Gene therapy vectors for the expression of preprodynorphin variants for the treatment of epilepsy

Abstract

The subject of the present invention is a delivery vector comprising a DNA sequence encoding a preprodynorphin or a preprodynorphin variant, wherein said delivery vector promotes expression of a prepro-peptide in a target cell, and wherein said delivery vector comprising said DNA sequence allows the release of a dynorphin or a dynorphin variant from the target cell on demand, and wherein said prepro-peptide is a preprodynorphin or a preprodynorphin variant, and wherein said prepro-peptide comprises a signal peptide, wherein said signal peptide is an N-terminal extension of a nascent polypeptide chain, and wherein said signal peptide mediates targeting of the protein to the lumen of the endoplasmic reticulum, and wherein said prepro-peptide comprises an N-terminal propeptide fragment at the C-terminus of said signal peptide, and wherein said N-terminal propeptide fragment comprises: i) elements DL and EX y The sorting motif containing L, especially the amino acid sequence DLX x EX y L, or ii) a sorting motif of a prepro-neuropeptide or protein, other than preprodynorphin, that sorts into large dense core vesicles, as defined herein, wherein in certain embodiments, 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, and wherein the N-terminal propeptide fragment consists of 16 to 90 amino acids, as well as corresponding peptides, DNA molecules, viral vectors, and particles comprising any of these.

Description

[Technical Field]

[0001] The present invention provides delivery vectors for transferring nucleic acid sequences encoding prepropeptides into cells in vitro, ex vivo, or in vivo. The present invention provides methods for delivering nucleic acid sequences to cells and methods for treating focal epilepsy.

[0002] Epilepsy, with a prevalence of 1–2% worldwide, is one of the most common neurological disorders (McNamarae et al., 1999), with focal epilepsies accounting for approximately 70% of cases. Among these, mesial temporal lobe epilepsy (mTLE) is the most frequent clinical manifestation. In mTLE, the focus is located in or near the hippocampus, which regulates learning, memory, and emotional control. mTLE is an acquired disorder frequently triggered by traumatic brain injury. Epileptic foci can also be caused by central nervous system infections, high fever, brain tumors, or vascular malformations. As the disease progresses, hippocampal sclerosis accompanied by neurological deficits can develop. These are the main clinical features of this subtype of mTLE (for a review, see Engel et al., 2001). Despite the introduction of a wide variety of antiepileptic drugs over the past few decades, the rate of drug-resistant epilepsy (30%–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 epileptic focus remains the definitive treatment option for patients whose epileptic focus is clearly defined and can be clearly separated from other critical CNS regions. The proximity of language centers to the hippocampal focus is a major contraindication to epilepsy surgery. However, even if epilepsy surgery is successful, there is no guarantee of permanent seizure freedom. Only up to 50% of patients remain seizure-free for at least one year after removal of the epileptic focus (Spencer et al., 2008).

[0003] Since the early 1980s, there have been data suggesting 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, deletion of the prodynorphin (pDyn) coding sequence in mice (Loacker et al., 2007) and the discovery of low levels of Dyn in humans due to mutations in the promoter region of the pDyn gene (Stogmann et al., 2002, Gambardella et al., 2003) have been associated with increased vulnerability to the development of epilepsy. In most animal models of temporal lobe epilepsy (TLE; including lateralized TLE and mTLE), cortical and hippocampal pDyn gene expression declines after an initial, brief peak of overexpression (for reviews, see Simonato et al., 1996, Schwarzer et al., 2009). This finding is consistent with a postictal, presumably short-term, increase in pDyn mRNA levels in hippocampal granule cells (Pirker et al., 2009). Previous studies have described a global decrease in Dyn immunoreactivity in surgically removed brain tissue from mTLE patients (de Lanerolle et al., 1997).

[0004] Dynorphins act preferentially on kappa opioid receptors (KORs). Despite the decline in endogenous Dyn, KORs remain available as drug targets under epileptic conditions, and experimental seizures can be suppressed by the application of KOR agonists (Tortella et al., 1988, Takahashi et al., 1990, Solbrig et al., 2006, Loacker et al., 2007, Zangrandi et al. 2016). Various selective KOR agonists administered by different routes produced time- and dose-dependent effects similar to those of phenytoin or phenobarbital treatment in epilepsy models (for a review, see Simonato et al., 1996). We previously demonstrated that unilateral injection of kainic acid in mice promotes hippocampal neuronal survival after an acute epileptic period (Schunk et al., 2011).

[0005] An object of the present invention is to provide a delivery vector for the transfer of a nucleic acid sequence encoding a prepropeptide or peptide containing a sequence that enables the propeptide (e.g., prodynorphin) to be packaged into vesicles, where the propeptide matures and an active substance, a dynorphin or dynorphin variant, is released with a series of action potentials that exceed a specific excitatory threshold. Therefore, an object of the present invention is to provide a delivery vector for the transfer of a nucleic acid sequence into cells in vitro, ex vivo, or in vivo. An object of the present invention is, in particular, a vector-based therapy for treating focal epilepsy using preprodynorphin or dynorphin or variants thereof. The delivery vector of the present invention, comprising a nucleic acid encoding preprodynorphin or prodynorphin or dynorphin or variants thereof, transduces neurons to express, process, store, and release preprodynorphin or prodynorphin or dynorphin or variants thereof, thereby resulting in activation of KOR in the epileptogenic focus and thereby inhibiting seizures.

[0006] The purpose of the present invention is to further develop AAV-based gene vectors for expressing preprodynorphin for the treatment of focal epilepsy. Here, AAV vectors are preferably used to express preprodynorphin. In the present invention, after transduction of neurons with the vector, preprodynorphin is expressed and processed into dynorphin peptides (dyn) of different types and lengths, which are released after stimulation by a trigger, which is high-frequency excitation.

[0007] The present invention is further based on the truncation of the ppDYN protein. To avoid interfering with the correct vesicle targeting within neurons and the correct processing into defined peptides, two strategies were employed.

[0008] First, we truncated the so-called N-peptide region of ppDyn. We found that this region contains a specific, conserved sorting motif. Next, for some neuropeptides (e.g., pPOMC or ppEnk), the signal and sorting sequences located at their N-termini are significantly shorter than those located at the N-terminus of ppDyn. Therefore, we constructed fusion proteins combining the N-terminus of a portion of such neuropeptide precursor molecules derived from pPOMC with the C-terminal sequence of ppDyn, encoding various active Dyn peptides (Figure 1).

[0009] Detailed Description of the Invention The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, and wherein the delivery vector promotes expression of the prepropeptide in the target cell; and wherein the delivery vector comprising the DNA sequence allows the dynorphin or dynorphin variant to be released from the target cell on demand, 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

[0010] wherein the prepropeptide is (i) an N-terminal propeptide fragment at the C-terminus of the signal peptide, and wherein the N-terminal propeptide fragment comprises elements DL and EX y The sorting motif containing L, especially 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 may independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in particular in a first sequence of e.g. 1 to 20 X, each X may individually be any amino acid, and in a second sequence of e.g. 1 to 10 X, each X may individually be any amino acid, as further defined herein); or wherein the prepropeptide is (ii) comprising, on the C-terminus of the signal peptide, an N-terminal propeptide fragment of a prepro-neuropeptide or protein other than preprodynorphin that is sorted into large dense core vesicles, wherein the N-terminal propeptide fragment comprises a sorting motif of the prepro-neuropeptide or protein that is sorted into large dense core vesicles, and wherein the N-terminal propeptide fragment consists of 16 to 90 amino acids; and

[0011] wherein the preprodynorphin or preprodynorphin variant comprises at least one of the following sequences selected from the group consisting of a, b, c, d, e, and f: a. DynA, SEQ ID NO: 2 (AA 207-223 of SEQ ID NO: 1; ppDyn), or a variant thereof consisting of the first 13 AAs (starting from the N-terminus), or a variant thereof consisting of the first 8 AAs (starting from the N-terminus), b. DynB (SEQ ID NO: 1; AA226-238 of ppDyn), which is SEQ ID NO: 3; c. Leumorphin, which is SEQ ID NO: 4 (SEQ ID NO: 1; AA226-254 of ppDyn); d. A variant of DynA of SEQ ID NO: 2 having at least 60% amino acid sequence identity within the first 8 AA (YGGFLRRI) from the N-terminus of SEQ ID NO: 2, i.e., having at least 60% amino acid sequence identity within the sequence YGGFLRRI contained in SEQ ID NO: 2; e. A variant of DynB of SEQ ID NO: 3 having at least 60% amino acid sequence identity within the first 8 AA (YGGFLRRQ) from the N-terminus of SEQ ID NO: 3, i.e., having at least 60% amino acid sequence identity within the sequence YGGFLRRQ contained in SEQ ID NO: 3; f. A variant of leumorphin of SEQ ID NO: 4 having at least 60% amino acid sequence identity within the first 8 AA (YGGFLRRQ) from the N-terminus of SEQ ID NO: 4, i.e., having at least 60% amino acid sequence identity within the sequence YGGFLRRQ contained in SEQ ID NO: 4.

[0012] In this context, 60% sequence identity is defined as follows: three of the first eight N-terminal amino acids may be removed or replaced with another amino acid. In the case of truncated peptide variants, the percent sequence identity of the shortened peptide is calculated. Introduction of additional amino acids is treated as a gap in the original sequence, and deletions are treated as gaps in the modified peptide in calculating sequence identity (YGGFLRRQ differs from YG-FLRRQ by one AA, but the AAs at positions 3, 4, 5, 6, and 7 do not differ). In either case, a variant of SEQ ID NO: 2 having at least 60% amino acid sequence identity from the N-terminus within the first eight amino acids may be a variant containing the sequence YGZFLRKZ, where each Z individually represents any amino acid, and K replaces R at position 7, preserving the peptidase recognition site (RK or RR).

[0013] In the present invention, "amino acid" refers to naturally occurring amino acids, and more particularly to standard amino acids, i.e. amino acids that are directly coded for by codons of the universal genetic code.

[0014] Throughout the present invention, "Z" in an amino acid sequence represents any of the naturally occurring amino acids, and in certain embodiments, "Z" may be selected from the group consisting of alanine, glycine, asparagine, glutamine, leucine, serine, valine, and isoleucine.

[0015] Preproneuropeptides other than preprodynorphin are known to those skilled in the art and are described, for example, in Zhang et al. Progress in neurobiology 90 (2010) 276-283: Prepro-neuropeptides or proteins other than preprodynorphin that are sorted into large dense core vesicles may be selected from the group including, but not limited to, preprosubstance P, pPOMC, pp tachykinin A, pp tachykinin B, pproopiomelanocortin, pp cholecystokinin, pp chromogranin B, calcitonin gene-related peptide (CGRP), preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, preproVIP, preproCCK, prepronociceptin, or preproNPY.

[0016] Preproneuropeptides or proteins that are sorted into large dense-core vesicles containing preprodynorphin have a signal peptide at their N-terminus, which directs their translocation to the endoplasmic reticulum (ER) as described above. Neuropeptides are expressed in neurons and secreted in response to physiological or pathological stimuli, meaning that neuropeptides are released as needed. Neuropeptide prohormones display a wide variety of sorting motifs, some of which contain the elements DL and EX. y The sorting motif containing L, especially the amino acid sequence DLX x EX y L (SEQ ID NO: 36), where 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 preproneuropeptides, such as ppneuropeptide Y, are also known as DLX. x EX y It contains a sorting motif other than L (SEQ ID NO: 36). In any case, the sorting is considered to be included in the N-terminal propeptide fragment consisting of 16 to 90 amino acids as defined above.

[0017] Sorting motif elements DL and EX yIt should be noted that the distance between L's can vary, and that it is particularly important that these elements are present in each amino acid sequence, and that sorting motifs are typically formed by the spatial proximity of these elements to one another. For the avoidance of doubt, the element DL refers to the amino acid sequence asparagine-leucine, and the element EX refers to the amino acid sequence asparagine-leucine. y L is the amino acid sequence glutamic acid-X y - refers to leucine, where X y are as defined herein. In certain embodiments, the elements DL and EX y L is the amino acid sequence in question, with DL···EX going from the N-terminus to the C-terminus of the sequence. y They appear in the order of L, in other words, DL is EX y It is located closer to the N-terminus than L.

[0018] In certain embodiments, the amino acid sequence DLX x EX y In a sorting motif consisting of L (SEQ ID NO: 36), x is an integer from 2 to 13, more particularly an integer from 5 to 10, more particularly an integer from 5 to 8, or 2, 11, or 13. In certain embodiments, the amino acid sequence DLX x EX y In a sorting motif consisting of the amino acid sequence DLX (SEQ ID NO: 36), y is an integer of 1 to 5. In a specific embodiment, x EX y In the sorting motif consisting of L (SEQ ID NO: 36), x is an integer of 2 to 13, more particularly an integer of 5 to 10, or x is 2, 5, 8, 10, or 13, and y is an integer of 1 to 5.

[0019] In some particular peptides, x is 2 (POMC), 11 (pDyn), or 13 (BDNF or TAC1). In some specific peptides, y is 1 to 2 (TAC1), 2 (POMC), 2 to 5 (BDNF), or 3 to 5 (pDyn).

[0020] According to the present invention, the delivery vector comprises a DNA sequence encoding a prepropeptide of dynorphin or a dynorphin variant. This means that the vector comprises a DNA sequence encoding a signal peptide fused to the propeptide. In one 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 prepropeptide to the lumen of the endoplasmic reticulum. The DNA sequence encoding the signal peptide may be the sequence of SEQ ID NO: 11. In another aspect of the present invention, the delivery vector comprises a DNA sequence encoding a propeptide fragment. In a particular aspect of the present invention, the propeptide fragment is the sequence of SEQ ID NO: 5.

[0021] An advantage of the delivery vectors of the present invention is the on-demand release of dynorphin or dynorphin variants. Specifically, this means that prodynorphin (or a prodynorphin variant) is packaged into vesicles, matures, and is released on demand upon high-frequency stimulation (e.g., stimulation ≥ 8 Hz), which occurs at the onset of a seizure. In particular, the on-demand release formulation thus provides preprodynorphin (or a preprodynorphin variant), which is packaged into vesicles, matures, and the active substance, dynorphin or a dynorphin variant, is released at an action potential frequency above a specific threshold. In other words, the "on-demand" release of dynorphin or a dynorphin variant from target cells is specifically referred to as "release upon high-frequency stimulation" and / or "release upon action potential frequencies above a specific threshold," since it occurs at the onset of a seizure. The specific threshold may be ≥ 6 Hz, ≥ 7 Hz in another embodiment, ≥ 8 Hz in another embodiment, or ≥ 9 Hz in another embodiment. This means that the on-demand release is triggered by an increase in neuronal firing frequency. The increase in neuronal firing frequency can be measured by EEG (electroencephalography) as spike trains, where "elevated" means that the frequency of spikes in the train as 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.

[0022] This means that the delivery vector promotes the expression of prepropeptides, thereby enabling the provision of dynorphin or dynorphin variants on demand. Such delivery vectors first express prepropeptides within neurons, where the resulting propeptides are sorted into large dense-core vesicles, where they are enzymatically processed and the resulting peptides are stored until release is triggered by sufficiently strong excitation, i.e., until release is initiated by an increase in the neuronal firing frequency, as described above. The Dyn peptide binds to presynaptic and / or postsynaptic KORs to activate G proteins, which, among other things, regulate ion channels to inhibit further amplification and spread of neuronal excitation. The first step is translation of the signal peptide of ppDyn, which directs ppDyn to the endoplasmic reticulum, from which prodynorphin is sorted into "large dense-core" vesicles (LDVs). Using existing neuronal mechanisms, prodynorphin is enzymatically processed into the mature peptide and transported to the axon terminal. LDVs are stored in the axon terminal and released in a stimulus-dependent manner. As mentioned above, high-frequency stimulation induces release, such as at the onset of a seizure, whereas low-frequency stimulation does not. This creates release on demand. The released Dyn peptide binds to pre- and / or postsynaptic KORs, which activate G proteins. The G proteins, among other things, regulate ion channels to attenuate further amplification and spread of neuronal excitation.

[0023] In other words, the composition for release on demand is a composition that releases a peptide having an agonistic effect on human KOR, derived from either a delivery vector, recombinant viral particle, liposome, or nanoparticle of the present invention, at the onset of a seizure in the subject. The onset of a seizure can be characterized by an increase in neuronal firing frequency, measurable by EEG (electroencephalography) as spike trains, where an increase 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.

[0024] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said N-terminal propeptide fragment consists of 16 to 90 amino acids, preferably 20 to 90 amino acids, preferably 30 to 90 amino acids.

[0025] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said N-terminal propeptide fragment at the C-terminus of the signal peptide is a modified propeptide fragment of ppDyn, wherein the unmodified propeptide fragment of ppDyn is SEQ ID NO:5: DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV and wherein the modification of said propeptide fragment of SEQ ID NO: 5 is x EX y The aim is to shorten the sequence while maintaining the sorting motif consisting of L (SEQ ID NO: 36).

[0026] A 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 said N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, which SEQ ID NO:6: DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLRKEQVKR It comprises or consists of:

[0027] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, which is SEQ ID NO:7: DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV or SEQ ID NO:8: DLGSKSVGEG PYSELAKLSG SFLRKEQV or SEQ ID NO: 9: DLGSKSVGEG PYSELAKLRK EQV or SEQ ID NO: 55: DLGSKSVGEG PYSELRKEQV It comprises or consists of:

[0028] One embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, which is SEQ ID NO:56: DLGSKSVGEG PYSELAKLSG SFLKKEQV It comprises or consists of:

[0029] Another embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, which is SEQ ID NO:57: DLGSKSVGEG PYSELAKL It comprises or consists of:

[0030] Another embodiment of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, which is SEQ ID NO:58: DLGSKSVGEG PYSEL It comprises or consists of:

[0031] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said N-terminal propeptide region C-terminal to the signal peptide is a modified hybrid propeptide fragment of ppDyn, wherein the unmodified propeptide fragment of ppDyn is SEQ ID NO:5: DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV and and wherein the modification of said propeptide fragment of SEQ ID NO: 5 is to replace a portion of SEQ ID NO: 5 with a propeptide or a fragment of a propeptide of a neuropeptide, wherein the modified propeptide fragment of ppDyn comprises: i) the elements DL and EX y At least one sorting motif containing L, in particular the amino acid sequence DLX x EX yL (SEQ ID NO: 36), or ii) a sorting motif of a prepro-neuropeptide or a protein that sorts into dense core vesicles, other than preprodynorphin, as further defined herein. For the avoidance of doubt, one skilled in the art can readily appreciate that in this particular context, a propeptide or fragment of a propeptide of a neuropeptide is clearly different from the unmodified propeptide fragment of ppDyn of SEQ ID NO: 5, since part of the sequence has been substituted.

[0032] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein said modified propeptide fragment is SEQ ID NO:10: MPRSCCSRSG ALLLALLLQ ASMEVRGWCL ESSQCQDLTT ESNLLECIRA CKP Includes.

[0033] The subject of the present invention is a delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the modified propeptide fragment comprises or consists of a propeptide fragment of preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, preproVIP, preproCCK, prepronociceptin, or preproNPY, wherein the propeptide fragment comprises the above-mentioned sorting motif or another sorting motif. For example, a different sorting motif has been proposed for ppNPY. 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 propeptide fragment is selected from the group consisting of any of SEQ ID NOs: 37 to 52.

[0034] These DNA sequences can be selected from DNA sequences encoding polypeptides from the group comprising: SEQ ID NO: 37: Preproenkephalin-N peptide MARFLTLCTW LLLLGPGLLA TVRAECSQDC ATCSYRLVRP ADINFLACVM ECEGKLPSLK IWETCKELLQ LSKPELPQDG TSTLRENSKP EESHLLA

[0035] SEQ ID NO: 38: Preproenkephalin-pDyn hybrid: MARFLTLCTW LLLLGPGLLA TVRAECSQDC ATCSYRLVRP ADINFLACVM ECEGKLPSLK IWETCKELLQ LSKPELPQDG TSTLRENSKP EESHLLAKRY GGFLRKYPKR SSEVAGEGDG DSMGHEDLYK RYGGFLRRIR PKLKWDNQKR YGGFLRRQFK VVTRSQEDPN AYSGELFDA

[0036] SEQ ID NO: 39: PreproNPY-N peptide MLGNKRLGLS GLTLALSLLV CLGALAEA

[0037] SEQ ID NO: 40: PreproNPY-pDyn hybrid: MLGNKRLGLS GLTLALSLLV CLGALEAKR YGGFLRKYPK RSSEVAGEGD GDSMGHEDLY KRYGGFLRRI RPKLKWDNQK RYGGFLRRQFK VVTRSQEDPN AYSGELFD

[0038] SEQ ID NO: 41: PreproBDNF-N peptide MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDHKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSM

[0039] SEQ ID NO: 42: PreproBDNF-pDyn hybrid: MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDHKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMKRYGGF LRKYPKRSSEV AGEGDGDSMG HEDLYKRYGG FLRRIRPKLK WDNQKRYGGF LRRQFKVVTR SQEDPNAYSG ELFD

[0040] SEQ ID NO: 43: Preprosomatostatin-N peptide MLSCRLQCALA ALSIVLALGC VTGAPSDPRL RQFLQKSLAA AAGKQELAKY FLAELLSEPN QTENDALEPE DLSQAAEQDE MRLELQR

[0041] SEQ ID NO: 44: Preprosomatostatin-pDyn hybrid: MLSCRLQCALA ALSIVLALGC VTGAPSDPRL RQFLQKSLAA AAGKQELAKY FLAELLSEPN QTENDALEPE DLSQAAEQDE MRLELQRKRY GGFLRKYPKR SSEVAGEGDG DSMGHEDLYK RYGGFLRRIR PKLKWDNQKR YGGFLRRQFK VVTRSQEDPN AYSGELFD

[0042] SEQ ID NO: 45: Preprotachykinin AN peptide MKILVALAVF FLVSTQLFAE EIGANDDLNY WSDWYDSDQI KEELPEPFEH LLQRIA

[0043] SEQ ID NO. 46: Preprotachykinin A-pDyn hybrid: MKILVALAVF FLVSTQLFAE EIGANDDLNY WSDWYDSDQI KEELPEPFEH LLQRIAKRYG GFLRKYPKRS SEVAGEGDGD SMGHEDLYKR YGGFLRRIRP KLKWDNQKRY GGFLRRQFKV VTRSQEDPNA YSGELFD

[0044] SEQ ID NO: 47: PreproVIP-N peptide MDTRNKAQLL VLLTLLSVLF SQTSAWPLYR APSALRLGDR IPFEGANEPD QVSLKEDIDM LQNALAENDT PYYDVSRNA

[0045] SEQ ID NO: 48: PreproVIP-pDyn hybrid: MDTRNKAQLL VLLTLLSVLF SQTSAWPLYR APSALRLGDR IPFEGANEPD QVSLKEDIDM LQNALAENDT PYYDVSRNAK RYGGFLRKYP KRSSEVAGEG DGDSMGHEDL YKRYGGFLRR IRPKLKWDNQ KRYGGFLRRQ FKVVTRSQED PNAYSGELFD

[0046] SEQ ID NO: 49: PreproCCK-N peptide MNSGVCLCVL MAVLAAGALT QPVPPADPAG SGLQRAEEAP RRQL

[0047] SEQ ID NO: 50: PreproCCK-pDyn hybrid MNSGVCLCVL MAVLAAGALT QPVPPADPAG SGLQRAEEAP RRQLKRYGGF LRKYPKRSSE VAGEGDGDSM GHEDLYKRYG GFLRRIRPKL KWDNQKRYGG FLRRQFKVVT RSQEDPNAYS GELFD

[0048] SEQ ID NO: 51: Prepronociceptin-N peptide MKVLLCDLLL LSLFSVFSS CQRDCLTCQE KLHPALDSFD LEVCILECEE KVFPSPLWTP CTKVMARSSW QLSPAAPEHV AAALYQPRAS EMQHL

[0049] SEQ ID NO: 52: Prepronociceptin-pDyn hybrid MKVLLCDLLL LSLFSSVFSS CQRDCLTCQE KLHPALDSFD LEVCILECEE KVFPSPLWTP CTKVMARSSW QLSPAAPEHV AAALYQPRAS EMQHLKRYGG FLRKYPKRSS EVAGEGDGDS MGHEDLYKRY GGFLRRIRPK LKWDNQKRYG GFLRRQFKVV TRSQEDPNAY SGELFD.

[0050] A subject of the present invention is a delivery vector comprising a DNA sequence encoding a preprodynorphin or preprodynorphin variant of the present invention, wherein the modified propeptide fragment is optionally flanked by peptidase recognition signals comprising K, R, KR, RK, or RR.

[0051] Peptidase (prohormone convertase) recognition signals are known to those skilled in the art and may be single or pairs of basic amino acids, preferably, but not limited to, K, R, KR, RK, or RR.

[0052] According to the above description, a specific preprodynorphin having a shortened modified propeptide fragment is as follows:

[0053] Array: 53 MAWQGLVLAA CLLMFPSTTA DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLRKEQVKR YGGFLRKYP K RSSEVAGEGD GDSMGHEDLY KRYGGFLRRI RPKLKWDNQK R YGGFLRRQF KVVTRSQEDP NAYSGELFDA .

[0054] The bolded amino acids may be those of the POMC sorting motif and are likely derived by analogy, the italicized amino acids represent DynA, the underlined amino acids represent leumorphin, and the bolded and underlined amino acids represent neoendorphin. Finally, the bolded and italicized amino acids represent the peptidase recognition signal.

[0055] According to the above description, a particular preprodynorphin having a hybrid modified propeptide fragment is as follows: Hybrid pPOMC-ppDyn (SEQ ID NO: 54): MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KPKEQVKR YG GFLRKYP KRS SEVAGEGDGD SMGHEDLYKR YGGFLRRIRP KLKWDNQKR Y GGFLRRQFKV VTRSQEDPNA YSGELFDA

[0056] Bold amino acids represent the amino acids of the POMC sorting motif, italic amino acids represent DynA, underlined amino acids represent leumorphin, and bold and underlined amino acids represent neoendorphin. Finally, bold and italic amino acids represent the peptidase recognition signal, and the non-peptide coding portion of ppDyn was replaced with a portion of pPOMC (shaded in gray).

[0057] 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 neurons of the central nervous system.

[0058] 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 principal neurons and GABAergic interneuron subtypes.

[0059] 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 is to be transported into the lumen of the endoplasmic reticulum.

[0060] 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 to be transported into the lumen of the endoplasmic reticulum, wherein a stretch of 5 to 16 amino acids tends to form a single alpha-helical structure.

[0061] The core of a signal peptide contains a stretch of hydrophobic amino acids (approximately 5–16 amino acids in length) that tends to form a single alpha helix, also called the "h-region." Furthermore, many signal peptides begin with a stretch of positively charged amino acids, which may help enforce the proper topology of the polypeptide during translocation, a rule known as the positive-inside rule. However, the amino acid sequences of signal peptides vary greatly, even within the group of prepro-neuropeptides.

[0062] The signal peptides described herein may exhibit several variable sequences. Non-limiting examples of common signal peptides and signal peptides of the present invention may be selected from the group including, but not limited to: MAWQGLVLAA CLLMFPSTTA (SEQ ID NO: 11) MARFLTLCTW LLLLGPGLLA TVRA (SEQ ID NO: 12) MLGNKRLGLS GLTLALSLLV CLGALAEA (SEQ ID NO: 13) MLSCRLQCAL AALSIVLALG CVTG (SEQ ID NO: 14) MKILVALAVF FLVSTQLFA (SEQ ID NO: 15) MRIMLLFTAI LAFSLA (SEQ ID NO: 16) MPRSCCSRSG ALLLALLLQA SMEVRG (SEQ ID NO: 17) MNSGVCLCVL MAVLAAGA (SEQ ID NO: 18) MKVLLCDLLL LSLFSSVFS (SEQ ID NO: 19) MQPTLLLSLL GAVGLAAVNS (SEQ ID NO: 20).

[0063] For the avoidance of doubt, the sorting motif and signal peptide may be derived from the same prepro-neuropeptide or protein that is sorted into large dense core vesicles, or from two different prepro-neuropeptides or proteins that are sorted into large dense core vesicles.

[0064] The subject of the present invention is a delivery vector, wherein said delivery vector provides for the on-demand release of a dynorphin or a dynorphin variant having an agonistic effect on the human kappa opioid receptor.

[0065] The subject of the present invention is a delivery vector, wherein said variants have at least 70% amino acid sequence identity within 8 amino acids of the first 8 amino acids from the N-terminus of SEQ ID NO: 2 (YGGFLRRI), the first 8 amino acids from the N-terminus of SEQ ID NO: 3 (YGGFLRRQ), or the first 8 amino acids from the N-terminus of SEQ ID NO: 4 (YGGFLRRQ), respectively.

[0066] The subject of the present invention is a delivery vector, said variants having at least 80% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

[0067] The subject of the present invention is a delivery vector, said variants having at least 90% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, respectively.

[0068] In a particular embodiment, a subject of the present invention is a delivery vector as described above, wherein said delivery vector comprises a plurality of 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.

[0069] This means, by way of example, that a delivery vector can contain a DNA sequence encoding SEQ ID NO: 2 twice, so that two molecules of a peptide according to SEQ ID NO: 2 are derived from one delivery vector.

[0070] Peptidase (prohormone convertase) recognition signals are known to those skilled in the art and may be single or pairs of basic amino acids, preferably, but not limited to, K, R, KR, RK, or RR.

[0071] In a particular embodiment, a subject of the present invention is a delivery vector as described above, wherein said delivery vector comprises a plurality of DNA sequences encoding SEQ ID NO: 2 and / or SEQ ID NO: 4 or variants thereof, wherein the sequences of SEQ ID NO: 2 and / or SEQ ID NO: 4 or variants thereof are flanked by peptidase recognition signals.

[0072] A subject of the present invention is a delivery vector, wherein said delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome, and in particular refers to a DNA sequence based on such a recombinant genome.

[0073] The delivery vectors produced according to the present invention are useful for in vitro, ex vivo, and in vivo delivery of nucleic acids to cells, and in particular, the delivery vectors can be advantageously used to deliver or transfer nucleic acids to animal, more preferably mammalian, cells.

[0074] Suitable vectors include viral vectors (e.g., retroviruses, lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, or herpes simplex viruses), lipid vectors, lipid nanoparticles, polylysine vectors, synthetic polyamino polymer vectors (which are used in conjunction with nucleic acid molecules such as plasmids), and the like.

[0075] 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 families: Adenoviridae, Adeno-associated Viridae (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, Corsicoviridae, Group Cryptic virus, and Cryptovirus group. Cryptovirus, Cucumovirus virus group Family ([PHgr]6 phage group); Cysioviridae; Group Carnation ringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae; Flaviviridae; Furovirusgroup); Group Germinivirus; Group Giardiavirus; Hepadnaviridae; Herpesviridae; Hordeivirus group; Illarvirus group; Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae; Luteovirus group; Marafivirus group; Maize chlorotic dwarf virus group; Icroviridae; Myoviridae; Necrovirus group; Nepovirus group group); Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnip yellow fleck virus group; Partitiviridae; Parvoviridae; Pea enation mosaic virus group; Phycodnaviridae, Picomaviridae, Plasmaviridae, Prodoviridae, Polydnaviridae, Potexvirus groupgroup, Potyvirus, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Group Rhizidiovirus, Siphoviridae, Sobemovirus group, SSV1 type phage, Tectiviridae, Tenuivirus, Tetraviridae, Group Tobamovirus, Group Tobravirus, Togaviridae, Group Tombusvirus, Group Tobovirus, Totiviridae, Group Tymovirus, and plant virus satellites. Protocols for generating recombinant viral vectors and using viral vectors for nucleic acid delivery are described in (Ausubel et al., 1989) and other standard laboratory manuals (e.g., Rosenzweig et al.(2007). Specific examples of viral vectors include those previously used to deliver nucleic acids, including, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAVs), and other parvoviruses, herpesviruses, and poxvirus vectors. As used herein, the term "parvovirus" encompasses the Parvoviridae family, including autonomous parvoviruses, densoviruses, and dependoviruses. The term adeno-associated virus (AAV) includes all vertebrate variants, particularly those derived from humans, primates, other mammals, birds, or snakes. Autonomous parvoviruses include members of the Parvovirus, Erythrovirus, Bocavirus, Densovirus, Iteravirus, and Contravirus genera. Examples of 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 other viruses classified as parvoviruses by the International Committee on Taxonomy of Viruses (ICTV). Other autonomous parvoviruses are known to those skilled in the art. See, for example, (Berns et al. 2013).

[0076] In one embodiment of the invention, the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome.

[0077] In one particular embodiment of the invention, said delivery vector further comprises a recombinant AAV vector, wherein preferably said vector is of a serotype of human or primate origin.

[0078] 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, or from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein said vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, snake AAV, ancestral AAV, AAV, The capsid is packaged within an AAV capsid selected from the group comprising: AV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid variants derived therefrom, or a chimeric AAV vector comprising capsid proteins derived from two or more, preferably two, of the aforementioned AAV serotype capsids. In certain embodiments, the capsid is a capsid selected from AAV serotypes 1 and / or 2, more particularly AAV1, and capsids derived therefrom (including AAV1P4, AAV1P5, or AAV2), and capsids derived therefrom (including AAV2-NN) (Borner et al. 2020; Pavlou et al. 2021; Challis et al. 2022; Naidoo et al. 2018; Hsu et al. 2020; Tordo et al. 2018).

[0079] In certain embodiments of the invention, the delivery vector comprises a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably from AAV serotype 2, or AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein said vector genome is selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, snake AAV, ancestral AAV, AAV-TT, AAVv66; AAV1 and are packaged into AAV capsids selected from the group including AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid variants derived therefrom, particularly AAV capsid variants of AAV serotype 1 or 2, more particularly AAV1, and into capsids derived therefrom (including AAV1P4, AAV1P5, or AAV2), and into capsids derived therefrom (including AAV2-NN) (Borner et al. 2020; Pavlou et al. 2021; Challis et al. 2022; Naidoo et al. 2018; Hsu et al. 2020; Tordo et al. 2018).

[0080] Chimeric vectors (also called mosaic vectors) and their production methods are known from the scientific literature [e.g., Hauck et al., (2003)] or are described in Noe et al. (2008) and During et al. (2003). In certain embodiments, such chimeric vectors may improve vector yield during the vector production process or delivery and allow binding to multiple cell surface molecules that function as receptors (see, for details, e.g., below). Furthermore, mosaic AAV capsids, particularly mixtures of AAV2 and AAV1, have been shown to enhance neurotropism and significantly reduce targeting to astrocytes or microglia during CNS delivery in rodents and non-human primates compared to AAV1-only capsids (Kimura et al. 2023). Chimeric vectors contain capsid proteins from two or more, usually two, different viral serotypes. The ratio between these different capsid proteins can be selected based on, for example, the desired cell targeting effect and / or manufacturability or desired AAV vector yield. For example, the ratio of capsid proteins from two AAV capsids in a chimeric vector can range from 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, referring to the protein ratio of the first AAV serotype capsid to the second AAV serotype capsid). In certain embodiments, such a chimeric vector comprises capsid proteins of AAV serotype 1 and capsid proteins of AAV serotype 2, e.g., capsids derived therefrom as detailed above and in the ratios detailed above, more specifically, capsids in which the ratio of AAV2 capsids to AAV1 capsids is about 90:10.

[0081] In certain embodiments of the invention, the delivery vector is a single-stranded (ssAAV) vector or a self-complementary vector (scAAV), also called a dimeric or double-stranded AAV vector (McCarty et al. 2001).

[0082] In certain embodiments of the present 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 including a promoter and / or enhancer that induce sufficient expression of the gene product of interest to achieve a therapeutic effect.

[0083] For example, the encoding nucleic acid may be operably linked to expression control elements (e.g., promoters, enhancers, other transcription / translation control signals, origins of replication, polyadenylation signals, and / or internal ribosome entry sites (IRES)). It will be further understood that various promoter / enhancer elements can be used depending on the desired level and tissue-specific expression. The promoter / enhancer may be constitutive or inducible depending on the desired expression pattern. The promoter / enhancer may be native or foreign, a naturally occurring sequence or a synthetic sequence. Foreign means that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced. Most preferred are promoter / enhancer elements that are functional in the target cell or subject to be treated. Mammalian promoter / enhancer elements are also suitable. Most preferred are promoter / enhancer elements that are active in human neurons but not active or less active in glial cells. The promoter / enhancer element can constitutively or inducibly express the transgene.

[0084] Examples of constitutive promoters include, but are not limited to, the beta-actin promoter, the cytomegalovirus promoter, the cytomegalovirus enhancer / chicken beta-actin hybrid promoter, and the Rous sarcoma virus promoter. Inducible expression control elements are commonly used in applications where it is desirable to provide controlled expression of heterologous nucleic acid sequences. Inducible promoter / enhancer elements for gene delivery include neuron-specific, brain-specific, muscle-specific (including cardiac, skeletal, and / or smooth muscle), liver-specific, bone marrow-specific, pancreas-specific, spleen-specific, and lung-specific promoter / enhancer elements. In certain embodiments, the promoter / enhancer functions in, and may be specific to, cells or tissues of the CNS. 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 synapsin (hSYN), phosphoglycerate kinase (PGK), or serotonin receptor promoters), glial cells (e.g., S100 or glutamine synthase promoters), and oligodendroglioma cells. Other promoters that have been shown to drive transcription in the CNS include, but are not limited to, the myelin basic protein (MBP) promoter (Tani et al., 1996) and the prion promoter (Loftus et al., 2002). Preferred are neuron-specific promoters that have significantly reduced, and preferably no, expression in glial cells.

[0085] Other inducible promoter / enhancer elements include drug-, hormone-, 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-repressible 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).

[0086] In certain embodiments of the invention, the promoter and / or enhancer is selected from the group comprising constitutively active promoters, such as CMV (cytomegalovirus immediate early gene enhancer / promoter), or CBA promoter (chicken beta actin promoter and human cytomegalovirus IE gene enhancer), or inducible promoters comprising gene switches, promoters from the tet operon, or neuron-specific promoters (preferably, but not limited to, human origin) from, for example, phosphoglycerate kinase (PGK), synapsin-1 (SYN), neuron-specific enolase (NSE).

[0087] In a specific embodiment of the present invention, the delivery vector further comprises a post-transcriptional regulatory element, preferably the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) or a truncated variant derived therefrom (Loeb et al. 1999; Choi et al. 2014). Other possible post-transcriptional regulatory elements are known to those skilled in the art.

[0088] A subject of the present invention is a recombinant viral particle or liposome or nanoparticle comprising a delivery vector according to the invention.

[0089] The subject of the present invention is a recombinant viral particle or liposome or nanoparticle, wherein said delivery vector further comprises a recombinant adeno-associated viral (AAV) vector genome, said rAAV vector genome being encapsulated within an AAV capsid, or said delivery vector further comprises a recombinant lentiviral vector genome and is packaged within a lentiviral particle.

[0090] For the sake of completeness, it will be apparent that the terms "encapsidated" and "packaged" as used herein with respect to viral particles are sometimes used interchangeably and refer to the polynucleotide (i.e., vector, genomic DNA, etc.) contained within the capsid or viral particle.

[0091] The subject of the present invention is also a recombinant gene therapy vector containing a heterologous therapeutic coding sequence, which is flanked by genetic elements for expression and virus-specific cis-elements for replication, genome packaging, genome integration, etc. The viral genome is packaged as a viral particle consisting of virus-specific proteins, as in the case of AAV. In the case of lentiviral vectors, the viral genome and virus-specific proteins (e.g., reverse transcriptase) are packaged in a lentiviral capsid, which is surrounded by a lipid bilayer in which the virus-specific proteins are embedded. Liposomes contain the above-mentioned nucleotide sequences or the entire DNA backbone, including all the regulatory elements of the gene therapy or delivery vector.

[0092] Examples of liposomes include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine N-[amino(polyethylene glycol)-2000], or DSPE-PEG2000-mal (1,2-distearoyl-sn-glycero-3-phosphoethanolamine N-[maleimide(polyethylene glycol)-2000], or variants containing sphingomyelin / cholesterol and phosphatidic acid.

[0093] In one particular 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 packaged in an AAV capsid.

[0094] Adeno-associated viruses (AAVs) have been developed as nucleic acid delivery vectors. For reviews, see (Muzyczka, 1992) and (Li and Samulski, 2020). AAVs are helper-dependent parvoviruses that require a helper virus, usually an adenovirus or a herpesvirus, for productive replication. AAVs are a growing family of at least 14 naturally occurring serotypes of human or primate origin. AAVs from other mammalian species, as well as avian and insect origins, have also been reported (see Berns et al., 2013). AAVs possess small icosahedral capsids, 18–26 nanometers in diameter, containing a single-stranded DNA genome 4–5 kilobases in length. AAVs encapsidate both AAV DNA strands, with either the sense or antisense DNA strand being incorporated into a single viral particle. The AAV genome contains two major open reading frames, encoding the rep and cap genes. rep encodes a family of overlapping nonstructural regulatory proteins. In AAV2, the most well-studied AAV prototype strain, Rep78 and Rep68 mRNAs are transcribed from the AAV p5 promoter (Stutika et al., 2015). Rep78 / 68 are required for AAV transcription, AAV DNA replication, and AAV integration into and rescue from the host cell genome. Rep52 and Rep40 are N-terminally truncated versions of Rep78 and Rep68 transcribed from a separate promoter, p19, and are required for packaging of newly synthesized AAV genomes into preformed AAV capsids. These are formed by three cap gene-derived proteins, VP1, VP2, and VP3. The cap ORF also encodes the assembly-enhancing proteins AAP and AAV egress facilitators called MAAPs. AAPs and MAAPs do not form part of the capsid (Sonntag et al. 2010; Elmore et al. 2021). The AAV ORF is flanked at both ends of the genome by inverted terminal repeats (ITRs), the length of which varies between AAV serotypes but is approximately 145 bp in AAV2, the first 125 bp of which can form a Y- or T-shaped double-stranded structure.The ITRs contain terminal resolution sites (trs), where the replicated, linear AAV genome is cleaved by Rep to form a unit-length ssAAV genome ready for packaging into AAV capsids. The ITRs are the minimal AAV sequences required in cis for DNA replication, packaging, genome integration, and rescue. These are the only sequences required within an AAV vector to ensure DNA replication and packaging of the AAV vector genome. A foreign gene flanked by the AAV-ITRs can be replicated and packaged into AAV capsids if 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) is deleted in one of the ITRs, so that the AAV genome is not cleaved by Rep at the affected end and remains as an uncleaved, double-stranded, self-complementary (sc) AAV genome.

[0095] AAV is one of the few viruses that can persist in vivo for months to years in non-dividing cells, such as neurons, muscle, liver, and heart. Wild-type AAV2 has been shown to integrate its genome into host cell genomes in a Rep78 / 68-dependent manner, with a preference for chromosomal loci that share DNA sequence homology with the so-called Rep binding sites that form part of the AAV-ITRs (Huser et al., 2014). In contrast, AAV vectors persist primarily as linear nuclear episomes. AAV vectors lacking the AAV rep and cap genes rarely integrate, and even when they do, they do not exhibit genome preference (Huser et al., 2014). Nevertheless, AAV has been shown to persist for long periods in non-dividing, post-mitotic cells, including neurons, making them ideal for CNS transduction and long-term gene-addition therapy of chronic diseases of inherited or acquired origin.

[0096] Typically, recombinant AAV vector (rAAV) genomes retain only the inverted terminal repeats (ITRs) in either native (ssAAV) or trs-deleted (scAAV) versions to maximize the size of the transgene that can be efficiently packaged by the vector. The structural and nonstructural protein coding sequences can be provided in trans from vectors such as plasmids, by stable integration of each gene into packaging cells, or by recombinant helper viruses such as HSV or baculovirus, as outlined in (Mietzsch, Grasse et al., 2014). Typically, rAAV vector genomes contain at least one AAV inverted terminal repeat (ITR), and more commonly, two AAV inverted terminal repeats, which are typically located at the 5' and 3' ends of the heterologous nucleotide sequence. AAV ITRs can be derived from any AAV, including serotypes 1-14. Because AAV2-derived ITRs can be cross-packaged into virtually any AAV serotype capsid, AAV2 ITRs in combination with AAV2 rep are primarily used. AAV terminal repeat sequences do not need to maintain wild-type terminal repeat sequences (e.g., wild-type sequences can be altered by insertions, deletions, truncations, or missense mutations) as long as they mediate the desired functions, such as DNA replication, viral packaging, integration, and / or proviral rescue. rAAV vector genomes generally range from about 70% to about 105% of the size of the wild-type genome and contain appropriate packaging signals as part of the AAV-ITRs. To facilitate packaging into AAV capsids, the entire vector genome (ITR to ITR) is preferably less than 5.2 kb, more preferably up to 4.8 kb, allowing the entire recombinant genome to be packaged into preformed AAV capsids. So-called dimeric or self-complementary AAV vectors (scAAV) have been developed to package double-stranded rather than single-stranded AAV genomes (McCarty et al., 2001). scAAVs result in enhanced AAV gene expression but reduced transgene capacity.The total packaging capacity is only 2.4 kb (ITR to ITR), which is sufficient for small genes or cDNAs, including those for neuropeptides.

[0097] Any suitable method known in the art can be used to generate AAV vectors expressing the nucleic acids of the present invention. AAV vector stocks can be generated by cotransfecting an ITR-flanked AAV vector genome plasmid expressing a transgene with an AAV rep / cap expression plasmid of the desired serotype and adenovirus-derived helper genes for AAV replication (Grimm et al., 2003; Xiao et al., 1998). AAV vectors can also be produced in mammalian or insect-derived packaging cell lines and / or in combination with recombinant helper viruses such as adenovirus, herpes simplex virus (HSV), another member of the herpesvirus family, or baculovirus, as reviewed and described in (Mietzsch, Grasse et al., 2014).

[0098] A subject of the present invention is a delivery vector comprising a DNA sequence encoding a preprodynorphin or a preprodynorphin variant as detailed herein.

[0099] The subject of the present invention is a delivery vector or recombinant viral particle or liposome or nanoparticle used to deliver nucleic acids to cells of the central nervous system, which comprises contacting the cells with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce a DNA sequence encoding preprodynorphin or a preprodynorphin variant into the cells, more particularly into the cell nucleus.

[0100] The delivery vectors of the present invention provide a means for delivering nucleic acid sequences to cells, preferably neurons, of the central nervous system. Delivery vectors can be used to transfer a nucleotide sequence of interest to cells in vitro (e.g., to produce a polypeptide in vitro) or for ex vivo gene therapy. The vectors are also useful in methods for delivering a nucleotide sequence to a subject in need thereof. In this way, the polypeptide is produced in vivo in the subject. The subject may need the polypeptide because they have a deficiency of the polypeptide, or because production of the polypeptide in the subject may provide some therapeutic benefit, as described further below, in a therapeutic or other manner.

[0101] In one particular embodiment of the method of delivering a nucleic acid to a cell of the central nervous system, preprodynorphin or a preprodynorphin variant is produced, processed, and the mature dynorphin peptide or variant thereof is released from the cell.

[0102] In one specific embodiment of the method for delivering nucleic acids to cells of the central nervous system, the method comprises contacting cells with the recombinant viral particles, liposomes, or nanoparticles described above under conditions sufficient to introduce a DNA sequence encoding preprodynorphin or a preprodynorphin variant into the cell nucleus.Conditions sufficient for the DNA sequence encoding preprodynorphin or a preprodynorphin variant to be introduced into cells are that the AAV capsid contacts the surface receptor and co-receptor of the host cell.AAV1 capsid binds to 2-3 sialic acid linked to N-acetylgalactosamine, followed by 1-4 N-acetylglucosamine, while AAV2 capsid binds to heparin sulfate proteoglycans on the cell surface, particularly 6-O- and N-sulfated heparin (Mietzsch, Broecker et al., 2014). AAV coreceptors, regardless of the presence of specific glycans, include FGFR-1, integrin aVb5, hepatocyte growth factor receptor (c-met), and the universal AAV receptor (AAVR, required for transduction by AAV1, AAV2, and other serotypes) (Pillay et al., 2016). AAVR directly binds to AAV particles and aids in their transport to the trans-Golgi network. AAV2 has been described to enter the nucleus using the nuclear pore complex, where it interacts with importin β alone or in complex with other import proteins (Nicolson and Samulski 2014). Most parvoviruses and AAV serotypes use similar mechanisms to enter the nucleus (Mattola et al. 2022), allowing AAV vectors to assemble within the cell nucleus.

[0103] A subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for use as a medicament.

[0104] A subject 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 the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject, or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, by activating human kappa opioid receptors in the epileptogenic focus to suppress seizures.

[0105] In particular, the delivery vectors or recombinant viral particles or liposomes or nanoparticles detailed herein can deliver DNA sequences encoding the preprodynorphin or preprodynorphin variants described herein, thereby promoting expression of the prepropeptide in target cells and enabling the on-demand release of dynorphin or dynorphin variants described herein from the target cells, thereby resulting in activation of human kappa opioid receptors in the epileptogenic focus.

[0106] The subject of the present invention is a delivery vector or a recombinant viral particle or a liposome or a nanoparticle for use in the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject by suppressing seizures through activation of human kappa opioid receptors in the epileptogenic focus and / or by on-demand release of peptides having an agonistic effect on human kappa opioid receptors in the epileptogenic focus, or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy.

[0107] A subject of the present invention is a delivery vector or recombinant viral particle or liposome or nanoparticle for use in the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein said vector or recombinant viral particle or liposome or nanoparticle is suitable for peripheral, or intracranial, or intracerebral, or intrathecal, or intraparenchymal administration.

[0108] A subject of the present invention is a delivery vector or recombinant viral particle or liposome or nanoparticle for use in the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein said delivery vector or recombinant viral particle or liposome or nanoparticle is administered intracerebrally, preferably intrafocally.

[0109] The subject of the present invention is an on-demand release pharmaceutical composition, a delivery vector or a recombinant viral particle or a liposome or a nanoparticle, and optionally a pharmaceutically acceptable carrier.

[0110] A subject of the present invention is a cell infected, preferably in vitro or ex vivo, with a delivery vector or a recombinant virus or a liposome or a nanoparticle.

[0111] The subject of the present invention is a method for treating a subject with focal epilepsy, in particular mesial temporal lobe epilepsy, or for preventing epileptic seizures in a subject suffering from focal epilepsy, wherein said method comprises administering to said subject a delivery vector recombinant viral particle or liposome or nanoparticle or pharmaceutical composition, wherein preferably said delivery vector or recombinant viral particle or liposome or nanoparticle encodes a prepropeptide which, after maturation and release, leads to activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures, and wherein preferably said delivery vector or recombinant viral particle or liposome or nanoparticle is administered intracerebrally, intraparenchyma, preferably into the focus.

[0112] Explanation of the following array:

[0113] SEQ ID NO: 1 (ppDyn) MAWQGLVLAA CLLMFPSTTA DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QVKRYGGFLR KYPKRSSEVA GEGDGDSMGH EDLYKRYGGF LRRIRPKLKW DNQKRYGG FLRRQFKVVT RSQEDPNAYS GELFDA Preprocessed human preprodynorphin expressed in the human brain.

[0114] SEQ ID NO: 2: DynA YGGFLRRIRPKLKWDNQ

[0115] SEQ ID NO: 3: DynB (rimorphin) YGGFLRRQFKVVT

[0116] SEQ ID NO: 4: Leumorphin YGGFLRRQFKVVTRSQEDPNAYSGELFDA

[0117] SEQ ID NO: 5: Unmodified propeptide fragment of ppDyn DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV

[0118] SEQ ID NO: 6: Modified propeptide fragment of ppDyn DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLRKEQVKR

[0119] SEQ ID NO: 7: Modified propeptide fragment of ppDyn DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV

[0120] SEQ ID NO: 8: Modified propeptide fragment of ppDyn DLGSKSVGEG PYSELAKLSG SFLRKE QV

[0121] SEQ ID NO: 9: Modified propeptide fragment of ppDyn DLGSKSVGEG PYSELAKLRKE QV

[0122] SEQ ID NO: 10: N-terminal portion of pPOMC MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KP

[0123] SEQ ID NO: 11: signal peptide of pp dynorphin MAWQGLVLAA CLLMFPSTTA

[0124] SEQ ID NO: 12: signal peptide of pp-enkephalin MARFLTLCTW LLLLGPGLLA TVRA

[0125] SEQ ID NO: 13: signal peptide of pp neuropeptide Y MLGNKRLGLS GLTLALSLLV CLGALAEA

[0126] SEQ ID NO: 14: signal peptide of pp somatostatin MLSCRLQCAL AALSIVLALG CVTG

[0127] SEQ ID NO: 15: signal peptide of pp tachykinin A MKILVALAVF FLVSTQLFA

[0128] SEQ ID NO: 16: signal peptide of pp tachykinin B MRIMLLFTAI LAFSLA

[0129] SEQ ID NO: 17: signal peptide of pProopiomelanocortin MPRSCCSRSG ALLLALLLQA SMEVRG

[0130] SEQ ID NO: 18: signal peptide of pp cholecystokinin MNSGVCLCVL MAVLAAGA

[0131] SEQ ID NO: 19: signal peptide of pp nociceptin MKVLLCDLLL LSLFSSVFS

[0132] SEQ ID NO: 20: signal peptide of pp chromogranin B MQPTLLLSLL GAVGLAAVNS

[0133] Sorting motif: SEQ ID NO: 36 DLX x EX y L Here, 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.

[0134] SEQ ID NO: 37: Preproenkephalin-N peptide MARFLTLCTW LLLLGPGLLA TVRAECSQDC ATCSYRLVRP ADINFLACVM ECEGKLPSLK IWETCKELLQ LSKPELPQDG TSTLRENSKP EESHLLA

[0135] SEQ ID NO: 38: Preproenkephalin-pDyn hybrid MARFLTLCTW LLLLGPGLLA TVRAECSQDC ATCSYRLVRP ADINFLACVM ECEGKLPSLK IWETCKELLQ LSKPELPQDG TSTLRENSKP EESHLLAKRY GGFLRKYPKR SSEVAGEGDG DSMGHEDLYK RYGGFLRRIR PKLKWDNQKR YGGFLRRQFK VVTRSQEDPN AYSGELFDA

[0136] SEQ ID NO: 39: PreproNPY-N peptide MLGNKRLGLS GLTLALSLLV CLGALAEA

[0137] SEQ ID NO: 40: PreproNPY-pDyn hybrid MLGNKRLGLS GLTLALSLLV CLGALEAKR YGGFLRKYPK RSSEVAGEGD GDSMGHEDLY KRYGGFLRRI RPKLKWDNQK RYGGFLRRQF KVVTRSQEDP NAYSGELFD

[0138] SEQ ID NO: 41: PreproBDNF-N peptide MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDHKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSM

[0139] SEQ ID NO: 42: PreproBDNF-pDyn hybrid MTILFLTMVI SYFGCMKAAP MKEANIRGQG GLAYPGVRTH GTLESVNGPK AGSRGLTSLA DTFEHVIEEL LDEDHKVRPN EENNKDADLY TSRVMLSSQV PLEPPLLFLL EEYKNYLDAA NMSMKRYGGF LRKYPKRSSE VAGEGDGDSM GHEDLYKRYG GFLRRIRPKL KWDNQKRYGG FLRRQFKVVT RSQEDPNAYS GELFD

[0140] SEQ ID NO: 43: Preprosomatostatin-N peptide MLSCRLQCAL AALSIVLALG CVTGAPSDPR LRQFLQKSLA AAAGKQELAK YFLAELLSEP NQTENDALEP EDLSQAAEQD EMRLELQR

[0141] SEQ ID NO: 44: Preprosomatostatin-pDyn hybrid MLSCRLQCAL AALSIVLALG CVTGAPSDPR LRQFLQKSLA AAAGKQELAK YFLAELLSEP NQTENDALEP EDLSQAAEQD EMRLELQRKR YGGFLRKYPK RSSEVAGEGD GDSMGHEDLY KRYGGFLRRI RPKLKWDNQK RYGGFLRRQF KVVTRSQEDP NAYSGELFD

[0142] SEQ ID NO: 45: Preprotachykinin AN peptide MKILVALAVF FLVSTQLFAE EIGANDDLNY WSDWYDSDQI KEELPEPFEH LLQRIA

[0143] SEQ ID NO: 46: Preprotachykinin A-pDyn hybrid MKILVALAVF FLVSTQLFAE EIGANDDLNY WSDWYDSDQI KEELPEPFEH LLQRIAKRYG GFLRKYPKRS SEVAGEGDGD SMGHEDLYKR YGGFLRRIRP KLKWDNQKRY GGFLRRQFKV VTRSQEDPNA YSGELFD

[0144] SEQ ID NO: 47: PreproVIP-N peptide MDTRNKAQLL VLLTLLSVLF SQTSAWPLYR APSALRLGDR IPFEGANEPD QVSLKEDIDM LQNALAENDT PYYDVSRNA

[0145] SEQ ID NO: 48: preproVIP-pDyn hybrid MDTRNKAQLL VLLTLLSVLF SQTSAWPLYR APSALRLGDR IPFEGANEPD QVSLKEDIDM LQNALAENDT PYYDVSRNAK RYGGFLRKYP KRSSEVAGEG DGDSMGHEDL YKRYGGFLRR IRPKLKWDNQ KRYGGFLRRQ FKVVTRSQED PNAYSGELFD

[0146] SEQ ID NO: 49: PreproCCK-N peptide MNSGVCLCVL MAVLAAGALT QPVPPADPAG SGLQRAEEAP RRQL

[0147] SEQ ID NO: 50: PreproCCK-pDyn hybrid MNSGVCLCVL MAVLAAGALT QPVPPADPAG SGLQRAEEAP RRQLKRYGGF LRKYPKRSSEV AGEGDGDSMG HEDLYKRYGG FLRRIRPKLK WDNQKRYGGF LRRQFKVVTR SQEDPNAYSG ELFD

[0148] SEQ ID NO: 51: Prepronociceptin-N peptide MKVLLCDLLL LSLFSVFSS CQRDCLTCQE KLHPALDSFD LEVCILECEE KVFPSPLWTP CTKVMARSSW QLSPAAPEHV AAALYQPRAS EMQHL

[0149] SEQ ID NO: 52: Prepronociceptin-pDyn hybrid MKVLLCDLLL LSLFSSVFSS CQRDCLTCQE KLHPALDSFD LEVCILECEE KVFPSPLWTP CTKVMARSSW QLSPAAPEHV AAALYQPRAS EMQHLKRYGG FLRKYPKRSS EVAGEGDGDS MGHEDLYKRY GGFLRRIRPK LKWDNQKRYG GFLRRQFKVV TRSQEDPNAY SGELFD

[0150] Shortened modified ppDyn: SEQ ID NO: 53 MAWQGLVLAA CLLMFPSTTA DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSGS FLRKEQVKR Y GGFLRKYP KR SSEVAGEGDG DSMGHEDLYK RYGGFLRRIR PKLKWDNQKR YGGFLRRQFK VVTRSQEDPN AYSGELFDA .

[0151] Hybrid pPOMC-ppDyn: SEQ ID NO: 54 MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KPKEQVKR YG GFLRKYP KRS SEVAGEGDGD SMGHEDLYKR YGGFLRRIRP KLKWDNQKR Y GGFLRRQFKV VTRSQEDPNA YSGELFDA

[0152] Modified propeptide fragment of ppDyn

[0153] SEQ ID NO: 55 DLGSKSVGEG PYSELRKEQV.

[0154] SEQ ID NO:56 DLGSKSVGEG PYSELAKLSG SFLKKEQV

[0155] SEQ ID NO:57 DLGSKSVGEG PYSELAKL

[0156] SEQ ID NO:58 DLGSKSVGEG PYSEL

[0157] SEQ ID NO: 59: ssAAV left ITR (145 bp) JPEG2025532800000001.jpg23160

[0158] SEQ ID NO: 60: ssAAV / scAAV right ITR (145 bp) JPEG2025532800000002.jpg21160

[0159] SEQ ID NO: 61: scAAV left ITR (Δtrs) (121 bp) JPEG2025532800000003.jpg23160

[0160] SEQ ID NO: 63: CBA promoter: CMV-enhancer, chicken beta actin promoter, chimeric intron (887 bp) JPEG2025532800000004.jpg92160

[0161] SEQ ID NO: 64: sCBA-promoter:CMV-enhancer, chicken beta actin promoter, chimeric intron (851 bp) JPEG2025532800000005.jpg90160

[0162] SEQ ID NO: 65: Human synapsin promoter (448 bp) JPEG2025532800000006.jpg49150

[0163] SEQ ID NO: 66: WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element (582 bp) JPEG2025532800000007.jpg59150

[0164] SEQ ID NO: 67: bGH polyA+: bovine growth hormone polyA+ signal sequence (208 bp) JPEG2025532800000008.jpg28150

[0165] SEQ ID NO: 68: SPA: synthetic polyA+ (49 bp) JPEG2025532800000009.jpg11126

[0166] SEQ ID NO: 69: ppDyn full-length cDNA codon-optimized 1 (765 bp) JPEG2025532800000010.jpg75150

[0167] SEQ ID NO: 70: ppDyn (576 bp) with shortened N-peptide codon-optimized 2 JPEG2025532800000011.jpg61150

[0168] SEQ ID NO: 71: ppDyn (399 bp) with truncated N-peptide codon-optimized 2 JPEG2025532800000012.jpg43150

[0169] SEQ ID NO: 72: ppDyn (519 bp) with shortened N-peptide codon-optimized 2 JPEG2025532800000013.jpg54150

[0170] SEQ ID NO: 73: ppDyn (417 bp) with the N-terminus from POMC fused to the C-terminal portion of pDyn codon-optimized 2 JPEG2025532800000014.jpg42150

[0171] SEQ ID NO: 74: ssAAV-pDyn (2820 bp) JPEG2025532800000015.jpg235138

[0172] SEQ ID NO: 75: scAAV-pDyn (2210 bp) JPEG2025532800000016.jpg203150

[0173] SEQ ID NO: 76: scAAV-pDyn (2603 bp) JPEG2025532800000017.jpg234148

[0174] SEQ ID NO: 77: scAAV-syn-pDyn (2164 bp) JPEG2025532800000018.jpg205150

[0175] SEQ ID NO: 78: scAAV-pDyn (2388 bp) JPEG2025532800000019.jpg218150

[0176] SEQ ID NO: 79: scAAV-pDyn (2270 bp) JPEG2025532800000020.jpg206150

[0177] SEQ ID NO: 80: scAAV-pDyn (2334 bp) JPEG2025532800000021.jpg212150

[0178] SEQ ID NO: 81: scAAV-pDyn (2447 bp) JPEG2025532800000022.jpg222150

[0179] Specific embodiments of the present invention are as follows.

[0180] 1. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the delivery vector promotes expression of the prepropeptide in the target cell; and wherein said delivery vector comprising said DNA sequence allows the dynorphin or dynorphin variant to be released from target cells as needed, 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 prepropeptide is (i) an N-terminal propeptide fragment at the C-terminus of the signal peptide, and wherein the N-terminal propeptide fragment comprises elements DL and EX y The sorting motif containing L, especially 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 may independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in particular in a first sequence of e.g. 1 to 20 X, each X may individually be any amino acid, and in a second sequence of e.g. 1 to 10 X, each X may individually be any amino acid, as further defined herein); or wherein the prepropeptide is (ii) comprising, on the C-terminus of the signal peptide, an N-terminal propeptide fragment of a prepro-neuropeptide or protein other than preprodynorphin that is sorted into large dense core vesicles, wherein the N-terminal propeptide fragment comprises a sorting motif of the prepro-neuropeptide or protein that is sorted into large dense core vesicles, and wherein the N-terminal propeptide fragment consists of 16 to 90 amino acids; wherein said preprodynorphin or preprodynorphin variant is provided by a vector comprising at least one of the following sequences selected from the group consisting of: a. DynA of 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. DynB, which is SEQ ID NO: 3; c. Leumorphin, which is SEQ ID NO: 4 d. A variant of DynA having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO:2; e. A variant of DynB having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 3; f. A variant of leumorphin having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO:4.

[0181] 2. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of embodiment 1, wherein the N-terminal propeptide fragment consists of 20 to 90 amino acids, preferably 30 to 90 amino acids.

[0182] 3. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of embodiment 1 or 2, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is a modified propeptide fragment of ppDyn, and wherein the unmodified propeptide fragment of ppDyn is: SEQ ID NO:5 DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV and wherein the modification of said propeptide fragment of ppDyn is shortened; or the modification is a replacement of a portion of SEQ ID NO: 5 with a propeptide of a neuropeptide or a fragment of a propeptide of a neuropeptide; wherein said modified propeptide fragment of ppDyn comprises: i) elements DL and EX y At least one sorting motif containing L, in particular the amino acid sequence DLX x EX y L (SEQ ID NO: 36), or ii) a sorting motif for said prepro-neuropeptide or protein that is sorted into large dense core vesicles other than prepro-dynorphin.

[0183] 4. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of any one of embodiments 1 to 3, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is: SEQ ID NO:6 DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLRKEQVKR A delivery vector which is a modified propeptide fragment of ppDyn comprising or consisting of:

[0184] 5. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of any one of embodiments 1 to 3, wherein the N-terminal propeptide fragment C-terminal to the signal peptide is: SEQ ID NO:7 DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV, or SEQ ID NO: 8 DLGSKSVGEG PYSELAKLSG SFLRKE QV, or SEQ ID NO: 9 DLGSKSVGEG PYSELAKLRKE QV A delivery vector which is a modified propeptide fragment of ppDyn comprising or consisting of:

[0185] 6. A delivery vector comprising a DNA sequence encoding a preprodynorphin or preprodynorphin variant according to embodiment 3, wherein the modification is a replacement of a portion of SEQ ID NO: 5 with a propeptide of a neuropeptide or a fragment of a propeptide of a neuropeptide; Here, the modified propeptide fragment of ppDyn comprises: i) elements DL and EX y At least one sorting motif containing L, in particular the amino acid sequence DLX x EX y L (SEQ ID NO: 36), or ii) a sorting motif for said prepro-neuropeptide or protein that is sorted into large dense core vesicles other than prepro-dynorphin.

[0186] 7. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of embodiment 6, wherein the modified propeptide fragment is: SEQ ID NO: 10 MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KP A delivery vector comprising or consisting of:

[0187] 8. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of embodiment 6, wherein the modified propeptide fragment comprises or consists of a propeptide fragment of preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, preproVIP, preproCCK, prepronociceptin, or preproNPY, wherein the propeptide fragment comprises a sorting motif, and in particular the propeptide fragment may be selected from the group comprising any of SEQ ID NOs: 37 to 52.

[0188] 9. A delivery vector comprising a DNA sequence encoding a preprodynorphin or preprodynorphin variant according to any one of embodiments 1 to 8, wherein the modified propeptide fragment is optionally flanked by peptidase recognition signals comprising K, R, KR, RK, or RR.

[0189] 10. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of embodiments 1 to 9, wherein the target cells are neurons of the central nervous system.

[0190] 11. A delivery vector comprising a DNA sequence encoding a preprodynorphin or 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 a precursor protein destined for the lumen of the endoplasmic reticulum.

[0191] 12. The signal peptide MAWQGLVLAA CLLMFPSTTA (SEQ ID NO: 11) MARFLTLCTW LLLLGPGLLA TVRA (SEQ ID NO: 12) MLGNKRLGLS GLTLALSLLV CLGALAEA (SEQ ID NO: 13) MLSCRLQCAL AALSIVLALG CVTG (SEQ ID NO: 14) MKILVALAVF FLVSTQLFA (SEQ ID NO: 15) MRIMLLFTAI LAFSLA (SEQ ID NO: 16) MPRSCCSRSG ALLLALLLQA SMEVRG (SEQ ID NO: 17) MNSGVCLCVL MAVLAAGA (SEQ ID NO: 18) MKVLLCDLLL LSLFSSVFS (SEQ ID NO: 19) MQPTLLLSLL GAVGLAAVNS (SEQ ID NO: 20) 12. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant according to any one of embodiments 1 to 11, selected from the group comprising:

[0192] 13. A delivery vector according to any one of embodiments 1 to 12, which provides for the on-demand release of a dynorphin or dynorphin variant having an agonistic effect on the human kappa opioid receptor.

[0193] 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 from the N-terminus of SEQ ID NO: 2 (YGGFLRRI), SEQ ID NO: 3 (YGGFLRRQ), or SEQ ID NO: 4 (YGGFLRRQ), respectively.

[0194] 15. A delivery vector according to any one of embodiments 1 to 14, wherein the variant has at least 80% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, respectively.

[0195] 16. A delivery vector according to any one of embodiments 1 to 15, wherein the delivery vector further comprises a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome.

[0196] 17. The delivery vector of any of embodiments 1 to 16, comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs) preferably derived from AAV serotype 2, or AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein the vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, snake 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 further AAV capsid mutants derived therefrom, or in a chimeric vector comprising capsid proteins from two or more, preferably two, of the foregoing AAV serotype capsids, and in certain embodiments, the capsid is from AAV serotype 1 and / or 2.

[0197] In certain embodiments, the delivery vector according to any one of embodiments 1 to 16 comprises a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably from AAV serotype 2, or AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein said vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. , 10, rh10, 11, 12, 13, 14, snake 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 further AAV capsid variants derived therefrom, preferably of AAV serotype 1 or 2.

[0198] 18. A delivery vector comprising a DNA sequence encoding a preprodynorphin or preprodynorphin variant according to any of the preceding embodiments.

[0199] 19. A recombinant viral particle or liposome or nanoparticle comprising a delivery vector according to any of the preceding embodiments.

[0200] 20. The recombinant viral particle, liposome, or nanoparticle of embodiment 19, wherein the delivery vector further comprises a recombinant adeno-associated viral (AAV) vector genome, and the rAAV vector genome is enclosed in an AAV capsid, or wherein the delivery vector further comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

[0201] 21. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any of embodiments 1 to 20 for use in delivering a nucleic acid to 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 sequence encoding preprodynorphin or a preprodynorphin variant into the cell.

[0202] 22. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 1 to 21 for use as a medicament.

[0203] 23. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any of embodiments 1 to 21 for use in the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject, or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle results in activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures.

[0204] It will be apparent to those skilled in the art that the delivery vectors or recombinant viral particles or liposomes or nanoparticles, as detailed herein, induce the production of the corresponding peptides, as further detailed herein, resulting in activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures.

[0205] 24. A DNA sequence selected from the group comprising SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73.

[0206] 25. A delivery vector comprising the DNA sequence according to embodiment 24.

[0207] 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.

[0208] 27. The delivery vector of embodiment 25 or 26, comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably derived from AAV serotype 2, or from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein the vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, snake AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, A delivery vector packaged within an AAV capsid selected from the group comprising AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid variants derived therefrom, or a chimeric vector, or a chimeric vector comprising mosaic capsid proteins derived from two or more, preferably two, of the aforementioned AAV serotype capsids, wherein in certain embodiments, the capsid is a capsid derived from AAV serotype 1 and / or 2. In certain embodiments, the delivery vector of embodiment 25 or 26 comprises a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably from AAV serotype 2, or from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or redesigned variants thereof, wherein said vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh1 0, 11, 12, 13, 14, snake 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 further AAV capsid variants derived therefrom, preferably packaged within two of the above AAV capsids, preferably AAV serotypes 1 and 2.

[0209] 28. A delivery vector described in any of embodiments 25 to 27, wherein the delivery vector further comprises at least one sequence selected from the group comprising 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.

[0210] 29. A delivery vector described in any of embodiments 25 to 28, wherein the delivery vector comprises a sequence selected from the group comprising 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.

[0211] 30. A recombinant viral particle, liposome or nanoparticle comprising a delivery vector according to any one of embodiments 25 to 29.

[0212] 31. The recombinant viral particle, liposome, or nanoparticle of embodiment 30, wherein the delivery vector further comprises a recombinant adeno-associated viral (AAV) vector genome, and the rAAV vector genome is enveloped in an AAV capsid, or the delivery vector further comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

[0213] 32. The delivery vector or recombinant viral particle or liposome or nanoparticle according to any of embodiments 25 to 31, for use in delivering nucleic acids to cells of the central nervous system, comprising contacting the delivery vector or recombinant viral particle or liposome or nanoparticle with a cell under conditions sufficient to introduce 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.

[0214] In certain embodiments, the DNA comprising a sequence selected from the group comprising SEQ ID NOs: 69 to 73 that is introduced into a cell is DNA selected from SEQ ID NOs: 74 to 81 (AAV genome), and in the case of a lentivirus, is DNA in which a sequence selected from the group comprising SEQ ID NOs: 69 to 73 and a regulatory sequence are embedded in the lentivirus genome.

[0215] 33. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of embodiments 25 to 32 for use as a medicament.

[0216] 34. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any of embodiments 25 to 33 for use in the treatment of epilepsy, particularly focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject, or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle results in activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures. [Brief explanation of the drawings]

[0217] [Figure 1] Figure 1: Overview of human ppDyncDNA truncations. [Figure 2] Figure 2: ELISA results measuring (A) dynorphin A (DynA) and (B) dynorphin B (DynB) content after intraparenchymal CNS transduction with AAV vectors expressing the indicated ppDyn variants. Variant A = truncated N-peptide retaining the sorting motif. Variant B = signal and N-peptide replaced with a POMC signal and sorting motif. Variant C = truncated N-peptide with the sorting motif deleted. ipsi = site of AAV transduction, contra = non-transduced (control) site. [Figure 3] Figure 3: Seizure suppression by two scAAV vector variants containing either a truncated pDyncDNA or an alternative signal and sorting sequence. Data represent N ± SEM (N = 3). [Figure 4]Figure 4: Overview of DNA sequence elements of AAV-pDyn vector variants. DNA sequence elements of the indicated AAV vectors are shown. Sequence numbering refers to that in the main text. Dark gray boxes: DNA sequence elements derived from AAV serotype 2. ITR = inverted terminal repeat; ΔITR = inverted terminal repeat with deleted terminal release site used in self-complementary (sc) AAV vectors. Light gray boxes: DNA sequence elements for promoters or other regulatory elements for gene expression. CBAprom. = cytomegalovirus (CMV) enhancer fused to chicken beta-actin promoter followed by a chimeric intron. sCBAprom. = truncated cytomegalovirus (CMV) enhancer fused to chicken beta-actin promoter followed by a chimeric intron. hSynprom. = human synapsin promoter. WPRE = woodchuck hepatitis virus posttranscriptional regulatory element; bGHpA+ = bovine growth hormone gene polyA signal sequence. SpA+ = synthetic polyA+ signal sequence. White boxes: DNA sequence elements of human ppDyncDNA. The ppDyncDNA sequence has been codon-optimized. Codon-optimized version 1 is used in SEQ ID NOs: 74 and 75. Codon-optimized version 2 is used in SEQ ID NOs: 76-80 and for the pDyn portion of SEQ ID NO: 81. Pre = DNA sequence of the signal sequence of ppDyn, pro = DNA sequence covering the N-peptide of ppDYN, pro1, pro2, and proDs = various shortened versions of "pro" referring to the N-peptide of ppDyn. POMC = cDNA sequence of the N-terminal portion of the neuropeptide POMC, spanning the pre- and pro-elements and replacing elements of ppDyn. pDyn = prodynorphin. [Figure 5]Figure 5: Reduction in seizure activity after injection of AAV-pDyn expressing SEQ ID NO: 70. Hpd, generalized seizures, and spike trains were measured at each time interval over 48 hours. Data are shown as % of pre-treatment seizure activity for Hpd and spike trains (left y-axis). Generalized seizures are shown as absolute number of seizures (right y-axis). N=7-9, p=0.0025 for Hpd, <0.0001 for spike trains, and 0.0001 for generalized seizures (one-way ANOVA). Mice show a significant reduction in drug-resistant focal seizures starting 7 days after treatment. Generalized seizures disappear almost completely after 4 weeks. [Figure 6] Figure 6: Reduction in seizure activity after injection of AAV-pDyn expressing SEQ ID NO: 77. HPD was measured at each time interval over 48 h. Data are shown as time spent in HPD. N=2-3. Mice show a significant reduction in drug-resistant focal seizures starting from day 10 after AAV delivery.

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Example

[0219] Example 1 Human ppDycDNA was truncated to enable packaging into scAAV vectors. 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 propeptide into large dense-core vesicles must be preserved. Alternatively, the entire N-terminal portion of the region encoding the active peptide can be replaced with a different signal peptide and sorting motif.

[0220] Example 2 Generation of mature dynorphin by two scAAV vector variants Mature dynorphin production by three scAAV vector variants containing truncated pDyn cDNA. The vectors were injected into the dorsal hippocampus of intact wild-type mice. Two weeks later, the hippocampi were excised, and dynorphin A (DynA) and dynorphin B (DynB) content was measured by ELISA as described by Agostinho et al. (2019) (see Figure 2). Mature peptides were produced exclusively in large dense-core vesicles, demonstrating correct sorting by truncated pDyn variants A (SEQ ID NO: 76) and B (SEQ ID NO: 81). A significant reduction in mature dynorphin production with variant C (SEQ ID NO: 80), which lacks the proposed sorting motif, demonstrates the importance of the proposed sorting motif.

[0221] Example 3 Seizure suppression by truncated pDyncDNA Two scAAV vector variants containing truncated pDyncDNA also suppressed seizures (Figure 3). The vectors were injected into the dorsal hippocampus of epileptic wild-type mice. Electroencephalograms were recorded and analyzed for hippocampal paroxysmal discharges (HPD), which represent drug-resistant focal seizures. Kainic acid treatment of animals, electrode implantation, and analysis were described in Widmann et al. (2022).

[0222] Example 7 Construction of AAV-pDyn vector variants AAV vectors were constructed in either ssAAV or scAAV formats, as shown in Figure 4. These consist of one left ITR sequence from AAV serotype 2 and one right ITR sequence (SEQ ID NOS: 59-61). AAV ITRs from alternative AAV serotypes or synthetic ITRs can be used as well.

[0223] The ITRs flank one of the heterologous gene expression cassettes shown (Figure 4). These cassettes consist of one of the promoter sequences (SEQ ID NOS: 63-65), a posttranscriptional regulatory element from woodchuck hepatitis virus (SEQ ID NOS: 66) (described by Loeb et al., Hum Gene Ther 10:2295-2305, 1999), a polyadenylation signal derived from the bovine growth hormone gene (SEQ ID NOS: 67) or a short synthetic poly(A) signal sequence (SEQ ID NOS: 68) (described by Levitt et al., Genes & Dev 3:1019-25, 1989), and the cDNA to be expressed.

[0224] The gene of interest is the cDNA sequence of human preprodynorphin (ppDyn), any of its variants shown (SEQ ID NOs: 69-72), or a fusion of the N-terminus of POMC with the C-terminal portion of prodynorphin lacking the signal sequence (pre) and N-peptide (pro) (SEQ ID NO: 73).

[0225] The cDNA was codon-optimized in two versions. Version 1 (SEQ ID NO: 69) is included in AAV SEQ ID NO: 74 and SEQ ID NO: 75. Codon-optimized version 2 (SEQ ID NOs: 70-73) was created to reduce the proportion of CpG sequence elements. Unmethylated CpG sequence elements are characteristic of bacterial DNA and represent pathogen-associated molecular patterns (PAMPs), which may activate the mammalian host's innate immune system and, under certain circumstances, may be problematic in the context of AAV gene therapy. A high CpG content in the transduced AAV genome may increase immune-mediated loss of transduced cells. The adverse effects of a high CpG content in the transduced AAV genome have also recently been demonstrated in the CNS after intraparenchymal / intracerebral AAV transduction (Suriano et al. 2021). Alternative codon-optimized strategies that combine enhanced transgene expression in human cells with sufficient reduction of unmethylated CpG elements in AAV-transduced genomes may be achieved through various DNA sequence modifications.

[0226] As shown in Figure 4, the complete AAV DNA sequence from ITR to ITR spanning all regulatory elements and the various ppDyn-derived transgenes is represented as a DNA sequence file (SEQ ID NOs: 74-81).

[0227] Any of the elements can be further exchanged; for example, the truncated ITRs of scAAV can be placed at the right end of the AAV genome instead of the left end, as shown here. Similarly, gene promoters can be exchanged and / or combined with one of the poly(A) signal sequences shown or with a poly(A) signal of a different origin. The WPRE element can be used as the full-length 582-bp element (SEQ ID NO: 66) shown. The WPRE is composed of subelements named gamma, alpha, and beta, in the order shown. A shorter version (WPRE2), displaying minimal gamma elements and partial alpha / beta elements, and WPRE3, displaying only minimal gamma and alpha elements (247 bp), have been described as being similarly active (Choi et al. 2014, incorporated herein by reference). The WPRE versions can be used interchangeably or not incorporated into the AAV genome at all.

[0228] Example 8 Functional testing of AAV vectors The AAV vectors of SEQ ID NOs: 76-78 and SEQ ID NO: 81 were functionally tested to generate fully processed mature dynorphin peptides (SEQ ID NOs: 76 and 81, Example 3), and to monitor focal seizure suppression in a TLE mouse model (SEQ ID NOs: 76, 77, and 78).

[0229] Reduction of different types of seizure activity after injection of AAV-pDyn expressing SEQ ID NO: 70. Hpd is shown in Figure 5. Hpd, generalized seizures, and spike trains were measured at various time intervals over 48 hours. Mice show a significant reduction in drug-resistant focal seizures starting from day 7 after treatment. Generalized seizures are almost completely eliminated after 4 weeks.

[0230] The reduction in HPD after injection of AAV-pDyn expressing SEQ ID NO:77 is shown in FIG. Mice show a marked reduction in drug-resistant focal seizures starting 10 days after AAV delivery.

Claims

1. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant, wherein the delivery vector promotes expression of the prepropeptide in the target cell, and wherein said delivery vector comprising said DNA sequence allows the dynorphin or dynorphin variant to be released from target cells as needed; 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 prepropeptide is (i) an N-terminal propeptide fragment at the C-terminus of the signal peptide, and wherein the N-terminal propeptide fragment comprises elements DL and EX ｙ Sorting motifs containing L, particularly 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 may independently be any amino acid (for the avoidance of doubt, this means that in the present invention, in particular in a first sequence of e.g. 1 to 20 X, each X may individually be any amino acid, and in a second sequence of e.g. 1 to 10 X, each X may individually be any amino acid, as further defined herein); or wherein the prepropeptide is (ii) comprising, on the C-terminus of the signal peptide, an N-terminal propeptide fragment of a prepro-neuropeptide or protein other than preprodynorphin that is sorted into large dense core vesicles, wherein the N-terminal propeptide fragment comprises a sorting motif of the prepro-neuropeptide or protein that is sorted into large dense core vesicles, and wherein the N-terminal propeptide fragment consists of 16 to 90 amino acids; and wherein the preprodynorphin or preprodynorphin variant is a vector comprising at least one of the following sequences selected from the group consisting of: a. DynA of 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. DynB, which is SEQ ID NO: 3; c. Leumorphin, which is SEQ ID NO: 4; d. A variant of DynA having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 2; e. A variant of DynB having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO: 3; f. A variant of leumorphin having at least 60% amino acid sequence identity within the first 8 amino acids from the N-terminus of SEQ ID NO:

4.

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

3. 3. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of claim 1 or 2, wherein the N-terminal propeptide fragment at the C-terminus of the signal peptide is a modified propeptide fragment of ppDyn, and wherein the unmodified propeptide fragment of ppDyn is: SEQ ID NO: 5 DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV and wherein the modification of said propeptide fragment of ppDyn is shortened; or the modification is a replacement of a portion of SEQ ID NO: 5 with a propeptide of a neuropeptide or a fragment of a propeptide of a neuropeptide; wherein said modified propeptide fragment of ppDyn comprises: i) elements DL and EX ｙ At least one sorting motif containing L, in particular the amino acid sequence DLX ｘ EX ｙ L (SEQ ID NO: 36), or ii) a sorting motif for said prepro-neuropeptide or protein that is sorted into large dense core vesicles other than prepro-dynorphin.

4. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of any one of claims 1 to 3, wherein the N-terminal propeptide fragment at the C-terminus of the signal peptide is SEQ ID NO: 6 DCLSRCSLCAVKTQDGPKPINPLICSLQCQAALLPSEEWERCQSFLSFFTPSTLGLNDKEDLGSKSVGEGPYSELAKLSGSFLRKEQVKR A delivery vector which is a modified propeptide fragment of ppDyn comprising or consisting of:

5. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of any one of claims 1 to 3, wherein the N-terminal propeptide fragment at the C-terminus of the signal peptide is SEQ ID NO:7 DLGSKSVGEG PYSELAKLSG SFLKELEKSK FLPSISTKEN TLSKSLEEKL RGLSDGFREG AESELMRDAQ LNDGAMETGT LYLAEEDPKE QV, or SEQ ID NO: 8 DLGSKSVGEG PYSELAKLSG SFLRKE QV, or SEQ ID NO: 9 DLGSKSVGEG PYSELAKLRKE QV A delivery vector which is a modified propeptide fragment of ppDyn comprising or consisting of:

6. 4. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of claim 3, wherein the modification is a replacement of a portion of SEQ ID NO: 5 with a propeptide of a neuropeptide or a fragment of a propeptide of a neuropeptide; wherein the modified propeptide fragment of ppDyn comprises: i) elements DL and EX ｙ At least one sorting motif containing L, in particular the amino acid sequence DLX ｘ EX ｙ L (SEQ ID NO: 36), or ii) a sorting motif for said prepro-neuropeptide or protein that is sorted into large dense core vesicles other than prepro-dynorphin.

7. 7. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of claim 6, wherein the modified propeptide fragment is: SEQ ID NO: 10 MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KP A delivery vector comprising or consisting of:

8. 7. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of claim 6, wherein the modified propeptide fragment comprises or consists of a propeptide fragment of preproenkephalin, preproBDNF, preprotachykinin, preprosomatostatin, preproVIP, preproCCK, prepronociceptin, or preproNPY, wherein the propeptide fragment comprises a sorting motif, and in particular the propeptide fragment may be selected from the group comprising any of SEQ ID NOs: 37 to 52.

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

10. A delivery vector comprising a DNA sequence encoding preprodynorphin or a preprodynorphin variant according to any one of claims 1 to 9, wherein the target cells are neurons in the central nervous system.

11. A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of 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 a precursor protein that is to become part of the lumen of the endoplasmic reticulum.

12. The signal peptide MAWQGLVLAA CLLMFPSTTA (SEQ ID NO: 11) MARFLTLCTW LLLLGPGLLA TVRA (SEQ ID NO: 12) MLGNKRLGLS GLTLALSLLV CLGALAEA (SEQ ID NO: 13) MLSCRLQCAL AALSIVLALG CVTG (SEQ ID NO: 14) MKILVALAVF FLVSTQLFA (SEQ ID NO: 15) MRIMLLFTAI LAFSLA (SEQ ID NO: 16) MPRSCCSRSG ALLLALLLQA SMEVRG (SEQ ID NO: 17) MNSGVCLCVL MAVLAAGA (SEQ ID NO: 18) MKVLLCDLLL LSLFSSVFS (SEQ ID NO: 19) MQPTLLLSLL GAVGLAAVNS (SEQ ID NO: 20) A delivery vector comprising a DNA sequence encoding the preprodynorphin or preprodynorphin variant of any one of claims 1 to 11, selected from the group comprising:

13. A delivery vector according to any one of claims 1 to 12, which provides for the on-demand release of dynorphin or a dynorphin variant having an agonistic effect on the human kappa opioid receptor.

14. The delivery vector of any one of claims 1 to 13, wherein the dynorphin variants have at least 70% amino acid sequence identity within the first eight amino acids from the N-terminus of SEQ ID NO: 2 (YGGFLRRI), the first eight amino acids from the N-terminus of SEQ ID NO: 3 (YGGFLRRQ), or the first eight amino acids from the N-terminus of SEQ ID NO: 4 (YGGFLRRQ).

15. 15. The delivery vector of any one of claims 1 to 14, wherein the variants have at least 80% amino acid sequence identity within the first 8 amino acids 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, further comprising a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome.

17. 17. The delivery vector of any of claims 1 to 16, comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably derived from AAV serotype 2, or AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rhlO, 11, 12, 13, 14, or redesigned variants thereof, wherein the vector genome is selected from the group consisting of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rhlO, 11, 12, 13, 14, snake AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV. A delivery vector packaged in an AAV capsid selected from the group comprising AAV2.CAP-MAC, AAV2.NN, or further in an AAV capsid mutant derived therefrom, preferably derived from AAV serotypes 1 and / or 2, or in a chimeric vector comprising capsid proteins derived from two or more, preferably two, of the aforementioned AAV serotype capsids, wherein in a particular embodiment, said capsid is a capsid derived from AAV serotype 1 and / or 2.

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

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

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

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

22. A 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. 22. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any one of claims 1 to 21 for use in the treatment of focal epilepsy, in particular mesial temporal lobe epilepsy, in a subject or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle results in activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures.

24. A DNA sequence selected from the group comprising 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 of claim 24.

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

27. 27. A delivery vector according to claim 25 or 26, comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITRs), preferably derived from AAV serotype 2, or from AAV serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, rhlO, 11, 12, 13, 14, or redesigned variants thereof, wherein the vector genome is derived from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rhlO, 11, 12, 13, 14, snake AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV. a delivery vector packaged within an AAV capsid selected from the group comprising AAV capsids selected from the group comprising AAV2.CAP-MAC, AAV2.NN, or further AAV capsid variants derived therefrom, or chimeric vectors comprising mosaic capsid proteins derived from two or more, preferably two, of the aforementioned AAV serotype capsids, wherein in certain embodiments, said capsid is a capsid derived from AAV serotype 1 and / or 2.

28. 28. The delivery vector of any of claims 25 to 27, wherein the delivery vector further comprises at least one sequence selected from the group comprising 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. 29. The delivery vector of any of claims 25 to 28, wherein the delivery vector comprises a sequence selected from the group comprising 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. 31. The recombinant viral particle, liposome, or nanoparticle of claim 30, wherein the delivery vector further comprises a recombinant adeno-associated viral (AAV) vector genome and the rAAV vector genome is enveloped in an AAV capsid, or the delivery vector further comprises a recombinant lentiviral vector genome and is packaged in a lentiviral particle.

32. 32. The delivery vector or recombinant viral particle or liposome or nanoparticle of any one of claims 25 to 31 for use in delivering nucleic acids to cells of the central nervous system, comprising contacting a cell with the delivery vector or recombinant viral particle or liposome or nanoparticle under conditions sufficient to introduce 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.

33. A 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. 34. A delivery vector or recombinant viral particle or liposome or nanoparticle according to any of claims 25 to 33 for use in the treatment of epilepsy, particularly focal epilepsy, especially mesial temporal lobe epilepsy, in a subject, or for use in the prevention of epileptic seizures in a subject suffering from focal epilepsy, wherein the delivery vector or recombinant viral particle or liposome or nanoparticle results in activation of human kappa opioid receptors in the epileptogenic focus, thereby suppressing seizures.

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