NEUROPEPTIDE-EXPRESSING VECTORS AND METHODS FOR THE TREATMENT OF EPILEPSY

Abstract

The present invention provides delivery vectors for transferring a nucleic acid sequence to a cell in vitro, ex vivo or in vivo. The present invention provides methods of delivering a nucleic acid sequence to a cell and methods of treating focal epilepsies.

Claims

1. A delivery vector comprising a DNA sequence encoding pre-prodynorphyin or pre-prodynorphin-variants and wherein said delivery vector drives expression of a pre-propeptide that is pre-prodynorphyin or a pre-prodynorphin-variant wherein said pre-propeptides comprise a signalpeptide and, whereas said pre-prodynorphyin or pre-prodynorphin-variants comprise at least one of the following sequences selected from the group: a. Dyn A that is SEQ Id No. 7 (AA 207-223 of SEQ ID No. 1; ppDyn) or a variant thereof consisting of the first 13 AA (first from the N-terminal end) or a variant thereof consisting of the first 8 AA (first from the N-terminal end) b. Dyn B that is SEQ ID No. 8 (AA 226-238 of SEQ ID No. 1; ppDyn) c. leumorphin that is SEQ ID No. 9 (AA 226-254 of SEQ ID No. 1; ppDyn) d. variants of Dyn A according to SEQ Id No.7 having an amino acid sequence identity of at least 60% within the first 8 AA counted from the N-terminus of SEQ ID No. 7 (YGGFLRRI (SEQ ID NO: 18)) i.e. having an amino acid sequence identity of at least 60% within the sequence YGGFLRRI (SEQ ID NO: 18) comprised in SEQ ID No. 7. e. variants of Dyn B according to SEQ ID No. 8 having an amino acid sequence identity of at least 60% within the first 8 AA counted from the N-terminus of SEQ ID No. 8 (YGGFLRRQ (SEQ ID NO: 19)) i.e. having an amino acid sequence identity of at least 60% within the sequence YGGFLRRQ (SEQ ID NO: 19) comprised in SEQ ID No. 8. f. variants of leumorphin according to SEQ ID No. 9 having an amino acid sequence identity of at least 60% within the first 8 AA counted from the N-terminus of SEQ ID No. 9 (YGGFLRRQ (SEQ ID NO: 19)), i.e. having an amino acid sequence identity of at least 60% within the sequence YGGFLRRQ (SEQ ID NO: 19) comprised in SEQ ID No. 9.

2. A delivery vector according to claim 1, wherein the variants have an amino acid sequence identity of at least 70% within the first 8 AA counted from the N-terminus of SEQ ID No. 7 (YGGFLRRI (SEQ ID NO: 18)), SEQ ID No. 8 (YGGFLRRQ (SEQ ID NO: 19)) or SEQ ID No. 9 (YGGFLRRQ (SEQ ID NO: 19)), respectively.

3. A delivery vector according to claim 1, wherein the variants have an amino acid sequence identity of at least 80% within the first 8 AA counted from the N-terminus of SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9, respectively.

4. A delivery vector according to claim 1 and wherein said delivery vector drives expression of a pre-propeptide that is pre-prodynorphyin or a pre-prodynorphin-variant wherein said pre-propeptide comprise a signalpeptide and, wherein said delivery vector comprises a DNA sequence encoding a pre-prodynorphin-variant that comprises at least one of the following sequences of variants selected from the group: TABLE-US-00012 a. SEQ ID No. 10 (YGZFLRRZRPKLKWDNQ) b. SEQ ID No. 11 (YGZFLRRZFKVVT) c. SEQ Id No. 12 (YGZFLRRZFKVVTRSQEDPNAYSGELFDA), wherein Z stands for any amino acid, and wherein at least one Z in a sequence according to a.; b. or c. is preferably substituted by another amino acid when compared to the wild-type sequence of said dynorphin fragment according to a sequence according to a.; b. or c.

5. A delivery vector according to claim 1 wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome.

6. A delivery vector according to claim 1 comprising a recombinant adeno-associated virus (AAV) vector genome, wherein said vector is a human serotype vector selected from the group comprising serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV or AAV capsid mutants derived thereof, preferably serotype 1 or 2.

7. A recombinant virus particle or a liposome, comprising a delivery vector according to claim 1.

8. The recombinant virus particle or liposome of claim 7, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome and said rAAV vector genome is encapsidated in an AAV capsid or wherein said delivery vector comprises in addition a recombinant lentivirus vector genome and is packaged in a lentivirus particle.

9. A method of delivering a nucleic acid to a cell of the central nervous system, comprising contacting the cell with the delivery vector or recombinant virus particle or liposome of claim 1 under conditions sufficient for the DNA sequence encoding pre-prodynorphyin or pre-prodynorphin-variants to be introduced into the cell.

10. A delivery vector or recombinant virus particle or liposome according to claim 1 for use as medicament.

11. A delivery vector or recombinant virus particle or liposome according to claim 1 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy whereby said delivery vector or recombinant virus particle or liposome provides activation of human Kappa Opiod Receptors in the epileptogenic focus, thereby inhibiting seizures.

12. A delivery vector or recombinant virus particle or liposome according to claim 1 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy whereby said delivery vector or recombinant virus particle or liposome provides activation of human Kappa Opiod Receptors in the epileptogenic focus, thereby inhibiting seizures whereby said delivery vector or recombinant virus particle or liposome leads to on-demand release of peptides with agonistic effects on human Kappa Opiod Receptors in the epileptogenic focus.

13. A delivery vector or recombinant virus particle or a liposome according to claim 1 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy, wherein said vector or recombinant virus particle is suitable for peripheral administration or for intracranial or for intracerebral or for intrathecal or for intraparenchymal administration.

14. A delivery vector or recombinant virus particle or a liposome according to claim 1 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy, wherein said delivery vector or recombinant virus particle or a liposome is applied intracerebral, preferred is applied focal.

15. A pharmaceutical release-on-demand composition delivery vector or recombinant virus particle or liposome according to claim 1, and optionally a pharmaceutically acceptable carrier.

16. A cell infected, preferably in vitro or ex vivo, with a delivery vector or recombinant virus or liposome particle according to claim 1.

17. A method of treating a subject with focal epilepsy in particular mesial temporal lobe epilepsy, or a method of preventing epileptic seizures in a subject that suffers from focal epilepsy: comprising administering a delivery vector, a recombinant virus particle, or a pharmaceutical composition as defined in claim 1 to the subject, whereby preferably said delivery vector or recombinant virus particle or liposome encode pre-propeptides, which after maturation and release provide activation of human Kappa Opiod Receptors in the epileptogenic focus, thereby inhibiting seizures, and wherein preferably said delivery vector or recombinant virus particle or a liposome is applied intracerebral, preferably applied focal.

18. Peptide with agonistic effects on human Kappa Opiod Receptors (KOR) derived from any of the delivery vectors according to claim 1, wherein preferably said peptide is selected from the group comprising the peptides having SEQ ID No.s 10, SEQ ID No.s 11, SEQ ID No.s 12, SEQ ID No.s 13, SEQ ID No.s 14 and SEQ ID No.s 15.

19. Peptide with agonistic effects on human Kappa Opiod Receptors (KOR) wherein said peptide is selected from the group comprising the peptides having SEQ ID No.s 10, SEQ ID No.s 11, SEQ ID No.s 12, SEQ ID No.s 13, SEQ ID No.s 14 and SEQ ID No.s 15.

20. A pharmaceutical release-on-demand composition comprising a peptide according to claim 18.

Description

EXAMPLES

Example 1

[0204] 1.1 AAV Constructs

[0205] Plasmids with the AAV2-ITR-flanked vector backbones contained the human CMV-IE gene enhancer followed by a truncated version of the chicken beta actin promoter. These drive expression of the full-length codon-optimized human ppdyn sequence (AA SEQ ID No. 1) or either of the variants (AA SEQ ID No. 2; SEQ ID No. 3) or a truncated variant (□GFP) or the full length form of EGFP. Gene expression was enhanced by the woodchuck hepatitis virus posttranslational enhancer element (WPRE) followed by a poly A.sup.+ signal sequence derived from bovine growth hormone. The AAV2 ITR was either in its wildtype configuration to yield AAV vectors with ssDNA genomes, or one ITR was truncated so that self-complementary AAV vectors with dsDNA genomes resulted (FIG. 1).

[0206] 1.2 AAV Vector Preparation

[0207] HEK 293 cells were seeded at 25-33% confluency in DMEM with 5% FCS. Cells were transfected 24 hr later by calcium phosphate cotransfection. AAV vectors were produced essentially as described elsewhere (Mietzsch, Grasse et al., 2014). In brief: The two-plasmid (pDG) cotransfection protocol using plasmids for AAV rep, cap, Ad5 helper genes, and the plasmid for rAAV expressing ppdyn (SEQ ID No. 1) or variants thereof (SEQ ID No. 2; SEQ ID No. 3) as described above. The medium was replaced 12 hr later by medium with reduced FCS content (2%). Cultures were harvested 72 hr after transfection by three freeze-thaw cycles for cell lysis. Crude lysates were digested with 250 U/ml benzonase (Merck) at 37° C. for 1 hr to degrade input and unpackaged plasmid DNA, centrifuged at 8,000 g for 30 min to pellet the cell debris.

[0208] 1.3 rAAV Purification

[0209] rAAV vectors were packaged in serotypes 1 or 2 capsids and were purified from benzonase treated, cleared freeze-thaw supernatants by one-step heparin sepharose chromatography or by AVB sepharose affinity chromatography using 1 ml prepacked HiTrap columns on an ÄKTA purifier (GE Healthcare) as follows: Freeze-thaw supernatants were diluted 1:1 in 1-phosphate-buffered saline (PBS) supplemented with 1 mM MgCl2 and 2.5 mM KCl (1.Math.PBSMK) before loading on the column at 0.5 ml/min. The column was washed with 20 ml of 1.Math.PBS-MK at a rate of 1 ml/min. AAV vectors were eluted with 0.1 M sodium acetate and 0.5 M NaCl pH 2.5 at a rate of 1 ml/min and neutralized immediately with 1/10 volume of 1 M Tris-HCl pH 10. Purified rAAV preparations were dialyzed against 1×PBS-MK using Slide-A-Lyzer dialysis cassettes (10,000 MWCO; Thermo Scientific) (Mietzsch, Grasse et al., 2014).

[0210] 1.4. Quantification of rAAV Vector Preparations

[0211] Highly purified rAAV vector preparations were digested with Proteinase K for 2 hr at 56° C. DNA was purified by repeated extractions with phenol and chloroform and precipitated with ethanol. Serial dilutions of capsid-released AAV genomes were analyzed by quantitative Light-Cycler PCR, using the Fast Start DNA Master SYBR Green kit (Roche). PCR primers were specific for the bovine growth hormone gene-derived polyA site of the vector backbone (5′-CTAGAGCTCGCTGATCAGCC-3′ and 5′-TGTCTTCCCAATCCTCCCCC-3′). The titer of the highly purified AAV preparations was measured as AAV-DNA containing genomic particles (gp)/ml (Mietzsch, Grasse et al., 2014).

Example 2

[0212] Animals

[0213] C57BL/6N wild-type and pDyn knockout (pDyn-KO) mice were investigated in this study. pDyn-KO mice were backcrossed onto the C57BL/6N background over 10 generations (Loacker et al., 2007). For breeding and maintenance, mice were group-housed with free access to food and water. Temperature was fixed at 23° C. and 60% humidity with a 12 h light-dark cycle (lights on 7 am to 7 pm). All procedures involving animals were approved by the Austrian Animal Experimentation Ethics Board in compliance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes ETS no.: 123, and the Canadian Council on Animal Care. Every effort was taken to minimise the number of animals used.

Example 3

[0214] Kainic Acid Injection and Electrodes Implantation

[0215] Male mice (12-14 weeks) were sedated with ketamine (160 mg/kg, i.p.; Graeub Veterinary Products, Switzerland) and then deeply anesthetised with sevoflurane through a precise vaporizer (Midmark, USA). Mice were injected with 50 nl of a 20 mM KA solution into the hippocampus (RC −1.80 mm; ML+1.80 mm; DV −1.60 mm) as previously described (Loacker et al., 2007). Two electrodes (one cortical and one depth electrodes) were implanted immediately after KA administration. Epoxylite-coated tungsten depth electrodes (diameter 250 μm; FHC, USA) were placed into the hippocampus aimed at the CA1 area (RC −1.80 mm; ML+1.80 mm; DV −1.60 mm). Surface electrodes were gold-plated screws placed into the skull on top of the motor-cortex (RC+1.70 mm; ML+1.6 mm with the bregma as a reference point) to monitor the generalisation of abnormal EEG activities. An additional surface electrode was placed on the cerebellum as ground and reference. Electrodes were secured in place with dental acrylate (Heraeus Kulzer GmbH, Germany).

Example 4

[0216] rAAV Injections

[0217] For experiments requiring rAAV administration, a guide cannula was implanted, which was attached to the hippocampal depth electrode. All animals received meloxicam (2 mg/kg) 20 minutes before surgery as an analgesic treatment. For the rAAVs injections animals were mildly anesthetized in a sevoflurane chamber during the time of the injection (20 minutes). The injection was made through the guide cannula with an injection pump at a flow of 0.1 μL/min, a total volume of 2 μL was injected.

Example 5

[0218] EEG Recoding and Analysis

[0219] The EEG was obtained using a wireless recording device (Neurologger, TSE, Germany) and automatically analysed using SciWorks Software (Datawave Technologies, USA). EEGs were filtered for epileptiform spikes defined as a high amplitude discharges (≥3× baseline) lasting less than 70 ms. Spike trains were defined as the occurrence of at least three spikes with a frequency higher than 1 Hz and lasting for at least 1 s (see FIG. 2). Spikes with lower frequencies were counted as inter-ictal spikes. Prolonged hippocampal paroxysmal discharges (hpd) were defined as spike trains lasting for a minimum of 10 sec (Zangrandi et al., 2016). Generalized seizures were assessed visually by co-appearance for high voltage EEG abnormalities in the hippocampal depth and motor-cortical surface electrode (see FIG. 3).

Example 6

[0220] Spatial Memory Testing (Barnes Maze)

[0221] To assess learning and memory of naive and treated animals the Barnes maze was used. the Barnes maze was executed at 60 lux on a flat circular table (diameter 100 cm) with 20 holes around its perimeter. Amongst those, only one allowed the mouse to exit the maze into an escape dark box. Visual clues were placed around the disk with an interval of 90°. The first day was the habituation day, mice were allowed to freely explore the maze during 5 minutes with the target hole open. The position of the escape box was kept constant during the entire experiment. When the mouse found the hole, the box was closed and the animal was kept in there for 2 minutes to let it associate the escape box as a secure place. If the animal didn't find the target hole during the 5 minutes, the animal was gently guided to the hole. Acquisition was made during the next 4 days. 3 trials of 3 minutes maximum were performed, as soon as the mouse find the hole, it was kept for 2 minutes inside. If the mice didn't find it after the 3 minutes it was gently guide to the hole. The primary errors done by the animal and the latency to find the hole were calculated and defined as learning criteria. The probe trial was executed one the sixth day. All the holes were closed and the mouse was free to explore the maze during 5 minutes. For evaluation, the board was divided in quadrants and the time spent in each quadrant was measured (see FIG. 5). The quadrant previously containing the open escape hole is referred to as Q1, this is flanked by Q2 and Q4, while Q3 is opposing Q1.

Example 7

[0222] Microdialysis

[0223] Microdialysis was performed on pDyn-KO animals which had received rAAV-pDyn injection into one hippocampus as described above 3 weeks before the microdialysis experiment. At the time of vector injection (RC −1.80 mm; ML+1.80 mm; DV −1.60 mm), animals were implanted with a stimulation electrode (RC −4.20 mm; ML+3.20 mm; DV −4.90 mm) and a guide cannula targeting the hilus of the rAAV-injected hippocampus. For microdialysis MAB-2 probes (SciPro, Sanborn, N.Y.) were placed into the hippocampus and flushed by artificial CSF (140 mM NaCl; 3.0 mM KCl; 1.25 mM CaCl.sub.2; 1.0 mM MgCl.sub.2; 1.2 mM Na.sub.2HPO.sub.4; 0.3 mM NaH.sub.2PO.sub.4; 3 mM glucose, pH 7.2) at a rate of 0.4 μL/min. ACSF was collected for 3×25 min followed by 25 min low frequency stimulation (300 μA; isolated 0.3 msec square pulses with 10 sec interval, ISO-STIM 01D, NPI, Tamm, Germany). After another 25 min baseline, 25 min of high frequency stimulation were performed (150 μA; trains of 0.3 msec square pulses 20 msec apart for 1 sec; trains were separated by 10 sec.).

Example 8

[0224] Dynorphin B Enzyme Immunoassay (EIA)

[0225] The content of processed Dynorphin B in the eluate of microdialysis experiments was measured by a specific Dyn B (SEQ ID No. 8) EIA (S-1429; Peninsula; San Carlos, Calif.), according to manufacturer's manual. In short, samples were incubated with the antiserum for 1 hour, followed by an overnight incubation with the Bt-tracer. On the second day, streptavidin-HRP was added for one hour after five washes with EIA buffer. After another 5 washes, samples were reacted with TMB solution for 5 minutes and then analysed on a plate reader a 450 nm. Dyn B (SEQ ID No. 8) content was analysed based on calibration samples run in parallel and expressed as ng/ml (see FIG. 4).

Example 9

[0226] Statistical Analysis

[0227] Following acquisition, electrophysiological recordings were viewed and analysed using pClamp 10.3 (Molecular devices). Prism 5 for Mac (version 5.0f) was used to perform a statistical analysis of in vivo experiments and to generate figures. For the statistical analysis, a one-way ANOVA with a Dunnett post hoc test was applied to in vivo experiments. For electrophysiology, the two-tailed, paired t-tests were applied for resting membrane potential analysis, and a one-way ANOVA was used to compare drug effects on IPSC. A p value less than 0.05 was considered significant. Data are presented as mean±standard error of the mean (SEM).

Example 10

[0228] Seizure Threshold

[0229] The seizure threshold is a measure for the susceptibility to develop seizures. The resistance against seizure-inducing agents or stimuli is used as readout in animals. Infusion of pentylenetetrazole, a GABA.sub.A receptor antagonist, into the tail vein of rodents is an accepted method to measure the seizure threshold. Anticonvulsant activity of substances or treatments can be demonstrated by an increased seizure threshold upon application. pDyn deficient mice display a lower seizure threshold than wild-type mice. This can be rescued by kappa opioid receptors agonists.

[0230] Seizure threshold was determined by pentylenetetrazole (PTZ) (see FIG. 6) tail-vein infusion on freely moving pDyn deficient mice at a rate of 100 μl/min (100 μg/ml PTZ in saline, pH 7.4). Infusion was stopped when animals displayed generalized clonic seizures. The seizure threshold dose was calculated from the infused volume in relation to body weight. Dyn B or modified variants thereof were dissolved in DMSO and diluted to the final dosages in saline containing final concentrations of 10% DMSO, 3% Tween 80. 3 μl of peptide-solution were applied 30 min. before tail-vein infusion under mild sevoflurane anaesthesia into the cisterna magna. The following peptides were tested: Dyn B=SEQ ID No. 8, Dyn B G3A=SEQ ID No. 11 wherein Z at position 3 is alanine, Dyn B G3A+Q8A=SEQ ID No. 11 wherein Z at position 3 is alanine and Z at position 8 of SEQ ID No. 11 is alanine, Dyn B Q8A=SEQ ID No. 11 wherein Z at position 8 is alanine.