-113 (1994) Cells from one plate at 80% confluence were transfected in suspension using the DOTAP transfection-reagent (Boehringer) with 30 μg of the ChAT expression vector and 3 or 6 μg of pcDNA RSV puro selection vector. Expression of the puromycin resistance gene was used to select stably transfected clones expressing a choline acetyltransferase activity. Puromycin (5 μg ml, Sigma) was added to the transfected cells 3 days after transfection and after about 3 weeks, independent clones were isolated and tested for ChAT activity. Control cells were transfected with 6 μg of the selection vector alone. In a second series of tests, one of the ChAT expressing cell lines (A24) was further transfected as described above with the VAChT construct (30 μg) and 3 days after traπfection, zeocin (Invitrogen) was used to select stable traπsfectants. After about 3 weeks, resistant cell lines were tested for the expression of VAChT using a binding assay with vesamicol as a specific ligand (Bahr & Parsons, 83 Proc. Natl. Acad. Sci. USA 2267-2270 (1986); Varoqui et al, 342 FEBS Lett. 97-102 (1994)). EXAMPLE 5 CHOLINE ACETYLTRANSFERASE ACTIVITY RBE4 stable transfectants were checked for ChAT activity by a procedure modified from Fonnum, 24 J. Neurochem. 407-409 (1975). Cells grown on 24 well - plates were washed 3 times with 1ml phosphate-buffered saline buffer (PBS) and removed with a scraper in 100 μl of a lysis buffer containing 200 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4) and 0.2% Triton. Cell debris was removed from the lysate by a brief centrifugation in a bench top centrifuge. An aliquot of the supernatant (10 μg protein) was transferred to Eppeπdorf tubes containing a mixture of [ 3 H] acetylCoA (4.4 Ci/mmol, New England Nuclear) and acetylCoA (15 μM) (Boehringer), 2 mM choline chloride and 0.2 mM serine salicylate (Sigma) in lysis buffer (total volume: 50 μl), and incubated for 45 or 60 min at 37°C. Incubation was stopped by addition of 500 μl of 10 M ice-cold sodium phosphate buffer, pH 7.4, containing 200 μM unlabelled acetylcholine. The [ 3 H] acetylcholine that has been synthesized was extracted with lOmg/ml of sodium tetraphenylboron in allyl cyanide while [ 3 H] cetylCoA remains in the aqueous phase, and radioactivity in the organic phase was counted. The amount of protein in the sample was measured by the Bradford method (Bradford, 72 Anal. Biochem. 248-254 (1976)) and activity was expressed in pmol [ 3 H] acetylcholine/μg protein x hr. EXAMPLE 6 [^H] VESAMICOL BINDING After several washings in PBS as described above, cells were harvested into 50 to 100 μl of ice -cold 50 mM sodium phosphate buffer, pH 7.4, containing 5 mM EDTA, a cocktail of protease inhibitors (1 mM phenylmethanesulfonyl fluoride (PMSF), 6 μg/ml leupeptin, 5 μg ml aprotinin, 10 μg ml pepstatin) and 2 M Chaps. Homogenization was completed by several passages through a 26-gauge needle and unbroken cells were eliminated by a low speed centrifugation as described above. Vesamicol binding was measured as previously described (Varoqui et al. 342 FEBS Lett. 97-102 (1994)) in the presence of 50 mM sodium phosphate buffer, pH 7.4, 2 mM Chaps and defined concentrations of L-[ 3 H] vesamicol (31 Ci/mmol, New England Nuclear). Bound [ 3 H] vesa icol was separated by vacuum filtration on glass fiber filters (Whatman GF/C). Unspecifϊc binding in the presence of 30 μM L-unlabelled vesamicol was subtracted from the total binding and specific binding was then expressed in pmol/ mg protein. EXAMPLE 7 EvMUNODETECTION OF ChAT AND VAChT Cell homogenates were lyophilized and solubilized in sodium dodecyl sulfate (SDS-PAGE) sample buffer (5% SDS, 50 mM Tris buffer, pH 6.8, 0.9 M sucrose) containing 50 mM L-dithiothreitol. Proteins from total cell lysate (100 μg/lane) were separated by SDS-PAGE electrophoresis (4-16%, Novex) and transferred to nitrocellulose membranes. Blots were probed with the goat polyclonal ChAT antibody (1/500, Chemicon) or with a rabbit anti- VAChT antibody raised against the C-terminus fragment of the Torpedo protein. Appropriate horseradish peroxidase - conjugated second antibodies were used and the complexes were visualized with a chemiluminescent system (ECL) using the manufacturer's instructions (Pierce). EXAMPLE 8 ACETYLCHOLINE RELEASE Cell lines were grown to about 80 % confluent in 25 cm 2 flask and acetylcholine st erases were inhibited by addition of 50 μM phospholine iodide 15 hr before the release test. For acetylcholine loading of control cells, 10 mM acetylcholine was also added to the culture medium as previously described {see for examples, Israel et al, 22 Neurochem. International 53-58 (1993), Israel et al, 36 europharmacol. 1789-1793 (1997)). After several washings with Dulbecco's modified Eagle's medium without choline (DMEM-0), cells were removed with a scraper into 2 ml of DMEM-0, and pelleted by centrifugation at 800xg for 8 min. Cells were resuspended in 100 μl DMEM-0 by repeated suckings through a 200 μl Gilson tip and kept at room temperature during the duration of the test. EXAMPLE 9 CHEMILUMINESCENT DETECTION OF ACh RELEASE Acetylcholine was measured by the chemiluminescent procedure of Israel & Lesbats, 37 J. Neurochem. 1475-1483 (1981); Israel & Lesbats, 39 J. Neurochem. 248-250 (1982)) in a release medium containing 300 μl of 120 mM Tris buffer, 120 mM sucrose, pH 8.6, 3 μl of acetylcholinest erase (1000 U/ml, Boehringer) and 15 μl of the chemiluminescent reaction mixture made of 100 μl of choline oxidase (250 U/ml, Wako), 50 μl of horseradish peroxidase type H (2 mg/ml, Sigma), 100 μl of lummol (1 mM in Tris buffer, pH 8.6, Merck). When acetylcholinesterase was omitted, only choline was detected. Intracellular acetylcholine levels were measured on a 3-5 μl aliquot by disrupting the cells with 0.4% Triton. Acetylcholine release was induced by calcium entry in the presence of the calcium ionophore A2 187 (Boehringer). Cells (50-300 pmol ACh) were added to the release medium containing 100 μM EGTA and 4 μM A23187. Release was then triggered by addition of 10 mM calcium. Internal calibration was done by adding a known amount of acetylcholine. EXAMPLE 10 ELECTROPHYSIOLOGAL DETECTION OF ACh RELEASE In parallel to chemically evoked release and using the same batch of cells, electrically evoked acetylcholine release was tested using spherical Xenopus myocytes (myoballs) as detector cells (Evers et al, 9 . Neurosci. 1523-1539, 1989 (1989); Dan & Poo, 359 Nature 733-736 (1992); Falk-Vairant et al, 63 (Supp) J. Neurochem. S90 (1994); Falk-Vairant et al, 45 . Neurosci. Res. 195-201 (1996)). Membrane currents from a myoball were recorded by the whole-cell patch-clamp method (Hamill et al, 391 Pflϋgers Arch 85-100 (1981)) in modified saline solution. After further disruption of the collagen fibers surrounding the cells by repeated passages through a 26 gauge needle, cells were laid onto the myoball culture dish. A single test cell was moved into contact with the recorded myoball with the help of a fine glass pipette and release was triggered by brief depolarizing pulses applied through this pipette. Membrane currents from the myoball were monitored with a patch-clamp amplifier (RK300, Biologic, FRANCE). Data were stored and analyzed with computer software (p-clamp and axotape, Axon Instruments; FIG.P, Biosoft). Results of EXAMPLES 3-10. ChAT activity in transfected cells. When RBE4 cells were transfected with the selection vector expressing puromycin resistance alone, a very low ChAT activity, if any, was detected (0.026 pmol [ H acetylcholine/μg protein x hr, n=6). FIG. 4A shows ChAT activity measured in standard conditions in some stable transfectants after co-transfection with a rat ChAT cDNA construct in a ratio of 10 (A) or 5 (B) with the selection vector. Sixty eight out of 78 puromycin-resistant independent clones had a high ChAT activity (2 and 8 were negative in A and B clones, respectively). Kinetics of ChAT activity was further studied in clone A24. FIG. 4B illustrates time and protein dependencies. ChAT activity in this clone reaches about 65 pmol/min x μg protein with a Km for acetylCoA around 5 μM. Irnmunoblot analysis of ChAT protein performed on A24 clone (lane 2) and a negative clone (lane 1) is shown on FIG. 5. Acetylcholine content and calcium-dependent acetylcholine release. The cellular Acetylcholine content was measured by a chemiluminescent procedure after disruption of the cells in the presence of 0.4% Triton. RBE4 expressing ChAT activity accumulates acetylcholine when exposed for 15 hr to an acetylcholiπesterase inhibitor (TABLE 1). However, the level of cellular acetylcholine content did not parallel the level of expressed ChAT activity. Calcium dependent release from RBE4 cells was studied using two parallel approaches. In the first one, acetylcholine release was monitored by a chemiluminescent method and triggered by the addition of calcium when the calcium ionophore A23 87 has been added to the reaction mixture. Transfectant cells are able to release the neurotransmitter they have synthesized and accumulated. FIG. 6A shows results from clone A24. The light emission elicited by calcium addition was calibrated by addition of a known amount of acetylcholine; no light emission was observed in the absence of acetylcholinesterase. Addition of Triton after release allows the determination of the residual content. At equal acetylcholine amounts during the test, acetylcholine release from the stable ChAT transfectant A24 appears very similar to the release from control cells artificially loaded with external acetylcholine (FIG. 6B). TABLE 2 shows the quantification of acetylcholine content and release in several clones. In parallel tests, acetylcholine release from a single cell stimulated by depolarizing pulses was monitored by electrophysiological procedures using aXenopus myoball as an acetylcholine detector cell. Using this system, we showed that stimulated transfected cells were able to generate rapid inward currents from the myocyte. FIG. 7 shows superimposed results of the currents evoked by consecutive stimuli applied to one cell of the transfectant clone A24. Similar results were obtained from loaded control cells (data not shown). The maximum amplitude of the inward evoked currents was 323.3 ± 63.5 pAin transfectant clone A24 (n=26) and 374.0 ± 161 (n^5) in control cells loaded with acetylcholine. FIG. 4B illustrates the chronological pattern of the peak currents. VAChT expression. A VAChT cDNA construct has been introduced in the stably transfected cell line A24 expressing the rat ChAT. Clones selected in zeocin containing media displayed a transient elongated morphology with long processes, seen at low density of cells (FIG. 8). Screening for VAChT expression of zeocin resistant clones was done using a binding assay with vesamicol as a specific ligand. No vesamicol binding was observed in untransfected control cells. FIG. 9 A illustrates the kinetics of vesamicol binding for the clone A24-56 (B max =2.9 pmol/mg protein; Kp = 16 mM). Irnmunoblot analysis of two clones for the VAChT protein (FIG. 9B; lane 1: A24-23; lane 2; A24-56; lane 3; parental A24) showed a characteristic pattern, with a broad band around 50-64 kilodaltons (kDa) (Varoqui et αl, 109 Progr. Brain Res. 83-95 (1996)). EXAMPLE 11 EXPRESSION OF SYNAPTIC PROTEINS IN RBE4 CELLS The intrinsic efficacy of RBE4 cells to release acetylcholine upon calcium challenge prompted us to look for the presence of specific synaptic proteins involved in the release machinery. We thus examined the expression pattern of several of these proteins in RBE4 cells and in ChAT/VAChT transfectants in control conditions as well as in the presence of differentiating agents. We showed that RBE4 cells endogenously express vesicular proteins (synaptobrevin (or VAMP), synaptotagmin, and SV2) and other proteins of the SNARE complex (SNAP23) but at different levels. Acetylcholine, either added in the external medium in control cells ox from endogenous origin in ChAT transfectants, was demonstrated to stimulate the expression of vesicular proteins. Similar results were observed in the presence of cyclic AMP. SNAP23, whose endogenous level is already high, does not appear to be further modulated by either of these agents (see, . FIG. 11). The results of this EXAMPLE are of particular importance for future use of ChAT/VAChT transgenes as a tool for improving altered cholinergic function in the central nervous system as they do show that not only some synaptic proteins are intrinsically expressed in RBE4 cells, but that expression of a set of these proteins is further challenged in ChAT transgenes and can be modulated by exogenous addition of differentiating agents. The foregoing description has been presented only for the purposes of illustration and is not intended to limit the invention to the precise form disclosed, but by the claims appended hereto.

Claims: WE CLAIM: 1. Mammalian non-neuronal cells that are modified to store and release a neurotransmitter. 2. The cells according to claim 1, wherein the cells are genetically modified to produce, store, and release the neurotransmitter. 3. The cells according to claim 1, wherein the cells are genetically modified by transfecting the cells with a gene that encodes an enzyme that causes production of a neurotransmitter in the cells. 4. The cells according to claim 3, wherein the cells have been further modified by transfecting the cells with a gene that encodes a vesicular transporter for the neurotransmitter in the cells. 5. The cells according to claim 4, wherein the vesicular transporter is VAChT. 6. The cells according to claim 3, wherein the cells have been further modified by transfecting the cells with a selection gene. 7. The cells according to claim 6, wherein selection gene is an antibiotic selection gene. 8. The cells according to claim 3, wherein the enzyme is choline acetyltransferase and the neurotransmitter is acetylcholine. 9. The cells according to claim 3, wherein the enzyme is tyrosine hydroxylase and the neurotransmitter is L-Dopa/dopamine. 0. The cells according to claim 3, wherein the enzyme is glutamic acid decarboxylase and the neurotransmitter is γ-aminobutyric acid. 11. The cells according to claim 3, wherein the enzyme is tryptophan hydroxylase and the neurotransmitter is serotonin. 12. The cells according to claim 1, wherein the cells are selected from the group consisting of mammalian endothehal cells, mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells, 13. The cells according to claim 12, wherein the mammalian brain endothehal cells, mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells are human brain endothehal cells, human retinal cells, human fibroblast cells, or human glial cells. 14. The cells accorciing to claim 12, wherein the endothehal cells are mammalian brain endothehal cells. 15. The cells according to claim 14, wherein the mammalian brain endothehal cells are human brain endothehal cells. 16. The cells according to claim 14, wherein the brain endothehal cells are RBE4 cells. 17. The cells according to claim 1, wherein the cells comprise a continuous cell line. 18. The cells according to claim 1, wherein the neurotransmitter is selected from the group consisting of acetylcholine, glutamate, γ-arntnobutyric acid (GABA), glycine, dopamine, norepinephrine, serotonin (5-hydroxytryptamiπe), or histamine. 19. A process for preparing recombinant mammalian non-neuronal cells that produce, store, and release neurotransmitters, comprising: transfecting mammalian non-neuronal cells with a gene that encodes an enzyme that causes production of a neurotransmitter. 20. The process according to claim 19, wherein the transfected cells are from a non-tumorigenic cloned cell line. 21 The process according to claim 19, wherein the cells have been further modified by transfecting the cells with a gene that encodes a vesicular transporter for the neurotransmitter in the cells. 22. The process according to claim 21, wherein the vesicular transporter is VAChT. 23. The process according to claim 19, wherein the cells have been further modified . by transfecting the cells with a selection gene. 24. The process according to claim 23, wherein selection gene is an antibiotic selection gene, 25. The process according to claim 19, wherein the enzyme is choline acetyltransferase and the neurotrammitter is acetylcholine. 26. The process according to claim 19, wherein the enzyme is tyrosine hydroxylase and the neurotransmitter is selected from the group consisting of L-Dopa and dopamine. 27. The process according to claim 19, wherein the enzyme is glutamic acid decarboxylase and the neurotransmitter is γ-aminobutyric acid. 28. The process according to claim 19, wherein the mammalian non-neuronal cells are human non-neuronal cells. s 29. The process according to claim 9, wherein the mammalian non-neuronal cells are selected from the group consisting of mammalian endothehal cells, mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells. 30. The process according to claim 29, wherein the mammalian endothehal cells, o mammalian retinal cells, mammalian fibroblast cells, or mammalian glial cells are human endothelial cells, human retinal cells, human fibroblast cells, or human glial cells. 31. The process according to claim 30, wherein the mammalian endothehal cells are 5 mammalian brain endothelial cells, 32. The process according to claim 31, wherein the mammalian brain endothelial cells are human brain endothelial cells. 0 33. The process according to claim 31, wherein the mammalian brain endothelial cells are RBE4 cells. 34. The process according to claim 19, wherein the transfected mammalian non-neuronal cells selectively express the enzyme, or selectively co-express the 5 enzyme and a second enzyme that causes co-release of a second neurotransmitter. 35. The process according to claim 34, wherein the transfected cells selectively release acetylcholine. 36. The process according to claim 35, wherein the transfected cells are brain endothelial cells. 37. The process according to claim 36, wherein the brain endothelial cells are RBE4 cells. 38. The process according to claim 34, wherein the transfected cells selectively release γ-aminobutyric acid. 39. The process according to claim 34, wherein the transfected cells selectively co-release acetylcholine and γ-aminobutyric acid. 40. The process according to claim 39, wherein the transfected cells are glial cells. 41. The process according to claim 34, wherein the transfected cells selectively co-release acetylcholine and glutamate. 42. The process according to claim 41, wherein the transfected cells are fibroblastic cells. 43. The process according to claim 34, wherein the transfected cells selectively release glutamate. 44. A method of treating a neurodegenerative disorder in a mammal, the method comprising: administering to a mammal in need of treatment mammalian non-neuronal cells that are modified to store and release a neurotransmitter. 45. The method according to claim 44, wherein the mammalian non-neuronal cells are genetically modified to produce, store, and release the neurotransmitter. 46. The method according to claim 44, wherein the mammalian non-neuronal cells are human non-neuronal cells. 47. The method according to claim 44, wherein the mammal is a human. 48. The method according to claim 44, wherein the neurotransmitter is selected from the group consisting of acetylcholine, glutamate, γ-aminobutyric acid (GABA), glycine, L-Dopa/dopamine, norepinephrine, serotonin (5-hydroxytryptamine) or histamine. 49. The method according to claim 48, wherein the mammal in need of treatment has a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, stroke, πeuronal cell death, and psychiatric disorders, including depression. 50. The method according to claim 48, wherein the mammal in need of treatment suffers from cholinergic deficiency, dementia, or Alzheimer's disease. 51. The method according to claim 48, wherein the neurotransmitter is selected from the group consisting of L-Dopa and dopamine. 52. The method according to claim 48, wherein the mammal in need of treatment suffers from Parkinson's disease. 53. The method according to claim 48, wherein the neurotransmitter is γ -aminobutyric acid. 54. The method according to claim 48, wherein the mammal in need of treatment suffers from Huntington's disease, epilepsy, or stroke. 55. The method according to claim 48, wherein the neurotransmitter is serotonin. 56. The method according to claim 48, wherein the mammal in need of treatment suffers from psychiatric disorders, including depression. 57. The method according to claim 48, wherein the cells are administered by direct iπtracranial injection, systemic I.V. injection, intra-arterial injection, and systemic intra-carotid injection. 58. The method according to claim 48, wherein the cells administered in biopolymers or capsules. 59. The method according to claim 48, wherein the cells are directed to a specific region of the brain by grafting. 60. A method of treating a neuromuscular disorder in a mammal, the method comprising administering to a mammal in need of treatment mammalian non-neuronal cells that are modified to store and release acetylcholine to the neuromuscular junction of the mammal. 61. The method according to claim 60, wherein the neuromuscular disorder is yasthenia gravis.

Inventors: COURAUD PIERRE-OLIVIER, ISRAEL MAURICE, TCHELINGERIAN JEAN-LEON, COURAUD, PIERRE-OLIVIER, ISRAEL, MAURICE, TCHELINGERIAN, JEAN-LEON
Applicants: NEUROTECH SA, NEUROTECH (S.A.)
Application year: 1999
Main classification: C12N5
IPC codes: A61K35, A61P25, C07K14