FI107737B - Plasmid for expression of the tyrosine hydroxylase gene in the brain - Google Patents

Description

107737

FIELD OF THE INVENTION This invention relates to gene therapy for neural or nonneural disorders, in particular Parkinson's disease. The invention provides an expression plasmid based on bovine type-1 papillomavirus with a therapeutic gene inserted for transfection into cells of a subject in vivo.

Background of the Invention

Gradual degeneration of dopaminergic neurons in the substantia nigra pars compacta occurs in Parkinson's disease, leading to the disappearance of dopaminergic nervous system in the Corpus striatum (Homykiewicz, 1982; Homykiewicz et al., 1986). Current treatment for Parkinson's disease is based on the administration of a dopamine precursor in combination with peripheral dopade carboxylase inhibition (DDC) (Birkmayer & Homykiewicz, 1961; Cotzias et al., 1967). In most patients, this treatment loses its effectiveness over time, and the treatment is also associated with a number of side effects (Chase et al., 1994; Nutt & Holford, 1996).

Tyrosine hydroxylase (TH) is a dopamine-producing enzyme that converts tyrosine into L-dopa, which is then produced by endogenous DDC to produce dopamine.

Since the brain is rich in other enzymes as well as tyrosine, another way to increase dopamine synthesis is to increase TH expression locally in the striatum (Elsworth & Roth, 1997). Indeed, it has been reported that transfection of striatal cells with a viral vector expressing the TH- • · 25 gene can lead to the production of TH messenger RNA and the synthesis of the corresponding · · · 1 .. protein in the striatum, so TH gene therapy may actually work for Parkinson's. · • · · in animal models of the disease. However, the results have been very variable (Jiao et al. 1993; - # During et al. 1994; Horellou et al. 1994; Cao et al. 1995; Geller et al. 1997; Jiao et al. Lundberg et al., 1996). In addition, * 1 1 30 toxic side effects have been observed with the use of some vector systems, which complicates the application of gene therapy in brain diseases and, .., the increased need to develop more useful vectors (Isacson, 1995; Sabel et al., 1995).

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The various BPV-1 based vectors described in the kernel are based on the entire BPV-1 genome or contain at least 69% of the viral genome - necessarily the 'early region' of the virus.

The BPV-1 expression plasmids of the present invention contain genes for viral replication-regulating proteins such that expression of these genes is driven by heterologous promoters, which makes expression of E1 and E2 proteins generally non-epithelial tissue cells possible.

Summary of the Invention

We have constructed and characterized a bovine type-1 papillomavirus (BPV-1) vector for expressing therapeutic genes in cells of a subject, particularly for expressing the tyrosine hydroxylase gene in brain cells.

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In other words, the general object of the present invention is a BPV-1 plasmid carrying a therapeutic gene for use in in vivo gene therapy of neural and nonneural disorders.

A method of expressing a therapeutic gene in cells of a subject is an object of the invention. Expression is achieved by infusing the described expression plasmid into cells of an individual * 1.

• · • · • «· • 1 • ♦; A specific subject of the present invention is a BPV-1 expression plasmid containing a? 25? Tyrosine hydroxylase gene for use in gene therapy for Parkinson's disease.

It is a further object of the present invention to provide a method of treating Parkinson's disease, thus. that a therapeutically effective amount of the BPV-1 expression plasmid is administered to a patient suffering from the disease.

• · «. 30 'The treatment of Parkinson's disease is achieved by expressing the tyrosine hydroxylase gene in an individual * · * 1; · 'In the brain such that the BPV-1 expression plasmid described in the invention is infused into a patient with a corpus striatum.

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Detailed Description of the Invention

The invention will be described in detail in the following experimental section with reference to the accompanying drawings, in which:

Figure IA shows the general structure of the pTkBPVTH expression plasmid. The transcription unit of pTkBPVTH contains the Tk (thymidine kinase) promoter, rat TH-cDNA and SV40 'early region' polyadenylation site. The BPV-E1 and E2 elements are required for expression of viral E1 and E2 proteins, which is necessary and sufficient for sustained extrachromosomal 10 replication.

Figure IB shows the general structure of the pTkBPVlacZ expression plasmid. The transcriptional unit is similar to pTkBPVTH, but the gene is IacZ.

Figure 2 shows transfection of pBPVTH plasmid in Cos-7 cell culture. Cells were transfected with 3 µg of pBPV-SRaTH (lane 1), pTkBPVTH (lane 2) or pure DNA (lanes 3 and 4) using electroporation technique. Cos-7 cells were lysed 48 h after transfection and TH enzyme expression was analyzed by Westem hybridization using rabbit polyclonal anti-TH antibodies.

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Figure 3 shows the effect of gene transfer of pTkBPVlacZ on the expression of β-galactosidase enzyme in the rat striatum. Transfection was infused with 50 ttg of pTkBPV-lacZ-5 ·. sense plasmid in 20 μΐ PB S buffer to rat striatum by infusion pump; at a rate of 1 μΐ / min. The β-galactosidase enzyme to be analyzed was detected using X-gal-· · · ·; 25 histochemistry and appears as a blue-green ambush around the infusion line (10x magnification).

*: **: Figures 4A, 4B, 4C and 4D illustrate the effect of pTkBPBTH gene therapy on TH-1 immunohistochemistry of striatum in a rat model of Parkinson's disease. TH expression in untranslated v · 30 and lesioned striatum is shown in the untransfected rat in Figures 4A (normal untransfected striatum) and 4B (transfected in transfected) and in transfected · ····························· • »· ....: striatum). Rat was dosed with 50 ttg of pTkBPVTH plasmid in the transfection solution, and 4,107,737 were infused with a microdialysis pump at a rate of 0.5 μΙ / min. For TH enzyme analysis, the brain was perfused and fixed with ice-cold PBS and PFA. After attachment, the brain was cut with cryostat into 15 µm slices and the TH enzyme was detected using anti-TH monoclonal antibodies and Vectastain immunohistochemistry kit as described in Materials and Methods (10x magnification).

Figure 5 shows the effect of pTkBPVTH gene transfer on apomorphine-stimulated spin behavior in an animal model of Parkinson's disease. Control rats were given antisense TH-cDNA plasmid and TH rats were given sense TH-cDNA plasmid. Plasmid pTkBPVTH-10 was diluted in 20-30 μ1: α3η PBS buffer and infused into the striatum with an infusion pump at a rate of 0.5-1 μΐ / min. The amount of transfected plasmid was 90 μ ^^^ΐι. Rotation results are percentages of control at different times as mentioned.

Experimental part

Materials and Methods Animals

Male Wistar rats were supplied by B&K (Sollentuna, Sweden) or the National Experimental Animal Center (Kuopio, Finland). The rats weighed 220-250 g at the start of the experiments and received. to drink tap water and eat rodent pellets freely throughout the study.

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The rats were kept at room temperature and the daily rhythm was 12:12 (light-skinned). Rats • · j. ^ five rats / cage were kept in plastic cages before one-sided lesions were made and * ·· one / cage after lesion.

• «· 25 • · ♦ ♦ ♦ ·. 1: ·. Compounds and Chemicals • · ·

Compounds and chemicals were obtained from different suppliers as follows. Apomorphine hydrochloride and 6-OH-dopamine (Research Biochemical Inc., MA, USA), X-gal (5-bromo-4-chloro-3-111; indolyl-β-galactopyranoside) and a mouse anti-TH antibody (Boehringer Mannheim, * · · 30 Germany), Vectastain ABC (Vector Laboratories, USA). pTkBPVlacZ and pTkBPVTH-. The expression plasmids were grown in E. coli DH5 cells and prepared by Qiagen. 1 with a purification kit (Qiagen, Germany) and CsCl isopycnic centrifugation. All other chemicals were of conventional analytical and molecular biological quality and were purchased from commercial sources.

Apomorphine-Induced Rotation Behavior S Lesion in Medial Forebrain Nucleus and Testing of Rotation Behavior

Male Wistar rats were made as described by Ungerstedt (1971) except for some modifications (Törmvall and Männistö, 1993). The rotameter (Coulboum Instruments, U.S.A.) records full spin in both right and left directions separately for eight rats simultaneously. Apomorphine (0.1 mg / kg) was administered subcutaneously to test for lesion success approximately 14 days after 6-OH-dopamine injection. The rats were allowed to adapt to the rotator a few minutes before the apomorphine injection. Rotation monitoring was started immediately after the injection of apomorphine.

BPV Tk (and SRa) Expression Plasmids Bovine papilloma virus type-1 (Bovine papilloma virus type-1 or BPV-1) plasmids were constructed to express the IacZ and TH genes. The construction of these plasmids has been based on the knowledge and know-how accumulated in recent years by papillomavirus replication mechanisms. For example, PCT Application No. WO 97/24451 describes recombinant BPV-1 vectors that have been the basis of the plasmids of the present invention.

Two-step cloning was used to prepare the plasmids. First, the IacZ or TH ···: 1 '* coding sequences were cloned into pUE83 having the Upstream Regulatory Region (URR) of BPV-1 with the minimal origin of replication. • · * · 1 · 25 (Minimal Origin of Replication, MO) and Minichromosome Maintenance Element (MME), Rous- • «* · * 1 Sarcoma Virus 5 ' -LTR promoter and rabbit β-globin intron and polyadenylation signal for message processing. Plasmid pGEMTH-3 containing rat TH-cDNA * · ... subcloned into pGEM-2 plasmid / wHI was obtained as a friendly donation from Dr. Kent E. Vrana (Wake Forest). University, NC, USA). The * 1 fragment contained in TH cDNA was digested from pGEMTH-3 with BamiH, and cloned at the only? 2? A / wHI site at the polyclonal site of PUE to give pUETH.

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An expression cassette encoding the IacZ or TH gene was introduced into pTkl .5 or pSRal4.2 plasmids at a single HindIII site.

In these plasmids, the strong SRa or weak HSV-1-Tk promoter controls transcription of the E1-5 gene. In addition, pSRaBPV and pTkBPV plasmids express the BPV-1 E2 protein, under the heterologous broad MoMLV LIR promoter, and contain a selectable marker for G418 resistance in eukaryotic and kanamycin resistance. Plasmids were grown in large numbers in E. coli -DH5 and purified by CsCl isopycnic centrifugation or Qiagen columns. Purified plasmids were dissolved in a suitable amount of buffer and stored at -20 ° C for gene transfer.

Gene transfer pTkBPVlacZ and pTkBPVTH plasmids were dissolved in phosphate buffered saline 15 (PBS, pH 7.4) at high concentrations (about 10 mg / ml) and infused into the striatum using a microdialysis pump (CMA 102, Carnegie Medio). The infusion cannulas were operated on one or three different coordinates in the striatum and placed 2 mm above the final coordinates, which were: 1) A 1.5, L 2, V -5; 2) A 0, L 3, V -5; 3) A 0, 4, V-6 (Paxinos and Watson, 1982). For transfection of striatum cells, 10-30 μΐ of BPV-20-containing transfection solution was infused at a rate of 0.5-2 μΐ / min for 10-30 minutes. The amount of plasmid infused ranged from 10 to 90 μg of DNA. Control rats were given a plasmid with antisense sequence and / or transfection solution alone. Apomorphine • * ·,. induced rotation was monitored weekly for up to four hours during the test period.

Histochemistry and Immunohistochemistry x1: For histochemical and immunohistochemical determination, rats were anesthetized with chloral hydrate (350 mg / kg, ip) and the brain was first perfused with 100 ml of ice-cold PBS buffer (0, 1 M PBS, pH 7.4) followed by 100 ml of 4% paraformaldehyde (4% PFA dissolved in · · · * ...: 0.1 M PBS, pH 7.4). After perfusion, rats were sacrificed and the brain was rapidly rescued: **: 30 and post-fixed at 21 + 4 ° C (4% PFA and 30% sucrose in 0.1 M PBS, pH 7.4).

The brain was then rapidly frozen at -70 ° C and stored frozen until the preparation of brain slices · * · ... Frozen brains were cast on Tissue Tek OCT media, and from the brain • *: · *: cryostat microtome (Bright 5030 Microtome, Instrument Company Ltd, 7 107737) was prepared.

Huntingdon, England) 12 μπι sections at -20 ° C and the sections were heat fixed to polylysine treated (PolyLysine ™ Microslides, Menzel Gläser, Germany) slides.

The finished brain sections were stored at -70 ° C until analysis. For immunohistochemical assays, brain sections were rinsed in PBS and incubated overnight at + 4 ° C with mouse monoclonal anti-TH antibodies (1: 500 dilution) in a humidified atmosphere. The brain slices were then rinsed in PBS and incubated with biotilylated equine anti-mouse antibodies and challenged with the biotin / avidin immunoperoxidase assay (Vectastain ABC kit). The slides were covered with xylene to protect the samples.

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When analyzing β-galactosidase expression, brain sections were incubated on X-gal substrate overnight at 37 ° C, and the enzyme activity was observed microscopically as a blue-green stain.

Finally, immunohistochemically and histochemically treated brain sections were analyzed

Olympus AX70 microscope and imaged with a SenSys digital camera, using Image-Pro Plus software (Media Cybernetics, L.P.) for Win95 / NT rigid system.

Results 20 >>>; To study the role of different promoters in the origin recog- • ·

; ·. nition factor) and viral helicase E1 expression, plasmids pSRaBPV were prepared

»· · And pTkBPV, and transfection efficiency was compared in Cos-7 and CV1-P cell cultures. pTkBPV ex-

: pression plasmids carrying the rat TH gene or the IacZ gene are shown in Figures 1A

• · 25 and IB. SRot plasmids have similar structures. In Cos-7 cells transfected with: pTkBPVTH (3 pg) by electroporation, TH expression was higher when

Expression of E1 was driven by the HSV-Tk promoter as the SRa promoter (Figure 2). Similar results were obtained when plasmid pSRotBPVlacZ and pTkBPVlacZ (2.5 µg) were transfected with Dosper liposomes into monkey CV1-P cells (Lampela et al., For publication 9 '). ': 30 submitted manuscripts). Therefore, pTkBPVlacZ- * * * * * and pTkBPVTH plasmids were used primarily for in vivo gene transfers.

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The functionality of the expression vector in vivo was confirmed in rat brain by transfecting pTk-BPVlacZ plasmid (50 µg) into non-lesioned control rats. PTkBPVlacZ vectors with either a sense or an antisense-lacZ coding sequence were infused into rat striatum, and the β-galactosidase enzyme was analyzed in situ by X-gal histochemistry. The dense effect of Posi-5 was seen as a blue-green depletion corresponding to transfection of the lacZ-sense cDNA (Figure 3). No effect was observed with antisense-lacZ plasmid or buffer alone. These results indicate that naked DNA infused in phosphate buffered saline can be used to express therapeutic genes in the brain.

Expression of 10 TH enzymes in the striatum was immunohistochemically analyzed in control and 6-OHDA-treated rats. TH expression was well evident on the non-lesioned side of the striatum in the rat brain (Figure 4A and 4C). Performing lesion in rat brain leads to loss of TH expression to a level corresponding to the background signal of immunohistochemical detection in the absence of primary antibody (cf. Figures 4A, 4B and 4C). This indicates that 6-OH dopamine-induced toxicity inactivates or kills most dopaminergic nigro-striatal neurons, leading to a lack of TH and dopamine synthesis in the striatum. This is also clearly reflected in the physiological response, which is evident in apomorphine-induced contralateral rotation in lesioned rats caused by unilateral dopamine-20 ergic nerve lesion and the resulting sensitization of ipsilateral postsynaptic dopamine receptors.

The effect of gene transfer on plasmid pTkBPVTH was evident by expression of TH enzyme (Figure 4D). Thus, normal TH expression (4A,? 25C) was observed in the non-lesional striatum. Striatum lesioned side, which was no transfer of the gene was not expressed TH • · · compression of l .. (Figure 4B), while lesioned pTkBPVTH expression vector transfected • • · · · 1 _ buffered in the striatum was a clear TH-staining (Figure 4D). TH expression was evident near the site where the DNA solution was placed, and expression was spread over a small area of brain tissue.

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The gene therapeutic effect of infusion of pTkBPVTH plasmid was reciprocally studied in • · • · * I 'lesioned rats, which were used as an experimental model of Parkinson's disease.

107737 pTkBPVTH sense or antisense construct (90 pg) was diluted in 20-30 μΐ PBS and infused at 0.5-2 μΐ / min. Rats were given either antisense cDNA (control rats) or sense-TH cDNA (TH rats). The effect of gene transfer was investigated by monitoring apomorphine-induced rotational behavior. The results of trans-5 transfections of the pTkBPVTH plasmid are shown in Figure 5. The numbers are the relative effects of TH-cDNA transfection on apomorphine-stimulated spin behavior.

Apomorphine-induced rotation was reduced by an average of 25-30% in three separate sets of experiments two and four weeks after gene transfer. No change in rotational behavior was observed when using antisense TH plasmid.

In the present invention, rat α-cDNA was ligated to the BPV expression plasmid and transfected into rat striatum in an animal model of Parkinson's disease, resulting in TH enzyme expression and partial recovery of rats from experimental Parkinson's disease.

Plasmid BPVTH was transfected into rat striatum in vivo by inducing naked DNA in rat normal or lesioned brain. We found that infusion of naked DNA into the brain can lead to the uptake of vector DNA into cells and the expression of genes in the brain, as evidenced by immunohistochemistry. In addition, the expression of pTkBPVTH-produced TH in the lesioned rat brain elicited a physiological response, which was observed as a change in contralateral rotational behavior when the rats were given a dopaminergic • · · ·

J1 'agonist, i.e.. apomorphine.

• • • • • • *. . The TkBPV expression plasmids and the gene transfer method used seemed promising for the development of physiological, nontoxic and safe gene therapy. In vitro gene transfer and expression were successful in the presence of serum, long transfection times, and low levels of DNA, and no significant cell death was observed under optimal transfection conditions.

....: In addition, BPV plasmids could also be transfected and expressed in the dividing sub- • ·. · · ·. in confluent cells and contact-inhibited confluent cells, of different origin • · · '. 30 cells of various tissues and species, i. monkey kidney fibroblasts and ... human neuroblastoma cells. Good expression of the reporter gene supports that the E2- • dependent chromatin binding ensures efficient location of the vector molecule in the nucleus and results in its efficient expression.

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We also experimented with gene transfer to rat brain using naked DNA, and found that both genes, i. IacZ and TH are moderately expressed in vivo in rat striatum. Expression of the therapeutic TH gene was also associated with moderate improvement in the animal model of Parkinson's disease. These results support that brain cells can take up exposed DNA, transport DNA to the nucleus, and express foreign genes attached to it. All of these properties are important under in vivo conditions when the number of proliferating cells in the tissue is quite small. The gene transfer method and BPV expression plasmids we use may thus be useful in gene therapy for Parkinson's disease.

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reference Publications

Birkmayer, W. and Homykiewicz, O. (1961). Der L-3,4-Dioxyphenylalanine (= DOPA) -Effect and Der Parkinson-Akinese. Vienna. Klin. Wochenschr., 73: 787-788.

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Cao, L, Zheng, Z.-C., Zhao, Y.-C., Jiang, Z.-H., Liu, Z.-G., Chen, S.-D., Zhou, C.-F ., and Liu, X.-Y. (1995). Gene Therapy of Parkinson's Disease Model Rat by Direct Injection of Plasmid DNA-Lipofectin Complex. Hum. Gen. Ther. 6, 1497-1501.

20 Chase, T.N., Engber, T.M. and Mouradian, M.M. (1994). Palliative and prophylactic benefits of continuously administered dopaminomimetics in Parkinson's disease. Neurology, 44: 15-18.

Cotzias, C.G., Van Woert, M.H. and SchifFer, L.M. (1967). Aromatic amino acids and 25 modifications of parkinsonism. N. Engl. J. Med., 276: 374-379.

...: During, M.J., Naegele, J.R., O'Malley K.L. and Geller, A.I. (1994). Long-Term Behavioral * Recovery in Parkinsonian Rats by an HSV Vector Expressing Tyrosine Hydroxylase.

Science 266, 1399-1403.

Ϊ1. 30 • · · *. . Elsworth, J.D. and Roth, R.H. (1997). Dopamine Synthesis, uptake, metabolism, and receptors: relevance to gene therapy for Parkinson's disease. Exp. Neurol., 144: 4-9.

Geller, A.I., During, M.J., Oh, Y.J., Freese, A. and O'Malley, K. (1995). AnHSV-1 Vector * · 1 1 35 Expressing Tyrosine Hydroxylase Causes Production and Release of L-DOPA from

Cultured Rat striatal cells. J. Neurochem. 64, 487-496.

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* 'Horellou, P., Vigne, E., Castel, M.-N., Bameoud, P., Colin, P., Perricaudet, M., Delaere, P.

*, ..! and Mallet, J. (1994). Direct intracerebral gene transfer of adenoviral vector expression *. 40 tyrosine hydroxylase in a rat model of Parkinson's disease. NeuroRepori 6, 49–53.

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Homykiewicz, O. (1982). Brain neurotransmitter changes in Parkinson's disease. In: .1 · 'Movement Disorders, Marsden, C.D. and Fahn, S. (Eds.), Butterworth Scientific, London, j '··· ss. 41-558.

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Homykiewicz, 0., Kish, S.J., Becker, L.E., Farley, I. and Shannak, K. (1986). Brain transmitters in dystonia musculorum deforms. N. Engl. J. Med., 315: 347-353.

Isacson, O. (1995). Behavioral Effects and Gene Delivery in a Rat Model of Parkinson's 5 Disease. Science 269, 856.

Jiao, S., Gurevich, V. and Wolff, J. (1993). Long-term correction of rat model of Parkinson's disease by gene therapy. Nature 362, 450-453.

10 Jiao, S., Gurevich, V. and Wolff, J. (1996). Retraction. Long-term correction of rat model of Parkinson's disease by gene therapy. Nature 380, 734.

Lundberg, C., Horellou, P., Mallet, J. and Björklund, A. (1996). Generation of DOPA-Producing Astrocytes by Retroviral Transduction of Human Tyrosine Hydroxylase Gene: 15 In Vitro Characterization and In Vivo Effects in the Rat Parkinson Model. Exp. Neurol. 139, 39-53.

Lampela, P., Ustav, E., Ustav, M., Männistö, P.T. and Raasmaja, A. (1999) Transfection of Bovine Papilloma Virus Plasmid Expression lacZ and Tyrosine Hydroxylase Genes in Primate Cell Cultures. Submitted for publication.

Nutt, J.G. and Hoiford, N.H. (1996). The response to levodopa in Parkinson's disease: Imposing Pharmacological law and order. Ann. Neurol., 39: 561-573.

25 Paxinos, G. and Watson, C. (1982). The Rat Brain in Stereotazic Coordinates. 1st edition Academic Press, London.

Sabel, B. A., Vick, A. and Holt, V. (1995). Neurotoxic reactions of CNS cells following gene transfer with defective herpes simplex virus (HSV-1) vector. NeuroReport 6,2447-2449.

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Tomwall, M. and Männistö, P. T. (1993). Effects of three types of catechol O-methylation ...: inhibitors on L-3,4, -dihydroxyphenylalanine-iridiated circulating behavior in rats. Eur. J.

.. Pharmacol 250, 77-84.

• · • · · • 35 Ungerstedt, (1971). Postsynaptic supersensitivity after 6-hydroxydopamine induced *. . degeneration of the nigro-striatal dopamine system. Acta Physiol. Scand 82 (Suppl. 367), 69–93.

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Claims (6)

107737 1. Bovine Papilloma Virus Type-1 (BPV-1) expression plasmid containing the 5th papilloma virus E1 and E2 genes for stable extrachromosomal replication, - minimal origin of papillomavirus replication (MO), - papillomavirus minichromosomal attachment element (MME), - nucleotide sequence encoding the tyrosine hydroxylase enzyme (TH). 10 The expression plasmid of claim 1, further comprising regulatory regions for controlling expression and transcription. The expression plasmid of claim 2, wherein the regulatory regions contain promoters that direct TH expression in cells. The expression plasmid of claim 2, wherein the regulatory regions include the upstream regulatory region (URR) of BPV-1 and the thymidine kinase (Tk) promoter. 20 Use of an expression plasmid according to any one of claims 1 to 4 in the preparation of a medicament for the gene therapy of * neural or nonneural diseases. ·· • · «·· • · *« · * · * · 25 Use according to claim 5, characterized in that the disease is Parkinson's disease. · * · • »· i« «· ♦ • · · *» »» * »» »» »» «107737 3