Gene therapy vectors expressing a proprenodenulin variant for

Abstract

The subject matter of the present invention is a delivery vector comprising a DNA sequence encoding pro-predynorphin or a variant of pro-predynorphin and wherein said delivery vector drives the expression of pro-predynorphin in a target cell and wherein said delivery vector comprising said DNA sequence is capable of releasing predynorphin or a variant of predynorphin from a target cell as needed, and wherein the pro-pro-peptide is pro-pro-peptide or a pro-pro-peptide variant and wherein the pro-pro-peptide comprises a signal peptide wherein the signal peptide is an N-terminal extension of a neonatal polypeptide chain and wherein the signal peptide mediates a protein targeting an endoplasmic network lumen, and wherein the pro-peptidogen comprises an N-terminal pro-peptidogen fragment on the C-terminus of the signal peptide and wherein the N-terminal pro-peptidogen fragment (i) comprises a sorting motif comprising elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL, or comprising ii) a sorting motif of pro-neuropeptidic or protein sorted into a dense core macrovesicle, except for pro-prodynorphin as defined herein, where in certain embodiments, x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X may independently be any amino acid, such as any amino acid, or any amino acid, such as any amino acid, or any amino acid, such as any amino acid, or any amino acid, such as any amino acid, or any amino acid, such as any amino acid, or any amino acid, such as any amino acid, or any amino acid. And wherein the N-terminal pro-peptidic fragment consists of 16 to 90 amino acids, as well as corresponding peptides, DNA molecular viral vectors and particles comprising any of these.

Description

Technical Field

[0001] The present invention provides a delivery vector for transferring a nucleic acid sequence encoding a pre-propeptide into a cell in vitro, ex vivo or in vivo. The present invention provides a method for delivering a nucleic acid sequence into a cell and a method for treating focal epilepsy. Background Art

[0002] With a prevalence of 1-2%, epilepsy is the most common neurological disease worldwide (McNamara et al., 1999), with focal epilepsy accounting for approximately 70%. Among them, mesial temporal lobe epilepsy (mTLE) is the most common clinical manifestation. In mTLE, the lesions are located in or near the hippocampus, which regulates learning, memory, and emotional control. mTLE represents an acquired disease that is usually induced by traumatic brain injury. In addition, central nervous system infections, high fever, brain tumors, or vascular malformations may lead to epileptic foci. As the disease progresses, hippocampal sclerosis may occur with neurological deficits. They represent the key clinical features of this mTLE subtype (for review, see Engel et al., 2001). Despite the introduction of a wide range of antiepileptic drugs over the past few decades, the incidence of drug-resistant epilepsy (30% to 70%) has not improved since Coatsworth's early studies in 1971 (Coatsworth et al., 1971, Loscher et al., 2011). To date, surgical resection of the epileptogenic focus remains the final treatment option for patients in whom the epileptogenic focus can be clearly defined and well distinguished from other critical central nervous system areas. Language centers close to hippocampal lesions represent a major contraindication to epilepsy surgery. But even if epilepsy surgery is possible, long-term seizure freedom is not guaranteed. At best, only 50% of patients remain seizure-free for at least one year after resection of the epileptogenic focus (Spencer et al., 2008).

[0003] Since the early 1980s, there has been evidence that the opioid, dynorphin (Dyn), acts as a modulator of neuronal excitability in vitro (Henriksen et al., 1982, Siggins et al., 1986). In line with this, the deletion of the prodynorphin (pDyn) coding sequence in mice (Loacker et al., 2007) and the low Dyn levels found in humans due to mutations in the promoter region of the pDyn gene (Stogmann et al., 2002, Gambardella et al., 2003) are associated with an increased susceptibility to the development of epilepsy. In most animal models of temporal lobe epilepsy (TLE; including epilepsy arising in the temporal lobe = lateral TLE and mTLE), cortical and hippocampal pDyn gene expression decreases after an initial transient peak of overexpression (reviewed in (Simonato et al., 1996, Schwarzer et al., 2009). This finding is consistent with the description of a putative transient postictal increase in pDyn mRNA levels in hippocampal granule cells (Pirker et al., 2009). Early studies have shown an overall reduction in Dyn immunoreactivity in surgically resected brain tissue from patients with mTLE (de Lanerolle et al., 1997).

[0004] Dynorphins preferentially act on kappa opioid receptors (KOR). Despite the reduction of endogenous Dyn, KOR can still be used as a drug target under epileptic conditions, and the application of KOR agonists can inhibit experimental epileptic seizures (Tortella et al., 1988, Takahashi et al., 1990, Solbrig et al., 2006, Loacker et al., 2007, Zangrandi et al. 2016). Various selective KOR agonists applied by different routes of administration produced time- and dose-dependent effects similar to those of epilepsy models treated with phenytoin or phenobarbital (for review, see Simonato et al., 1996). It has been previously demonstrated that activation of KOR promotes the survival of hippocampal neurons after the acute epileptic period after unilateral injection of kainic acid in mice (Schunk et al., 2011). Summary of the invention

[0005] The object of the present invention is to provide a delivery vector to transfer a nucleic acid sequence encoding a pre-pro-peptide or a peptide comprising a sequence capable of packaging the pro-peptide (e.g., prodynorphin) into a vesicle, wherein the pro-peptide undergoes maturation and releases an active substance as a dynorphin or a dynorphin variant after a series of action potentials exceeding a certain excitation threshold. Therefore, the object of the present invention is to provide a delivery vector for transferring a nucleic acid sequence into a cell in vitro, ex vivo or in vivo. In particular, the object of the present invention is a vector-based therapy for treating focal epilepsy with pre-prodynorphin or dynorphin or a variant thereof. The delivery vector of the present invention comprising a nucleic acid encoding pre-prodynorphin or prodynorphin or dynorphin or a variant thereof will transduce neurons, express, process, store and release pre-prodynorphin or prodynorphin or dynorphin or a variant thereof, thereby providing activation of KORs in the epileptogenic focus, thereby inhibiting epileptic seizures.

[0006] The purpose of the present invention is to further develop AAV-based gene vectors for expressing pre-prodynorphin for treating focal epilepsy. Here, AAV vectors are preferably used for the expression of pre-prodynorphin. In the present invention, after vector transduction of neurons, pre-prodynorphin is expressed and processed into dynorphins (dyn) of different types and lengths, which are released after high-frequency trigger stimulation.

[0007] The present invention is further based on truncation of the ppDYN protein. In order to avoid interference with correct vesicle targeting or correct processing into defined peptides in neurons, two strategies were used:

[0008] First, the so-called N-peptide region of ppDyn was shortened. Here, it was found that this region contains a specific sorting motif, which was preserved. Secondly, for some neuropeptides (e.g., pPOMC or ppEnk), these N-terminally located signal sequences and sorting sequences are significantly shorter than those located in the N-terminus of ppDyn. Therefore, fusion proteins were constructed that combined the N-terminus of a part of such neuropeptide precursor molecules derived from pPOMC with the C-terminal sequence of ppDyn encoding various active Dyn peptides ( Figure 1 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Overview of the shortening of the human ppDyn cDNA.

[0010] Figure 2: Results of ELISA measuring (A) dynorphin A (DynA) and (B) dynorphin B (DynB) levels in the brain parenchyma after CNS transduction of AAV vectors expressing the indicated ppDyn variants. Variant A = shortened N-peptide retaining the sorting motif. Variant B = signal and N-peptide replaced by POMC signal and sorting motif. Variant C = shortened N-peptide with deleted sorting motif. ipsi = AAV transduction site, contra = non-transduction (control) site.

[0011] Figure 3 : Inhibition of epileptic seizures by two scAAV vector variants containing shortened pDyn cDNA or with alternative signal and sorting sequences. Data represent N±SEM (N=3).

[0012] Figure 4 : Overview of DNA sequence elements of AAV-pDyn vector variants.

[0013] Shown are DNA sequence elements of the displayed AAV vectors. SEQ ID numbers refer to those in the main body of the text.

[0014] Dark grey boxes: DNA sequence elements derived from AAV serotype 2. ITR = inverted terminal repeat, ΔITR = inverted terminal repeat with deleted terminal resolution sites as used in self-complementary (sc) AAV vectors.

[0015] Light grey boxes: DNA sequence elements of promoters or other regulatory elements of gene expression. CBA promoter = cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter followed by a chimeric intron. sCBA promoter = shortened cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter followed by a chimeric intron. hSyn promoter = human synapsin promoter. WPRE = woodchuck hepatitis virus posttranscriptional regulatory element, bGH pA+ = poly A+ signal sequence of the bovine growth hormone gene. SpA+ = synthetic poly A+ signal sequence.

[0016] White boxes: DNA sequence elements of the human ppDyn cDNA. The ppDyn cDNA sequence is codon optimized. Codon optimized version 1 was used for SEQ ID No. 74 and SEQ ID No. 75. Codon optimized version 2 was used for SEQ ID No. 76 to 80 and for the pDyn part of SEQ ID No. 81. Pre = DNA sequence of the ppDyn signal sequence, pro = DNA sequence covering the N-peptide of ppDYN, pro1, pro2 and proDs = different abbreviated versions of "pro" for the N-peptide of ppDyn. POMC = cDNA sequence of the N-terminal part of the neuropeptide POMC spanning the pre and pro elements and replacing those of ppDyn. pDyn = prodynorphin.

[0017] Figure 5 : Reduced epileptic activity after injection of AAV-pDyn expressing Seq ID No.70. Hpd, generalized epileptic seizures, and spike cohorts were measured within 48 hours of each time interval. The data are shown as the percentage of pre-treatment epileptic activity for Hpd and spike cohorts (left y-axis). Generalized epileptic seizures are given as the absolute number of seizures (right y-axis, N=7-9, p=0.0025 for Hpd, p<0.0001 for spike cohorts, and p=0.0001 for generalized epileptic seizures (one-way ANOVA). Starting from 7 days after treatment, the mice had a significant reduction in drug-resistant focal epileptic seizures. After 4 weeks, generalized epileptic seizures were almost completely eliminated.

[0018] Figure 6 : Reduction of epileptic activity after injection of AAV-pDyn expressing Seq ID No. 77. HPD was measured over 48 h at each time interval. Data are shown as time spent in HPD. N=2-3. Starting 10 days after AAV delivery, mice had a significant reduction in drug-resistant focal epileptic seizures. DETAILED DESCRIPTION

[0019] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, and

[0020] wherein the delivery vector drives expression of the prepropeptide in the target cell, and

[0021] wherein the delivery vector comprising the DNA sequence is capable of releasing dynorphin or a dynorphin variant from a target cell as desired, and

[0022] wherein the prepropeptide is preprodynorphin or a preprodynorphin variant, and

[0023] wherein the prepropeptide comprises a signal peptide, wherein the signal peptide is an N-terminal extension of the nascent polypeptide chain and wherein the signal peptide mediates targeting of the protein to the lumen of the endoplasmic reticulum, and,

[0024] wherein the pre-propeptide comprises (i) an N-terminal pro-peptide fragment at the C-terminus of the signal peptide and wherein the N-terminal pro-peptide fragment comprises a peptide containing elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), wherein x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X can independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in a first sequence of, e.g., 1 to 20 Xs, each X can individually be any amino acid, and in a second sequence of, e.g., 1 to 10 Xs, each X can individually be any amino acid, in particular as further defined herein), or wherein the pre-propeptide comprises (ii) a pre-pro-neuropeptide or an N-terminal pro-peptide fragment of a protein sorted into dense-core large vesicles other than pre-prodynorphin at the C-terminus of the signal peptide, and wherein the N-terminal pro-peptide fragment comprises the sorting motif of the pre-pro-neuropeptide or protein sorted into dense-core large vesicles, and wherein the N-terminal pro-peptide fragment consists of 16 to 90 amino acids, and

[0025] wherein the pre-prodynorphin or pre-prodynorphin variant comprises at least one of the following sequences selected from the group consisting of a, b, c, d, e, f:

[0026] a. Dyn A, which is SEQ ID No. 2 (AA 207-223 of SEQ ID No. 1; ppDyn) or a variant thereof consisting of the first 13 AAs (the first 13 starting from the N-terminus) or a variant thereof consisting of the first 8 AAs (the first 8 starting from the N-terminus)

[0027] b. Dyn B, which is SEQ ID No. 3 (AA 226-238 of SEQ ID No. 1; ppDyn)

[0028] c. Leumorphin, which is SEQ ID No. 4 (AA 226-254 of SEQ ID No. 1; ppDyn)

[0029] d. A variant of Dyn A according to SEQ ID No.2, which has at least 60% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No.2 (YGGFLRRI), that is, it has at least 60% amino acid sequence identity within the sequence YGGFLRRI contained in SEQ ID No.2.

[0030] e. A variant of Dyn B according to SEQ ID No.3, which has at least 60% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No.3 (YGGFLRRQ), that is, it has at least 60% amino acid sequence identity within the sequence YGGFLRRQ contained in SEQ ID No.3.

[0031] f. A variant of leuproreceptin according to SEQ ID No.4, which has at least 60% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No.4 (YGGFLRRQ), that is, it has at least 60% amino acid sequence identity within the sequence YGGFLRRQ contained in SEQ ID No.4.

[0032] In the present context, 60% sequence identity is defined as follows: 3 of the first 8 N-terminal amino acids can be removed or replaced by another amino acid. In the case of truncated peptide variants, the percentage of sequence identity of the shortened peptide is calculated. The introduction of additional amino acids is considered as a gap in the original sequence, and deletions are considered as gaps in the modified peptide for calculating sequence identity (YGGFLRRQ differs from YG-FLRRQ by only 1 AA, although the AAs at positions 3, 4, 5, 6 and 7 are now different). In any case, a variant of SEQ ID No. 2 having at least 60% amino acid sequence identity in the first 8 AAs starting from the N-terminus can be a variant comprising the sequence YGZFLRKZ, wherein each Z individually represents any amino acid and K replaces R again at position 7, retaining the peptidase recognition site (RK or RR).

[0033] In the present invention, "amino acid" refers to naturally occurring amino acids, more specifically, they are typical amino acids, ie amino acids directly encoded by the codons of the universal genetic code.

[0034] Throughout the present invention, "Z" in the amino acid sequence represents any naturally occurring amino acid. In a specific embodiment, "Z" can be selected from the group consisting of alanine, glycine, asparagine, glutamine, leucine, serine, valine and isoleucine.

[0035] Preproneuropeptides other than preprodynorphin are known to the person skilled in the art and are described, for example, in Zhang et al., Progress in neurobiology 90 (2010) 276-283:.

[0036] The preneuropeptides or proteins other than predynorphin that are sorted into dense core large vesicles can be selected from the group including but not limited to: pre-pro-Substance P, pPOMC, pre-tachykinin A, pre-tachykinin B, pre-opiomelanocortin, pre-cholecystokinin, pre-chromogranin B, calcitonin gene-related peptide (CGRP), pre-proEnkephalin, pre-proBDNF, pre-proTachykinin, pre-proSomatostatin, pre-pro-VIP, pre-pro-CCK, pre-proNociceptin or pre-proNPY.

[0037] Prepro-neuropeptides or proteins, including preprodynorphin, that are sorted into dense core large vesicles have a signal peptide at their extreme N-terminus that directs their translocation into the endoplasmic reticulum (ER), as described above. Neuropeptides are expressed in neurons and are secreted in response to physiological or pathological stimuli, meaning that they are released on demand. Neuropeptide prohormones exhibit a wide variety of sorting motifs. Some of these have a signal peptide containing the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), wherein x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X can independently be any amino acid as described above. Other preneuropeptides, such as preneuropeptide Y, contain a sequence different from DLX x EX y Another sorting motif other than L (SEQ ID No. 36). In any case, the sorting should be comprised in said N-terminal pro-peptide fragment consisting of 16 to 90 amino acids as defined above.

[0038] It should be noted that the elements DL and EX of the sorting motif yThe distance between L can vary and is particularly relevant if these elements are present in the respective amino acid sequences; the sorting motif is usually formed by the spatial proximity of said elements to one another. For the avoidance of doubt, element DL refers to the amino acid sequence asparagine-leucine, while element EX y L refers to the amino acid sequence Glutamic acid-X y -leucine, where X y As defined herein. In certain embodiments, elements DL and EX y L to DL…EX y The order in which L appears in the amino acid sequence in question, reading from the N-terminus to the C-terminus of the sequence, in other words, DL is more likely to be present than EX. y L is closer to the N terminus.

[0039] In certain embodiments, in the case of a polypeptide consisting of the amino acid sequence DLX x EX y L (SEQ ID No. 36), x is an integer from 2 to 13, more specifically from 5 to 10, more specifically from 5 to 8, or alternatively, x is 2, 11 or 13. In certain embodiments, in the amino acid sequence DLX x EX y In the sorting motif consisting of L (SEQ ID No. 36), y is an integer from 1 to 5. In certain embodiments, in the sorting motif consisting of the amino acid sequence DLX x EX y L (SEQ ID No. 36), x is an integer from 2 to 13, more specifically 5 to 10, or alternatively, x is 2, 5, 8, 10 or 13, and y is an integer from 1 to 5.

[0040] In certain specific peptides, x is 2 (POMC), 11 (pDyn) or 13 (BDNF or TAC1).

[0041] In certain specific peptides, y is 1-2 (TAC1), 2 (POMC), 2-5 (BDNF), or 3-5 (pDyn).

[0042] According to the present invention, the delivery vector comprises a DNA sequence encoding a pre-propeptide of a dynorphin or a dynorphin variant. This means that the vector comprises a DNA sequence encoding a signal peptide fused to the pro-peptide. As an aspect, the present invention provides a delivery vector for transferring a nucleic acid into a cell, the delivery vector comprising a segment encoding a signal peptide that targets the pre-propeptide to the lumen of the endoplasmic reticulum. The DNA sequence encoding the signal peptide may be a sequence according to SEQ ID No.: 11. In another aspect of the present invention, the delivery vector comprises a DNA sequence encoding a pro-peptide fragment. In a specific aspect of the present invention, the pro-peptide fragment is a sequence according to SEQ ID No.5.

[0043] The advantage of the present delivery vector is that dynorphin or dynorphin variant is released on demand. Specifically, this means that prodynorphin (or a variant of prodynorphin) is packaged in a vesicle, undergoes maturation, and is released as needed under high-frequency stimulation (e.g., stimulation ≥8Hz) during an epileptic seizure. Therefore, in particular, the release-on-demand formulation provides pre-prodynorphin (or a variant of pre-prodynorphin), which is then packaged in a vesicle, undergoes maturation, and releases the active substance as dynorphin or dynorphin variant when the action potential frequency exceeds a certain threshold. In other words, dynorphin or dynorphin variant is released "on demand" from target cells, particularly meaning "release at high-frequency stimulation" and / or "release at an action potential frequency exceeding a certain threshold" that occurs during an epileptic seizure. The specific threshold can be a threshold of ≥6Hz, in another embodiment, ≥7Hz, in another embodiment, ≥8Hz, in another embodiment, ≥9Hz. This means that the release on demand is triggered by an increase in the firing frequency of neurons. The increased neuronal firing frequency can be measured by EEG (electroencephalography) as a spike train, and "increased" means that the frequency of spikes in the train measured by EEG in the subject is ≥6 Hz, in another embodiment, ≥7 Hz, in another embodiment, ≥8 Hz, and in another embodiment, ≥9 Hz.

[0044] This means that the present delivery vector drives the expression of pre-pro peptides, thereby being able to provide dynorphin or dynorphin variants on demand, because this delivery vector first expresses pre-pro peptides in neurons, where the produced pro peptides are sorted into dense core large vesicles, where they are enzymatically processed and the derived peptides are stored until sufficiently strong stimulation leads to their release, i.e., the release is triggered by an increase in the firing frequency of neurons as described above. The Dyn peptide binds to presynaptic and / or postsynaptic KORs, which activate G proteins and, among other things, modulate ion channels to inhibit further amplification and spread of neuronal excitation. Translation of the ppDyn signal peptide is the initial step, directing ppDyn to the endoplasmic reticulum, from which pro-dynorphin is sorted into "dense core large vesicles" (LDVs). Using existing mechanisms in neurons, pro-dynorphin is enzymatically processed into mature peptides and transported to axon terminals. LDVs are stored in axon terminals and released in a stimulus-dependent manner. High frequency stimulation, such as during epileptic seizures, as described above, induces release, while low frequency stimulation does not. This results in a release-on-demand situation. The released Dyn peptide binds to presynaptic and / or postsynaptic KORs, which activates G proteins, which among other things modulate ion channels, to inhibit further amplification and spread of neuronal excitation.

[0045] In other words, the release-on-demand composition is a composition that releases a peptide having an agonistic effect on human KOR when the subject has an epileptic seizure, the peptide being derived from any of the delivery vectors or recombinant viral particles or liposomes or nanoparticles according to the present invention. An epileptic seizure may be characterized by an increased neuronal firing frequency, which may be measured by EEG (electroencephalography) as a spike train, an increase being a frequency of spikes in a train measured by EEG in the subject of ≥6 Hz, in another embodiment, ≥7 Hz, in another embodiment, ≥8 Hz, in another embodiment, ≥9 Hz.

[0046] Subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide fragment consists of 16 to 90 amino acids, preferably 20 to 90 amino acids, preferably 30 to 90 amino acids.

[0047] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn, wherein the unmodified pro-peptide fragment of ppDyn is

[0048] SEQ ID No.5:

[0049]

[0050] And wherein the modification of the original peptide fragment of SEQ ID No.5 is shortening while maintaining the amino acid sequence DLX x EX y L (SEQ ID No. 36).

[0051] The subject of the present invention is a delivery vector comprising a DNA sequence encoding a preprodynorphin or a preprodynorphin variant according to the invention, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.6:

[0052] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence:

[0053] SEQ ID No:7 DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKENTLSKSLEEKL RGLSDGFREGAESELMRDAQ LNDGAMETGTLYLAEEDPKE QV or SEQ ID No 8 DLGSKSVGEGPYSELAKLSGSFLRKEQV or SEQ ID No 9 DLGSKSVGEG PYSELAKLRK EQV or SEQ ID No.55 DLGSKSVGEG PYSELRKEQV.

[0054] One embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.:56 DLGSKSVGEG PYSELAKLSG SFLKKEQV

[0055] Another embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.:57 DLGSKSVGEG PYSELAKL

[0056] Another embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.:58 DLGSKSVGEG PYSEL

[0057] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal pro-peptide region on the C-terminus of the signal peptide is a modified hybrid pro-peptide fragment of ppDyn, wherein the unmodified pro-peptide fragment of ppDyn is SEQ ID No.5

[0058] and wherein the modification of the pro-peptide fragment of SEQ ID No.5 is to replace a portion of SEQ ID No.5 with a pro-peptide or pro-peptide fragment of a neuropeptide, wherein the modified pro-peptide fragment of ppDyn i) comprises at least one of the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), or ii) comprises a sorting motif of said pre-pro-neuropeptide or a protein other than pre-pro-dynorphin as further defined herein which is sorted into dense core large vesicles. For the avoidance of doubt: A person skilled in the art will readily appreciate that in this particular context, a pro-peptide or pro-peptide fragment of a neuropeptide is clearly different from an unmodified pro-peptide fragment of ppDyn of SEQ ID No. 5, as a portion of the sequence will be replaced.

[0059] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the modified pro-peptide fragment comprises SEQ ID No.10:

[0060] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the modified pro-peptide fragment comprises or consists of a pro-peptide fragment of preproenkephalin, pre-proBDNF, preprotachykinin, preprosomatostatin, pre-pro-VIP, pre-pro-CCK, preproorphanin or pre-proNPY, wherein the pro-peptide fragment comprises the sorting motif as described above or another sorting motif. For example, for ppNPY, different sorting motifs are proposed. In particular, the subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the modified pro-peptide fragment is selected from the group comprising any one of the following sequences: SEQ ID No: 37 to 52.

[0061] These DNA sequences may be selected from DNA sequences encoding polypeptides selected from the group consisting of: SEQ ID No.37: Preproenkephalin-N-peptide SEQ ID No.38: Preproenkephalin-pDyn hybrid: SEQ ID No.39: pre-pro-NPY-N-peptide SEQ ID No.40: pre-pro-NPY-pDyn hybridization: SEQ ID No.41: pre-pro BDNF-N-peptide SEQ ID No.42: pre-pro BDNF-pDyn hybridization: SEQ ID No.43: Prosomatostatin-N-peptide SEQ ID No.44: Prosomatostatin-pDyn hybrid: SEQ ID No.45: Pretachykinin A pro-N-peptide SEQ ID No.46: Preprotachykinin A-pDyn hybrid: SEQ ID No.47: pre-pro-VIP-N-peptide SEQ ID No.48: pre-pro-VIP-pDyn hybridization: SEQ ID No.49: pre-pro CCK-N-peptide SEQ ID No.50: pre-pro CCK-pDyn hybrid SEQ ID No.51: Preproorphanin-N-peptide SEQ ID No.52: Preproorphanin-pDyn hybrid

[0062] Subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to the invention, wherein the modified pro-peptide fragment is optionally flanked by peptidase recognition signals comprising K, R, KR, RK or RR.

[0063] Peptidase (prohormone convertase) recognition signals are known to those skilled in the art and may be single or paired basic amino acids, preferably but not limited to K, R, KR, RK or RR.

[0064] According to the above description, a specific pre-prodynorphin having a shortened modified pro-peptide fragment may be the following: SEQ:53

[0065] The bold amino acids may represent amino acids of the POMC sorting motif and are derived from analogs, the italic amino acids represent Dyn A, the underlined amino acids represent leuprophanin, and the bold and underlined amino acids represent neoendorphin. Finally, the bold italic amino acids represent peptidase recognition signals.

[0066] According to the above description, the specific pre-prodynorphin having hybrid modified pro-peptide fragment may be the following:

[0067] Hybridization pPOMC-ppDyn (SEQ ID No.54):

[0068] The bold amino acids represent the amino acids of the POMC sorting motif, the italic amino acids represent Dyn A, the underlined amino acids represent leuproreceptin, and the bold and underlined amino acids represent neoenkephalin. Finally, the bold italicized amino acids represent the peptidase recognition signal, and the non-peptide encoding part of ppDyn is replaced by part of pPOMC (grey shading).

[0069] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the target cells are neuronal cells of the central nervous system.

[0070] One embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the target cells are a subtype of principal neurons and GABAergic interneurons.

[0071] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the signal peptide is a short peptide sequence of 10 to 30 amino acids at the N-terminus of the precursor protein that will enter the lumen of the endoplasmic reticulum.

[0072] One embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the signal peptide is a short peptide sequence of 10 to 30 amino acids at the N-terminus of the precursor protein that will enter the lumen of the endoplasmic reticulum, wherein a stretch of 5 to 16 amino acids tends to form a single alpha helical structure.

[0073] The core of the signal peptide contains a stretch of hydrophobic amino acids (approximately 5 to 16 amino acids in length); it has a tendency to form a single alpha helix, also known as the "h-region". In addition, many signal peptides begin with a stretch of positively charged amino acids, which may help to enforce the proper topology of the polypeptide during translocation by the so-called positive-inside rule. However, even within the prepro-neuropeptide group, there is a great deal of variation in the amino acid sequence of signal peptides.

[0074] Signal peptides as described herein can exhibit many different sequences. Non-limiting examples of general signal peptides and signal peptides under the present invention can be selected from, but not limited to, the following groups:

[0075] For the avoidance of doubt, according to the present invention, the sorting motif and the signal peptide may be derived from the same pre-pro-neuropeptide or protein that is sorted into dense core large vesicles, or from two different pre-pro-neuropeptides or proteins that are sorted into dense core large vesicles.

[0076] The subject of the present invention is a delivery vehicle, wherein said delivery vehicle results in the on-demand release of a dynorphin or a dynorphin variant having an agonistic effect on the human kappa opioid receptor.

[0077] The subject of the present invention is a delivery vector, wherein the variant has at least 70% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No. 2 (YGGFLRRI), SEQ ID No. 3 (YGGFLRRQ) or SEQ ID No. 4 (YGGFLRRQ), respectively.

[0078] Subject of the present invention is a delivery vector, wherein the variant has at least 80% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, respectively.

[0079] Subject of the present invention is a delivery vector, wherein the variant has at least 90% amino acid sequence identity within the first 8 AA starting from the N-terminus of SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, respectively.

[0080] In a specific embodiment, the subject of the present invention is a delivery vector as described above, wherein the delivery vector comprises multiple DNA sequences encoding SEQ ID No. 2, SEQ ID No. 3 and / or SEQ ID No. 4 or variants thereof, wherein the sequences according to SEQ ID No. 2, SEQ ID No. 3 and / or SEQ ID No. 4 or variants thereof are flanked by peptidase recognition signals.

[0081] This means, as an example, that the delivery vector may comprise twice the DNA sequence encoding SEQ ID No. 2, so that two molecules of the peptide according to SEQ ID No. 2 will be derived from one delivery vector.

[0082] The peptidase (prohormone convertase) recognition signal is known to those skilled in the art and can be a single or a pair of basic amino acids, preferably but not limited to K, R, KR, RK or RR.

[0083] In a specific embodiment, the subject of the present invention is a delivery vector as described above, wherein the delivery vector comprises multiple DNA sequences encoding SEQ ID No. 2 and / or SEQ ID No. 4 or variants thereof, wherein the sequences according to SEQ ID No. 2 and / or SEQ ID No. 4 or variants thereof are flanked by peptidase recognition signals.

[0084] The subject of the present invention is a delivery vector, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentiviral genome, in particular a DNA sequence based on such a recombinant genome.

[0085] The delivery vector prepared according to the present invention can be used to deliver nucleic acids into cells in vitro, ex vivo and in vivo. Specifically, the delivery vector can be advantageously used to deliver or transfer nucleic acids into animal cells, more preferably mammalian cells.

[0086] Suitable vectors include viral vectors (e.g., retrovirus, lentivirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus or herpes simplex virus), lipid vectors, lipid nanoparticles, polylysine vectors, synthetic polyamino polymer vectors for use with nucleic acid molecules, such as plasmids, and the like.

[0087] Any viral vector known in the art can be used in the present invention. Examples of such viral vectors include, but are not limited to, vectors derived from the following: Adenoviridae; Adeno-associated virus (AAV), Birnaviridae; Bunyaviridae; Caliciviridae, Capillovirus group; Carlavirus group; Carmovirus virus group; Group Caulimovirus; Closterovirus Group; Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2 phage group; Corcicoviridae; Group Crypticvirus; group Cryptovirus; Cucumovirus family; virus groupFamily)([PHgr]6phage group); Cysioviridae; Group Carnation ringspot; Group Dianthovirus virus group; Group Broad bean wilt; Group Fabavirus virus group; Family Filoviridae; Family Flaviviridae; Family Furovirus group; Group Germinivirus; Group Giardiavirus; Family Hepadnaviridae; Family Herpesviridae; Family Hordeivirus virus group; Family Illarvirus virus group group); Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae;Luteovirus group; Marafivirus virus group; Maize chlorotic dwarf virus group; Icroviridae; Myoviridae; Necrovirus group; Nepovirus virus group; Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnip yellowfleck virus group; Partitiviridae; Parvoviridae; Pea enation mosaic virus group group; Phycodnaviridae; Picomaviridae; Plasmaviridae; Prodoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae; Reoviridae; Retroviridae; Rhabdoviridae; Group Rhizidiovirus; Siphoviridae; Sobemovirus group; SSV 1-Type Phages; Tectiviridae; Tenuivirus; Tetraviridae; Group Tobacco Mosaic Virus Tobamovirus; Group Tobravirus; Togaviridae; Group Tombusvirus; Group Tobovirus; Totiviridae; Group Tymovirus;And plant virus satellites. The scheme of producing recombinant viral vectors and using viral vectors for nucleic acid delivery can be learned in (Ausubel et al., 1989) and other standard laboratory manuals (e.g., Rosenzweig et al., 2007). Specific examples of viral vectors are those previously used to deliver nucleic acids, including, for example, retroviruses, slow viruses, adenoviruses, adeno-associated viruses (AAV) and other parvoviruses, herpesviruses and poxvirus vectors. As used herein, the term "parvovirus" includes parvoviridae, including autonomous parvoviruses, densoviruses and dependent viruses. The term adeno-associated virus (AAV) includes all vertebrate variants, particularly vertebrate variants of human, primate, other mammals, avian or serpentine origin. Autonomous parvoviruses include members of the genus Parvovirus, genus Erythrovirus, genus Bocavirus, genus Densovirus, genus Iteravirus and subfamily Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, HI parvovirus, muscovy duck parvovirus, bocavirus, bufavirus, tusavirus, and B19 virus, as well as any other virus classified as a parvovirus by the International Committee on Taxonomy of Viruses (ICTV). Other autonomous parvoviruses are known to those skilled in the art. For example, see (Berns et al., 2013). ;

[0088] In one embodiment of the present invention, the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentiviral genome.

[0089] In a specific embodiment of the present invention, the delivery vector further comprises a recombinant AAV vector, wherein preferably, the vector is of a serotype of human or primate origin.

[0090] The subject of the present invention is a delivery vector comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs) preferably derived from AAV serotype 2, optionally derived from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14 or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV), AAV-TT, AAVv66; AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, or a chimeric AAV vector comprising two or more, preferably two, capsid proteins derived from the above-mentioned AAV serotype capsids; in a specific embodiment, the capsid is a capsid derived from AAV serotype 1 and / or 2, further specifically AAV1 and its derived capsids (including AAV1P4, AAV1P5) or AAV2 and its derived capsids (including AAV2-NN) ( et al., 2020; Pavlou et al., 2021; Challis et al., 2022; Naidoo et al., 2018; Hsu et al., 2020; Tordo et al., 2018).

[0091] In a specific embodiment of the invention, the delivery vector comprises a recombinant adeno-associated virus (AAV) vector genome comprising an inverted terminal repeat (ITR) sequence, which is preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66; AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants or chimeras derived therefrom, in particular AAV serotype 1 or 2, further in particular AAV1 and its derived capsids (including AAV1P4, AAV1P5) or AAV2 and its derived capsids (including AAV2-NN) ( et al., 2020; Pavlou et al., 2021; Challis et al., 2022; Naidoo et al., 2018; Hsu et al., 2020; Tordo et al., 2018).

[0092] Chimeric vectors (also called mosaic vectors) and methods for their production are known from the scientific literature, for example from Hauck et al., (2003), or as shown in Noè et al., (2008), and During et al., (2003). In certain embodiments, such chimeric vectors can increase the vector yield during vector production or delivery, and can allow binding on multiple cell surface molecules that serve as receptors (for example, see below for details). In addition, mosaic AAV capsids, in particular mixtures of AAV2 and AAV1, have been shown to result in enhanced neurotropism when delivered to the central nervous system of rodents and non-human primates, but with greatly reduced targeting to astrocytes or microglia, when compared to capsids with only AAV1 (Kimura et al., 2023). Chimeric vectors contain capsid proteins from more than one, usually two different viral serotypes. The ratio between these different capsid proteins can be selected according to the desired cell targeting effect and / or manufacturability or the desired AAV vector yield; for example, the ratio of the chimeric vector comprising capsid proteins from two AAV capsids can be in the range of 90:10 to 10:90; 80:20 to 20:80; 70:30 to 30:70; 60:40 to 40:60; or about 50:50 (in each case meaning the ratio of proteins of the first AAV serotype capsid to the second AAV serotype capsid). In a specific embodiment, such a chimeric vector comprises a capsid protein of AAV serotype 1 and a capsid protein of AAV serotype 2, e.g., a capsid derived therefrom and in the ratios as detailed above, more specifically, a ratio of AAV2 capsid to AAV1 capsid of about 90:10.

[0093] In a specific embodiment of the invention, the delivery vector is a single-stranded (ssAAV) vector or a self-complementary vector (scAAV), also known as a dimeric or duplex AAV vector (McCarty et al., 2001).

[0094] In a specific embodiment of the invention, the delivery vector is a delivery vector as described above, wherein the DNA sequence encoding preprodynorphin or a preprodynorphin variant is operably linked to expression control elements, which include promoters and / or enhancers, which induce sufficient expression of the gene product of interest to obtain a therapeutic effect.

[0095] For example, the encoding nucleic acid can be operably combined with expression control elements (such as promoters, enhancers, other transcription / translation control signals, replication origins, polyadenylation signals and / or internal ribosome entry sites (IRES), etc.). It should also be understood that various promoter / enhancer elements can be used, depending on the desired level and tissue-specific expression. Promoters / enhancers can be constitutive or inducible, depending on the desired expression pattern. Promoters / enhancers can be natural or exogenous, and can be natural sequences or synthetic sequences. So-called exogenous means that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced. Promoter / enhancer elements that work in target cells or subjects to be treated are most preferred. Mammalian promoter / enhancer elements are also preferred. Most preferably, promoter / enhancer elements that are active in human neurons but not or to a lesser extent in glial cells are not active. Promoter / enhancer elements can express transgenes constitutively or inducibly.

[0096] Exemplary constitutive promoters include, but are not limited to, β-actin promoter, cytomegalovirus promoter, cytomegalovirus enhancer / chicken β-actin hybrid promoter and Rous sarcoma virus promoter. Inducible expression control elements are commonly used in applications where it is desired to provide overexpression regulation of heterologous nucleic acid sequences (multiple). Inducible promoter / enhancer elements for gene delivery include neuron-specific, brain-specific, muscle-specific (including cardiac muscle, skeletal muscle and / or smooth muscle), liver-specific, bone marrow-specific, pancreas-specific, spleen-specific and lung-specific promoter / enhancer elements. In a specific embodiment, promoter / enhancer has function in cells or tissues of the central nervous system, and may even be specific to cells or tissues of the central nervous system. Such promoters / enhancers include, but are not limited to, promoters / enhancers that function in the eye (e.g., retina and cornea), neurons (e.g., neuron-specific enolase, AADC, human synaptophysin (hSYN), phosphoglycerate kinase (PGK) or serotonin receptor promoters), glial cells (e.g., S100 or glutamine synthetase promoters), and oligodendrocytes. Other promoters that have been shown to induce transcription in the central nervous system include, but are not limited to, myelin basic protein (MBP) promoters (Tani et al., 1996) and prion promoters (Loftus et al., 2002). Preferred are neuron-specific promoters that show significantly reduced expression, preferably no expression, in glial cells.

[0097] Other inducible promoter / enhancer elements include drug-inducible, hormone-inducible, and metal-inducible elements, as well as other promoters regulated by exogenously supplied compounds, including but not limited to the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO 98 / 10088); the ecdysone-inducible insect promoter (No et al., 1996); the tetracycline repressor system (Gossen and Bujard, 1992); the tetracycline inducible system (Gossen et al., 1995); see also (Harvey et al., 1998); the RU486 inducible system (Wang, DeMayo et al., 1997); (Wang, Xu et al., 1997); and the rapamycin inducible system (Magari et al., 1997).

[0098] In a particular embodiment of the present invention, the promoter and / or enhancer is selected from the group comprising: a constitutively active promoter, such as CMV (cytomegalovirus immediate early gene enhancer / promoter) or CBA promoter (chicken β-actin promoter and human cytomegalovirus IE gene enhancer), or an inducible promoter, comprising a gene switch, a tet operator-derived promoter, or a neuron-specific promoter derived from, for example, phosphoglycerate kinase (PGK), synapsin-1 (SYN), neuron-specific enolase (NSE), preferably of, but not limited to, human origin.

[0099] In a specific embodiment of the present invention, the delivery vector further comprises a post-transcriptional regulatory element, preferably a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a shortened variant thereof (Loeb et al., 1999; Choi et al., 2014). Other possible post-transcriptional regulatory elements are known to those skilled in the art.

[0100] Subject of the present invention are recombinant viral particles or liposomes or nanoparticles comprising the delivery vector according to the invention.

[0101] The subject of the present invention is a recombinant viral particle or liposome or nanoparticle, wherein the delivery vector additionally comprises a recombinant adeno-associated virus (AAV) vector genome and the rAAV vector genome is encapsulated in an AAV capsid, or wherein the delivery vector additionally comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

[0102] For the sake of completeness, it is apparent that as used herein, the terms encapsulation and packaging with respect to viral particles are used interchangeably in some cases and refer to the polynucleotide (ie, vector, genomic DNA, etc.) contained within the capsid or viral particle.

[0103] In addition, the subject of the present invention is a recombinant gene therapy vector comprising an exogenous therapeutic coding sequence, which is flanked by genetic elements for its expression and viral-specific cis elements for its replication, genome packaging, genome integration, etc. The viral genome is packaged as a viral particle, consisting of virus-specific proteins, such as in the case of AAV. In the case of a lentiviral vector, the viral genome and virus-specific proteins (such as reverse transcriptase, etc.) are encapsulated in a lentiviral capsid. They are encapsulated by a lipid bilayer, and the virus-specific proteins are embedded in the lipid bilayer. The liposome contains the above-mentioned nucleotide sequence or the entire DNA backbone, including all regulatory elements of the gene therapy or delivery vector.

[0104] Examples of liposomes include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[amino(polyethylene glycol)-2000]), or DSPE-PEG2000-mal (1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[maleimide(polyethylene glycol)-2000]) or variants comprising sphingomyelin / cholesterol and phosphatidic acid.

[0105] In a specific embodiment of the invention, the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome, and the recombinant AAV (rAAV) vector genome is encapsidated in an AAV capsid.

[0106] Adeno-associated virus (AAV) has been developed as a nucleic acid delivery vector. For review, see (Muzyczka, 1992)(Li and Samulski, 2020). AAV is a helper-dependent parvovirus that requires a helper virus, usually an adenovirus or herpes virus for productive replication. AAV represents a growing family of at least 14 naturally occurring serotypes of human or primate origin. AAVs of other mammalian species or of avian or insect origin have been described (see Berns et al., 2013). AAV has a small icosahedral capsid with a diameter of 18-26 nanometers and contains a single-stranded DNA genome of 4-5 kilobases in length. AAV encapsidates two AAV DNA strands, and the sense or antisense DNA strand is integrated into a single virion. The AAV genome carries two major open reading frames, encoding the genes rep and cap. Rep encodes an overlapping family of nonstructural regulatory proteins. In the most well-studied prototype strain of AAV, AAV2, mRNAs for Rep78 and Rep68 are transcribed from the AAV p5 promoter (Stutika et al., 2015). Rep78 / 68 are required for AAV transcription, AAV DNA replication, AAV integration into the host cell genome, and rescue from it. Rep52 and Rep40 represent N-terminally truncated versions of Rep78 and Rep68 transcribed from a separate promoter, p19, and are required for encapsidation of newly synthesized AAV genomes into preformed AAV capsids. These are formed by the three cap gene-derived proteins VP1, VP2, and VP3. The cap ORF also encodes AAP, an assembly-enhancing protein, and an AAV egress-promoting factor called MAAP. AAP and MAAP do not form part of the capsid (Sonntag et al., 2010; Elmore et al., 2021). The AAV ORFs are flanked by inverted terminal repeats (ITRs) at either end of the genome. The length of these varies between AAV serotypes, in AAV2 these consist of approximately 145 bp, the first 125 bp of which are able to form a Y-shaped or T-shaped duplex structure. The ITRs include the terminal resolution sites (trs), where the replicated concatemeric AAV genome is nicked by Rep to form a unit length of ssAAV genome ready for packaging into AAV capsids. ITRs represent the minimal AAV cis sequences required for DNA replication, packaging, genome integration and rescue. Only these must be retained in the AAV vector to ensure DNA replication and packaging of the AAV vector genome.Foreign genes flanked by AAV-ITRs will be replicated and packaged into AAV capsids, provided that the AAV genes rep and cap are expressed in trans in the selected packaging cells (Muzyczka, 1992). In the case of scAAV, the terminal resolution site (trs) in one of the ITRs is deleted, so that the AAV genome cannot be cleaved by Rep at the affected end, thereby remaining as an unresolved duplex, self-complementary (sc) AAV genome.

[0107] AAV is one of the few viruses that can persist for months or even years in non-dividing cells in vivo, including neurons, muscle, liver, heart, etc. Wild-type AAV2 has been shown to integrate its genome into the host cell genome in a Rep78 / 68-dependent manner, preferring chromosomal sites with DNA sequence homology to the so-called Rep binding sites that form part of the AAV-ITR (Hüser et al., 2014). In contrast, AAV vectors are mostly present as concatemeric nuclear episomes. AAV vectors lacking the AAV genes rep and cap rarely integrate, and if they do, have no genomic preference (Hüser et al., 2014). Nevertheless, long-term persistence of AAV has been demonstrated in non-dividing, post-mitotic cells, including neurons, making AAV vectors ideal for CNS transduction and long-term gene addition therapy of chronic diseases of genetic or acquired origin.

[0108] Typically, the recombinant AAV vector (rAAV) genome will retain only the inverted terminal repeat (ITR) sequences of its native (ssAAV) or trs-deleted (scAAV) version to maximize the size of the transgene that the vector can effectively package. Structural and non-structural protein coding sequences can be provided in trans, for example, by stable integration of the respective genes into packaging cells, from a vector such as a plasmid, or in a recombinant helper virus such as HSV or baculovirus, as reviewed by Mietzsch, Grasse et al., 2014. Typically, the rAAV vector genome contains at least one AAV inverted terminal repeat (ITR), more typically two AAV inverted terminal repeats, which are typically located at the 5' end and 3' end of the heterologous nucleotide sequence. AAV ITRs can be derived from any AAV, including serotypes 1-14. Since AAV2-derived ITRs can be cross-packaged into almost any AAV serotype capsid, the combination of AAV2 ITRs and AAV2 rep is mainly used. AAV terminal repeats do not need to maintain wild-type terminal repeats (e.g., wild-type sequences can be altered by insertions, deletions, truncations, or missense mutations), as long as the terminal repeats mediate the desired functions, such as DNA replication, viral packaging, integration, and / or provirus rescue, etc. The rAAV vector genome typically spans about 70% to about 105% of the wild-type genome size and includes an appropriate packaging signal as part of the AAV-ITR. For ease of packaging into the AAV capsid, the size of the entire vector genome (from ITR to ITR) is preferably less than 5.2 kb, more preferably up to 4.8 kb, to allow the entire recombinant genome to be packaged into a preformed AAV capsid. So-called dimers or self-complementary AAV vectors (scAAV) have been developed to package double-stranded rather than single-stranded AAV genomes (McCarty et al., 2001). scAAV leads to enhanced AAV gene expression, but at the expense of reduced transgenic capacity. The total packaging capacity is only 2.4 kb (from ITR to ITR), which is sufficient to accommodate small genes or cDNAs, including those for neuropeptides.

[0109] Any suitable method known in the art can be used to prepare an AAV vector expressing a nucleic acid of the present invention. AAV vector stocks can be produced by co-transfecting a plasmid expressing an AAV vector genome flanked by ITRs of a transgene with an AAV rep / cap expression plasmid of the desired serotype and an adenovirus-derived AAV replication helper gene (Grimm et al., 2003; Xiao et al., 1998). AAV vectors can also be produced in packaging cell lines of mammalian or insect origin and / or in combination with recombinant helper viruses, such as adenovirus, herpes simplex virus (HSV), another member of the herpes virus family, or baculovirus, as reviewed and discussed by Mietzsch, Grasse et al., 2014.

[0110] Subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant as described in detail herein.

[0111] The subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for delivering a nucleic acid into a cell of the central nervous system, comprising contacting the cell with the delivery vector or the recombinant viral particle or the liposome or the nanoparticle under conditions sufficient to introduce a DNA sequence encoding preprodynorphin or a preprodynorphin variant into the cell, more specifically into the cell nucleus.

[0112] The delivery vector of the present invention provides a method for delivering a nucleic acid sequence to a cell (preferably a neuron) of the central nervous system. The delivery vector can be used to transfer a nucleotide sequence of interest to a cell in vitro, for example, to produce a polypeptide in vitro or for in vitro gene therapy. The carrier is also useful in the method of delivering a nucleotide sequence to a subject in need thereof. In this way, the polypeptide can therefore be produced in vivo in the subject. The subject may need a polypeptide because the subject lacks a polypeptide, or because producing a polypeptide in the subject may produce some therapeutic effects, as a method of treatment or other methods, which will be further explained below.

[0113] In a specific embodiment of the method of delivering a nucleic acid to a cell of the central nervous system, processed preprodynorphin or a preprodynorphin variant is produced and mature dynorphin or a variant thereof is released from the cell.

[0114] In a specific embodiment of the method for delivering nucleic acid to a cell of the central nervous system, the method comprises contacting the cell with the above-described recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant into the cell nucleus. The conditions sufficient to introduce a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant into the cell are contact of the AAV capsid with host cell surface receptors and co-receptors. The AAV1 capsid binds 2-3 sialic acids linked to N-acetylgalactosamine and then binds 1-4-linked N-acetylglucosamine, while the AAV2 capsid binds heparin sulfate proteoglycans on the cell surface, particularly 6-O- and N-sulfated heparin (Mietzsch, Broecker et al., 2014). AAV coreceptors include FGFR-1, integrin aVb5, hepatocyte growth factor receptor (c-met), and the universal AAV receptor. AAVR is required for transduction with AAV1, AAV2, and other serotypes, independent of the presence of specific glycans (Pillay et al., 2016). AAVR binds directly to AAV particles and facilitates transport to the trans-Golgi network. AAV2 has been described to enter the nucleus using the nuclear pore complex, thereby interacting with importin-β alone or in complex with other import proteins (Nicolson and Samuelski 2014). Most parvovirus and AAV serotypes use similar nuclear entry mechanisms (Mattola et al., 2022), and AAV vectors are assembled in the nucleus.

[0115] Subject of the invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for use as a medicament.

[0116] Subject matter of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle as detailed herein for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject suffering from focal epilepsy by activating human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures.

[0117] Specifically, the delivery vector or recombinant viral particle or liposome or nanoparticle as described in detail herein is capable of delivering a DNA sequence encoding pre-prodynorphin or pre-prodynorphin variant as described herein, which in turn drives the expression of pre-pro-peptide in target cells, enabling the dynorphin or dynorphin variant to be released from the target cells as required, thereby leading to the activation of human kappa opioid receptors in the epileptic focus.

[0118] The subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for inhibiting epileptic seizures by activating human kappa opioid receptors in the epileptogenic focus and / or for preventing epileptic seizures in a subject with focal epilepsy by releasing on demand a peptide having an agonistic effect on human kappa opioid receptors in the epileptogenic focus.

[0119] The subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject suffering from focal epilepsy, wherein the vector or the recombinant viral particle or the liposome or the nanoparticle is suitable for peripheral administration or for intracranial administration or for intracerebral administration or for intrathecal administration or for intraparenchymal administration.

[0120] The subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for use in treating focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject, or for preventing epileptic seizures in a subject suffering from focal epilepsy, wherein the delivery vector or the recombinant viral particle or the liposome or the nanoparticle is applied to the brain, preferably focally.

[0121] The subject of the present invention is a pharmaceutical composition, a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for release on demand, and optionally a pharmaceutically acceptable carrier.

[0122] Subject of the invention is a cell infected with a delivery vector or a recombinant virus or a liposome or a nanoparticle, preferably in vitro or ex vivo.

[0123] The subject of the present invention is a method for treating a subject with focal epilepsy, in particular mesial temporal lobe epilepsy, or a method for preventing epileptic seizures in a subject with focal epilepsy, comprising administering to the subject a delivery vector, a recombinant viral particle or a liposome or a nanoparticle, or a pharmaceutical composition, wherein preferably, the delivery vector or the recombinant viral particle or the liposome or the nanoparticle encodes a propeptide which, after maturation and release, activates human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures, and wherein preferably the delivery vector or the recombinant viral particle or the liposome or the nanoparticle is applied intracerebrally or intraparenchymally, preferably focally.

[0124] Description of the following sequence: SEQ ID No.1 (ppDyn)

[0125] Preprocessed human prodynorphin expressed in the human brain. SEQ ID No.2Dyn A SEQ ID No.3Dyn B (dynorphin) SEQ ID No.4: Leu-enkephalin SEQ ID No.5: Unmodified pro-peptide fragment of ppDyn SEQ ID No.6: Modified pro-peptide fragment of ppDyn SEQ ID No.7 Modified pro-peptide fragment of ppDyn SEQ ID No.8 Modified pro-peptide fragment of ppDyn SEQ ID No.9 Modified pro-peptide fragment of ppDyn SEQ ID No.10: N-terminal portion of pPOMC SEQ ID No.11: Signal peptide of preprodynorphin SEQ ID No.12: Signal peptide of preproenkephalin SEQ ID No.13: Signal peptide of pre-neuropeptide Y (ppneuropeptide Y) SEQ ID No.14: Signal peptide of prosomatostatin SEQ ID No.15: Signal peptide of preprotachykinin A SEQ ID No.16: Signal peptide of preprotachykinin B SEQ ID No.17: Signal peptide of prepro-opiomelanocortin SEQ ID No.18: Signal peptide of ppcholecystokinin SEQ ID No.19: Signal peptide of preproorphanin SEQ ID No.20: Signal peptide of prepro-chromogranin B Sorting motif: SEQ ID No.36 DLX x EX y L wherein x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X can independently be any amino acid. SEQ ID No.37: Preproenkephalin-N-peptide SEQ ID No.38: Proenkephalin-pDyn hybrid SEQ ID No.39: pre-pro-NPY-N-peptide SEQ ID No.40: pre-pro-NPY-pDyn hybrid SEQ ID No.41: pre-pro BDNF-N-peptide SEQ ID No.42: pre-pro BDNF-pDyn hybrid SEQ ID No.43: Prosomatostatin-N-peptide SEQ ID No.44: Prosomatostatin-pDyn hybrid SEQ ID No.45: Pretachykinin A pro-N-peptide SEQ ID No.46: Preprotachykinin A-pDyn hybrid SEQ ID No.47: pre-pro-VIP-N-peptide SEQ ID No.48: pre-pro-VIP-pDyn hybrid SEQ ID No.49: pre-pro CCK-N-peptide SEQ ID No.50: pre-pro CCK-pDyn hybrid SEQ ID No.51: Preproorphanin-N-peptide SEQ ID No.52: Preproorphanin-pDyn hybrid Shortened modified ppDyn SEQ ID No.53 Hybridization pPOMC-ppDyn: SEQ ID No 54 Modified pro-peptide fragment of ppDyn SEQ ID No. 55 SEQ ID No.:56 SEQ ID No.:57 SEQ ID No.:58 SEQ ID No.59: ssAAV left ITR (145 bp) SEQ ID No.60: ssAAV / scAAV right ITR (145bp) SEQ ID No.61: scAAV left ITR (Δtrs) (121bp) SEQ ID No.63: CBA promoter: CMV-enhancer, chicken β-actin promoter, chimeric intron (887 bp) SEQ ID No.64: sCBA-Promoter: CMV-Enhancer, Chicken β-Actin Promoter, Chimeric Intron (851 bp) SEQ ID No.65: Human synapsin promoter (448 bp) SEQ ID No.66: WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element (582 bp) SEQ ID No.67: bGH poly A+: bovine growth hormone poly A+ signal sequence (208 bp) SEQ ID No.68: SPA: synthetic poly A+ (49 bp) SEQ ID No.69: ppDyn full-length cDNA optimized codon 1 (765 bp) SEQ ID No.70: ppDyn with shortened N-peptide optimized codon 2 (576 bp) SEQ ID No.71: ppDyn with shortened N-peptide optimized codon 2 (399 bp) SEQ ID No.72: ppDyn with shortened N-peptide optimized codon 2 (519 bp) SEQ ID No.73: ppDyn (417 bp) derived from the N-terminus of POMC fused to the C-terminal portion of pDyn optimized codon 2 SEQ ID No.74: ssAAV-pDyn(2820bp) SEQ ID No.75: scAAV-pDyn(2210bp) SEQ ID No.76: scAAV-pDyn(2603bp) SEQ ID No.77:scAAV-syn-pDyn(2164bp) SEQ ID No.78: scAAV-pDyn(2388bp) SEQ ID No.79: scAAV-pDyn(2270bp) SEQ ID No.80: scAAV-pDyn(2334bp) SEQ ID No.81: scAAV-pDyn(2447bp)

[0126] A specific embodiment of the invention is

[0127] 1. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant,

[0128] wherein the delivery vector drives expression of the prepropeptide in the target cell, and

[0129] wherein the delivery vector comprising the DNA sequence is capable of releasing the dynorphin or dynorphin variant from the target cell as desired, and

[0130] wherein the prepropeptide is preprodynorphin or a preprodynorphin variant, and

[0131] wherein the prepropeptide comprises a signal peptide, wherein the signal peptide is an N-terminal extension of the nascent polypeptide chain and wherein the signal peptide mediates targeting of the protein to the lumen of the endoplasmic reticulum, and,

[0132] wherein the pre-propeptide comprises (i) an N-terminal pro-peptide fragment at the C-terminus of the signal peptide, and wherein the N-terminal pro-peptide fragment comprises a peptide sequence comprising elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX yL (SEQ ID No. 36), wherein x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X can independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in a first sequence of, for example, 1 to 20 Xs, each X can individually be any amino acid, and in a second sequence of, for example, 1 to 10 Xs, each X can individually be any amino acid, in particular as further defined herein), or wherein the pre-propeptide comprises (ii) a pre-pro-neuropeptide or an N-terminal pro-peptide fragment of a protein sorted into dense-core large vesicles other than pre-pro-dynorphin at the C-terminus of the signal peptide, and wherein the N-terminal pro-peptide fragment comprises the sorting motif of the pre-pro-neuropeptide or protein sorted into dense-core large vesicles, and wherein the N-terminal pro-peptide fragment consists of 16 to 90 amino acids, and

[0133] wherein the pre-prodynorphin or pre-prodynorphin variant comprises at least one of the following sequences selected from the group consisting of:

[0134] a. Dyn A, which is SEQ ID No. 2 or a variant thereof consisting of the first 13 amino acids from the N-terminus or a variant thereof consisting of the first 8 amino acids from the N-terminus

[0135] b. Dyn B, which is SEQ ID No. 3

[0136] c. Leuco-enkephalin, which is SEQ ID No.4

[0137] d. A variant of Dyn A, said variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2,

[0138] e. a Dyn B variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 3,

[0139] f. A variant of leuproreceptin, said variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No.4.

[0140] 2. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to embodiment 1, wherein the N-terminal pro-peptide fragment consists of 20 to 90 amino acids, preferably 30 and 90 amino acids.

[0141] 3. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to embodiment 1 or 2, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn, wherein the unmodified pro-peptide fragment of ppDyn is SEQ ID No.5

[0142] and wherein the modification of said pro-peptide fragment of ppDyn is shortening;

[0143] Or the modification is to replace part of SEQ ID No. 5 with the pro-peptide or pro-peptide fragment of the neuropeptide;

[0144] wherein the modified pro-peptide fragment of ppDyn i) comprises at least one element comprising the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), or ii) a sorting motif comprising said pre-pro-neuropeptide or a protein other than pre-pro-dynorphin that is sorted into dense core large vesicles.

[0145] 4. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to any one of embodiments 1 to 3, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.6

[0146] 5. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of embodiments 1 to 3, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.7 or SEQ ID No.8 or SEQ ID No 9

[0147] 6. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to embodiment 3, wherein the modification is to replace a portion of SEQ ID No. 5 with a pro-peptide or a pro-peptide fragment of a neuropeptide; wherein the modified pro-peptide fragment of ppDyn i) comprises at least one comprising the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), or ii) a sorting motif comprising said pre-pro-neuropeptide or a protein other than pre-pro-dynorphin that is sorted into dense core large vesicles.

[0148] 7. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to embodiment 6, wherein the modified pro-peptide fragment comprises or consists of the following sequence SEQ ID No.10:

[0149] 8. A delivery vector according to embodiment 6 comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the modified pro-peptide fragment comprises preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, pre-pro-VIP, prepro-CCK, preproorphanin or preproNPY or consists of them, wherein the pro-peptide fragment comprises a sorting motif, specifically, the pro-peptide fragment can be selected from the group including any one of SEQ ID No: 37 to 52.

[0150] 9. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to embodiments 1 to 8, wherein the modified pro-peptide fragment is optionally flanked by a peptidase recognition signal comprising K, R, KR, RK or RR.

[0151] 10. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of embodiments 1 to 9, wherein the target cell is a neuronal cell of the central nervous system.

[0152] 11. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of embodiments 1 to 10, wherein the signal peptide is a peptide sequence of 10 to 30 amino acids at the N-terminus of the precursor protein that will enter the lumen of the endoplasmic reticulum.

[0153] 12. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of embodiments 1 to 11, wherein the signal peptide is selected from the group comprising:

[0154] 13. The delivery vector of any one of embodiments 1 to 12, wherein the delivery vector results in the on-demand release of a dynorphin or a dynorphin variant having an agonistic effect on the human kappa opioid receptor.

[0155] 14. A delivery vector according to any one of embodiments 1 to 13, wherein the dynorphin variant has at least 70% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2 (YGGFLRRI), SEQ ID No. 3 (YGGFLRRQ) or SEQ ID No. 4 (YGGFLRRQ), respectively.

[0156] 15. The delivery vector of any one of embodiments 1 to 14, wherein the variant has at least 80% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, respectively.

[0157] 16. The delivery vector of any one of embodiments 1 to 15, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentiviral genome.

[0158] 17. The delivery vector according to any one of embodiments 1 to 16, comprising a recombinant adeno-associated virus (AAV) vector genome, the vector genome comprising inverted terminal repeats (ITRs), which are preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14 or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, or a chimeric vector comprising two or more, preferably two, derived capsid proteins from the above-mentioned AAV serotype capsids; in a specific embodiment, the capsid is a capsid derived from AAV serotype 1 and / or 2.

[0159] In certain embodiments, the delivery vector according to embodiments 1 to 16 comprises a recombinant adeno-associated virus (AAV) vector genome comprising an inverted terminal repeat (ITR) sequence, which is preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, preferably derived from AAV serotype 1 or 2.

[0160] 18. A delivery vector according to any one of the preceding embodiments comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant.

[0161] 19. A recombinant viral particle, liposome or nanoparticle comprising the delivery vector according to any one of the preceding embodiments.

[0162] 20. A recombinant viral particle, liposome or nanoparticle according to embodiment 19, wherein the delivery vector also comprises a recombinant adeno-associated virus (AAV) vector genome and the rAAV vector genome is encapsulated in an AAV capsid, or wherein the delivery vector also comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

[0163] 21. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 1 to 20, for delivering a nucleic acid into a cell of the central nervous system, comprising contacting the cell with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce the DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant into the cell.

[0164] 22. The delivery vector or the recombinant viral particle or the liposome or the nanoparticle according to any one of embodiments 1 to 21 for use as a medicament.

[0165] 23. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 1 to 21, for treating focal epilepsy in a subject, particularly mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject with focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle activates human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures.

[0166] It is apparent to those skilled in the art that, as described in detail herein, the delivery vector or recombinant viral particle or liposome or nanoparticle activates human kappa opioid receptors in the epileptogenic focus by inducing the production of the corresponding peptide, thereby inhibiting epileptic seizures, as further described in detail herein.

[0167] 24. A DNA sequence selected from the group consisting of SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72 and SEQ ID No. 73.

[0168] 25. A delivery vector comprising the DNA sequence according to embodiment 24.

[0169] 26. The delivery vector of embodiment 25, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome.

[0170] 27. The delivery vector according to embodiment 25 or 26, comprising a recombinant adeno-associated virus (AAV) vector genome, the vector genome comprising inverted terminal repeats (ITRs), which are preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14 or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants or chimeric vectors derived therefrom, or a chimeric vector comprising two or more, preferably two, capsid proteins derived from the above-mentioned AAV serotype capsids; in a specific embodiment, the capsid is a capsid derived from AAV serotype 1 and / or 2.

[0171] In certain embodiments, the delivery vector according to embodiment 25 or 26 comprises a recombinant adeno-associated virus (AAV) vector genome, the vector genome comprising inverted terminal repeats (ITRs), which are preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14 or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, preferably two of the above-mentioned AAV capsids, preferably AAV serotype 1 or 2.

[0172] 28. The delivery vector of any one of embodiments 25 to 27, wherein the delivery vector further comprises at least one sequence selected from the group consisting of SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, and SEQ ID No. 68.

[0173] 29. The delivery vector of any one of embodiments 25 to 28, wherein the delivery vector comprises a sequence selected from the group consisting of SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 78, SEQ ID No. 79, SEQ ID No. 80, and SEQ ID No. 81.

[0174] 30. A recombinant viral particle, liposome or nanoparticle comprising the delivery vector according to any one of embodiments 25 to 29.

[0175] 31. A recombinant viral particle, liposome or nanoparticle according to embodiment 30, wherein the delivery vector also comprises a recombinant adeno-associated virus (AAV) vector genome and the rAAV vector genome is encapsulated in an AAV capsid, or wherein the delivery vector also comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

[0176] 32. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 25 to 31, for delivering a nucleic acid into a cell of the central nervous system, comprising contacting the cell with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce a DNA comprising a sequence selected from the group consisting of SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72 and SEQ ID No. 73 into the cell.

[0177] In a specific embodiment, the DNA comprising a sequence selected from the group including SEQ ID Nos. 69-73 to be introduced into the cell is DNA extracted from SEQ ID Nos. 74-81 (AAV genome), and in the case of a lentivirus, is a DNA in which a sequence selected from the group including SEQ ID Nos. 69-73 and a regulatory sequence are embedded in the lentiviral genome.

[0178] 33. The delivery vector or the recombinant viral particle or the liposome or the nanoparticle according to any one of embodiments 25 to 32 for use as a medicament.

[0179] 34. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 25 to 33, for treating epilepsy in a subject, particularly focal epilepsy, particularly mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject with focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle activates human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures.

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[0181] Example

[0182] Example 1

[0183] Shorten the human ppDyn cDNA so that it can be packaged into the scAAV vector. Some amino acids in the region between the signal peptide and the region encoding the active peptide can be removed. However, the sorting motif responsible for packaging the original peptide into dense core large vesicles needs to be retained. Alternatively, the entire N-terminal part of the region encoding the active peptide can be replaced with a different signal peptide and sorting motif.

[0184] Example 2

[0185] Production of mature dynorphin by two scAAV vector variants

[0186] Production of mature dynorphins by three scAAV vector variants containing shortened pDyn cDNA. The vectors were injected into the dorsal hippocampus of naive wild-type mice. Two weeks later, the hippocampi were excised and the content of dynorphin A (DynA) and dynorphin B (DynB) was measured by ELISA as described in Agostinho et al., (2019), see Figure 2. The production of mature peptides occurred only in large dense-core vesicles, demonstrating the correctness of the sorting of shortened pDyn variants A (SEQ ID No. 76) and B (SEQID No. 81). The significant reduction in mature dynorphins using variant C (SEQ ID No 80) lacking the proposed sorting motif indicates the importance of the proposed sorting motif.

[0187] Example 3

[0188] Suppression of epileptic seizures by shortened pDyn cDNA

[0189] Seizure suppression by two scAAV vector variants containing shortened pDyn cDNA ( Figure 3 ). The vector was injected into the dorsal hippocampus of epileptic wild-type mice. EEG was recorded and analyzed for hippocampal paroxysmal discharges (HPD), which represent drug-resistant focal epileptic seizures. Widmann et al., (2022) describe the treatment of animals with kainic acid, electrode implantation, and analysis.

[0190] Example 7

[0191] Construction of AAV-pDyn vector variants

[0192] AAV vectors are constructed in ssAAV or scAAV formats, such as Figure 4 As shown. They consist of a left ITR sequence derived from AAV serotype 2 and a right ITR sequence (SEQ ID No. 59 to 61). AAV ITRs or synthetic ITRs of alternative AAV serotypes can be used similarly.

[0193] The ITRs flank any of the heterologous gene expression cassettes displayed ( Figure 4). These cassettes consist of one of the promoter sequences (Seq ID No. 63 to 65), a post-transcriptional regulatory element of woodchuck hepatitis virus (Seq ID No. 66) (such as Loeb et al., Hum Gene Ther 10: 2295-2305, 1999), a polyadenylation signal (derived from the bovine growth hormone gene (SEQ ID No. 67) or a short synthetic poly A signal sequence (SEQ ID No. 68)) (such as Levitt et al., Genes & Dev 3: 1019-25, 1989) and the cDNA to be expressed.

[0194] The gene of interest is the cDNA sequence of human preprodynorphin (ppDyn), any of its displayed variants (SEQ ID No. 69 to 72), or a fusion of the N terminus of POMC with the C terminal part of prodynorphin lacking its signal sequence ((pre) and N peptide (pro) (SEQ ID No. 73).

[0195] The cDNA was codon optimized in two versions. Version 1 (SEQ ID No. 69) was included in AAV SEQ ID No. 74 and SEQ ID No. 75. Codon-optimized version 2 ((SEQ ID Nos. 70 to 73) was generated to reduce the percentage of CpG sequence elements. Unmethylated CpG sequence elements are a hallmark of bacterial DNA and represent pathogen-associated molecular patterns (PAMPs) that can activate the innate immune system in mammalian hosts and may be a problem in AAV gene therapy in some cases. The high CpG content of the transduced AAV genome may be associated with an increased probability of immune-mediated loss of transduced cells. Recently, adverse effects of the high CpG content of the transduced AAV genome have also been shown in the central nervous system after intraparenchymal / intracerebral AAV transduction (Suriano et al., 2021). Through different DNA sequence changes, alternative codon optimization strategies can be achieved to combine enhanced transgene expression in human cells with a sufficient reduction of unmethylated CpG elements of AAV transduction.

[0196] like Figure 4 As shown, the complete AAV DNA sequence from ITR to ITR spans all regulatory elements and different ppDyn-derived transgenes and is represented as a DNA sequence file (SEQ ID Nos. 74 to 81).

[0197] Any of the elements can be further interchanged, for example, the truncated ITR of scAAV can be located at the right end of the AAV genome instead of the left end, as shown here. Similarly, gene promoters can be interchanged and / or combined with any of the poly A signal sequences shown or poly A signals from different sources. The WPRE element can be used as the full-length 582bp element shown (Seq ID 66). The WPRE consists of subelements named γ, α, and β in the given order. A shorter version (WPRE2) showing minimal γ and partial α / β elements and WPRE3 showing only minimal γ and α elements (247bp) are described as having similar activities (Choi et al., 2014, incorporated herein by reference). Versions of the WPRE can be used interchangeably or not incorporated into the AAV genome at all.

[0198] Example 8

[0199] Functional testing of AAV vectors

[0200] Functional testing of AAV vectors of SEQ ID Nos. 76 to 78 and SEQ ID No. 81 was performed. Fully processed mature dynorphins were produced (Seq ID Nos. 76 and 81; Example 3) and suppression of focal epileptic seizures was monitored in a TLE mouse model (SEQ ID Nos. 76, 77, 78).

[0201] After injection of AAV-pDyn expressing Seq ID No.70, different types of epileptic activity were reduced. Figure 5 As shown. Hpd, generalized seizures, and spike cohorts were measured over a 48-hour period at each time interval. Starting 7 days after treatment, drug-resistant focal seizures were significantly reduced in mice. After 4 weeks, generalized seizures were almost completely eliminated.

[0202] After injection of AAV-pDyn expressing Seq ID No.77, the reduction of HPD was as shown in Figure 6 Starting 10 days after AAV delivery, mice showed a significant reduction in drug-resistant focal epileptic seizures.

Claims

1. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the delivery vector drives expression of the prepropeptide in the target cell, and wherein the delivery vector comprising the DNA sequence is capable of releasing dynorphin or a dynorphin variant from a target cell as desired, and wherein the prepropeptide is preprodynorphin or a preprodynorphin variant, and wherein the prepropeptide comprises a signal peptide, wherein the signal peptide is an N-terminal extension of the nascent polypeptide chain and wherein the signal peptide mediates targeting of the protein to the lumen of the endoplasmic reticulum, and, wherein the pre-propeptide comprises (i) an N-terminal pro-peptide fragment at the C-terminus of the signal peptide and wherein the N-terminal pro-peptide fragment comprises a peptide containing elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), wherein x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X can independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in a first sequence of, e.g., 1 to 20 Xs, each X can individually be any amino acid, and in a second sequence of, e.g., 1 to 10 Xs, each X can individually be any amino acid, in particular as further defined herein), or wherein the pre-propeptide comprises (ii) a pre-pro-neuropeptide or an N-terminal pro-peptide fragment of a protein sorted into dense-core large vesicles other than pre-pro-dynorphin at the C-terminus of the signal peptide, and wherein the N-terminal pro-peptide fragment comprises the sorting motif of the pre-pro-neuropeptide or protein sorted into dense-core large vesicles, and wherein the N-terminal pro-peptide fragment consists of 16 to 90 amino acids, and wherein the preprodynorphin or preprodynorphin variant comprises at least one of the following sequences selected from the group consisting of: a. Dyn A, which is SEQ ID No. 2 or a variant thereof consisting of the first 13 amino acids from the N-terminus or a variant thereof consisting of the first 8 amino acids from the N-terminus b. Dyn B, which is SEQ ID No. 3 c. Leuco-enkephalin, which is SEQ ID No.4 d. A variant of Dyn A, said variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2, e. A variant of Dyn B, said variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 3, f. A variant of leuproreceptin, said variant having at least 60% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No.

4.

2. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to claim 1, wherein the N-terminal pro-peptide fragment consists of 20 to 90 amino acids, preferably 30 and 90 amino acids.

3. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to claim 1 or 2, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn, wherein the unmodified pro-peptide fragment of ppDyn is SEQ ID No. 5 DCLSRCSLCAVKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKEDLGSKSVGEG PYSELAKLSGSFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREGAESELMRDAQLNDGAMETGT LYLAEEDPKE QV and wherein the modification of the ppDyn pro-peptide fragment is shortening; Or the modification is to replace part of SEQ ID No.5 with the pro-peptide or pro-peptide fragment of the neuropeptide; wherein the modified pro-peptide fragment of ppDyn i) comprises at least one element comprising the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), or ii) a sorting motif comprising said pre-pro-neuropeptide or a protein other than pre-pro-dynorphin that is sorted into dense core large vesicles.

4. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 3, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.6 DCLSRCSLCAVKTQDGPKPINPLICSLQCQAALLPSEEWERCQSFL SFFTPSTLGLNDKEDLGSKSVGEGPYSELAKLSGSFLRKEQVKR 5. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 3, wherein the N-terminal pro-peptide fragment at the C-terminus of the signal peptide is a modified pro-peptide fragment of ppDyn comprising or consisting of the following sequence: SEQ ID No.7 DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKENTLSKSLEEKL RGLSDGFREGAESELMRDAQ LNDGAMETGTLYLAEEDPKE QV or SEQ ID No.8 DLGSKSVGEG PYSELAKLSG SFLRKE QV or SEQ ID No 9 DLGSKSVGEG PYSELAKLRKE QV 6. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to claim 3, wherein the modification is to replace a portion of SEQ ID No. 5 with a pro-peptide or a pro-peptide fragment of a neuropeptide; wherein the modified pro-peptide fragment of ppDyn i) comprises at least one comprising the elements DL and EX y The sorting motif of L, in particular, consists of the amino acid sequence DLX x EX y L (SEQ ID No. 36), or ii) a sorting motif comprising said pre-pro-neuropeptide or a protein other than pre-pro-dynorphin that is sorted into dense core large vesicles.

7. The delivery vector comprising a DNA sequence encoding pre-prodynorphin or a pre-prodynorphin variant according to claim 6, wherein the modified pro-peptide fragment comprises or consists of SEQ ID No. 10MPRSCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKP.

8. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to claim 6, wherein the modified pro-peptide fragment comprises preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, pre-pro-VIP, prepro-CCK, preproorphanin or preproNPY or consists of the same, wherein the pro-peptide fragment comprises the sorting motif, in particular, the pro-peptide fragment can be selected from the group including any one of SEQ ID No: 37 to 52.

9. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 8, wherein the modified pro-peptide fragment is optionally flanked by a peptidase recognition signal comprising K, R, KR, RK or RR.

10. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 9, wherein the target cell is a neuronal cell of the central nervous system.

11. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 10, wherein the signal peptide is a peptide sequence of 10 to 30 amino acids at the N-terminus of precursor proteins that will enter the lumen of the endoplasmic reticulum.

12. The delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 11, wherein the signal peptide is selected from the group comprising: MAWQGLVLAACLLMFPSTTA(SEQ ID No.11) MARFLTLCTWLLLLGPGLLATVRA(SEQ ID No.12) MLGNKRLGLSGLTLALSLLVCLGALAEA(SEQ ID No.13) MLSCRLQCALAALSIVLALGCVTG(SEQ ID No.14) MKILVALAVFFLVSTQLFA(SEQ ID No.15) MRIMLLFTAILAFSLA(SEQ ID No.16) MPRSCCSRSGALLLALLLQASMEVRG(SEQ ID No.17) MNSGVCLCVLMAVLAAGA(SEQ ID No.18) MKVLLCDLLLLSLFSSVFS(SEQ ID No.19) MQPTLLLSLLGAVGLAAVNS (SEQ ID No. 20).

13. The delivery vehicle according to any one of claims 1 to 12, wherein the delivery vehicle results in the on-demand release of a dynorphin or a dynorphin variant having an agonistic effect on the human kappa opioid receptor.

14. The delivery vector according to any one of claims 1 to 13, wherein the dynorphin variant has at least 70% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2 (YGGFLRRI), SEQ ID No. 3 (YGGFLRRQ) or SEQ ID No. 4 (YGGFLRRQ), respectively.

15. The delivery vector according to any one of claims 1 to 14, wherein the variant has at least 80% amino acid sequence identity within the first 8 amino acids starting from the N-terminus of SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, respectively.

16. The delivery vector of any one of claims 1 to 15, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentiviral genome.

17. The delivery vector according to any one of claims 1 to 16, comprising a recombinant adeno-associated virus (AAV) vector genome, the vector genome comprising inverted terminal repeats (ITRs), which are preferably derived from AAV serotype 2, optionally derived from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14 or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, preferably derived from AAV serotype 1 or 2, or a chimeric vector comprising two or more, preferably two, capsid proteins derived from the above-mentioned AAV serotype capsids; in a specific embodiment, the capsid is a capsid derived from AAV serotype 1 and / or 2.

18. A delivery vector according to any one of the preceding claims comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant.

19. A recombinant viral particle or liposome or nanoparticle comprising a delivery vector according to any one of the preceding claims.

20. The recombinant viral particle, liposome or nanoparticle of claim 19, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome and the rAAV vector genome is encapsulated in an AAV capsid, or wherein the delivery vector further comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

21. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 1 to 20 for delivering a nucleic acid into a cell of the central nervous system, comprising contacting the cell with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce the DNA sequence encoding preprodynorphin or a preprodynorphin variant into the cell.

22. The delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 1 to 21 for use as a medicament.

23. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 1 to 21 for treating focal epilepsy in a subject, particularly mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject with focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle activates human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures.

24. A DNA sequence selected from the group consisting of SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72 and SEQ ID No.

73.

25. A delivery vector comprising the DNA sequence according to claim 24.

26. The delivery vector of claim 25, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentiviral genome.

27. The delivery vector of claim 25 or 26, comprising a recombinant adeno-associated virus (AAV) vector genome comprising an inverted terminal repeat (ITR) sequence, preferably derived from AAV serotype 2, optionally derived from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or a heavily engineered variant thereof, wherein the vector genome is packaged in an AAV capsid selected from the group consisting of: AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN or other AAV capsid mutants derived therefrom, or a chimeric vector comprising mosaic capsid proteins derived from two or more, preferably two, of the above-mentioned AAV serotype capsids; in a specific embodiment, the capsid is a capsid derived from AAV serotype 1 and / or 2.

28. A delivery vector according to any one of claims 25 to 27, wherein the delivery vector further comprises at least one sequence selected from the group consisting of SEQ ID No.59, SEQ ID No.60, SEQ ID No.61, SEQ ID No.63, SEQ ID No.64, SEQ ID No.65, SEQ ID No.66, SEQ ID No.67 and SEQ ID No.

68.

29. The delivery vector according to any one of claims 25 to 28, wherein the delivery vector comprises a sequence selected from the group consisting of SEQ ID No.74, SEQ ID No.75, SEQ ID No.76, SEQ ID No.77, SEQ ID No.78, SEQ ID No.79, SEQ ID No.80 and SEQ ID No.

81.

30. A recombinant viral particle, liposome or nanoparticle comprising the delivery vector according to any one of claims 25 to 29.

31. A recombinant viral particle, liposome or nanoparticle according to claim 30, wherein the delivery vector also comprises a recombinant adeno-associated virus (AAV) vector genome and the rAAV vector genome is encapsulated in an AAV capsid, or wherein the delivery vector also comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

32. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 25 to 31, for delivering a nucleic acid into a cell of the central nervous system, comprising contacting the cell with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce into the cell a DNA comprising a sequence selected from the group consisting of SEQ ID No.69, SEQ ID No.70, SEQ ID No.71, SEQ ID No.72 and SEQ ID No.

73.

33. The delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 25 to 32 for use as a medicament.

34. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 25 to 33 for treating epilepsy in a subject, particularly focal epilepsy, particularly mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject with focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle activates human kappa opioid receptors in the epileptogenic focus, thereby inhibiting epileptic seizures.

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