GENE EXPRESSION SYSTEM AND REGULATION THEREOF

Abstract

The present invention relates to a novel gene expression system comprising: a) a first nucleotide sequence encoding a fusion polypeptide of: a1) a destabilizing domain (DD) based on DHFR, and a2) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and b) a second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof. The invention also relates to use of this gene expression system together with a ligand binding to a destabilizing domain (DD) based on dihydrofolate reductase (DHFR) for treatment of diseases associated with a reduced dopamine level, such as Parkinson's disease.

Claims

1. A gene expression system comprising: a first nucleotide sequence encoding a fusion polypeptide of: a) a destabilizing domain (DD), and b) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and a second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof.

2-4. (canceled)

5. A gene expression system according to claim 1, wherein said gene expression system comprises two vectors each containing one expression cassette, wherein: the expression cassette in the first vector comprises the first nucleotide sequence and a first promoter sequence operably linked to the first nucleotide sequence, and the expression cassette in the second vector comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence.

6. A gene expression system according to claim 1, wherein said gene expression system comprises one vector comprising both the first nucleotide sequence and the second nucleotide sequence, wherein the vector comprises either: i) one expression cassette, wherein ia) a promotor is operably linked to either the first or the second nucleotide sequence, and wherein the nucleotide sequence to which the promotor is linked to the other of the first and second nucleotide sequence via a translation initiating nucleotide sequence, such as an internal ribosome entry site (IRES); or ib) a promotor is operably linked to either the first or the second nucleotide sequence and wherein the nucleotide sequence to which the promotor is linked to the other of the first and the second nucleotide sequence via a 2A peptide; or ii) two expression cassettes, wherein one expression cassette comprises the first nucleotide sequence and a first promoter sequence operably linked to first nucleotide sequence, and the other expression cassette comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence; or iii) a fusion polypeptide of the first nucleotide sequence and the second nucleotide sequence.

7-13. (canceled)

14. A method of treating a disease or condition associated with a reduced dopamine level comprising administering a gene expression system and a ligand binding to a destabilizing domain (DD) to a patient in need thereof, wherein said gene expression system comprises: a first nucleotide sequence encoding a fusion polypeptide of: a) a DD, and b) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and a second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof.

15. A method of treating a disease or condition associated with a reduced dopamine level in a patient that previously has been subject to gene therapy using a ligand binding to a destabilizing domain (DD), whereby a gene expression system comprising: a first nucleotide sequence encoding a fusion polypeptide of: a) a DD, and b) a GTPcyclohydrolase 1 (GCH1) polypeptide, or a biologically active fragment or variant thereof; and a second nucleotide sequence encoding a tyrosine hydroxylase (TH) polypeptide, or a biologically active fragment or variant thereof, has been administered to the brain of the patient.

16. The method of claim 15, wherein the treatment involved controlling the DOPA synthesis in the brain of the patient.

17. The method of claim 14, wherein said gene expression system comprises two vectors each containing one expression cassette, wherein: the expression cassette in the first vector comprises the first nucleotide sequence and a first promoter sequence operably linked to the first nucleotide sequence, and the expression cassette in the second vector comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence.

18. The method of claim 15, wherein said gene expression system comprises two vectors each containing one expression cassette, wherein: the expression cassette in the first vector comprises the first nucleotide sequence and a first promoter sequence operably linked to the first nucleotide sequence, and the expression cassette in the second vector comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence.

19. The method of claim 14, wherein said gene expression system comprises one vector comprising both the first nucleotide sequence and the second nucleotide sequence, wherein the vector comprises either: i) one expression cassette, wherein ia) a promotor is operably linked to either the first or the second nucleotide sequence, and wherein the nucleotide sequence to which the promotor is linked to the other of the first and second nucleotide sequence via a translation initiating nucleotide sequence, such as an internal ribosome entry site (IRES); or ib) a promotor is operably linked to either the first or the second nucleotide sequence and wherein the nucleotide sequence to which the promotor is linked to the other of the first and the second nucleotide sequence via a 2A peptide; or ii) two expression cassettes, wherein one expression cassette comprises the first nucleotide sequence and a first promoter sequence operably linked to first nucleotide sequence, and the other expression cassette comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence; or iii) a fusion polypeptide of the first nucleotide sequence and the second nucleotide sequence.

20. The method of claim 15, wherein said gene expression system comprises one vector comprising both the first nucleotide sequence and the second nucleotide sequence, wherein the vector comprises either: i) one expression cassette, wherein ia) a promotor is operably linked to either the first or the second nucleotide sequence, and wherein the nucleotide sequence to which the promotor is linked to the other of the first and second nucleotide sequence via a translation initiating nucleotide sequence, such as an internal ribosome entry site (IRES); or ib) a promotor is operably linked to either the first or the second nucleotide sequence and wherein the nucleotide sequence to which the promotor is linked to the other of the first and the second nucleotide sequence via a 2A peptide; or ii) two expression cassettes, wherein one expression cassette comprises the first nucleotide sequence and a first promoter sequence operably linked to first nucleotide sequence, and the other expression cassette comprises the second nucleotide and a second promoter sequence operably linked to the second nucleotide sequence; or iii) a fusion polypeptide of the first nucleotide sequence and the second nucleotide sequence.

21. The method of claim 14, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

22. The method of claim 15, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

23. The method of claim 17, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

24. The method of claim 18, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

25. The method of claim 19, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

26. The method of claim 20, wherein said ligand binding to a DD is trimethoprim (TMP) or an analogue or derivative thereof.

27. The method of claim 14, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

28. The method of claim 15, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

29. The method of claim 17, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

30. The method of claim 18, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

31. The method of claim 19, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

32. The method of claim 20, wherein said disease or condition is selected from the group consisting of idiopathic or genetic forms of Parkinson's disease, Parkinsonism and related disorders, schizophrenia, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, and restless legs syndrome (RLS).

33. A gene expression system according to claim 1, wherein the destabilizing domain (DD) is based on DHFR.

34-35. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] In the examples below, references are made to the accompanying figures on which:

[0130] FIG. 1 illustrates DOPA measurements in medium from 293 cells transfected with plasmids encoding different combinations of the DD regulated constructs. Transfection with solely TH or GCH1 or a combination of both (A). Combining constitutively expression of TH or GCH1 and with DD regulated versions of either TH and GCH1 coupled to both N- and C-terminal side of the enzymes (B or C, respectively). All conditions run as triplicates.

[0131] FIG. 2 shows striatal extracellular levels of HVA in anesthetized animals that received a regulated vector combination with DD coupled to either GCH1 (TH+DD-GCH1; n=2) or TH (DD-TH+GCH1; n=2) in a pilot microdialysis experiment. In addition, the figure shows data from animals that received the constitutively expressed vector (TH+GCH1; n=3) from the microdialysis experiment in the main study. Six baseline samples were collected before infusion of NSD-1015 via the probe for 2 h followed by two hours infusion of NSD-1015 and TMP. All animals received oral administration of TMP (2 mg/ml) prior the microdialysis experiment.

[0132] FIG. 3 illustrates the experimental design for the long-term behavioral assessment study (Experiment 2). Eighty-nine Sprague Dawley rats received 6-OHDA injections in the medial forebrain bundle and were then screened and pre-scored with amphetamine induced rotations, corridor test and stepping test. Rats were then selected based on the severity of the lesion-induced behavioral impairments and allocated into two vector treatment groups and one lesion control group. In addition, 11 rats were included in the study as intact control group. Ten weeks after the lesion, 9 animals were injected with a mixture of two vectors encoding for constitutively active TH and GCH1 genes (depicted as TH+GCH1 group), while another 19 were injected with a vector mix in which TH expression was combined with a regulated GCH1 expression (depicted as TH+DD-GCH1). All rats were then followed with repeated behavioral assessment with corridor and stepping test over a 33-week period. At 15 weeks post-AAV injection, 10 out of 19 animals in the TH+DD-GCH1 group received trimethoprim (TMP) in their drinking water. TMP was administered in three concentrations0.5, 1.0 and 2.0 mg/mlfor 6 weeks each in a dose-escalation study design. After 33 weeks all animals were terminated and brain tissue taken for histological processing.

[0133] FIG. 4 shows the results from Experiment 1 where striatal DOPAC, HVA and 5-HIAA levels were measured in awake-freely moving animals using online microdialysis. All animals were TMP-nave at the start of the experiment. After the baseline sampling was done over 1 hour duration (5 samples every 12 min), 20 M TMP-lactate (dissolved in ringer solution) was administered via the probe using reverse microdialysis principle and maintained throughout the rest of the experiment. Panel A shows 1-hour average data from baseline (BL) and 6- and 12-hours after start of TMP infusion in the TH+DD-GCH1 (n=7), TH+GCH1 (n=3), Les-Sham (n=2) and intact control (n=4) groups. DOPAC levels from animals in the TH+DD-GCH1 group (n=7) are plotted individually (black lines in panel B), while the group averages of intact and lesion controls as well as the constitutively active TH+GCH1 treatment group are presented for reference (gray lines). Data traces present in panel B are floating point averages of two data points from consecutive samples plotted over a 13-hour period of observation.

[0134] FIG. 5 illustrates motor behavioral assessment in Experiment 2 of the animals that was performed using the stepping (A,B) and corridor (C) test paradigms over 33-week follow-up period in 5 phases of the experiment as indicated in the X-axis. The stepping test results are presented in the forehand (A) and the backhand direction (B). Three measurements were conducted post AAV at 6, 9 and 12 weeks before the introduction of TMP in drinking water at three doses from 0.5 mg/ml to 1 and 2 mg/ml every six weeks. During this period 2 tested were performed with 3 weeks intervals. Note that during the post-AAV assessment period, the TH+DD-GCH1 group (open circles) was an average of 19 animals, while in the TMP dose-escalation phase of the study 9 animals were followed without TMP (open circles) and 10 were followed with TMP (gray circles).

[0135] FIG. 6 illustrates regulation of transgenic GTP cyclohydrolase 1 (GCH1) fused with destabilized dihydrofolate reductase (DHFR) (depicted as DD-GCH1) in Experiment 2. Animals treated with constitutively expressed TH+GCH1 showed robust expression of transgenic human GCH1 in the striatum (B). Note that this antibody does not cross-react with rodent GCH1 and thus no immunoreactivity is observed in intact animals (A). In the absence of TMP, TH+DD-GCH1 treated rats have very faint or negative GCH1 or DHFR immunoreactivity (C,G), whereas in animals receiving TMP the immunoreactivity of GCH1, as well as DHFR, is increased to readily detectable levels (D,H). At 2 mg/ml dose level (as shown in these panels) GCH1 expression is similar to constitutive expression group (compare B and D). Scale bar represents 100 m.

[0136] FIG. 7 shows high magnification images obtained from the striatum from animals in Experiment 2 showing part of the transduction area stained using TH immunoreactivity, cresyl violet (CV), NeuN, and glial markers Iba1 and ED1 (equivalent to human CD68) in columns from left to right. Each experimental group is represented in rows. Scale bar in Y represents 100 m and applies to all panels.

[0137] FIG. 8 shows high magnification images obtained from the globus pallidus from animals in Experiment 2 stained using TH immunoreactivity, cresyl violet (CV), NeuN, and glial markers Iba1 and ED1 (equivalent to human CD68) in columns from left to right. Each experimental group is represented in rows. Scale bar in Y represents 100 m and applies to all panels.

[0138] FIG. 9 contains Table 1, which shows striatal DOPA, DOPAC and HVA metabolites measured using online microdialysis in anesthetized rats from Experiment 1. Baseline (BL) levels are shown followed by measurements after 2 h infusion of NSD-1015 (AADC inhibitor) and 2 h infusion of NSD+TMP via the sampling probe. Note that the values shown are averages (SD) and the TH+DD-GCH1 group included in the study were treated with either 0.5 (n=4) or 2.0 (n=5) mg/ml TMP for 2 weeks prior sampling. Intact (n=3), Les-Sham (n=2), TH+GCH1 (n=3).

[0139] FIG. 10 shows GCH1 immunoreactivity in two non-human primates injected with AAV5-CBA-DD-GCH1 vectors. One of the monkeys was treated with TMP systemically (A-D), while the other was followed as control (E-H). Panels A and E represent injections at the highest dose tested (1.2E14 vg/ml), B and F are from areas targeted using a 3-fold diluted vector, whereas C and G are 9-fold and D and H are 27-fold dilutions of the highest dose.

EXAMPLES

[0140] The entire study described in this document was conducted in separate parts, starting with an in vitro test followed by a pilot microdialysis experiment. Based on these results, two long-term experiments, denoted Experiment 1 and Experiment 2, were performed. Thirty-six animals were used in Experiment 1, where 4 groups of rats were subjected to an in vivo online microdialysis using one of two protocols as described below. Experiment 2 was designed to assess the long term behavioral effects of the regulated gene expression system tested in this study and included a total of 38 animals (selected from a total of 89 rats with 6-OHDA lesions) with validated severe and stable motor behavioral impairments and 11 intact control rats. The selection criteria used for inclusion in the second study was >6 net turns/min ipsilateral to the lesion side after challenge with amphetamine (2.5 mg/kg), <5% left retrievals in the corridor test, and no left forehand adjusting steps. Collectively these three measures marks animals with severe impairments induced by dopamine depletion. The details of the long-term study timeline are presented in FIG. 3.

[0141] The pilot microdialysis experiment included four rats with validated 6-OHDA lesions (>6 ipsilateral net turns/min).

Subjects

[0142] One hundred fifty nine female Sprague-Dawley rats (Charles River, Schweinfurt, Germany) weighing 225-250 g were used in this study. Animals were housed 2-3 per cage under a 12 h light/12 h dark cycle with free access to food and water except during assessment with corridor test (as described below). All experimental procedures were approved by the Ethical Committee for use of Laboratory Animals in the Lund-Malmo region.

Surgical Procedures

[0143] Anesthesia was induced by fentanyl citrate (Fentanyl, Apoteksbolaget, Sweden) and medetomidine hydrochloride (Dormitor, Apoteksbolaget, Sweden) injected i.p. at doses of 6 ml/kg (300 mg/kg and 0.3 mg/kg, respectively). Animals were placed in a stereotactic frame (Stoelting, Wood Dale, Ill.) and intracerebral injections were made with a Hamilton syringe (Hamilton, Bonaduz, Switzerland) fitted with a glass capillary. The anteroposterior (AP) and mediolateral (ML) coordinates were calculated from bregma and the dorsoventral (DV) coordinates from the dural surface, according to the atlas of Watson and Paxinos.

6-OHDA Lesions

[0144] Fourteen g free base 6-OHDA (Sigma-Aldrich AB, Sweden) was dissolved in ascorbate-saline (0.02%), resulting in a concentration of 3.5 g/l, and injected into the right medial forebrain bundle using the following coordinates: AP: 4.4 mm; ML: 1.2 mm and DV: 7.8 mm with the tooth bar set to 2.4 mm. The injection speed was constant at a speed of 1 l/min and the needle was kept in place for 3 min before it was slowly retracted.

AAV Vector Injections

[0145] Vector preparations of TH+GCH1 or TH+DD-GCH1 were injected at two sites in the striatum with two deposits along each tract. In addition, in the pilot microdialysis experiment, a vector combination of DD-TH and GCH1 was injected in two rats with the same parameters as described here. In total 5 l vector was injected per animal, distributed by 1.5 l in the ventral and 1.0 l in the dorsal deposit in each site. A pulled glass capillary (outer diameter 60-80 m) was mounted on a Hamilton syringe with a 22-gauge needle to the minimize tissue damage and improve accuracy. The injection coordinates were: (1) AP: +1.0 mm; ML: 2.8 mm and DV: 4.5, 3.5 mm and (2) AP: 0.0 mm; ML: 4.0 mm and DV: 5.0, 4.0 mm with the tooth bar set to 2.4 mm. The injection speed was kept constant at 0.4 l/min and the needle was kept in place for 1 min after the ventral and 3 min after the dorsal deposit. Animals in the intact and lesion control groups underwent sham surgery by drilling a burr hole at the corresponding position in the skull but without penetrating the dura.

AAV Vectors

[0146] The viral vectors used in this study were AAV serotype 5 with ITR sequences from serotype 2, and all transgenes were driven by the chicken beta actin (CBA) promoter, which includes a rabbit gamma globulin intron and a cytomegalovirus (CMV) enhancer, and terminated with an early SV40 poly-A sequence. The two transgenes were human TH and GCH1. Regulation of GCH1 and TH expression was achieved by coupling a destabilizing domain (DD) derived from E. coli dihydrofolate reductase (DHFR) to the N- and C-terminal side of the proteins. Generation of the controllable DHFR domains has been described in detail earlier (Iwamoto et al., 2010).

[0147] Two vector combinations were used in the in vivo studies (i.e., pilot study, Experiment 1 and Experiment 2); TH and GCH1 constitutively expressed (group denoted TH+GCH1) and constitutive expression of TH combined with regulated GCH1 (DD-GCH1) (group denoted TH+DD-GCH1). All combinations were prepared in DPBS mixed at 5:1 ratio of TH or DD-TH over GCH1 or DD-GCH1. The final titers of the vectors used in Experiment 1 and Experiment 2 for TH+GCH1 and TH+DD-GCH1 were 1.9E+14 gc/ml (resulting in 9.5E11 gc injected) and 1.8E+14 gc/ml (resulting in 9.0E11 gc injected), respectively.

[0148] AAV vectors were produced in HEK-293 cells grown in tissue culture flasks for adherent cells (BD Falcon) to about 60-80% confluence. Transfection was achieved with the calcium-phosphate method and included equimolar amounts of transfer and helper plasmid DNA (pDP5 encoding for the AAV5 capsid proteins). The cells were incubated for 3 days before harvesting with PBS-EDTA. They were then centrifugated (1000g for 5 min at 4 C.), re-suspended with lysis buffer (50 mM Tris, 150 mM NaCl, pH 8.5) and lysed by freeze-thawing cycles with dry ice/ethanol baths. The lysate was treated with benzonase (Sigma-Aldrich AB, Sweden) and then purified by centrifugation to remove cellular debris (4500g for 20 min. at 4 C.) followed by ultracentrifugation (1.5 h at 350 000g at 18 C.) in a discontinuous iodixanol gradient (Zolotukhin et al., 1999) and then by ion-exchange chromatography using an Acrodisc Mustang Q membrane device (Pall Life Sciences). Briefly, the Mustang Q membranes were preconditioned according to the manufacturer's instructions with a final wash with a low salt buffer (20 mM Tris, 15 mM NaCl, pH 8.0). The virus suspension was diluted threefold in the same low salt buffer, before initiating the purification. Addition of the virus to the membranes was followed by a wash with the same low salt buffer. The virus was eluted from the membranes using a high salt buffer (20 mM Tris, 250 mM NaCl, pH 8.0). The virus suspension was then buffer exchanged approximately hundredfold by adding DPBS buffer (Life technologies) and concentrated with a centrifugation filter device (Millipore Amicon Ultra 100 kDa MWCO) at 2000g and 18 C. Dilutions of viruses were done using the same DPBS buffer. The titers of the vector preparations were determined with TaqMan quantitative PCR using primers targeting the ITR sequence promoter (Aurnhammer et al., 2012).

In Vitro Study

[0149] HEK 293 cells were transfected with plasmids encoding DD regulated TH and GCH1, fused either on the N- or C-terminal side using Lipofectamine according to the product protocol (Life Technologies). The regulated plasmid construct was combined with either constitutively expressed TH or GCH1 in a ratio of 5:1 in favor of TH/DD-TH/TH-DD over GCH1/DD-GCH1/GCH1-DD. Six hours after transfection the culture medium was substituted with medium containing 1E-5 M TMP dissolved in 0.01% DMSO. After 24 hours, samples of the culture medium were aspirated and processed for HPLC analysis for DOPA levels. Each plasmid combination was performed as triplicates and an average was calculated.

Oral TMP Administration

[0150] Nine of nineteen TH+DD-GCH1 treated animals in behavioral part of the experiment (Experiment 2) received oral TMP suspension (Meda AB, Solna, Sweden) in their drinking water 15 weeks post-AAV injection. TMP was administered in three different doses in 6-week intervalsstarting concentration 0.5 mg/ml, 1.0 mg/ml at 21 weeks, and finally 2.0 mg/ml at 28 weeks (FIG. 5). Drinking behavior was closely monitored in a subset of animals, which enabled approximation of the corresponding dose, 22.82.8, 40.96.2, and 87.114.6 mg TMP/kg/day, respectively. In addition, in the microdialysis pilot all animals received 2.0 mg/ml oral TMP prior the experiment.

Behavioral Tests

[0151] Amphetamine-induced rotation test was used as an initial screen to exclude animals with incomplete dopaminergic lesion and was performed five weeks after 6-OHDA surgeries. Animals received injections of D-amphetamine sulfate (2.5 mg/kg, i.p., Apoteksbolaget, Sweden) and their full left and right body turns were quantified over 90 minutes using automated rotometer bowls (AccuScan Instruments Inc., Columbus, Ohio). The cut-off value for net ipsilateral rotational asymmetry score was 6 full body turns/min.

[0152] Corridor test was first described by Dowd and colleagues (Dowd et al., 2005), and measures lateralized sensory neglect. Briefly, the rat was placed in the end of a corridor (150723 cm) with ten adjacent pairs of cups filled with 5 sugar pellets evenly distanced along the floor of the corridor. Animals were allowed to explore the corridor freely. An investigator blinded to the group identity directly quantified retrievals; defined as each time the rat poked its nose into a unique cup, regardless of if it ate any pellets. Revisits in the same cup were not scored unless a retrieval was made from another cup in between. All rats were tested until 20 retrievals were made or the test duration exceeded 5 min. Before testing, all rats were placed in an empty corridor for 5 minutes to reduce novelty of the environment. The rats were food restricted the day prior and during the two to three days of testing. Results were calculated as an average of the contralateral retrievals (left) and presented as percentage of total retrievals.

[0153] Stepping test, developed by Schallert and colleagues (Schallert et al., 1979) and modified by Olsson et al (Olsson et al., 1995) was employed in this study. In brief, a blinded investigator assessed forelimb use by holding the rat with two hands only allowing one forepaw to touch the table surface. The investigator then moved the rat sideways over a defined distance of 90 cm with a constant speed over 4-5 sec and scored the amount of steps in both forehand and backhand direction for each forelimb. Each direction was scored twice on each testing day and the average score was calculated over 3 days.

Online Microdialysis

[0154] In Experiment 1, two microdialysis protocols were employed in separate groups of animals. In total 36 animals were used for this part of the experiment. The first protocol was performed in four groups of TMP-nave animals, namely the TH+DD-GCH1 (n=7), TH+GCH1 (n=4), Les-Sham (n=4) and intact controls (n=4) groups.

[0155] All animals were surgically implanted with a probe guide, which was cemented to the skull two days prior to the actual sampling. This was achieved with two screws fastened to the skull without penetrating the dura and drilling at the position of the vector injections i.e., AP: +0.5 mm; ML: 3.7 mm and DV: 1.7 mm with the tooth bar set to 2.4 mm. The DV coordinate was calculated so the membrane of the probe was positioned in the center of the transduction. A tether screw was then placed on the positioned to later hold the tether and then dental cement was added to fixate all components to the skull bone. The animal was given analgesia after the surgery and allowed to recover for at least two days before the experiment. At the day of the experiment, the animal was briefly sedated with isofluorane to easily be able to remove the guide dummy in the probe guide, insert the sampling probe and then attach the tether to the screw. The rat was placed in the testing cylinder where it had free access to food and water throughout the experiment. Following an equilibration period of 90 min baseline samples over 60 min (5 samples at 12 min intervals). The ringer solution of artificial CSF was changed to ringer containing 2E-5 M TMP lactate salt (Sigma-Aldrich AB, Sweden) so that the next sample became the first time bin when TMP was infused to the brain via reverse MD. This approach resulted in a precise measure of the time of initiation and controlled exposer of TMP over several hours following this time point. The animal was allowed to freely move in the test chamber for an additional 12 h and the dialysates were instantly injected and analyzed with a HPLC coupled to the outlet of the OMD system while the samples were collected every 12 min. The dialysates were then analyzed by HPLC with the Alexys monoamine analyzer system (Antec Leyden, The Netherlands) consisting of a DECADE II detector and VT-3 electrochemical flow cell. DA and metabolites were detected with a mobile phase consisting of 50 mM citric acid, 8 mM NaCl, 0.05 mM EDTA, 15% methanol, 700 mg/I 1-octanesulfonic acid sodium salt, at pH 3.15, with 1 mm50 mm column with 3 mm particle size (ALF-105) at a flow rate of 90 ml/min. Peak identification and quantification was conducted using the Clarity chromatographic software package (DataApex, Prague, Czech Republic).

[0156] The second microdialysis protocol in Experiment 1 (identical microdialysis methods and protocols were used in the pilot study) was performed in anaesthetized animals was performed in TH+DD-GCH1 treated animals that received oral TMP administration in their drinking water at least 2 weeks prior sampling; either 0.5 mg/ml (n=4) or 2 mg/ml (n=5) (same TMP emulsion that was administered to the animals followed with behavior tests). In addition, this experiment also included animals from the TH+GCH1 (n=3), Les-Sham (n=2) and intact control (n=3) groups. Probe placement was calculated to position the membrane of the probe in the center of the transduction area in striatum, which corresponded to the coordinates: AP: +0.5 mm; ML: 3.7 mm and DV: 5.7 mm with the tooth bar set to 2.4 mm. After 90 min equilibration, baseline samples were collected before 1E-5 M NSD-1015 (Sigma-Aldrich, St. Louis, Mo., USA) was administered via the probe in the ringer solution for 2 h. This was then followed by a 2 h administration of 2E-5 M TMP lactate salt in addition to 1E-5 M NSD-1015 in the ringer solution. Samples were analyzed readily as described for the first microdialysis experiment. After the last sample was collected the animal was terminated and brain tissue taken for histology.

Histological Analysis

[0157] After the last behavioral assessment point all animals were anaesthetized by an injection of 1.2 ml sodium pentobarbital (i.p., Apoteksbolaget, Sweden) and then transcardially perfused with 50 ml room temperature saline followed by 250 ml ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer adjusted to pH 7.4, at a 50 ml/min rate. The brains were then dissected and post-fixated in 4% PFA for 24 hours before cryoprotection in 25% sucrose for 24-48 hours. The fixed brains were cut in coronal orientation at a thickness of 35 m on a semi-automated freezing microtome (Microm HM 450) and collected in 8 and 6 (striatum and substantia nigra, respectively) series and stored in anti-freeze solution (0.5 M sodium phosphate buffer, 30% glycerol and 30% ethylene glycol) at 20 litnu C. Immunohistochemistry was performed using antibodies further processing, (AR, Rogers, Freez-Pel 1:2000 rabbit IgG, 0-40101P) raised against TH 1:2000 mouse IgG, Z3138 MCA) 1GCHAbD Serotec, Oxford, UK), AADC (AB1569, rabbit IgG, 1:500, Millipore, Billerica, Mass.), NeuN (MAB377, mouse IgG 1:500, Millipore), IBA1 (019-19741, rabbit IgG 1:1000, Wako, Richmond, Va.), ED1 (MCA341-R, mouse IgG 1:200, Serotec, Oxford, UK), and DHFR (custom made, Rabbit IgG, 1:50 000). Incubation with biotinylated secondary antibodies (BA1000, goat anti-rabbit 1:200 and BA2001, horse anti-mouse 1:200, Vector Laboratories, Burlingame, Calif.) was followed by a second 1-hour incubation with avidin-biotin peroxidase solution (ABC Elite, Vector Laboratories, Burlingame, Calif.). The staining was visualized using 3,3-diaminobenzidine in 0.01% H.sub.202.

Primate Studies

Animals and Housing

[0158] All animal studies were conducted according to the European (EU Directive 86/609/EEC) and the French regulations (authorization n A 92-032-02). The animal facilities are authorized by local veterinarian authorities and comply with Standards for Humane Care and Use of Laboratory Animals of the Office of Laboratory Animal Welfare (OLAWn.sup.o#A5826-01) for CEA laboratories.

[0159] Experiments were conducted on 2 male cynomolgus monkeys (Macaca fascicularis) supplied by Noveprim (Mauritius Island) of 4 and 6 years of age and weighing 5.8 and 3.8 kg. Experimental protocols and appropriate animal care procedures were authorized by special Decrees of the French. All efforts were made to minimize animal suffering and animal care was supervised by veterinarians and animal technicians skilled in the healthcare and housing of non-human primates. All NHPs were housed under standard environmental conditions (12-hour light-dark cycle, temperature: 221 C. and humidity: 50%) with free access to food and water. NHPs received diet (containing less than 1% folate per 10 kg of food) from the beginning of the experimental protocol.

The MPTP Model of PD

[0160] Parkinsonism was induced by systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP (Sigma, St Louis, Mo., USA) as previously described (Aron Badin et al, 2013). Briefly, non-human primates (NHPs) were exposed to daily intramuscular injections of 0.25 mg/kg MPTP for 7 consecutive days and cycles of MPTP intoxication were repeated with MPTP-free washout periods between cycles until a stable parkinsonian state was achieved. NHPs were scored daily on a scale of 0-14 according to relevant clinical scales used in PD patients and primates looking at posture, dystonia, tremor, and akinesia (Papa & Chase, 1996; Obeso et al, 2000). Parkinsonism was considered satisfactory based on the clinical scores and the presence of significant and stable reduction in spontaneous locomotor activity (by at least 80% compared to baseline) that lasted at least one month.

Imaging Studies

[0161] Magnetic resonance imaging (MRI)

[0162] MRI was performed on all NHPs shortly before or after the baseline PET scan in order to allow precise determination of regions of interest for PET analysis and the coordinates for surgical delivery of viral constructs.

[0163] NHPs were anesthetized with 10:1 mg/kg ketamine:xylazine and placed in the magnet in a sphinx position, fixed by mouth and ear bars to a stereotactic MRI-compatible frame (M2E, France). Once in the magnet, NHPs were heated by a hot air flux and their temperature and respiration parameters monitored remotely.

[0164] MRI was performed on a 7 Tesla horizontal system (Varian-Agilent Technologies, USA) equipped with a gradient coil reaching 100 mT/m (300 s rise time) and a circular radiofrequency 1H coil (12 cm inner diameter). T2-weighted images were acquired using a fast spin-echo sequence with the following parameters: TR=4750 ms, effective TE=62 ms, acquisition time=16 min, FOV=115115 mm and matrix=256256 resulting in a 450450 m in plane resolution, 40 coronal slices, slice thickness=1 mm. T2*-weighted images were acquired using a multiple gradient echo sequence with the following parameters: TR=2000 ms, effective TE=20 ms, acquisition time=8 min30 s, flip angle=40, with identical geometric parameters as the T2-weighted images.

Total scan duration: 35 minutes.

Surgery

Surgically-Placed Gastrostomy (SPG)

[0165] In the interest of refining the procedure of daily oral delivery of the antibiotics and in order to minimize the stress induced by this manipulation, the NHP receiving TMP was equipped with a subcutaneous chamber connected to the stomach with a small catheter. The SPG device, consists of an injection port (X-Port, BARD Access Systems, France) with a self-sealing silicone septum (base 22.6 mm28.2 mm, internal volume 0.6 ml) with attachable 8 Fr. Groshong radio-opaque silicon catheter (50 cm long, internal diameter 1.5 mm, volume 0.6 ml). In particular, a Groshong valve, positioned at the end of the catheter, helps to prevent gastric juice reflux into the port/catheter system. All materials are biocompatible. Only non-coring needles were used (22 gauge, 2.5 cm long, BARD Access Systems, France) to puncture the silicone septum of the port, minimizing the risk of damaging it (Fante et al, 2012).

[0166] Anesthesia was induced by intramuscular injections of 10:1 mg/kg ketamine:xylazine and maintained under propofol (1 mg/kg/hour) throughout the procedure. An oral antibiotic treatment was administered before surgery (amoxicillin and clavulanic acid, 45 mg/kg/daily and 6 mg/kg/daily) and repeated daily for 5 post-operative days. Following a midline substernal laparotomy (about 3 cm long), a subcutaneous pouch was fashioned on the upper left side of the abdomen, then in the left antero-lateral site of the rib cage where the port was to be placed. The anterior wall of the stomach was identified and exteriorized. Using the Seldinger technique, the catheter was inserted in the gastric lumen through its anterior face, between the gastric body and the antrum, equidistant and 7-8 cm from the lesser and greater gastric curvatures. Then the catheter was anchored to the gastric wall with a purse-string suture (resorbable Vicryl 3/0) and passed through the left muscle layers of the anterior abdominal wall, 1-2 cm from the costal arch and about 2 cm from the midline incision. Pexy between the stomach and the abdominal wall around the catheter exit site was performed with 4 resorbable Vicryl 3/0 stitches. The catheter was connected to the port that was then inserted subcutaneously in the thoracic subcutaneous pouch and anchored to the external fascia of the rib cage (resorbable Vicryl 3/0), enabling a stable attachment and good usability when the port needle was used. The port was tested and the midline was sutured in a double layer (single suture, resorbable Vicryl 2/0).

Intracerebral Microinjections

[0167] NHPs were induced by intramuscular injections of 10:1 mg/kg ketamine:xylazine and maintained anesthetised with propofol (1 mg/kg/hour) throughout the procedure. NHPs were placed in a dedicated MRI-compatible stereotactic frame with the head resting on a mouth bar, fixed by blunt ear bars. Temperature was maintained at 37 C. using a feed-back coupled heating blanket, and the respiratory rate, pO2, pCO2, cardiac rhythm and blood pressure were continuously monitored. All injections were performed using a dedicated Hamilton syringe and a 26G sterile needle. A midline incision was performed on the head and skin and muscle were retracted in order to access the skull. A surgical drill (point 0.280, 30000 rm) was used to open 8 holes through the skull without piercing the dura matter. Baseline MRI images were used to calculate all injection targets in the caudate (AC+1, AC+4) and the (AC & AC-4) and 20 L of virus were delivered per site in a single deposit at a rate of 1 L/minute using an injection micropump (KDS30, France). Each NHP received bilateral intra-striatal injections of an AAV5-CBA-DD-GCH1 (add 1.2E14 vg/ml).

[0168] Each caudate and putamen was injected twice with the same concentration of virus. The caudate nucleus in the left hemisphere received the highest concentration and 3-fold dilution was used for the putamen on the same side, whereas the right hemisphere was injected with two 9 and 27-fold dilutions in the caudate and putamen, respectively. NHPs were housed in A2 biosafety level facilities for 3 weeks following viral injection.

[0169] Blood and CSF were collected on the day of surgery and at euthanasia in order to evaluate the presence of viral antigens.

TMP Administration and Monitoring

[0170] TMP administration begun at 1 month post-injection, which allowed for the viral vector to reach high expression level in the different brain regions targeted. TMP was administered to only one of the two NHPs using the SPG device. TMP was administered daily for 2 months at a constant dose of 20 mg/kg. After administration of the appropriate dose, the catheter and chamber were rinsed with 10 ml of distilled water. The treated primate was weighted weekly to adjust the dose if necessary. TMP was kept in the dark at room temperature throughout the experiment.

[0171] Blood samples were collected on the day of surgery and at 1 and 2 months after TMP administration in order to measure folates and TMP. All NHPs were anesthetized 1.5 hours after TMP administration in order to avoid interfering with the absorption of the TMP molecule. TMP levels in blood were measured by liquid chromatography with UV detection (Phatophy, France).

Euthanasia & Post-Mortem Studies

[0172] Before euthanasia NHPs were deeply anesthetized and blood and CSF were collected 1.5 h after TMP administration in the case of one primate. At the end of the experimental protocol, monkeys were euthanized by a lethal dose of pentobarbital delivered before transcardial perfusion with ice-cold 0.9% NaCl. The brains were extracted and placed in a dedicated NHP brain matrix on ice (M2E, France) bearing 2 mm subdivisions in the antero-posterior axis of the caudate and putamen in order to extract punches for biochemistry on certain brain slices and for immunohistochemistry in other slices.

[0173] Two blocks were subdivided into three 2 mm-thick slices that were placed on a petri dish on ice to obtain punches of 3 mm . Samples were weighed and immediately frozen on dry ice.

[0174] All brains were post-fixed for 4 days in 4% paraformaldehyde and then cryo-preserved by immersion into sucrose-containing phosphate buffer gradients with increasing concentrations (5-10-20%) for 3 days at a time. Brains were then sliced into 40 m-thick slices and floating slices were stained with an antibody against GTP cyclohydrolase 1 enzyme, as detailed elsewhere in this document.

Statistical Analysis

[0175] In FIG. 5, non-parametric statistics were performed with Mann-Whitney U for between group comparisons and Friedman's test followed by Wilcoxin signed ranks test to assess the effect of TMP dose. Statistical comparisons were conducted with the SPSS statistical package version 21 (SPSS Inc., Chicago, Ill.).

Results

Reconstitution of DOPA, Dopamine Metabolites by the Regulated Gene Expression System

[0176] The inventors designed the first part of the study to test different plasmid combinations in an in vitro setting to determine the function, capacity and basal activity of the DD regulation system when fused with TH and GCH1 genes. For this purpose, they studied N- and C-terminal fusion peptides of the two enzymes. The readout measurement was DOPA levels in the culture medium from 293 cells transfected with the different plasmid combinations of the various constructs tested (FIG. 1). Transfection with plasmids constitutively expressing GCH1 enzyme alone did not result in any measurable DOPA production as were the mock transfected cells. Expression of TH resulted in low but detectable DOPA production compared to when both TH and GCH1 enzymes were expressed in the cells suggesting that in this cell line optimal TH activity required additional GCH1 activity that needed to be supplied exogenously (FIG. 1A). Combining constitutively expressed TH and DD-GCH1 resulted in efficient DOPA production when the DD was coupled on the N-terminal side of the enzyme and in the presence of TMP. In the absence of TMP, DOPA levels were similar to what were observed in TH only transfected cells. Coupling the DD to the C-terminally to GCH1 was detrimental since this construct did not support enhanced DOPA synthesis (FIG. 1B). Regulation of TH by coupling the DD on the N-terminal side gave DOPA levels comparable to the constitutively expressed vector combination in presence of TMP (C). Coupling DD to the C-terminal side of TH appeared not to be regulatable as it resulted in high levels of DOPA in both the presence and absence of TMP. These in vitro results suggested that combining DD on the N-terminal side was the most favorable placement and the lowest basal activity and leakage was achieved by controlling the TH enzyme.

[0177] Notably DD-TH combined with constitutively expressed GCH1 appeared to be working well, as in the presence of TMP, DOPA levels were at the same level as TH+GCH1 combination showing that in vitro this system displayed the full dynamic range as would be predicted based on earlier results reported in literature prior to performing the experiments.

[0178] Next, the functionality of the TH and GCH1 N-terminally coupled to DD was tested in an in vivo microdialysis experiment (FIG. 2). First, animals injected with DD-TH+GCH1 were tested with online microdialysis (OMD) to measure the vector-derived DA metabolites in the brain. These animals received oral TMP emulsion in their drinking water prior to the OMD measurements. The experiment was conducted to compare the efficacy and dynamic range of the TMP controlled gene expression systems as compared with the constitutive constructs (i.e., TH+GCH1). Surprisingly, we found that contrary to observations in cell lines, and the results that would have been anticipated by skilled person, treatment with the DD-TH+GCH1 vector combination resulted in 5-fold lower levels of HVA, as compared with the constitutively active vectors. Next, we tested whether TH combined with DD-GCH1 would have the same limitation as the DD-TH+GCH1 combination. Unexpectedly, the inventors found that the DD-mediated control of GCH1 stability and therefore the availability of BH4 was very effective. This was exemplified in two ways: First the production of DOPA from DD-GCH1 in the presence of TMP was as high as the condition when constitutively expressed genes are used. Secondly, in the absence of TMP, the DOPA levels were not different from the appropriate control group suggesting that the behaviour of this system in the brain was different from the a priori anticipated outcome. It was both tightly controlled and had the full dynamic. The inventors designed two long-term in vivo studies, denoted below as Experiment 1 and Experiment 2.

[0179] Experiment 1 was designed to validate the functionality of controlled DOPA synthesis in the striatum obtained by gene therapy incorporating a destabilized domain based on DHFR (DD) coupled to GCH1 gene in combination with constitutively expressed TH (TH+DD-GCH1). For this purpose, the inventors used two online microdialysis (OMD) study protocols. First, in anesthetized rats they determined (1) steady state production of DOPA, DOPAC and HVA under baseline conditions; (2) total DOPA synthesis capacity after inhibition of AADC in the striatum of animals where stabilization of the GCH1 protein was induced with oral administration of TMP, thus permitting synthesis of BH.sub.4 that can activate the TH enzyme (Table 1 in FIG. 9). In intact rats, DOPAC and HVA levels in the extracellular space were abundant (typically between 1 and 2 M), while DOPA concentrations were below reliable quantification limit of the system (2 nM). In rats with complete 6-OHDA lesion of the ascending dopaminergic projections of the medial forebrain bundle, the DOPAC and HVA concentrations fell to 1.0-25.8 nM, representing more than 99% depletion on the average. In animals treated with the TH+GCH1 constitutively active vectors, both DOPAC and HVA levels were increased (to values about 15-40% of intact controls. Notably, there was a readily detectable DOPA present in the extracellular space. Similarly, in the group treated with TH+DD-GCH1 and maintained under TMP activation, the DOPA, DOPAC and HVA levels were increased above the lesion baseline, although the DOPA concentrations appeared lower than the constitutively active group.

[0180] In order to explore the maximum capacity of the enzymatic machinery obtained from transgenic expression of the human TH enzyme, the second protocol followed the initial baseline period with addition of NSD-1015 to block the AADC enzyme. Consequently, the levels of DOPAC and HVA declined while the DOPA accumulated in all vector treated animals, suggesting that the newly synthesized DOPA was continuously converted to DA by the endogenously present residual AADC enzyme and contribute to generation of DOPAC and HVA measured in the samples obtained during the OMD experiment. Notably, the results obtained with addition of TMP to the ringer solution used as the dialysate did not further increase the DOPA levels suggesting that the system was fully activated with the oral TMP given to the animals during the 2 weeks prior to the OMD experiment. Finally, the inventors have tested two oral TMP doses (0.5 and 2.0 mg/ml in drinking water) and found that both were effective in reaching a similar biochemical reconstitution under these experimental conditions.

[0181] After establishing that the controlled expression system had comparable efficacy in the steady state on long-term TMP administration, the inventors turned their attention to the transition from a baseline state in the absence of TMP to the activated state upon introduction of the ligand. To be able to perform an OMD analysis in an analogous way, while anticipating a longer sampling period, the inventors adapted the measurements to be carried out in awake and freely moving animals and monitored the dialysis samples over 15-17 hours continuously and quantified levels of DOPAC, HVA and as a control also 5HIAA, the metabolite of serotonin (data from 13 hours sampling presented in FIG. 4A). As in the first protocol performed in anesthetized rats, DOPAC and HVA levels were severely depleted in the complete 6-OHDA lesioned group accounting for about 0.5% of values compared with intact controls. In rats treated with the AAV vector mix expressing the TH and GCH1 proteins constitutively (TH+GCH1 group), the DOPAC and HVA levels were about 30% and 80% of intact baseline, respectively. Notably, the TH+DD-GCH1 group (nave to TMP) had very low baseline levels of both DOPAC and HVA, corresponding to about 2 to 4% of normal values and slightly higher when compared with lesion controls. Addition of 20 M TMP in the dialysis solution resulted in gradual accumulation of DOPAC and HVA that at the 12 h mark were comparable to the constitutive expression group, i.e., reached to approximately 25 and 50% of normal values in intact animals as compared with about 70 and 60% in the TH+GCH1 group, respectively (FIG. 4A). Interestingly, the animals in the TH+DD-GCH1 group varied from each other in that 4 of 7 showed HVA levels within the same range of constitutive TH+GCH1 and intact groups at the 12 h point, while 2 cases had a lower value and one was about 2-fold higher than the intact average value (FIG. 4B).

[0182] Collectively, these data established that DD-GCH1 mediated controlled DOPA production worked best in this widely used animal model of PD with equal efficiency to constitutively active constructs and far better than the results obtained with the DD-TH vector.

Behavioral Recovery in TH+DD-GCH1 Treated Rats in Response to Oral Administration of TMP

[0183] In Experiment 2, the inventors assessed long-term motor recovery in lesioned animals treated with active vectors using the stepping and corridor tests and compared them to lesion and intact controls (See FIG. 3 for experimental design). Intact nave rats (n=1; black squares in FIG. 5) perform on average 9-11 steps in the forehand direction and 11-13 steps in the backhand direction in the stepping test (FIG. 5A,B). The normal performance in the corridor test is equal number of nose pokes in lids (retrievals) positioned on the left and right side of the corridor (FIG. 5C). The 6-OHDA lesioned rats (n=9; open squares in FIG. 5) used in this study have a severe impairment in all three parameters; i.e., make few or no adjusting steps and have a near complete bias to taking pellets from the lid on the right side, ipsilateral to the lesion. Treatment with TH+GCH1 (n=10; black circles in FIG. 5) resulted in near complete recovery in the stepping test already 6 weeks post-AAV and this effect was maintained throughout the rest of the study (FIG. 5A,B). In the corridor test, animals in this group showed an initial overcompensation at the 6-week assessment point where they almost exclusively made left retrievals (FIG. 5C). This effect was decreased at the following assessment time points but the animals continued to display an approximate 75% left side bias for the remaining time of the study until 33 weeks post-AAV injection.

[0184] Animals that received the TH+DD-GCH1 (n=19) vector showed a very limited or no recovery during the baseline assessment at 6, 9 and 12 weeks (open circles in FIG. 5). During this assessment period, the inventors observed that this group behaved similar to lesion controls and their own baseline measurements prior to vector treatment suggesting that in the absence of TMP, i.e., when GCH1 enzyme is destabilized and presumably BH.sub.4 levels are low. This was in contrast to the constitutively active TH+GCH1 group that showed clear signs of functional changes in both behavioral tests. The follow-up period of 3 months allowed us to demonstrate that the DD-regulated delivery system was tightly controlled and could be maintained without any functional consequences to the animals over long-term.

[0185] Fifteen weeks post-AAV, half of the animals in this group (n=10; gray circles in FIG. 5) started receiving oral TMP in their drinking water while the remaining rats (n=9; open circles in the TMP dose escalation phase) continued receiving drinking water without TMP. Three concentrations of TMP were administered to the animals in escalating doses between 0.5, 1 and 2 mg/ml, each lasting 6 weeks. Based on this concentration and the amount of water consumed per day, the TMP dose per rat was estimated to be 22.82.8, 40.96.2 and 87.114.6 mg TMP/kg/day, respectively. Behavioral assessment was performed at three and six weeks with each TMP dose. During the 0.5 mg/ml TMP administration phase TH+DD-GCH1 group showed partial recovery already at the first assessment point at 3 weeks, in contrast to animals that received the same vector but no TMP or lesion controls (p<0.001 in all three parameters tested). When the animals received a TMP dose of 1 and 2 mg/ml the therapeutic effect in the stepping test increased gradually to a magnitude comparable to the TH+GCH1 group and were not different from intact controls in corridor test. Notably, at the second assessment of the 1 mg/ml TMP dose, an overcompensation effect was seen in the corridor test (approximately 90% retrievals on the left, previously neglected side) similar to the observations in the TH+GCH1 group at 6 weeks after transduction (FIG. 5C). This effect was transient and was not observed in the following test 3 weeks later, despite the fact that the dose of TMP was increased to 2 mg/ml.

Oral TMP Administration Stabilizes DHFR-Coupled GCH1 and Rescues TH Expression

[0186] After the last behavioral assessment, brain tissue from all animals was processed for histological analysis. The expression of the GCH1 transgene was documented using an antibody specific to the human protein (with no cross reactivity to the rodent species; see lack of staining in the untreated side, FIG. 6A). As expected this analysis showed that there was a robust expression in both the constitutively active TH+GCH1 group (FIG. 6B) and the TH+DD-GCH1 group receiving the TMP ligand (FIG. 6D), while those animals that were maintained without TMP showed minimal or no immunoreactivity to GCH1 protein (FIG. 6C). Using an antibody directed to the DD, the inventors were able to confirm that the GCH1 immunoreactivity in the TH+DD-GCH1 group was accompanied with a matching staining for the DHFR (FIG. 6H) and that the same antibody stained only few cells in the group that did not receive TMP (FIG. 6G).

[0187] With respect to the expression of the TH enzyme, 6-OHDA lesion removes the endogenous dopaminergic fiber terminals in the striatum essentially completely (compare FIG. 7A with F), which can also be appreciated as loss pre-terminal axon projections seen transiting the globus pallidus (GP) (compare FIG. 8A with F). Expression of the human TH transgene from the AAV vector introduces a new TH enzyme pool within the striatal neurons (FIG. 7K) as well as the GP (FIG. 8K). Interestingly in the TH+DD-GCH1 group that did not receive TMP, the absence of GCH1 immunoreactivity was associated with low level of TH expression observed only in a few cells (FIG. 7P and FIG. 8P). This was in contrast to animals in the TH+DD-GCH1 group that received TMP, which showed similarly robust TH expression that were seen in animals in the constitutively active TH+GCH1 group (compare panels K and U in FIGS. 7 and 8).

[0188] These observations suggested that the tight regulation of DOPA production and functional effects from DD-GCH1 vector was achieved due to two levels of control; first, directly on the stability of the DD-fused CGH1 enzyme, resulting in control of BH.sub.4 levels, second and indirectly, stability of the TH enzyme (in addition to its biological activity) via the availability of BH.sub.4 itself.

[0189] Finally, the inventors assessed any potential toxic effects of transgene expression by staining serial sections from all groups of animals with either CV to see all cellular profiles, NeuN to assess the total neuronal profiles, Iba1 and ED1 antibodies for evidence of microglial activation. CV and NeuN stained specimens at the level of striatum and GP (second and third columns in FIGS. 7 and 8) showed no clear evidence of cell loss or perivascular hypercellularity and no apparent alterations were seen in NeuN-positive profiles either. No clear alterations in microglial morphologies were noted in the Iba1 stained specimens and abundance of ED1 profiles were only minimally increased suggesting that the activation of the neuroinflammatory processes were of low grade (compare groups within column 4 and 5 in FIGS. 7 and 8).

Translation of the Tunable Gene Expression Concept to Non Human Primates

[0190] The studies in rodents were followed up with a proof-of-concept experiment in a non-human primate model of Parkinson's disease induced by systemic MPTP intoxication. Once the stabel parkinsonian state was established, two monkeys were dosed with AAV5-CBA-DD-GCH1 vectors at 4 increasing doses between (5.0E12 and 1.2E14 vg/ml) in each of the two sides of the brain using caudate nucleus and putamen as separate sites for injections. One of the monkeys was then treated with TMP to stabilize the DD-GCH1 fusion protein, while the other one was maintained without any TMP. At the end of the followup period, both monkeys were killed and tissue processed for histological documentation of gene expression in the brain. FIG. 10 shows the GCH1 immunoreactive cells in the caudate nucleus and putamen. Panels A-D display activation of the transgene in the target nuclei. The strong induction of immunoreactivity after TMP treatment in this brain shows that GCH1 protein expression can be controlled in the non-human primate brain via systemically administered TMP. In the absence of TMP the residual staining is seen only with the highest dose and in small number of cells, while in the three other doses there is essentially no/minimal specific staining detected.

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