WO2001048232A1

WO2001048232A1 - A plasmid for the expression of a therapeutic gene in a tissue

Info

Publication number: WO2001048232A1
Application number: PCT/FI2000/001140
Authority: WO
Inventors: Atso Raasmaja; Mart Ustav; Pekka T. MÄNNISTÖ
Original assignee: Atso Raasmaja; Mart Ustav; Maennistoe Pekka T
Priority date: 1999-12-23
Filing date: 2000-12-22
Publication date: 2001-07-05
Also published as: FI19992771A; FI107737B; AU2518601A

Abstract

The present invention relates to gene therapy of neural or non-neural diseases, especially Parkinson's disease. The invention provides a Bovine Papilloma Virus type-1 expression plasmid carrying at lest one therapeutic gene to be transfected in vivo into the cells of a subject. In specific, the invention provides a BPV-1 expression plasmid carrying a tyrosine hydroxylase gene, to be transfected into corpus striatum of a patient for the treatment of Parkinson's disease.

Description

A plasmid for the expression of a therapeutic gene in a tissue

Field of the invention

The present invention relates to gene therapy of neural or non-neural diseases, especially Parkinson's disease. The invention provides a Bovine PapiUoma Virus type-1 expression plasmid carrying at least one therapeutic gene to be transfected in vivo into the cells of a subject.

Background of the invention

Parkinson's disease is characterised by a progressive degeneration of the dopaminergic neurons in the subs tan - tia nigra pars compacta, which results in loss of the dopaminergic innervation of the corpus striatum (Horny- kiewicz, 1982; Hornykiewicz et al . , 1986). The current therapy of Parkinson's disease is administration of a dopamine precursor together with a peripheral inhibition of dopa decarboxylase (DDC) (Birkmayer & Hornykiewicz, 1961; Cotzias et al . , 1967) . In most patients, this treatment loses efficiency over time and has a plethora of adverse effects (Chase et al . , 1994; Nutt & Holford, 1996) .

Tyrosine hydroxylase (TH) is a rate limiting enzyme in the dopamine biosynthesis, converting tyrosine to L-dopa which is then used by endogenous DDC (dopadecarboxylase) to synthesise dopamine. Since there is a surplus of other enzymes as well as tyrosine in the brain, an alternative strategy to enhance dopamine synthesis is to increase the local level of expression of the TH enzyme in the striatum (Elsworth & Roth, 1997) . Indeed, it has been reported that a transfection of striatal cells with viral vectors expressing TH gene could lead to an increased TH mRNA expression and respective protein synthesis in the stria- turn, suggesting that the TH gene therapy may really work in animal models of Parkinson's 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 . , 1996; Lundberg et al . ,

1996) . In addition, toxic side-effects have been observed in the use of some vector systems, hampering the applica- tions of gene therapy in brain diseases and increasing the need for development of more useful vectors (Isacson, 1995; Sabel et al . , 1995).

The various BPV-1-based vectors described in the literat- ure use the whole BPV-1 genome, or at least 69% of the viral genome - essentially the early region of the virus.

The BPV-1 expression plasmids according to the present invention use the same replication proteins as the virus itself, but the expression of these proteins is directed by heterologous promoters, therefore allowing expression of El and E2 genes of the virus, ubiquitously in the cells of different origin from the epithelial tissue.

Summary of the invention

We have constructed and characterised a Bovine PapiUoma Virus type-1 (BPV-1) vector for expression of therapeutic genes in the cells of a subject, especially tyrosine hydroxylase gene in brain cells.

Consequently, a general object of the present invention is to provide a BPV-1 expression plasmid comprising at least one therapeutic gene or nucleotide sequence, to be used in in vivo gene therapy of neural or non-neural diseases . A method of expressing a therapeutic gene in the cells of a subject is one object of the invention. The expression is achieved by administering the expression plasmid according to the invention into the cells of the subject.

A specific object of the present invention is a BPV-1 expression plasmid comprising papilloma virus El and E2 genes for stable extrachromosomal replication, a minimal origin of replication of a papilloma virus (MO) , a mini- chromosomal maintenance element of a papilloma virus (MME) , and a tyrosine hydroxylase gene, to be used in gene therapy of Parkinson's disease.

A further object of the invention is the use of the expression plasmid of the invention, comprising at least one therapeutic gene or nucleotide sequence, for the preparation of a medicament for gene therapeutic treatment of a neural or non-neural disease.

A still further object of the present invention is a method of treating Parkinson's disease, wherein a thera- peutically effective amount of the BPV-1 expression plasmid is administered to a patient suffering from said disease .

The treatment of Parkinson's disease is achieved by expressing tyrosine hydroxylase gene in the brain of a subject, which is effected by infusing into the corpus striatum of the subject the BPV-1 expression plasmid according to the invention.

Detailed description of the invention

The invention will be described in more detail in the following experimental section of this specification, referring to the enclosed drawings, wherein FIG. 1A shows the general structure of pTkBPVTH expression plasmid. The transcription unit in pTkBPVTH contains the Tk (tymidine kinase) promoter, the rat TH cDNA and the SV40 early region polyadenylation site. The BPV El and BPV E2 elements are required to express the viral proteins El and E2 which are necessary and sufficient for stable extrachromosomal replication.

FIG. IB shows the general structure of pTkBPVlacZ expression plasmid. The transcription unit is similar to that of pTkBPVTH, except the gene of interest (lacZ) .

FIG. 2 shows the transfection of pBPVTH plasmid in the Cos-7 cell cultures. The cells were transfected by an electroporation technique with 3 μg of BPV-SR TH plasmid (lane 1) , pTkBPVTH plasmid (lane 2) or only plain carrier DNA (lane 3 and 4) . The Cos-7 cells were lysed 48 h post- transfection and the expression of TH enzyme was analysed using Western hybridization and rabbit polyclonal anti TH antibodies .

FIG. 3 shows the effect of pTkBPVlacZ gene transfection on the expression of β-galactosidase enzyme in the rat striatum. The transfection of 50 μg pTkBPV plasmid containing the sense coding sequence for the lacZ gene was performed into the rat striatum using an infusion pump in 20 μl of PBS buffer with a flow rate of 1 μl/min. The presence of β-galactosidase enzyme was detected with the X-gal histochemistry and can be seen as the blue-green staining around the infusion site (10 x magnification) .

FIGS 4A, 4B, 4C and 4D show the effect of the pTkBPVTH gene therapy on the striatal TH immunohistochemistry in the rat model of Parkinson's disease. The TH expression in the unlesioned and lesioned striatum from the non- transfected rat is shown in Fig. 4A (normal unlesioned striatum) and 4B (lesioned nontransfected striatum) , and from the transfected rat in Fig. 4C (unlesioned transfected striatum) and 4D (lesioned transfected striatum) . The rat received 50 μg of pTkBPVTH plasmid dissolved in transfection medium, administered with the microdialysis pump in a flow rate of 0.5 μl/min. For the analysis of TH enzyme expression, the brain was perfused and fixed using an ice-cold PBS and PFA. After fixation, the brain was cut into the 15 μm sections with a cryostat, and the TH enzyme was detected using the monoclonal anti-TH antibodies and Vectastain immunohistochemical kit as described in Materials and Methods (10 x magnification) .

FIG. 5 shows the effect of pTkBPVTH gene transfection on the apomorphine-stimulated turning behaviour in the rat model of Parkinson's disease. Control rats received the antisense THcDNA and TH rats the sense THcDNA. The pTkBPVTH plasmid was diluted in 20-30 μl of PBS and infused into the striatum with an infusion pump in a flow rate of 0.5-1 μl/min. The amount of transfected plasmid was 90 μg/striatum. The values are per cent of control at different times as indicated.

The expression 'a therapeutic gene or nucleotide sequence', used in this specification and the appended claims, is intended to illustrate the possibility that a nucleotide sequence and a gene are not necessarily identical, i.e. a functional nucleotide sequence can be a certain part of a gene, or the nucleotide sequence may comprise fragments not included in a native gene.

The TkBPV expression plasmids and the method of gene delivery, as described herein, showed promise as of a physiological, nontoxic and safe method for gene therapy. The in vi tro transfection and expression were successful in the presence of serum, using a long exposure time and a low amount of DNA, and no significant cell damage was detected when optimal transfection conditions were used. Moreover, the BPV plasmids could be transfected and ex- pressed also as well in dividing subconfluent cells as in contact-inhibited confluent cells of different origin and species, i.e., monkey kidney fibroblasts and human neuro- blastoma cells. The good expression level of the reporter indicates that E2 dependent chromatin attachment of the origin containing plasmid would provide efficient com- partmentalization of the vector molecule in the cell nucleus leading to its efficient expression.

We also tried the transfection of the plain DNA into the brain of the rats and found that both studied genes, lacZ and TH, were modestly expressed in vivo in the rat striatum. The expression of therapeutic TH gene was also associated with a moderate recovery from Parkinson's disease in the animal model. These results indicate that the brain cells could take up the plain DNA, deliver that into the nucleus and express the foreign genes. All these characteristics are important in in vivo conditions, when the percentage of the proliferating cells in the tissue would be quite low. Therefore, our method of gene delive- ry using BPV expression plasmids may be useful in the gene therapy of Parkinson's disease.

Besides TH gene, the pBPV plasmid of the invention can also be used for the transfection and expression of other genes in the gene therapy of Parkinson's disease, alone or in combination with TH gene. This is evident based on the results with TH gene, which is encoding one of the enzymes involved in the synthesis of catecholamines . The effect of pBPVTH plasmid was studied in the gene therapy of experimental Parkinson's disease. It was shown that the transfection and expression of pBPVTH plasmid was associated with the therapeutic effect in the rat model of Parkinson's disease as seen as the decreased apomor- phine-induced turning behaviour.

Furthermore, the transfection of pBPVTH plasmid led to the expression of TH enzyme in the lesioned striatum of the unilaterally lesioned rat indicating that the observed recovery from experimental Parkinson's disease was due to the synthesis of new TH enzyme encoded by the transfected pBPVTH plasmid. Because the therapeutic effect of the gene therapy was related to the enhancement of dopaminergic stimulation in the unhealthy striatum, it is obvious that also other genes and/or gene combinations with therapeutic effects on the dopaminergic mechanisms could be used in a similar way for the gene therapy of Parkinson's disease. Therefore, it is suggested that pBPV plasmids containing at least one gene encoding therapeutic proteins, e.g. dopamine or other catecholamine syn- thetising enzymes and/or growth factors, can be used for the gene therapy of Parkinson's disease.

The pBPV plasmid can be used for the gene therapy also in other cells and tissues than the brain and the striatum. This is evident because the transfection and expression of pBPV plasmids has been efficient in vi tro in several types of cells from different origin, i.e. neural and nonneural, secondary and primary, non-mammalian and mammalian, including human cells. It has also been shown that the pBPV plasmids could be transfected using different nonviral transfection methods and reagents, leading to the efficient expression of insert genes in the host cells. Especially the transfection of pBPVTH has been associated with the expression of TH enzyme and production of catecholamines in different types of cells. Therefore, it is suggested that the pBPV plasmids could be used for the gene therapy in general, including different types of diseases. Experimental section

Materials and methods

Animals

Male Wistar rats were purchased from B & K (Sollentuna, Sweden) or from the National Animal Center (Kuopio, Finland) . The rats weighed 220 to 250 g at the beginning of the study, and had free access to tap water and rodent pellets throughout the study. The rats were housed at room temperature with the 12:12 light: dark schedule. The rats were kept in plastic cages in groups of five/cage before the unilateral lesion and one/cage after operation.

Drugs and chemicals

Apomorphine hydrochloride and 6-OH-dopamine were obtained from Research Biochemical Inc. (MA, U.S.A.). X-gal (5- bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and mouse anti-TH antibodies were purchased from Boehringer Mannheim (Germany) and Vectastain ABC kit was from Vector Laboratories (U.S.A.). pTkBPVlacZ and pTkBPVTH expression plasmids were grown up in the E. coli DH5 and prepared using Qiagen purification kits (Qiagen, Germany) or CsCl isopycnic centrifugation. All other chemicals were of common analytical and molecular biological grade and obtained from general commercial sources .

Apomorphine-induced circling behavior Lesion on the medial forebrain bundle and testing of turning behavior of the male Wistar rats was done according to Ungerstedt (1971) with some modifications (Torn- wall and Mannistό, 1993) . The rotameter (Coulbourn Instruments, U.S.A.) registered right and left full turns separately from eight rats at the same time. Apomorphine (0.1 mg/kg) was given subcutaneously to test the success of the lesioning about 14 days after 6-OH-dopamine infusion. The rats were allowed to adapt to the bowls for a few minutes before apomorphine injections. The monitoring of the circling was started immediately after apomorphine injection.

BPV Tk (and SRoc) expression plasmids

Bovine papilloma virus type-1 (BPV-1) plasmids were constructed to express lacZ and TH genes. The construc- tion of these plasmids was based on the knowledge accumulated over the last years about the replication mechanisms of papillomaviruses . For instance in PCT application No. WO 97/24451 recombinant BPV-1 vectors are described, which form a basis for the expression plasmids constructed in this invention.

Two-step cloning was applied for construction of the plasmids. First, the lacZ or TH coding sequences were cloned into the pUE83 plasmid containing BPV-1 Upstream Regulatory Region (URR) comprised of Minimal Origin of replication (MO) and Minichromosome Maintenance Element (MME) , Rous Sarcoma Virus 5 ' -LTR promoter for directing the expression of gene of interest and rabbit β-globin intron and polyadenylation signal for processing of the message. The pGEMTH-3 plasmid containing the rat TH cDNA, which was subcloned into the BaϋiHI site of the pGEM-2 plasmid, was a kind gift from Dr. Kent E. Vrana (Wake Forest University, NC, U.S.A.). The TH cDNA-containing fragment was digested from the pGEMTH-3 plasmid with Ba HI, and cloned into the unique Ba UI site of the polycloning site of the pUE plasmid, resulting in the pUETH plasmid. The expression cartridge encoding the lacZ or TH was transferred into the plasmid pTkl .5 or pSR l4.2 into the unique HindiII site.

In these plasmids, the strong SRa or the weak HSV-l Tk promoter controls the transcription of El gene. In addi- tion, the pSR BPV and pTkBPV plasmids express the E2 protein of the BPV-1 under the control of the heterologous wide range MoMLV LTR promoter and contain a selection marker for G418 resistance in the eukaryotic, and kanamycin resistance in the bacterial cells. The plasmids were grown in E. coli DH5 strain in large quantities and purified using the CsCl isopycnic centrifugation or Qiagen columns. The purified plasmids were dissolved in appropriate buffer and stored at -20°C until used for gene transfections .

Gene transfection pTkBPVlacZ and pTkBPVTH plasmids were dissolved in the phosphate buffered saline (PBS, pH 7.4) at high concen- tration (up to 10 mg/ml) and different amounts of DNA were infused into striatum as indicated using a micro- dialysis pump (CMA 102, Carnegie Medicin, Sweden). Cannu- las were inserted 2 mm above the one or three different coordinates of the final infusion sites: 1) A 1.5, L 2, V -5; 2) A 0, L 3, V -5, and 3) A 0, L 4, V -6 according to Paxinos and Watson (1982) . To transfect striatal cells, 10-30 μl of transfection medium containing the BPV plasmid was infused with a flow rate of 0.5-2 μl per min for 10-30 min. The infused amount of plasmids varied from 10 to 90 μg of DNA. Control rats received plasmids containing antisense sequences and/or plain transfection medium. Apomorphine-induced (0.1 mg/kg s.c.) rotation was monitored for 4 hours weekly during the experimental period.

Histochemistry and immunohistochemistry

For histochemistry and immunohistochemistry, the rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.), and the brains were perfused using 100 ml of ice- cold PBS (0.1 M PBS, pH 7.4) followed by 100 ml of 4% paraformaldehyde (4% PFA in 0.1 M PBS, pH 7.4) . After perfusion, the rats were decapitated and the brains were quickly removed and postfixed for 2 h at 4 EC (4% PFA and 30% sucrose in 0.1 M PBS, pH 7.4) . Then, the brains were quickly frozen at -70°C until used for preparing of brain slices. The frozen brains were embedded in Tissue Tek OCT embedding compound, and 12 μm brain sections were prepa- red at -20°C using a microtome cryostat (Bright 5030

Microtome, Instrument Company Ltd, Huntingdon, England) and thaw-mounted on polylysine-coated slides (PolyLysi- ne™ Microslides, Menzel Glaser, Germany) .

The mounted brain sections were stored at -70°C until analysed. For immunohistochemistry, the brain sections were rinsed in PBS and incubated overnight at 4°C with mouse monoclonal anti-TH antibodies (1:500 dilution) in a humid atmosphere. Then, the brain sections were rinsed in PBS and incubated with biotinylated horse-antimouse antibodies and stained using the biotin/avidin immunoperoxi- dase procedure (Vectastain ABC kit) . The slides were coverslipped with xylene.

For analysis of β-galactosidase expression, the brain slices were incubated in the presence of X-gal substrate overnight at 37°C, and the enzymatic activity was detected microscopically as a blue-green staining.

Finally, the brain sections from immunohistochemical and histochemical assays were analysed with Olympus AX70 microscope and SenSys digital camera using the Image-Pro Plus program for Win95/NT (Media Cybernetics, L.P.).

Results

In order to study the role of different promoters driving the expression of origin recognition factor and viral helicase El, the pSR BPV and pTkBPV plasmids were con- structed and the efficacy of the transfections in the

Cos-7 and CV1-P cell cultures were compared. The expression plasmids pTkBPV for rat TH gene and lacZ gene are

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body (compare Figs. 4A, 4B and 4C) . This shows that the 6-OH-dopamine induced toxicity inactivates or kills most of the dopaminergic nigrostriatal neurons leading to the absence of TH and the synthesis of dopamine in the stria- turn. This is clearly reflected also in the physiological effect, demonstrating the apomorphine-induced contralate- ral rotation of the rats owing to the unilateral loss of dopaminergic nerves and the subsequent supersensitivity of the ipsilateral postsynapse dopamine receptors.

The effect of pTkBPVTH gene transfection was seen in the expression of TH enzyme (Fig. 4D) . Thus, in the unlesioned striatum, the normal TH expression was detected (Fig. 4A, 4C) . There was no expression in the lesioned striatum without any TH gene transfection (Fig. 4B) , whereas a clear TH staining was detected when the striatum was transfected with the pTkBPVTH expression vectors (Fig. 4D) . The expression of the TH was detected near the site of administration of the DNA solution and showed little spreading of the expression in the brain tissue.

The gene therapeutic effect of the infusion of the pTkBPVTH plasmid was studied in the unilaterally lesioned rats which would provide an experimental model of Parkin- son's disease. pTkBPVTH sense or antisense construct (90 μg) diluted in 20-30 μl of PBS was infused with a flow rate of 0.5-2 μl/min. Rats received either an antisense TH cDNA (control rats) or a sense TH cDNA (TH rats) , and the effect of gene transfection was monitored using the apomorphine-induced turning behaviour. The results with the transfections of the pTkBPVTH into the striatum are summarized in Fig. 5. Values represent the relative effect of TH cDNA transfection on the apomorphine-stimulated turning behaviour.

There was an average decrease of 25-30 % in the mean apomorphine-induced turning behaviour in three different studies at 2 and 4 weeks after the gene transfection. No change was observed in the turning behaviour, when the antisense TH plasmid was used.

In the present invention the rat TH cDNA was subcloned into the BPV expression plasmid and transfected into the striatum in the rat model of Parkinson's disease leading to the expression of TH enzyme and partial recovery of the rats from the experimental Parkinson's disease.

The BPV TH plasmid was transfected into the rat striatum in vivo by infusion of the plain DNA into the normal or lesioned rat brain. We found that the plain DNA infusion into the rat brain could result in the uptake of the vector DNA and expression of the genes in the brain, as shown by immunohistochemistry. In addition, the physiological response on the expression of TH from the pTkBPVTH in the lesioned rat brain was detected by a measurement of the change in the contralateral turning behavior in response to dopaminergic agonist, i.e., apomorphine.

References

Birkmayer, W. and Hornykiewicz, 0. (1961). Der L-3,4- Dioxyphenylalanin (=DOPA) -Effekt bei der Parkinson-Akine- se. Wien . Klin . Wochenschr. , 73: 787-788.

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 Disease Model Rat by Direct Injection of Plasmid DNA-Lipofectin Complex. Hum . Gen .

Ther. 6, 1497-1501.

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 modification of parkinsonis . N. Engl . J. Med. , 276: 374-379.

During, M.J., Naegele, J.R., O'Malley K.L. and Geller, A.I. (1994). Long-Term Behavioural Recovery in Parkin- sonian Rats by an HSV Vector Expressing Tyrosine Hydroxy- lase. Science 266, 1399-1403.

Elsworth, J.D. and Roth, R.H. (1997) . Dopamine synthesis, uptake, metabolism, and receptors: relevance to gene therapy of Parkinson's disease. Exp . Neurol . , 144: 4-9.

Geller, A.I., During, M.J., Oh, Y.J., Freese, A. and O'Malley, K. (1995) . An HSV-1 Vector Expressing Tyrosine Hydroxylase Causes production and Release of L-DOPA from Cultured Rat striatal cells. J. Neurochem . 64, 487-496.

Horellou, P., Vigne, E., Castel, M.-N., Barneoud, P., Colin, P., Perricaudet, M., Delaere, P and Mallet, J. (1994) . Direct intracerebral gene transfer of adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease. NeuroReport 6, 49-53.

Hornykiewicz, 0. (1982). Brain neurotransmitter changes in Parkinson's disease. In: Movement Disorders, Marsden, CD. and Fahn, S. (Eds.), Butterworth Scientific, London, pp. 41-558.

Hornykiewicz, 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, 0. (1995). Behavioural Effects and Gene Delivery in a Rat Model of Parkinson's 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. Jiao, S., Gurevich, V. &_ 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 Bjόrklund, A. (1996) . Generation of DOPA-Producing Astrocytes by Retro- viral Transduction of Human Tyrosine Hydroxylase Gene: In Vitro Characterization and In Vivo Effects in the Rat Parkinson Model. Exp. Neurol . 139, 39-53. Lampela, P., Ustav, E., Ustav, M., Mannistδ, P.T. and Raasmaja, A. (1999) Transfection of Bovine Papilloma Virus Plasmids Expressing lacZ and Tyrosine Hydroxylase Genes in Primate Cell Cultures . Submitted Nutt, J.G. and Holford, N.H. (1996) . The response to levodopa in Parkinson's disease: imposing pharmacological law and order. Ann . Neurol . , 39: 561-573.

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Claims

1. A Bovine Papilloma Virus type-1 (BPV-1) expression plasmid, comprising - a papilloma virus El and E2 genes for stable extra- chromosomal replication,

- a minimal origin of replication of a papilloma virus (MO) ,

- a minichromosomal maintenance element of a papilloma virus (MME) , and

- at least one therapeutic gene or nucleotide sequence

2. The expression plasmid according to claim 1, comprising one therapeutic gene, which is a tyrosine hydroxylase (TH) gene.

3. The expression plasmid according to claim 1, comprising several therapeutic genes or nucleotide sequences, one of which is a TH gene.

4. The expression plasmid according to any one of the claims 1 to 3 , which further comprises regulatory regions for controlling expression, transcription and/or replication.

5. The expression plasmid according to claim 4, wherein the regulatory regions comprise promoters driving the gene expression in cells.

6. The expression plasmid according to claim 4, wherein the regulatory regions comprise

- a BPV-1 Upstream Regulatory Region (URR) and

- a tymidine kinase (Tk) promoter.

7. Use of the expression plasmid according to any one of the claims 1 to 6 for the preparation of a medicament for gene therapeutic treatment of a neural or non-neural disease .

8. Use according to claim 7, wherein the disease is Parkinson's disease.

9. A method of expressing at least one therapeutic gene or nucleotide sequence in the cells of a subject, comprising administering the expression plasmid according to any one of the claims 1 to 6 into the cells of said subject .

10. A method of expressing tyrosine hydroxylase gene in the brain of a subject, comprising infusing the expression plasmid according to claim 2 into the corpus striatum of the subject.

11. A method of treating Parkinson's disease, comprising administering a therapeutically effective amount of the expression plasmid according to any one of the claims 2 to 6 to a patient suffering from said disease.

12. The method according to claim 11, wherein the amount administered is from 10 to 90 μg of plasmid DNA.

13. The method according to claim 12, wherein the amount administered is 50 μg of plasmid DNA.

14. The method according to any one of the claims 9 to 13, wherein the administration is carried out by infusing a buffer solution of the expression plasmid into the corpus striatum of the patient.

15. A method for delivery of a tyrosine hydroxylase gene to the brain cells of a subject, comprising administering into the striatal cells of said subject an expression plasmid according to any one of the claims 2 to 6, where- in the gene is delivered to the brain cells of said subject .