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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13045—Special targeting system for viral vectors

Definitions

• the present invention relates to recombinant expression vectors carrying a gene encoding a therapeutically active polypeptide, which gene is under transcriptional control of a ubiquitin promoter.
• the invention also relates to the use of a ubiquitin promoter to direct in vivo expression of therapeutic genes after transfer of such genes to the central nervous system.
• Therapeutically active polypeptides may be delivered to the central nervous system (CNS) by implantation of polypeptide producing cells, or by injection of viral vectors capable of infecting brain cells and directing the expression of a therapeutically active polypeptide encoded by the viral vector in the brain cells.
• CNS central nervous system
• primary and immortalised cells have been successfully used for gene transfer applications.
• primary cells of neuronal origin e.g. glia cells and astrocytes
• non-neuronal origin e.g. fibroblasts, myoblasts and hepatocytes.
• Such cells may not survive for longer periods in the CNS unless they are immortalised.
• Intracerebral grafting of foetal tissue for the treatment of neurological disorders have also been investigated.
• stem cells may be used.
• cerebral endothelial cell Another cell type which has been proposed as gene transfer vehicle is the cerebral endothelial cell.
• the direct implantation of immortalised and genetically transformed cerebral endothelial cells into various parts of the brain has been disclosed. It has also been suggested to deliver therapeutic agents by infecting endothelial cells of blood vessels located in the brain with a viral vector, as a result of intravascular administration of the vector to the host near the site of infection (WO 96/22112).
• a broad range of viral vectors such as including adenovirus vectors (WO 95/26408), adeno-associated virus vectors (WO 95/34670), herpes virus vectors (Glorioso et al. Sem. Virol. 1992 3 265-276), vaccinia virus vectors, and retroviral vectors, including systems based on HIV, has been suggested as delivery vehicles for therapeutic genes to the CNS.
• Such vectors can be administered to the CNS by the intravenous and the intracranial route, and by administration to the cerebrospinal fluid.
• WO 95/09654 discloses a method for the treatment of adverse conditions of the CNS by administration of producer cells containing a retroviral vector to the cerebrospinal fluid.
• the producer cells produce retroviral particles which is capable of transducing cells present in the nervous system.
• Intracerebral implantation of encapsulated cells producing a therapeutic peptide has also been suggested.
• Ubiquitin proteins are "house hold proteins", essential for the catabolism of all cellular proteins. The structure of ubiquitin proteins is highly conserved during evolution.
• the ubiquitin promoter is a constitutive promoter which controls the expression of ubiquitin proteins. A number of transfection experiments have shown that this promoter is as strong as, or even stronger than the commonly used viral promoters. Furthermore, heterologous genes under the control of the ubiquitin promoter are actively transcribed in every cell line that has been transfected with this promoter (more than 20, including several cell lines of neuronal origin). In transgenic mice, the human ubiquitin C promoter (UbC) have been shown to direct in vivo expression of heterologous genes in several types of tissues, including brain-tissue (Nucleic Acids Research 1996 24 (9) 1787-1788).
• the therapeutic gene In order to obtain efficient expression in the CNS, the therapeutic gene must be put under transcriptional control of a promoter which is active in the CNS.
• viral promoters are down-regulated in vivo in mammalian cells. If efficient transfer of a therapeutic polypeptide to the CNS is to be obtained with a viral vector, it is should be replaced the with a promoter which will provide stable and efficient expression.
• the ubiquitin promoter has proven to direct stable high level expression of heterologous genes in in vitro experiments. However, there is no indication of its efficiency in vivo.
• the present invention is directed to the use of an ubiquitin promoter for stable and efficient expression of genes encoding therapeutically active polypeptides in the CNS.
• An ubiquitin promoter of present invention includes the human ubiquitin promoter, which is indistinguishable from the endogenous ubiquitin promoter and should therefore not be susceptible to the in vivo down-regulation observed for viral promoters.
• the present invention is directed towards a recombinant expression vector comprising a gene encoding a therapeutically active polypeptide, which gene is under transcriptional control of an ubiquitin promoter.
• the invention provides an eukaryotic cell, transfected with the eukaryotic expression vector of the invention.
• the invention provides a packaging cell line comprising the retroviral expression vector of the invention and one or more nucleotide constructs encoding the proteins required for the genome of the retroviral vector to be packaged.
• the invention provides a method of producing a retroviral particle by culturing the packaging cell line of the invention, and a retroviral particle obtained according to this method.
• the invention provides an eukaryotic cell, e.g. an immortalised cerebral endothelial cell line, infected by a retroviral particle obtained by the method of this invention.
• the invention is directed towards the use of the recombinant expression vector of the invention for the manufacture of a pharmaceutical composition, useful for the treatment of a neurological disease or disorder.
• the invention provides a method for the treatment of a living body suffering from a neurological disease or disorder which is responsive to a therapeutically active polypeptide, which method comprises administering to the living body, a retroviral particle obtained by the method of the invention, encoding the therapeutically active polypeptide; or implanting into the living body, the cells of the invention, producing the therapeutically active polypeptide.
• Fig. 1 shows a graphic illustration of a UbC promoter containing plasmid based expression vector, pUbilz, obtained according to Example 2.
• the present invention resides in the use of a ubiquitin promoter to direct in vivo expression of therapeutic genes after transfer of such genes to the central nervous system.
• the invention provides a recombinant expression vector comprising a gene encoding a therapeutically active polypeptide, which gene is under transcriptional control of an ubiquitin promoter.
• the recombinant expression vector of the invention may be any vector suitable for transferring a gene under transcriptional control of a ubiquitin promoter to mammalian cells.
• the recombinant expression vector is an eukaryotic expression vector or a recombinant viral expression vector.
• Genes can be transferred into cells using a variety of means including calcium phosphate precipitation [Graham et al., Virol. 1973 52 456-467; Wigler et al., Cell 1979 777-785], electroporation [Neumann et al., EMBO J. 1982 841-845], microinjection [Graessmann et al., Meth.
• Viral vectors provide an efficient means of transferring genes into cells both in vivo and in vitro.
• the efficiency of viral gene-transfer is due to the fact that transfer of DNA is an essential part of the natural life cycle of viruses and that DNA transfer is a receptor-mediated process.
• viral systems including retrovirus, adenovirus, adeno-associated virus, vaccinia virus and herpes virus have been developed as in vivo therapeutic gene transfer vectors for gene therapy of CNS disorders.
• Viral vectors have also been used to transform various forms of cells such as astrocytes, fibroblast cells and cerebral endothelial cells which is thereafter implanted into the CNS. Further WO 96/06942 describe genetically altered T-cells which enter the CNS and may be used as gene transfer vehicles.
• the recombinant viral expression vector of the invention may be any viral expression vector suited for in vivo transfer and expression of genes.
• Preferred viral expression vectors include retroviral vectors, recombinant adenovirus vectors, recombinant adeno-associated virus vectors, vaccinia virus vectors and recombinant herpes virus vectors.
• Recombinant retroviral gene delivery methods are the most extensively used due in part to: (1) the efficient entry of genetic material (the vector genome) into replicating cells; (2) an active, efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) the potential to target particular cellular subtypes through control of the vector target cell binding and the tissue specific control of gene expression; (5) a general lack of pre-existing host immunity; and substantial knowledge and clinical experience which has been gained with such vectors.
• the recombinant vector carrying a therapeutic gene under transcriptional control of an ubiquitin promoter is therefore a retroviral vector.
• the retroviral genome consists of an RNA molecule with the structure R-U5- gag-pol-env-U3-R. During the process of reverse transcription, the U5 region is duplicated and placed at the right hand end of the generated DNA molecule, whilst the
• U3 region is duplicated and placed at the left hand end of the generated DNA molecule.
• the resulting terminal structure U3-R-U5 is called LTR (Long Terminal
• the U3 region at the left hand end of the provirus harbours the promoter that is used to drive expression after infection has occurred. This promoter drives the synthesis of an RNA transcript initiating at the boundary between the left hand U3 and
• the RNA is packaged into retroviral particles and transported into the target cell to be infected.
• the RNA genome is reverse transcribed as described above.
• Retroviral vector systems used for the generation of recombinant retroviral particles consist of two components.
• the retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins (gag, pol and/or env) have been replaced by therapeutic genes and/or marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.
• the second component is a cell line that produces large quantities of the viral proteins (gag, pol and/or env), however lacks the ability to produce replication competent virus.
• This cell line is known as the packaging cell line and consists of a cell line transfected with one or more plasmids carrying the genes (gag, pol and/or env) enabling the modified retroviral vector to be packaged.
• the vector plasmid is transfected into the packaging cell line.
• the modified retroviral genome including the inserted therapeutic and marker genes is transcribed from the vector plasmid and packaged into modified retroviral particles (recombinant viral particles).
• This recombinant virus is then used to infect target cells in vitro or in vivo and the vector genome and any carried marker or therapeutic genes become integrated into the target cell's DNA.
• a cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells.
• the DNA of the vector carrying the therapeutic and marker genes is integrated in the cell's DNA as a provirus and can now be expressed in the infected cell.
• WO-A1 -9607748 describes the principle and construction of a new type of retroviral vector.
• the ProCon-vector plasmid the right-hand (3') U3 region is altered, but the normal left-hand (5') U3 structure is maintained; the vector can be normally transcribed into RNA utilising the normal retroviral promoter located within the left- hand (5') U3 region upon its introduction into packaging cells.
• the generated RNA will only contain the altered right-hand (3') U3 structure.
• this altered U3 structure will be present in both Long Terminal Repeat at either end of the retroviral structure.
• any promoter including the human ubiquitin promoter, can be inserted and this promoter is then utilised exclusively in the target cell for expression of linked sequences encoding therapeutic polypeptides.
• DNA segments homologous to one or more cellular sequences can also be inserted into the polylinker for the purposes of gene targeting, by homologous recombination.
• the retroviral vectors of the invention need not be of the ProCon type, but can be any conventional retroviral vector carrying therapeutic genes under transcriptional control of the an ubiquitin promoter.
• Such vectors include Self-lnactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (WO-A 1-94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (WO-A1 -89/11539). In both of these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the therapeutic gene.
• the retroviral vectors of the invention are based preferably either on a BAG vector [Price, Turner J D, and Cepko C; Proc. Natl. Acad. Sci. USA 1987 84 156-160) or an LXSN vector [Miller A D, & Rossman G J; Biotechni ⁇ ues 1989 7 980-990].
• Eukaryotic Expression Vectors include a BAG vector [Price, Turner J D, and Cepko C; Proc. Natl. Aca
• the recombinant eukaryotic expression vector of the invention may be any eukaryotic expression vector suited for transferring a gene under transcriptional control of a ubiquitin promoter to mammalian cells.
• Preferred eukaryotic expression vector of the invention are pTEJ-4, pTEJ-8, or pUbilZ.
• the eukaryotic expression vector should contain sequences which facilitate the prokaryotic propagation along with two eukaryotic transcription units.
• Prokaryotic sequences include a bacterial resistance gene under the transcriptional control of the prokaryotic EM7 promoter and a bacterial origin of DNA replication.
• the eukaryotic transcription unit responsible for eukaryotic selection employ the SV 40 early promoter to drive the expression of a resistance gene and a polyadenylation signal.
• the present expression vectors contain one of the following prokaryotic/eukaryotic selection markers: neomycin, hygromycin, pyromycin or zeocin resistance gene allowing the selection of bacterial clones under EM7 promoter and cellular clones under SV 40 early promoter.
• the second eukaryotic transcription unit contain the UbC promoter, a polyadenylation signal, and finally a polylinker for insertion of nucleotide sequences encoding the protein in question (as an example see pUbilZ, of Example 2).
• the ubiquitin promoter contemplated according to the present invention is an ubiquitin promoter derived from any convenient origin, e.g. derived from the 5' end
• the ubiquitin promoter is of human origin, in particular the human ubiquitin C promoter (UbC).
• the ubiquitin promoter of the invention is a human ubiquitin promoter having the sequence presented as SEQ ID NO: 1 in the attached sequence listing.
• a therapeutically active polypeptide of the invention may be any peptide, polypeptide or protein that is capable of ameliorating neurological disorders.
• the therapeutically active polypeptide of the invention is a Nerve Growth Factor (NGF); a Fibroblast Growth Factor (FGF), in particular an acidic or a basic Fibroblast Growth Factor (aFGF or bFGF); an Insulin-like Growth Factor, in particular IGF I or IGF II; an Endothelial Growth Factor (EGF), in particular a Vascular Endothelial Growth and Permeability Factor (VEGPF); a member of the Transforming Growth Factor (TGF) superfamily, including a Transforming Growth Factor- ⁇ and - ⁇ (TGF ⁇ and TGF ⁇ ); a Glial Derived Neurotrophic Factor (GDNF); a Ciliary Neurotrophic Factor (CNTF); a Brain Derived Neurotrophic Factor (BDNF); a Neurotrophin, in particular Neurotrophin 3, 4/5, 6 or 7; a Neuturin (NTN); a Percipin; a Tumor Necrosis Factor (TNF), in
• the therapeutically active polypeptide of the invention may be employed in order to treat diseases or disorders in the brain and central nervous system.
• diseases and disorders include ischemic strokes; angiogenesis; metabolic diseases of the brain; axonal injury; spinal cord injury; Alzheimer's disease; Amyothropic Lateral Sclerosis (ALS); Parkinson's disease; Huntington's disease; motor neuron diseases; central nervous system infections; epilepsy; post polio syndrome; mucopolysaccharidoses (MPS), in particular MPS types I to VII; lipidoses; in particular Gaucher's disease; Lesch-Nyhan syndrome; X-linked ADL; metachromatic leukodystrophy; Krabbe's disease; Charcot-Marie-Tooth disease; Fragile X; epilepsies; Down's syndrome; phenylketonuria; degenerative disorders; and mental disorders.
• MPS mucopolysaccharidoses
• the eukaryotic expression vectors and the recombinant retroviral vectors according to the invention may in addition to the therapeutic gene carry a gene encoding a marker.
• the marker may in particular be proteins like ⁇ -galactosidase, neomycin, alcohol dehydrogenase, luciferace, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, secreted alkaline phosphatase, or green or blue fluoroscent proteins (GFP).
• the vector of the invention may be used to transduce cells to secrete the therapeutic polypeptide in vivo after being implanted into the CNS.
• cells producing the therapeutically active polypeptide are generated by transfection with a plasmid vector
• recipient cells e.g. immortalised neural stem cells, immortalised cerebral endothelial cell, or other immortalised cells compatible with the CNS.
• transfection with a viral vector are used for transplantation of the packaging cell, several cells may be used, e.g. immortalised neural stem cells, immortalised cerebral endothelial cell, or other immortalised cell compatible with CNS.
• immortalised neural stem cells e.g. immortalised neural stem cells, immortalised cerebral endothelial cell, or other immortalised cell compatible with CNS.
• human cells may be used, e.g. immortalised neural stem cells, immortalised cerebral endothelial cell, or fibroblast-like human cell, e.g. HEK 293 or HeLa
• the packaging cell line of the invention can be selected from an element of the group consisting of psi-2 [Mann R, Mulligan R C, & Buttimore D; CeN 1983 33 153- 159], psi-Crip [Danos O & Mulligan R C; Proc. Natl. Acad. Sci. USA 1988 85, 6460-
• the packaging cell line is made from human cells, e.g. HT1080 cells (WO-A1-9621014), HEK 293, thereby allowing production of recombinant retrovirus that are capable of surviving inactivation by human serum.
• the present invention resides in the use of a ubiquitin promoter to direct in vivo expression of therapeutic genes after transfer of such genes to the central nervous system. Accordingly, in a particular embodiment, the invention is directed to the use of the recombinant expression vector of the invention for the manufacture of a pharmaceutical composition, useful for the treatment of a neurological disease or disorder.
• the pharmaceutical composition of the invention preferably is a composition suited for injection or implantation into the human brain. Such compositions may be provided in the form of vials of frosen cells, optionally provided in a freeze medium in suspension.
• compositions suited for injection or implantation may be prepared by the skilled person according to conventional methods (see e.g. Scientia Medicinalis. 1997, Schering AG, ISSN 1433-190X).
• the pharmaceutical compounds of the present invention may thus be formulated for in ampoules, pre-filled syringes, small volume infusion containers, etc.
• the compositions may take such forms as suspensions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
• the invention provides a method for the treatment of a living body suffering from a neurological disease or disorder which is responsive to a therapeutically active polypeptide, which method comprises administering to the living body a retroviral particle obtained by the method of the invention, encoding the therapeutically active polypeptide; or by implanting into the living body, cells of the invention, producing the therapeutically active polypeptide.
• the neurological disorders contemplated according to the present invention are e.g.
• ischemic stroke angiogenesis; metabolic disease of the brain; axonal injury; spinal cord injury; Alzheimer's disease; Amyothropic Lateral Sclerosis (ALS); Parkinson's disease; Huntington's disease; motor neuron disease; central nervous system infections; epilepsy; post polio syndrome; mucopolysaccharidoses (MPS), in particular MPS types I to VII; lipidosis, in particular Gaucher's disease; Lesch-Nyhan syndrome; X-linked ADL; metachromatic leukodystrophy; Krabbe's disease; Charcot- Marie-Tooth disease; Fragile X; epilepsy; Down's syndrome; phenylketonuria; 5 degenerative disorders; or mental disorders.
• MPS mucopolysaccharidoses
• the human UbC promoter was cloned into a modified version of pcDNA3.1/Zeo.
• the unmodified pcDNA3.1/Zeo is commercial available from Invitogen.
• the modified version is smaller than the parent vector, since the ampicillin gene (from position 3933 to 5015) and a sequence from position 2838 to 3134 were removed.
• the ampicillin gene from position 3933 to 5015
• a sequence from position 2838 to 3134 were removed.
• EGFP Enhanced Green Fluorescence Protein
• HiB5 cells 25 14 embryo hipocampus [Refranz et al; Cell 1991 66 713-729].
• the HiB5 cells were transfected with Lipid 6 from Invitrogen according to manufactories recommendations.
• GDNF The gene encoding GDNF was cloned using conventional techniques or as described in WO 93/06116 and inserted into the pUbilZ expression vector. Clones of Immortalised neural stem cells (rat and human) and immortalised cerebral endothelial cells (rat or human) expressing therapeutic genes have been generated. Clones will be studied in short-term experiments in order to establish the ability of each cell line to secrete the introduced neurotrophic factor (e.g. NGF, GDNF, NTN or CNTF) in vivo in sufficiently high amounts to produce full neuroprotective effects in established models.
• the introduced neurotrophic factor e.g. NGF, GDNF, NTN or CNTF
• the cells will be implanted into the septum in rats subjected to a unilateral fimbria-fornix lesion, and the magnitude of cholinergic cell loss in the medial septal nucleus will be established 2 weeks after operation. From previous studies on NGF-secreting rat HiB5 and RBE cells it is known that an implant of 2 x 10 5 cells with an estimated in vivo secretion rate of 50 ng NGF/day will be able to provide complete protection in this model.
• GDNF and NTN secreting cell lines cells will be implanted in the substantia nigra and striatum, first, in C57 mice treated one week later with a single injection of MPTP (40 mg/kg s.c). The loss of dopamine neurones in the nigra and the reduction in dopamine levels and tyrosine hydroxylase immunoreactivity in the striatum will be analysed 4 weeks after injection.
• nigral neurones will be pre-labelled with the retrograde tracer FluoroGold from the striatum according to the procedure established in the Swedish laboratory, in order to provide an accurate estimation of the magnitude of lesion induced nigral cell death.
• transplants of cells expressing the reporter gene, EGFP will be used as controls. Since the minimum number of cells necessary to produce a full neuroprotective effect has not been established for GDNF and NTN, different cell numbers, from 2-8x10 5 cells, will be tested.
• CNTF and NGF cells will be evaluated by their protective effect against quinolinic acid lesion (200 nmol) of striatal medium size projection neurones. This type of lesion induces complete neuronal death in 2 days to one week. The cells will be transplanted prior to the lesion and neuroprotection assessed one week post lesion, determining neuroprotection in the striatum, and preservation of innervation of target territories in the SNr.
• the long-term functional effects and the stability of transgene expression will be studied in animal models relevant to the three clinical conditions, Parkinson's disease, Huntington ' s chorea and dementia. Three animal models will be used to study long-term functional effects of the therapeutic cell lines.
• Rats with unilateral intrastriatal 6-hydroxydopamine lesions will receive implants of GDNF or NTN producing cells, either into the substantia nigra, 3 days after the lesion (i.e. prior to the onset of cell death), or into the striatum, 4 weeks after the lesion (at the time when dopamine neuron degeneration is well advanced).
• the ability of the cell implants to reverse the Parkinsonian condition in tests of spontaneous motor behaviour (stepping and paw use tests) and to prevent the progressive neuro- degeneration long-term will be studied over a 6 month period.
• NGF-secreting human neural and endothelial cells will be implanted into the nucleus basalis and septum in aged cognitively impaired rats (22-24 month old Sprague-Dawley rats) following the protocol used in previous studies on NGF secreting rat HiB5 cells in the Lund laboratory. Groups of young unimpaired rats, transplanted identically, will be studied in parallel. The ability of the cells to reverse the age dependent learning and memory impairment in the water maze test and the age-dependent cholinergic neuronal atrophy in basalis and septum will be studied over 1 and 6 months. EGFP expressing cells will be used as control.
• the neuroprotective effects of the NGF or CNTF secreting human cell lines will be studied in rats receiving injections of quinolinic acid into the striatum, as above.
• the cells will be implanted one week before the lesion, and the ability of the cells to block the excitotoxic striatal damage, as well as to prevent the development of impairments in motor behaviours, will be studied both short-term (4 days and 1 month) and long-term (6 months). Long-term expression of the transgene will be evaluated as above.
• an UbC containing ProCon Vector and Production of Retroviral Particles by Packaging Cells In a preferred embodiment of the invention immortalised cerebral endothelial cells are transformed by a ProCon vector as described above carrying the gene encoding GDNF under transcriptional control of the human ubiquitin promoter.
• the vector for transformation of the cerebral endothelial cells may be obtained by insertion of the human ubiquitin promoter prepared by PCR from the plasmid pTEJ-8 described in FEBS Letters. 1990 267 (2) 289-294, into the polylinker of the ProCon vector described in WO 96/07748.
• a GDNF encoding gene may be cloned using conventional techniques or as described in WO 93/06116 and inserted into the body of the ProCon vector.
• Retroviral particles are produced by introducing the ProCon vector obtained as above into a packaging cell line followed by isolation of the retroviral particles produced.
• Immortalised cerebral endothelial cells may then be infected by the retroviral particles produced by the packaging cell line.
• genes encoding GDNF or NGF may be introduced into rat cerebral endothelial cells (RBE) by transfection with an eukaryotic expression vector.
• the eukaryotic expression vector may be constructed by insertion of GDNF cDNA or NGF cDNA into the polylinker of the plasmid pTEJ-8 or pUbMZ.
• the resulting vector may be used to transfect immortalised cerebral endothelial cells of rat or human origin.
• the genetically transformed immortalised endothelial cells may hereafter be implanted into the CNS of an individual suffering from Parkinson's disease or another disease susceptible to treatment with GDNF.
• the immortalised neural stem cells or immortalised endothelial cells may be implanted into the striatum of the host but may also be implanted into other parts of the brain.
• polylinker is used for a short stretch of artificially synthesised DNA which carries unique restriction sites allowing the easy insertion of any promoter or DNA segment.
• GAGCGCAGCA AAATGGCGGC TGTTCCCGAG TCTTGAATGG AAGACGCTTG TGAGGCGGGC 960 TGTGAGGTCG TTGAAACAAG GTGGGGGGCA TGGTGGGCGG CAAGAACCCA AGGTCTTGAG 1020

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