SCHWARZER CHRISTOPH (AT)
HÜSER DANIELA (DE)
WO1998010088A1 | 1998-03-12 |
EP3472196A1 | 2019-04-24 |
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C76014WO BOEHMERT & BOEHMERT Claims 1. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants and ^ wherein said delivery vector drives expression of a pre-propeptide in a target cell, and ^ wherein said delivery vector comprising said DNA sequence enables the release of dynorphin or dynorphin-variants from the target cell on demand, and ^ wherein said pre-propeptide is pre-prodynorphin or a pre-prodynorphin-variant and ^ wherein said pre-propeptide comprises a signal peptide, wherein the signal peptide is a N- terminal extension of a nascent polypeptide chain and wherein said signal peptide mediates protein targeting to the lumen of the endoplasmatic reticulum, and, ^ wherein said pre-propeptide comprises either (i) a N-terminal pro-peptide fragment on the C-terminal end of said signal peptide and wherein said N-terminal pro-peptide fragment comprises a sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (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 may independently be any amino acid (for the avoidance of doubt, this means that in the present invention in the first sequence of e.g. 1 to 20 X, each X may individually be any amino acid, and in the second sequence of e.g.1 to 10 X, each X may individually be any amino acid, particularly as further defined herein), or wherein said pre-propeptide comprises (ii) a N-terminal pro-peptide fragment of a pre-pro-neuropeptide or protein sorted to large dense core vesicles, other than pre-prodynorphin, on the C-terminal end of said signal peptide and wherein said N-terminal pro-peptide fragment comprises a sorting motif of said pre-pro-neuropeptide or protein sorted to large dense core vesicles, and wherein said N-terminal pro-peptide fragment consists of 16 to 90 amino acids, and ^ wherein said pre-prodynorphyin or pre-prodynorphin-variants comprise at least one of the following sequences selected from the group: a. Dyn A that is SEQ ID No.2 or a variant thereof consisting of the first 13 amino acids from the N-terminal end or a variant thereof consisting of the first 8 amino acids from the N- terminal end b. Dyn B that is SEQ ID No.3 c. leumorphin that is SEQ ID No. 4 d. variants of Dyn A , said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No.2, e. variants of Dyn B, said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No.3, f. variants of leumorphin, said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 4. 2. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to claim 1, wherein said N-terminal pro-peptide fragment consists of 20 to 90 amino acids, preferably 30 and 90 amino acids. 3. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to claim 1 or 2, wherein said N-terminal pro-peptide fragment on the C-terminal end 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 ppDyn is a shortening; or the modification is a replacement of parts of SEQ ID No.5 with a propeptide of or a fragment of a propeptide of a neuropeptide; wherein the modified propeptide fragment of ppDyn i) comprises at least one sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (SEQ ID No.36) or ii) comprises a sorting motif of said pre-pro- neuropeptide or protein sorted to large dense core vesicles other than pre-prodynorphin. 4. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-3, wherein said N-terminal pro-peptide fragment on the C- terminal end of the signal peptide is a modified propeptide fragment of ppDyn that comprises or consists of SEQ ID No.6 DCLSRCSLCAVKTQDGPKPINPLICSLQCQAALLPSEEWERCQSFLSFFTPSTLGLNDKED LGSKSVGEGPYSELAKLSGSFLRKEQVKR 5. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-3, wherein said N-terminal pro-peptide fragment on the C- terminal end of the signal peptide is a modified propeptide fragment of ppDyn that comprises or consists of 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 6. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to claim 3, wherein the modification is a replacement of parts of SEQ ID No. 5 with a propeptide of or a fragment of a propeptide of a neuropeptide; wherein the modified propeptide fragment of ppDyn i) comprises at least one sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (SEQ ID No.36) or ii) comprises a sorting motif of said pre-pro-neuropeptide or protein sorted to large dense core vesicles other than pre-prodynorphin. 7. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to claim 6 wherein the modified propeptide fragment comprises or consists of SEQ ID No.10 MPRSCCSRSGALLLALLLQASMEVRGWCLESSQCQDLTTESNLLECIRACKP 8. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to claim 6 wherein the modified propeptide fragment comprises or consists of a propeptide fragment of preproEnkephalin, preproBDNF, preproTachykinin, prepro-Somatostatin, pre-pro-VIP, prepro-CCK, preproNociceptin or preproNPY, wherein said propeptide fragment comprises said sorting motif, in particularly said propeptide fragment maybe selected from the group comprising of any of SEQ ID Nos: 37 to 52. 9. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-8 wherein the modified propeptide fragment is optionally flanked by peptidase recognition signals comprising K, R, KR, RK or RR. 10. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-9, wherein the target cells are neuronal cells of the central nervous system. 11. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-10, wherein the signal peptide is a peptide sequence of from 10 to 30 amino acids at the N-terminal end of precursor-proteins that are destined into the lumen of the endoplasmatic reticulum. 12. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of claims 1-11, wherein the signal peptide is selected from the group comprising: MAWQGLVLAACLLMFPSTTA (SEQ ID No. 11) MARFLTLCTWLLLLGPGLLATVRA (SEQ ID No.12) MLGNKRLGLSGLTLALSLLVCLGALAEA (SEQ ID No.13) MLSCRLQCALAALSIVLALGCVTG (SEQ ID No.14) MKILVALAVFFLVSTQLFA (SEQ ID No.15) MRIMLLFTAILAFSLA (SEQ ID No. 16) MPRSCCSRSGALLLALLLQASMEVRG (SEQ ID No.17) MNSGVCLCVLMAVLAAGA (SEQ ID No.18) MKVLLCDLLLLSLFSSVFS (SEQ ID No.19) MQPTLLLSLLGAVGLAAVNS (SEQ ID No.20) 13. A delivery vector according to any of claims 1-12, wherein said delivery vector leads to release-on- demand of dynorphins or dynorphin-variants with agonistic effects on human Kappa Opioid Receptors. 14. A delivery vector according to any of claims 1-13, wherein the dynorphin variants have an amino acid sequence identity of at least 70 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 2 (YGGFLRRI), SEQ ID No. 3 (YGGFLRRQ) or SEQ ID No. 4 (YGGFLRRQ), respectively. 15. A delivery vector according to any of claims 1-14, wherein the variants have an amino acid sequence identity of at least 80 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 2, SEQ ID No.3 or SEQ ID No.4, respectively. 16. A delivery vector according to any of claims 1 to 15, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome. 17. A delivery vector according to any of claims 1-16 comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITR) preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof, preferably of AAV serotype 1 or 2 or a chimeric vector comprising capsid proteins derived from two or more, preferably two of the aforementioned AAV serotype capsids; in particular embodiments, the capsids are capsids derived from AAV serotype 1 and/or 2. 18. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of the preceding claims. 19. A recombinant virus particle or a liposome or nanoparticle, comprising a delivery vector according to any of the preceding claims. 20. The recombinant virus particle or liposome or nanoparticle of claim 19, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome and said rAAV vector genome is encapsidated in an AAV capsid or wherein said delivery vector comprises in addition a recombinant lentivirus vector genome and is packaged in a lentivirus particle. 21. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 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 virus particle or liposome or nanoparticle under conditions sufficient for the DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants to be introduced into the cell. 22. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 1-21 for use as medicament. 23. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 1-21 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy whereby said delivery vector or recombinant virus particle or liposome or nanoparticle provides activation of human Kappa Opioid Receptors in the epileptogenic focus, thereby inhibiting seizures. 24. DNA sequence selected from the group comprising the following sequences: 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 a DNA sequence according to claim 24. 26. A delivery vector according to claim 25, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome. 27. A delivery vector according to claim 25 or 26 comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITR), preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof, or a chimeric vector comprising mosaic capsid proteins derived from two or more, preferably two of the aforementioned AAV serotype capsids; in particular embodiments, the capsids are capsids derived from AAV serotype 1 and/or 2. 28. A delivery vector according to any of claims 25-27, wherein said delivery vector comprises in addition 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. A delivery vector according to any of claims 25-28, wherein said delivery vector comprises a sequence selecetd 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 virus particle or a liposome or nanoparticle, comprising a delivery vector according to any of claims 25-29. 31. The recombinant virus particle or liposome or nanoparticle according to claim 30, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome and said rAAV vector genome is encapsidated in an AAV capsid or wherein said delivery vector comprises in addition a recombinant lentivirus vector genome and is packaged in a lentivirus particle. 32. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 25-31 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 virus particle or liposome or nanoparticle under conditions sufficient for a DNA comprising a sequence selected from the group comprising the SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, and SEQ ID No. 73 to be introduced into the cell. 33. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 25-32 for use as medicament. 34. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of claims 25-33 for use in treating epilepsy in a subject, in particular focal epilepsy, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy, wherein said delivery vector or recombinant virus particle or liposome or nanoparticle provides activation of human Kappa Opioid Receptors in the epileptogenic focus, thereby inhibiting seizures. |
SEQ ID No.63: CBA Promoter: CMV-enhancer, chicken beta-actin promoter, chimeric intron (887bp) SEQ ID No.64: sCBA-Promoter: CMV-enhancer, chicken-beta actin promoter, chimeric intron (851bp)
SEQ ID No.65: Human synapsin-promoter (448bp) SEQ ID No.66: WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element (582bp) SEQ ID No.67: bGH polyA+: Bovine growth hormone poly A+ signal sequence (208bp)
SEQ ID No.68: SPA: Synthetic poly A+ (49bp) SEQ ID No.69: ppDyn full-length cDNA codon-optimized1 (765bp) SEQ ID No.70: ppDyn with shortened N-peptide codon-optimized2 (576bp)
ID No.71: ppDyn with shortened N-peptide codon-optimized2 (399bp) SEQ ID No.72: ppDyn with shortened N-peptide codon-optimized2 (519bp) SEQ ID No.73: ppDyn with N-terminus from POMC fused to C-terminal part of pDyn codon-optimized2 (417bp)
SEQ ID No.74: ssAAV-pDyn (2820bp)
SEQ ID No.75: scAAV-pDyn (2210bp) 5 SEQ ID No.76: scAAV-pDyn (2603bp)
SEQ ID No.77: scAAV-syn-pDyn (2164bp) 5 SEQ ID No.78: scAAV-pDyn (2388bp) 5 SEQ ID No.79: scAAV-pDyn (2270bp) 5 SEQ ID No.80: scAAV-pDyn (2334bp) 5 SEQ ID No.81: scAAV-pDyn (2447bp)
Particular embodiments of the present invention are 1. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants and ^ wherein said delivery vector drives expression of a pre-propeptide in a target cell, and ^ wherein said delivery vector comprising said DNA sequence enables the release of dynorphin or dynorphin-variants from the target cell on demand, and ^ wherein said pre-propeptide is pre-prodynorphin or a pre-prodynorphin-variant and ^ wherein said pre-propeptide comprises a signal peptide, wherein the signal peptide is a N- terminal extension of a nascent polypeptide chain and wherein said signal peptide mediates protein targeting to the lumen of the endoplasmatic reticulum, and, ^ wherein said pre-propeptide comprises either (i) a N-terminal pro-peptide fragment on the C- terminal end of said signal peptide and wherein said N-terminal pro-peptide fragment comprises a sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (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 may independently be any amino acid (for the avoidance of doubt, this means that in the present invention in the first sequence of e.g. 1 to 20 X, each X may individually be any amino acid, and in the second sequence of e.g. 1 to 10 X, each X may individually be any amino acid, particularly as further defined herein), or wherein said pre-propeptide comprises (ii) a N-terminal pro-peptide fragment of a pre-pro-neuropeptide or protein sorted to large dense core vesicles, other than pre-prodynorphin, on the C-terminal end of said signal peptide and wherein said N-terminal pro-peptide fragment comprises a sorting motif of said pre-pro-neuropeptide or protein sorted to large dense core vesicles, and wherein said N-terminal pro-peptide fragment consists of 16 to 90 amino acids, and ^ wherein said pre-prodynorphyin or pre-prodynorphin-variants comprise at least one of the following sequences selected from the group: a. Dyn A that is SEQ ID No. or a variant thereof consisting of the first 13 amino acids from the N-terminal end or a variant thereof consisting of the first 8 amino acids from the N- terminal end b. Dyn B that is SEQ ID No.3 c. leumorphin that is SEQ ID No. 4 d. variants of Dyn A , said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No.2, e. variants of Dyn B, said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No.3, f. variants of leumorphin, said variants having an amino acid sequence identity of at least 60 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 4. 2. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiment 1, wherein said N-terminal pro-peptide fragment consists of 20 to 90 amino acids, preferably 30 and 90 amino acids. 3. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiment 1 or 2, wherein said N-terminal pro-peptide fragment on the C- terminal end 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 ppDyn is a shortening; or the modification is a replacement of parts of SEQ ID No.5 with a propeptide of or a fragment of a propeptide of a neuropeptide; wherein the modified propeptide fragment of ppDyn i) comprises at least one sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (SEQ ID No. 36) or ii) comprises a sorting motif of said pre-pro- neuropeptide or protein sorted to large dense core vesicles other than pre-prodynorphin. 4. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of embodiments 1 to 3, wherein said N-terminal pro-peptide fragment on the C-terminal end of the signal peptide is a modified propeptide fragment of ppDyn that comprises or consists of SEQ ID No.6 DCLSRCSLCA VKTQDGPKPI NPLICSLQCQ AALLPSEEWE RCQSFLSFFT PSTLGLNDKE DLGSKSVGEG PYSELAKLSG SFLRKEQVKR 5. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of embodiments 1 to 3, wherein said N-terminal pro-peptide fragment on the C-terminal end of the signal peptide is a modified propeptide fragment of ppDyn that comprises or consists of 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 comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiment 3, wherein the modification is a replacement of parts of SEQ ID No. 5 with a propeptide of or a fragment of a propeptide of a neuropeptide; wherein the modified propeptide fragment of ppDyn i) comprises at least one sorting motif comprising the elements DL and EXyL, in particular a sorting motif consisting of the amino acid sequence DLXxEXyL (SEQ ID No.36) or ii) comprises a sorting motif of said pre-pro-neuropeptide or protein sorted to large dense core vesicles other than pre-prodynorphin. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiment 6 wherein the modified propeptide fragment comprises or consists of SEQ ID No.10: MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KP A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiment 6 wherein the modified propeptide fragment comprises or consists of a propeptide fragment of preproEnkephalin, preproBDNF, preproTachykinin, prepro- Somatostatin, pre-pro-VIP, prepro-CCK, preproNociceptin or preproNPY, wherein said propeptide fragment comprises a sorting motif, in particular said propeptide fragment maybe selected from the group comprising of any of SEQ ID Nos: 37 to 52. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to embodiments 1-8 wherein the modified propeptide fragment is optionally flanked by peptidase recognition signals comprising K, R, KR, RK or RR. 10. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of embodiments 1-9, wherein the target cell are neuronal cells of the central nervous system. 11. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of embodiments 1-10, wherein the signal peptide is a peptide sequence of from 10 to 30 amino acids at the N-terminal end of precursor-proteins that are destined into the lumen of the endoplasmatic reticulum. 12. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of embodiments 1-11, wherein the signal peptide is selected from the group comprising: 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) 13. A delivery vector according to any of embodiments 1-12, wherein said delivery vector leads to release-on-demand of dynorphins or dynorphin-variants with agonistic effects on human Kappa Opioid Receptors. 14. A delivery vector according to any of embodiments 1-13, wherein the dynorphin variants have an amino acid sequence identity of at least 70 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 2 (YGGFLRRI), SEQ ID No. 3 (YGGFLRRQ) or SEQ ID No. 4 (YGGFLRRQ), respectively. 15. A delivery vector according to any of embodiments 1-14, wherein the variants have an amino acid sequence identity of at least 80 % within the first 8 amino acids from the N-terminal end of SEQ ID No. 2, SEQ ID No.3 or SEQ ID No.4, respectively. 16. A delivery vector according to any of embodiments 1-15, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome. 17. A delivery vector according to any of embodiments 1-16 comprising a recombinant adeno- associated virus (AAV) vector genome comprising inverted terminal repeats (ITR) preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof or a chimeric vector comprising capsid proteins derived from two or more, preferably two of the aforementioned AAV serotype capsids; in particular embodiments, the capsids are capsids derived from AAV serotype 1 and/or 2. In certain embodiments, the delivery vector according to embodiment 1-16 comprises a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITR) preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof, preferably of AAV serotype 1 or 2. 18. A delivery vector comprising a DNA sequence encoding pre-prodynorphin or pre-prodynorphin- variants according to any of the preceding embodiments. 19. A recombinant virus particle or a liposome or nanoparticle, comprising a delivery vector according to any of the preceding embodiments. 20. The recombinant virus particle or liposome or nanoparticle of embodiment 19, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome and said rAAV vector genome is encapsidated in an AAV capsid or wherein said delivery vector comprises in addition a recombinant lentivirus vector genome and is packaged in a lentivirus particle. 21. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 1-20 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 virus particle or liposome or nanoparticle under conditions sufficient for the DNA sequence encoding pre-prodynorphin or pre- prodynorphin-variants to be introduced into the cell. 22. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 1-21 for use as medicament. 23. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 1 to 21 for use in treating focal epilepsy in a subject, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy whereby said delivery vector or recombinant virus particle or liposome or nanoparticle provides activation of human Kappa Opioid Receptors in the epileptogenic focus, thereby inhibiting seizures. It is apparent to the skilled person that, as detailed herein, the delivery vector or recombinant virus particle or liposome or nanoparticle provides activation of human Kappa Opioid Receptors in the epileptogenic focus, thereby inhibiting seizures, through inducing production of corresponding peptides, as further detailed herein. 24. DNA sequence selected from the group comprising the following sequences: 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 a DNA sequence according to embodiment 24. 26. A delivery vector according to embodiment 25, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome or a recombinant lentivirus genome. 27. A delivery vector according to embodiment 25 or 26 comprising a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITR), preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof or a chimeric vector or a chimeric vector comprising capsid proteins derived from two or more, preferably two of the aforementioned AAV serotype capsids; in particular embodiments, the capsids are capsids derived from AAV serotype 1 and/or 2. In certain embodiments, the delivery vector according to embodiment 25 or 26 comprises a recombinant adeno-associated virus (AAV) vector genome comprising inverted terminal repeats (ITR), preferably derived from AAV serotype 2, alternatively from AAV serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, or reengineered variants thereof, wherein said vector genome is packaged in an AAV capsid selected from the group comprising AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, 11, 12, 13, 14, serpentine AAV, ancestral AAV, AAV-TT, AAVv66, AAV1P4, AAV1P5, AAV-PHP.B, AAV-PHP.eB, AAV2-HBKO, AAV.CAP-B10, AAV.CAP-MAC, AAV2.NN, or further AAV capsid mutants derived thereof, preferably two of the aforementioned AAV capsids, preferably of AAV serotype 1 and 2. 28. A delivery vector according to any of embodiments 25-27, wherein said delivery vector comprises in addition 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. A delivery vector according to any of embodiments 25-28, wherein said delivery vector comprises a sequence selecetd 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 virus particle or a liposome or nanoparticle, comprising a delivery vector according to any of embodiments 25-29. 31. The recombinant virus particle or liposome or nanoparticle according to embodiment 30, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome and said rAAV vector genome is encapsidated in an AAV capsid or wherein said delivery vector comprises in addition a recombinant lentivirus vector genome and is packaged in a lentivirus particle. 32. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 25-31 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 virus particle or liposome or nanoparticle under conditions sufficient for a DNA comprising a sequence selected from the group comprising the SEQ ID No.69, SEQ ID No.70, SEQ ID No. 71, SEQ ID No.72, and SEQ ID No. 73 to be introduced into the cell. In particular embodiments, the DNA comprising a sequence selected from the group comprising the SEQ ID Nos. 69-73 to be introduced into the cell is a DNA spected from SEQ ID Nos. 74-81 (AAV genomes), and, in the case of lentiviruses, a DNA wherein a sequence selected from the group comprising the SEQ ID Nos.69-73 and regulatory sequences are embedded in a lentivirus genome. 33. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 25-32 for use as medicament. 34. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of embodiments 25-33 for use in treating epilepsy in a subject, in particular focal epilepsy, in particular mesial temporal lobe epilepsy, or for use in preventing epileptic seizures in a subject that suffers from focal epilepsy, wherein said delivery vector or recombinant virus particle or liposome or nanoparticle provides activation of human Kappa Opioid Receptors in the epileptogenic focus, thereby inhibiting seizures.
Figure Description Figure 1: Overview of the shortening of the human ppDyn cDNA. Figure 2: Results of an ELISA to measure the content of (A) dynorphin A (DynA) and (B) dynorphin B (DynB) after intraparenchymal CNS transduction of AAV vectors expressing the indicated ppDyn variants. Variant A = shortened N-peptide preserving the sorting motif. Variant B = signal and N-Peptide replaced by POMC signal and sorting motif. Variant C = shortened N-peptide with deleted sorting motif. ipsi = site of AAV transduction, contra = non-transduced (control) site. Figure 3: Seizure suppression by two scAAV vector variants containing shortened pDyn cDNA or with alternative signal and sorting sequence. Data represent N± SEM (N=3). Figure 4: Overview of DNA-sequence elements of AAV-pDyn vector variants. Displayed are the DNA sequence elements of the displayed AAV vectors. SEQ ID numbering refers to the numbering in the body of the text. Dark grey box: DNA sequence elements derived from AAV serotype 2. ITR= inverted terminal repeat, ^ITR= inverted terminal repeat with deleted terminal resolution site as used in self-complementary (sc)AAV vectors. Light grey box: DNA sequence elements for promoters or other regulatory elements of gene expression. CBA prom. = cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter followed by a chimeric intron. sCBA prom. = shortened cytomegalovirus (CMV) enhancer fused to chicken beta- actin promoter followed by a chimeric intron. hSyn prom. = human synapsin promoter. WPRE = woodchuck hepatitis virus posttranscriptional regulatory element, bGH pA+ = polyA+ signal sequence of the bovine growth hormone gene. SpA+ = synthetic poly A+ signal sequence. White box: DNA sequence elements for the human ppDyn cDNA. ppDyn cDNA sequences are codon- optimized. Codon optimized version 1 is used for SEQ ID No.74 and SEQ ID No.75. Codon optimized version 2 is used for SEQ ID No.76 to 80, and for the pDyn part of SEQ ID No.81. Pre = DNA sequence for the signal sequence of ppDyn, pro = DNA sequence covering the N-peptide of ppDYN, pro1, pro2, and proDs = different shortened versions of “pro” referring to the N-peptide of ppDyn. POMC= cDNA sequence of the N-terminal part of neuropeptide POMC spanning the pre and pro elements and replacing those of ppDyn. pDyn= prodynorphin. Figure 5: Reduction of seizure activity after injection of AAV-pDyn expressing Seq ID No. 70. Hpds, generalized seizures and spike trains were measured over periods of 48 hours each time-interval. Data are shown as % of pretreatment seizure activity for Hpds and spike trains (left y-axis). Generalized seizures are given as absolute number of seizures (right y-axis. N=7-9, p = 0.0025 for Hpds, < 0.0001 for spike trains and 0.0001 for generalized seizures (one-way ANOVA). Mice show a significant reduction of drug-resistant focal seizures starting from 7 days after treatment. Generalized seizures are almost completely abolished after 4 weeks. Figure 6: Reduction of seizure activity after injection of AAV-pDyn expressing Seq ID No. 77. HPDs were measured over periods of 48 hours each time-interval. Data are shown as time spent in HPDs. N = 2-3. Mice show a marked reduction of drug-resistant focal seizures starting from 10 days after AAV delivery.
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Examples Example 1 Shortening of the human ppDyn cDNA to enable packing into scAAV vectors. Some amino acids in the region between signal peptide and the region coding for active peptides can be removed. However, a sorting motif responsible for packing the propeptide into large dense core vesicles needs to be conserved. Alternatively, the entire part N-terminal to the region coding for active peptides can be replaced by a different signal peptide and sorting motif. Example 2 Production of mature dynorphins by two scAAV vector variants Production of mature dynorphins by three scAAV vector variants containing shortened pDyn cDNA. The vectors were injected into the dorsal hippocampus of naive wild-type mice. After 2 weeks the hippocampi were resected and the content of dynorphin A (DynA) and dynorphin B (DynB) was measured by ELISA as described in Agostinho et al. (2019), see Figure 2. The production of mature peptides occurs only in large dense core vesicles, proving the correctness of sorting of the shortened pDyn variants A (SEQ ID No.76) and B (SEQ ID No.81). The marked reduction of mature dynorphins applying variant C (SEQ ID No 80), which lacks the proposed sorting motif, indicates the importance of the proposed sorting motif. Example 3 Seizure suppression by shortened pDyn cDNA Seizure suppression by two scAAV vector variants containing shortened pDyn cDNA (Fig. 3). The vectors were injected into the dorsal hippocampus of epileptic wild-type mice. EEGs were recorded and analyzed for hippocampal paroxysmal discharges (HPD), representing drug-resistant focal seizures. The treatment of animals with kainic acid, the implantation of electrodes and the analysis is described in Widmann et al. (2022). Example 7 Construction of AAV-pDyn vector variants AAV vectors were constructed in the ssAAV or scAAV format as displayed in Fig. 4. They are composed of one AAV serotype 2 derived left and one right ITR sequence (SEQ ID No.59 to 61). AAV ITRs of alternative AAV serotypes or synthetic ITRs may be used similarly. The ITRs flank any of displayed heterologous gene expression cassettes (Fig. 4). These cassettes are composed of one of the promoter sequences (Seq ID No. 63 to 65), a posttranscriptional regulatory element from woodchuck hepatitis virus (Seq ID No. 66) (as described in Loeb et al. Hum Gene Ther 10:2295-2305, 1999), a polyadenylation signal, either derived from the bovine growth hormone gene (SEQ ID No.67) or a short synthetic polyA signal sequence (SEQ ID No.68) (as described in Levitt et al. Genes & Dev 3:1019-25, 1989) and the cDNA to be expressed. The gene of interest is the cDNA sequence of human preprodynorphin (ppDyn), any of the displayed variants thereof (SEQ ID No.69 to 72), or a fusion of the N-terminus of POMC with the C-terminal part of prodynorphin devoid of its signal-sequence (pre) and N-peptide (pro) (SEQ ID No.73). The cDNAs are codon-optimized in two versions. Version 1 (SEQ ID No.69) is contained in AAV SEQ ID No. 74 and SEQ ID No. 75. Codon optimized version 2 ((SEQ ID No. 70 to 73) was generated to reduce the percentage of CpG sequence elements. Unmethylated CpG sequence elements are hallmarks of bacterial DNA and represent pathogen-associated molecular patterns (PAMP) which may activate the innate immune system in a mammalian host and could be an issue in the context of AAV gene therapies under certain circumstances. High CpG content of transduced AAV genomes may be associated with an increased probability of immune-mediated loss of transduced cells. Adverse effects of high CpG content of transduced AAV genomes have recently been shown also in the CNS after intraparenchymal/ intracerebral AAV transduction (Suriano et al. 2021). Alternative codon optimization strategies to combine enhanced transgene expression in human cells with sufficient reduction of AAV-transduced unmethylated CpG elements may be achieved by different DNA sequence alterations. The complete AAV DNA sequences from ITR to ITR spanning all regulator elements and the different ppDyn-derived transgenes as displayed in Fig 4 and are represented as DNA sequence files (SEQ ID No. 74 to 81). Any of the elements may be further interchanged, e.g. the truncated ITR of scAAV may be positioned at the right instead of the left end of the AAV genome as displayed here. Likewise, the gene promoters may be interchanged and/or combined with any of the displayed poly A signal sequence, or a polyA signal of different origin. The WPRE element may be used as full-length 582bp element (Seq ID 66) as displayed. WPRE is composed of subelements named gamma, alpha, beta, in the given order. Shorter version (WPRE2) diplaying a minimal gamma and partial alpha/beta element and WPRE3 displaying only minimal gamma and alpha elements (247bp) were described to be similarly active (Choi et al.2014, incorporated herein by reference). The versions of WPRE may be used interchangeably or may not be incorporated into the AAV genome at all. Example 8 Functional testing of AAV vectors AAV vectors of SEQ ID No. 76 to 78 and SEQ ID No. 81 were tested functionally. Fully processed, mature dynorphin peptides were produced (Seq ID No. 76 and No. 81; Example 3) and suppression of focal seizures were monitored in the TLE mouse model SEQ ID No.76, 77, 78). The reduction of distinct types of seizure activity after injection of AAV-pDyn expressing Seq ID No. 70. Hpds are shown in Figure 5. Hpds, generalized seizures and spike trains were measured over periods of 48 hours each time-interval. The mice show a significant reduction of drug-resistant focal seizures starting from 7 days after treatment. Generalized seizures are almost completely abolished after 4 weeks. The reduction of HPDs after injection of AAV-pDyn expressing Seq ID No.77 is shown in Figure 6. The mice show a marked reduction of drug-resistant focal seizures starting from 10 days after AAV delivery.
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