WO1999041381A1 - Neuroserpin - Google Patents

Abstract

There are described pharmaceutical and diagnostic applications of neuroserpins, in particular human neuroserpin. Neuroserpins are valuable agents in the treatment of disorders of the nervous system, in particular the central nervous system. They are very useful in the treatment of stroke and for the development of drugs.

Description

NEUROSERPIN

Cross References to Related Applications

This application claims the priority of U.S. patent application 09/023,129, filed February 13, 1998, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention concerns neuroserpins for the use as pharmaceuticals and diagnostic agents.

BACKGROUND ART

Nervous system related disorders, in particular central nervous system related disorders, are getting greater importance, be it due to the enhanced average age of the people, be it due to enhanced numbers of injured people due to the enhanced occurence of potential dangers .

Although there is a great interest in getting more knowledge about nervous system and nervous system involved disorders, such as pain development, regeneration of injured nerve activity etc., and in particular about healing such disorders or injuries, or at least ameliorating the state of a patient suffering from such disorders or injuries, there is still a need for pharmaceutical and diagnostic preparation in said field.

A structural class of nervous system active proteins denominated serpins is well known. So far, more than 60 members of the serpin family of proteins have been identified and characterized; see P.A. Patson and P.G. . Gettins, Thrombosis and Haemostasis 72, pages 166- 179 (1994). As members of the serpine family e.g. chicken, mouse and human neuroserpins have been described (see T. Osterwalder et al . Neuroserpin, an axonally secreted serine protease inhibitor, The EMBO Journal , Vol . 15, No . 12, pages 2944-2953 (1996); S. P. Schrimpf et al . Human Neuroserpin (PI12) : cDNA Cloning and Chromosomal Localization to 3q26, Geno ics , Vol . 39, pages 1-8 (1997) ) .

Although in view of the presence of neuroserpin in the nervous system its influence on some nervous system specific processes has to be assumed, nothing is known so far about neuroserpin related disorders .

Thus there still exists a need for pharmaceutical and diagnostic preparations for disorders with close connection to processes in the nervous system, in particular processes in the central nervous system.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide an effective (active) substance for the treatment of disorders of the nervous system, in particular of the central nervous system, as well as pharmaceutical and diagnostic preparations comprising such an effective substance. Said effective substance is neuroserpin or a neuroserpin like protein, or a nucleic acid sequence encoding such a protein, in particular a DNA sequence, be it a genomic one or a cDNA.

A DNA sequence of the present invention is a DNA sequence encoding a polypeptide product having at least part of the primary structural conformation of that of neuroserpin, said DNA sequence being selected from a) the DNA sequences of Tables I and II, b) DNA sequences which hybridize under stringent conditions to the protein coding regions of the DNA sequences of Tables I or II or respective genomic sequences, c) sequences that but for the degeneracy of the genetic code would hybridize to the DNA sequences defined under a) and b) , d) DNA sequences that encode alleles and/or mutants of the neuroserpins of figures I and II, e) parts of the sequences defined under a) to d) that encode a protein with protease inhibitory activity, and f) the complementary strands of the sequences defined above, for the use as pharmaceutical or diagnostic agent .

Table I: Human neuroserpin

GCGGAGCACA GTCCGCCGAG CACAAGCTCC AGCATCCCGT CAGGGGTTGC AGGTGTGTGG 60

GAGGCTTGAA ACTGTTACAA T ATG GCT TTC CTT GGA CTC TTC TCT TTG CTG 111 Met Ala Phe Leu Gly Leu Phe Ser Leu Leu

-15 -10

GTT CTG CAA AGT ATG GCT ACA GGG GCC ACT TTC CCT GAG GAA GCC ATT 159 Val Leu Gin Ser Met Ala Thr Gly Ala T r Phe Pro Glu Glu Ala lie -5 1 5 10

GCT GAC TTG TCA GTG AAT ATG TAT AAT CGT CTT AGA GCC ACT GGT GAA 207

Ala Asp Leu Ser Val Asn Met Tyr Asn Arg Leu Arg Ala Thr Gly Glu 15 20 25

GAT GAA AAT ATT CTC TTC TCT CCA TTG AGT ATT GCT CTT GCA ATG GGA 255

Asp Glu Asn lie Leu Phe Ser Pro Leu Ser lie Ala Leu Ala Met Gly 30 35 40 ATG ATG GAA CTT GGG GCC CAA GGA TCT ACC CAG AAA GAA ATC CGC CAC 303 Met Met Glu Leu Gly Ala Gin Gly Ser Thr Gin Lys Glu He Arg His 45 50 55

TCA ATG GGA TAT GAC AGC CTA AAA AAT GGT GAA GAA TTT TCT TTC TTG 351 Ser Met Gly Tyr Asp Ser Leu Lys Asn Gly Glu Glu Phe Ser Phe Leu 60 65 70

AAG GAG TTT TCA AAC ATG GTA ACT GCT AAA GAG AGC CAA TAT GTG ATG 399 Lys Glu Phe Ser Asn Met Val Thr Ala Lys Glu Ser Gin Tyr Val Met 75 80 85 90 AAA ATT GCC AAT TCC TTG TTT GTG CAA AAT GGA TTT CAT GTC AAT GAG 447

Lys He Ala Asn Ser Leu Phe Val Gin Asn Gly Phe His Val Asn Glu 95 100 105

GAG TTT TTG CAA ATG ATG AAA AAA TAT TTT AAT GCA GCA GTA AAT CAT 495

Glu Phe Leu Gin Met Met Lys Lys Tyr Phe Asn Ala Ala Val Asn His 110 115 120 GTG GAC TTC AGT CAA AAT GTA GCC GTG GCC AAC TAC ATC AAT AAG TGG 543 Val Asp Phe Ser Gin Asn Val Ala Val Ala Asn Tyr He Asn Lys Trp 125 130 135

GTG GAG AAT AAC ACA AAC AAT CTG GTG AAA GAT TTG GTA TCC CCA AGG 591 Val Glu Asn Asn Thr Asn Asn Leu Val Lys Asp Leu Val Ser Pro Arg 140 145 150

GAT TTT GAT GCT GCC ACT TAT CTG GCC CTC ATT AAT GCT GTC TAT TTC 639 Asp Phe Asp Ala Ala Thr Tyr Leu Ala Leu He Asn Ala Val Tyr Phe 155 160 165 170

AAG GGG AAC TGG AAG TCG CAG TTT AGG CCT GAA AAT ACT AGA ACC TTT 687

Lys Gly Asn Trp Lys Ser Gin Phe Arg Pro Glu Asn Thr Arg Thr Phe 175 180 185

TCT TTC ACT AAA GAT GAT GAA AGT GAA GTC CAA ATT CCA ATG ATG TAT 735 Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gin He Pro Met Met Tyr 190 195 200

CAG CAA GGA GAA TTT TAT TAT GGG GAA TTT AGT GAT GGC TCC AAT GAA 783 Gin Gin Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu 205 210 215 GCT GGT GGT ATC TAC CAA GTC CTA GAA ATA CCA TAT GAA GGA GAT GAA 831 Ala Gly Gly He Tyr Gin Val Leu Glu He Pro Tyr Glu Gly Asp Glu 220 225 230

ATA AGC ATG ATG CTG GTG CTG TCC AGA CAG GAA GTT CCT CTT GCT ACT 879 He Ser Met Met Leu Val Leu Ser Arg Gin Glu Val Pro Leu Ala Thr 235 240 245 250

CTG GAG CCA TTA GTC AAA GCA CAG CTG GTT GAA GAA TGG GCA AAC TCT 927 Leu Glu Pro Leu Val Lys Ala Gin Leu Val Glu Glu Trp Ala Asn Ser 255 260 265

GTG AAG AAG CAA AAA GTA GAA GTA TAC CTG CCC AGG TTC ACA GTG GAA 975

Val Lys Lys Gin Lys Val Glu Val Tyr Leu Pro Arg Phe Thr Val Glu 270 ^' 275 280

CAG GAA ATT GAT TTA AAA GAT GTT TTG AAG GCT CTT GGA ATA ACT GAA 1023

Gin Glu He Asp Leu Lys Asp Val Leu Lys Ala Leu Gly He Thr Glu 285 290 295 ATT TTC ATC AAA GAT GCA AAT TTG ACA GGC CTC TCT GAT AAT AAG GAG 1071 He Phe He Lys Asp Ala Asn Leu Thr Gly Leu Ser Asp Asn Lys Glu 300 305 310

ATT TTT CTT TCC AAA GCA ATT CAC AAG TCC TTC CTA GAG GTT AAT GAA 1119 He Phe Leu Ser Lys Ala He His Lys Ser Phe Leu Glu Val Asn Glu 315 320 325 330

GAA GGC TCA GAA GCT GCT GCT GTC TCA GGA ATG ATT GCA ATT AGT AGG 1167 Glu Gly Ser Glu Ala Ala Ala Val Ser Gly Met He Ala He Ser Arg 335 340 345 ATG GCT GTG CTG TAT CCT CAA GTT ATT GTC GAC CAT CCA TTT TTC TTT 1215

Met Ala Val Leu Tyr Pro Gin Val He Val Asp His Pro Phe Phe Phe 350 355 360

CTT ATC AGA AAC AGG AGA ACT GGT ACA ATT CTA TTC ATG GGA CGA GTC 1263

Leu He Arg Asn Arg Arg Thr Gly Thr He Leu Phe Met Gly Arg Val 365 370 375 ATG CAT CCT GAA ACA ATG AAC ACA AGT GGA CAT GAT TTC GAA GAA CTT 1311

Met His Pro Glu Thr Met Asn Thr Ser Gly His Asp Phe Glu Glu Leu 380 385 390 394

TAAGTTACTT TATTTGAATA ACAAGGAAAA CAGTAACTAA GCACATTATG TTTGCAACTG 1371

GTATATATTT AGGATTTGTG TTTTACAGTA TATCTTAAGA TAATATTTAA AATAGTTCCA 1431

GATAAAAACA ATATATGTAA ATTATAAGTA ACTTGTCAAG GAATGTTATC AGTATTAAGC 1491

TAATGGTCCT GTTATGTCAT TGTGTTTGTG TGCTGTTGTT TAAAATAAAA GTACCTATTG 1551 AACATGTGAA AAAAAAAAAA AAAAAA 1577

Table II: Murine neuroserpin

CGGCACGAGA TCCGGAGCAG TCTCAGCCTG CCCAGCATCC TCTCCAGCAT CCCGAGCGGG 60

GATTGCAGGT GTGAAGGAGA CTTGAAACCA TCCCATC ATG ACT TAC CTT GAA CTG 115

Met Thr Tyr Leu Glu Leu -15

CTT GCT TTG CTG GCC TTG CAA AGT GTG GTG ACA GGG GCA ACG TTC CCA 163 Leu Ala Leu Leu Ala Leu Gin Ser Val Val Thr Gly Ala Thr Phe Pro -10 -5 1 5

GAT GAA ACC ATA ACT GAG TGG TCA GTG AAC ATG TAT AAC CAC CTT CGA 211 Asp Glu Thr He Thr Glu Trp Ser Val Asn Met Tyr Asn His Leu Arg 10 15 20 GGC ACC GGG GAA GAT GAA AAC ATT CTC TTC TCT CCA CTA AGC ATT GCC 259 Gly Thr Gly Glu Asp Glu Asn He Leu Phe Ser Pro Leu Ser He Ala 25 30 35

CTT GCG ATG GGA ATG ATG GAG CTT GGG GCT CAA GGA TCT ACT AGG AAA 307 Leu Ala Met Gly Met Met Glu Leu Gly Ala Gin Gly Ser Thr Arg Lys 40 45 50

GAA ATC CGC CAT TCA ATG GGA TAT GAG GGT CTG AAA GGT GGT GAA GAA 355 Glu He Arg His Ser Met Gly Tyr Glu Gly Leu Lys Gly Gly Glu Glu 55 60 65 70

TTT TCT TTC CTG AGG GAT TTT TCT AAT ATG GCC TCT GCC GAA GAA AAC 403 Phe Ser Phe Leu Arg Asp Phe Ser Asn Met Ala Ser Ala Glu Glu Asn 75 80 85

CAA TAT GTG ATG AAA CTT GCC AAT TCG CTC TTT GTA CAA AAT GGA TTT 451 Gin Tyr Val Met Lys Leu Ala Asn Ser Leu Phe Val Gin Asn Gly Phe 90 95 100 CAT GTC AAT GAG GAA TTC TTG CAA ATG CTG AAA ATG TAC TTT AAT GCA 499

His Val Asn Glu Glu Phe Leu Gin Met Leu Lys Met Tyr Phe Asn Ala

105 110 115

GAA GTC AAC CAT GTG GAC TTC AGT CAA AAT GTG GCT GTG GCT AAC TCC 547

Glu Val Asn His Val Asp Phe Ser Gin Asn Val Ala Val Ala Asn Ser

120 125 130 ATC AAT AAA TGG GTG GAG AAT TAT ACA AAC AGT CTG TTG AAA GAT CTG 595 He Asn Lys Trp Val Glu Asn Tyr Thr Asn Ser Leu Leu Lys Asp Leu 135 140 145 150

GTG TCT CCG GAG GAC TTT GAT GGT GTC ACT AAT TTG GCC CTC ATC AAT 643 Val Ser Pro Glu Asp Phe Asp Gly Val Thr Asn Leu Ala Leu He Asn

155 160 165

GCT GTA TAT TTC AAA GGA AAC TGG AAG TCT CAG TTT AGA CCT GAA AAT 691 Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser Gin Phe Arg Pro Glu Asn 170 175 180

ACC AGA ACT TTC TCC TTC ACG AAA GAT GAT GAA AGT GAA GTG CAG ATT 739 Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gin He 185 190 195

CCA ATG ATG TAT CAA CAA GGA GAA TTT TAT TAT GGT GAA TTT AGT GAT 787 Pro Met Met Tyr Gin Gin Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp 200 205 210 GGA TCC AAT GAG GCT GGT GGT ATC TAC CAA GTC CTT GAG ATA CCC TAT 835 Gly Ser Asn Glu Ala Gly Gly He Tyr Gin Val Leu Glu He Pro Tyr 215 220 225 230

GAG GGA GAT GAG ATC AGC ATG ATG CTG GCA CTG TCC AGA CAG GAA GTC 883 Glu Gly Asp Glu He Ser Met Met Leu Ala Leu Ser Arg Gin Glu Val

235 240 245

CCA CTG GCC ACA CTG GAG CCT CTG CTC AAA GCA CAG CTG ATC GAA GAA 931 Pro Leu Ala Thr Leu Glu Pro Leu Leu Lys Ala Gin Leu He Glu Glu 250 255 260

TGG GCA AAC TCT GTG AAG AAA CAA AAG GTG GAA GTG TAC TTG CCC AGG 979 Trp Ala Asn Ser Val Lys Lys Gin Lys Val Glu Val Tyr Leu Pro Arg 265 270 275

TTC ACT GTG GAA CAG GAA ATT GAT TTA AAA GAC ATC TTG AAA GCC CTT 1027 Phe Thr Val Glu Gin Glu He Asp Leu Lys Asp He Leu Lys Ala Leu 280 285 290 GGG GTC ACT GAA ATT TTC ATC AAA GAT GCA AAT TTG ACT GCC ATG TCA 1075 Gly Val Thr Glu He Phe He Lys Asp Ala Asn Leu Thr Ala Met Ser 295 300 305 310

GAT AAG AAA GAG CTG TTC CTC TCC AAA GCT GTT CAC AAG TCC TGC ATT 1123 Asp Lys Lys Glu Leu Phe Leu Ser Lys Ala Val His Lys Ser Cys He

315 320 325

GAG GTT AAT GAA GAA GGG TCA GAA GCC GCT GCA GCC TCC GGA ATG ATT 1171 Glu Val Asn Glu Glu Gly Ser Glu Ala Ala Ala Ala Ser Gly Met He 330 335 340

GCG ATT AGT AGG ATG GCT GTG CTG TAC CCT CAG GTT ATT GTC GAC CAT 1219 Ala He Ser Arg Met Ala Val Leu Tyr Pro Gin Val He Val Asp His 345 350 355 CCA TTT CTC TAT CTT ATC AGG AAC AGG AAA TCT GGC ATA ATC TTA TTC 1267

Pro Phe Leu Tyr Leu He Arg Asn Arg Lys Ser Gly He He Leu Phe 360 365 370

ATG GGA CGA GTC ATG AAC CCT GAA ACA ATG AAT ACA AGT GGC CAT GAC 1315

Met Gly Arg Val Met Asn Pro Glu Thr Met Asn Thr Ser Gly His Asp 375 380 385 390 TTT GAG GAA CTT TAAATGACGA CGTTTGAGTA CAAAGAAAGC AGGAACAAAG 1367 Phe Glu Glu Leu 394

CACATTATGT TTGCAAGTGG TATATATTTA GGATTTCTGT TTTATAGTGT TACTTAGGGA 1427

AATATTTAAA TAGTTCTGGA TAGTAGTAAT CCATGTGACC TATAAGTTAG CCTGTCAAAA 1487

GCTGTTATCA GTATAAAGAG TATGGTCCCA TTGTGTCATT GTGTCTGGTG TGCTGCTGTT 1547 TAAAATAAAA GTACATATTG AAACTGTGAA CCACTTTTTT TCATTTTGAA AGTAGTTGTA 1607

GTCTATACAA TACTATGTCT GAGATTTGAA ACCTATGCTG TTTCTTTAGG AATTGTAGTA 1667

AAATGATCCT ACAAGGCAAA ATGTAGAAAC TGTTGTTTCT GAGTTTCTTC ATAATCATGC 1727

AGAATCAAAC ACCAAAGTAA GCAACATACA TATATATATA TAATAAGCAA TACTGTGAAG 1787

GGGAGGCCAA AAGGCAGAGA AATTGAGATT GTTATTTAGT GTGGCATTCC ATGACAAAAG 1847 ATTTAGGAGG AAATGTGGGA TATGTAAGAC CCATAGATGT ATATTTTGTA TATCTGTAGT 1907

ATTATACTTT TAATTTATTA AAGTATAACT CTTTTATTTA TTTTTAAAAG TTTCCTGTGA 1967

ACCAATATGC CACATGACTC TACTAGCAAG TTCAGATATC TCATTAGCTA TTCTGGATGA 2027

CATCAAGAGG CCTCATGGAG GGAATCCCGT GTACCATTTA CGTTTTAGTG ATTTTTTGTG 2087

ATGTTCACAC AAAGATGAAA TCACATTGTT GCACACTCTC TAGACTATAT CCAAGAAAGG 2147 CATCAAGTGG TACATTGGTG TGCCAGGAAA ATAGATGTAA TTACTTTATT AAAAAAGTTC 2207

CTGGTATTGT GCATCATATG GAATCAGTGC TGCTTAAACT TAGTACGTCC TGCTGACACC 2267

TGGTCACTTA TTACAAATAT AGGTTCTTAT CCAGGATGTC TAAGGTAGAG TGGGAACCAC 2327

AGCTTTCTAT CATTACTGAC ATCCAAATGA TGCCGCAGAT ATCTGACCAT AGCCTTTGCT 2387

GAGAGTCCCT TGGGTTGCAA _TGTCGTACTT GAAGTCAGCC TCACATTTTC ACAGACTGAG 2447 ATTGGAGAGA TGAGGGTGCA GGGAGGAGAT AATCTACACT AGTGATACGA TGCCTTTGTC 2507

AAGCACTGGT GTGATCTCGA AGTATTCTAG TACACACTCT AGATAAATTC TTCTGTACAT 2567

TACAACACTT GAAATGCAGT CGTTAAAAAT ATGGAGACAT TTATAGGCAA TACCCATGAA 2627

AGAATTTATG ACTATCCGAG GACACAGTAC TTAACAATGA ATCTTTTACA GCTTATATTT 2687

TCAGAGGACT TGTAGTTTAT TCATAAATCT TCATGTTATT GTACAATAGT GCTCTTGTTT 2747 TCATTTATAA TTTATGAAGC TGAGATGCTG GTGTTAATTC AGTGTTCACA TTCTCTGCTA 2807

AGAACAGTCT TTATCTCTGT ATCCTTCTTG TTAATATGAC ATCTATAGCT ATATCTATAT 2867

GTTCATTAGT TAAACAAATG TATGGCCTGT AAGGAAGAAT AAACATTATT ATGCAATCAT 2927

GTAAAAAAAA AAAAAAA 2944 DNA sequences of the present invention are sequences that code for proteins having the function of protease inhibitory activity, but also DNA sequences with defects the proteins expressed thereon not having said activity. The first type of sequences is very much preferred for the use as pharmaceutical.

DNA sequences of the present invention as defined above shall be understood to also include splice variants of neuroserpins. Under stringent conditions hybridizing sequences in general are sequences with at least about 80 % identity, preferably about 90 % identity and most preferred 100 % identity. Such sequences comprise sequences encoding amino acid sequences having protease inhibitory activity as well as such sequences that encode amino acid sequences without protease inhibitory activity, in particular such sequences that for small defects have lost said activity.

A protein of the present invention is a protein with an amino acid sequence as encoded by the above defined DNA sequences, in particular the sequences set forth in Tables I or II or alleles or mutants thereof . Preferred amino acid sequences are those having protease inhibitory activity, or mutants thereof having said activity, for the use as pharmaceutical or diagnostic agent. Amino acid sequences with no or reduced activity are of interest in diagnostic and in drug screening.

The present invention furthermore concerns pharmaceutical and diagnostic compositions, that comprise such a DNA sequence .

A pharmaceutical composition of the present invention can also comprise as an at least one active substance (ingredient) a protein as defined above. Also such a pharmaceutical composition can furthermore comprise at least one further active compound, e.g. a compound that increases the protease inhibitory activity of said above defined protein, or in that it prolongs the time of presence of such a protein at its place of action in the body.

The present invention furthermore encompasses a pharmaceutical composition that comprises as an at least one active compound a substance which enhances or inhibits the transcription of a mRNA derived from a DNA as defined above, or in that it enhances or inhibits the translation of such a DNA. The present invention as well concerns a pharmaceutical composition, that comprises as an at least one active compound a compound that reduces or increases the protease inhibitory activity of a protein as defined above, or in that it shortens or prolongs the time of presence of such a protein at its place of action in the body.

The DNA sequence or proteins defined above are suitable for the treatment of nervous system disorders, in particular in order to prevent, ameliorate or cure disorders of the nervous system due to at least one protease, in particular proteases selected from the group consisting of tissue-type plasminogen activator, abbreviated as tPA, urokinase-type plasminogen activator, abbreviated as uPA, or plasmin. Most preferably the present invention concerns such DNA sequence or protein for the minimization of the tissue destruction in stroke.

By a preparation comprising such DNA sequence or protein, the minimization of the tissue destruction in stroke including brain infarction and ischemia, intracerebral hemorrhage, and subarrachnoid hemorrhage, as for example by exerting a protecting effect on the cells of the so-called penumbra zone surrounding the necrotic tissue can be obtained. Other disorders where an effective substance or preparation can be used, be it as pharmaceutical, be it as diagnostic agent include as a suitable selection the treatment of tissue destruction in traumatic brain injury, as for example by exerting a protective effect on the cells of the so-called penumbra zone surrounding the necrotic tissue, the prevention, amelioration or cure of negative effects caused by neurodegenerative diseases, or neuroinflammatory diseases, as for example multiple sclerosis , the reduction or prevention of negative effects on brain tissue caused by epileptic seizures, the rescue of endangered neurons, as for example neurons endangered by hypoxia and ischemia, excitotoxicity, neuroinflammatory diseases and processes, epileptic seizures, and cancerous neoformations , the axonal regeneration and/or restoration of synaptic integrity and functions, the prevention, amelioration, or cure of retinal disorders, as for example retinal degeneration and retinal neoangiogenesis , the cell death of cells of the nervous system, in particular a cell death in connection with damages of the nervous tissue, for example infarct of the brain and ischemic stroke, or hemorrhage of the brain, or trauma of the brain, and/or a cell death in connection with damages of the nervous tissue, which occur due to lack of oxygen or glucose or due to intoxication, and/or a cell death in connection with epileptic seizures, and/or a cell death in connection with neurodegenerative diseases and inherited genetic deficiencies of the nervous system, the regeneration of injured, damaged, underdeveloped, or maldeveloped brain tissue and/or nervous tissue, the reorganization of the brain or nervous areas that have remained intact after brain and/or nerve injuries or after the destruction or damage of brain areas, the prevention, amelioration, or cure of pathological pain syndromes, the amelioration or cure of disorders in the field of disorders of the psychic wellness, or the psychosomatic state of health, as for example nervosity or „ inner unrest", disorders in the field of the emotional functions, as for example states of anxiety, the prevention, amelioration or cure of psychiatric disorders, in particular psychiatric disorders in the field of schizophrenia and schizophrenia-like disorders, including chronic schizophrenia, chronic schizo-affective disorders, unspecific disorders, including acute and chronic schizophrenia of various symptomatologies, as for example severe, non-remitting „Kraepelinic" schizophrenia, or as for example the DSM-III-R-prototype of the schizophrenialike disorders, including episodic schizophrenic disorders, including delusionic schizophrenia-like disorders, including schizophrenia-like personality disorders, as for example schizophrenia-like personality disorders with mild symptomatics , including schizotypic personality disorders, including the latent forms of schizophrenic or schizophrenia-like disorders, including non-organic psychotic disorders, and/or in the field of the endogenic depressions or in the field of manic or manic-depressive disorders, the treatment of tumors such as prevention or reduction of the growth, the expansion, the infiltration and the metastasis of primary and metastatic tumors, inhibition of the formation of new blood vessels or neoangiogenesis, in particular the treatment of brain tumors or tumors of the retina.

The present invention also concerns the amelioration of the learning and memory functions in healthy persons, as well as in persons with reduced learning and memory functions. In one additional aspect, the present invention concerns a method for the production of proteins as defined above, that is characterized in that suitable host procaryotic and eucaryotic cells, in particular mammalian cells, are transfected with a DNA sequence as defined above in a vector ensuring the expression of said DNA sequence, and in that said transfected cells are cultured under suitable conditions allowing expression of said protein. The DNA sequences and/or the proteins defined above can furthermore be used as means for the screening of drugs against neuroserpin involving disorders, but also active ingredients such as transcription enhancers or reducers and activity enhancers or reducers. Furthermore, the present invention also comprises the use of a sequence as defined above as a means to produce antigens or as antigen for the production of antibodies.

Such antibodies can e.g. be antibodies that inhibit or promote the protease inhibitory function or antibodies that can be used for immunohistochemical studies .

The present invention also regards transgenic animals comprising an exogenous DNA sequence as defined above. Such animals are suitable for the study of diseases and the test of active substances as defined above

Such animals are in particular mammals (excluding man), such as mice. Still a further aspect of the present invention concerns the use of a DNA sequence as defined above for the inactivation or the mutation of the corresponding endogenous gene by means of gene targeting techniques . Such gene targeting techniques are for example the elimination of the gene in the mouse through homologous recombination or the replacement of the gene by a mutated form thereof .

A DNA sequence as defined above can, within the scope of the present invention, also be used for the preparation of a diagnostic preparation for the diagnostic of disorders due to defects or alterations in the genomic sequence comprising a coding sequence similar to but not identical with one of the coding sequences shown in Tables I or II. The nucleic acid sequences of the present invention are of great interest in gene therapeutical applications in humans and in animals, as for example as parts of gene therapy vectors, such as biological and synthetic vectors, or as parts of artificial chromosomes.

MODES FOR CARRYING OUT THE INVENTION

Neuroserpin is known to be expressed predominantly in the brain; the expression in the brain takes place nearly exclusively in the neurons.

The neuroserpins of primary interest for the present invention are neuroserpin of the human (sequence of Table I) , neuroserpin of the mouse (sequence of Table II) .

The identity of the amino acid sequences of the mature proteins (part of the proteins shown in Tables I and II) amounts to 88%.

Both, the neuroserpin of the human (see Table I) and the neuroserpin of the mouse (see Table II) have a coding sequence of 1230 nucleotides. The coded peptide in both sequences has a length of 410 amino acids and contains a signal peptide of 16 amino acids. The mature protein in both sequences is composed of 394 amino acids. A particularly high degree of similarity is found in the segment forming the reactive site loop.

The reactive site loop between the amino acid positions 327 and 360 of the two sequences has the following sequence:

Pi Pi' EVNEEGSEAAAVSGMIAISR MAVLYPQVIVDHPF Partial sequence of the sequence of the Table I

EVNEEGSEAAAASGMIAISR MAVLYPQVIVDHPF Partial sequence of the sequence of the Table II.

The capital letters represent standard single letter codes for amino acids . The gap between R and M of the two sequences marks the location of the scissile bond of the reactive site loop. The amino acids denoted PI and PI', which are flanking the putative scissile bond, are identical in both sequences (Pi: Arg346; PI': Met347). The above mentioned segment including the Pl/Pl' site exhibits only one position out of 34 in which the amino acids of the human neuroserpin (sequence of the Table I) and the murine neuroserpin (sequence of the Table II) are not identical. The non-identical amino acids are Val338 and Ala338 and, thus, represent a conservative substitution (printed underscored) .

The coded proteins of the sequences of Tables I and II are known to be in vi tro potent inhibitors of the serine proteases tissue-type plasminogen activator (tPA) , urokinase-type plasminogen activator (uPA) , and plasmin. The protease inhibitory function of the neuroserpins is specific. No measurable inhibition of thrombin has been found (see St.R. Krueger et al . , Expression of Neuroserpin, an Inhibitor of Tissue Plasminogen Activator, in the Developing and Adult Nervous System of the Mouse, The Journal of Neuroscience, 17 (23 ) , 8984-8996 (1997); T. Osterwalder et al . , The

Axonally Secreted Serine Protease Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin, The Journal of Biological Chemistry, Vol . 4 , Issue of January 23 , 2312-2321 , 1998) . Enzyme kinetic studies with tissue-type plasminogen activator revealed that the inhibitory activity of the neuroserpins is approximately 100 times faster than that of protease nexin-I .

Neuroserpins are unique when compared with the previously known serpins in that they are expressed almost exclusively in neurons.

In the scope of the present invention it could now be shown that neuroserpin has also an in vivo activity making it a very useful tool for the diagnostic and therapy of protease involving disorders of the nervous system, in particular of the central nervous system.

It is known that the expression of the neuroserpins during neural development starts at the beginning of the time range in which restructuration processes of synapses are observed, that in the adult nervous system, their expression is predominant in brain regions in which synapse plasticity occurs, and that a particularly high expression of neuroserpins is found in the cerebral cortex, the hippocampus, and the amygdala of the mouse.

In the deeper structures of the brain, in the brain stem, and in the spinal cord of the adult mouse, a weaker expression of the neuroserpins is found. In the adult peripheral nervous system, the neuroserpins are expressed in a subpopulation of the sensory ganglia neurons. The gene expression pattern of the neuroserpins in the brain is extremely interesting, because these molecules are expressed in the adult nervous system predominantly in neurons of those regions that are thought to play an important role in learning and in memory functions.

The gene expression pattern of the neuroserpins in the cerebral cortex is extremely interesting, because a reduction of the cellular differentiation in the cerebral cortex has been found to be associated with schizophrenia.

Another prominent characteristic of the neuroserpins consists therein that they are secreted by neurons . This fact - together with the function as a protease inhibitor and the expression pattern in the developing and adult brain - suggests that the neuroserpins play a role in the regulation of the extracellular proteolysis in brain areas which are involved in the processing and storage of learned behaviors, learned emotions, or memory contents.

Together with the recently found evidence for a role of extracellular proteases, in particular tissue- type plasminogen activator, in neural plasticity (see Frey et al., J. Neurosci. 16, pages 2057-2063, 1996;

Huang et al . , Proc. Natl. Acad. Sci. USA 93, pages 699- 704, 1996), the expression pattern allows the assumption that the protease inhibitory activity of neuroserpin has a role in learning and memory operations, for example operations which are involved in the processing and storage of learned behaviors, learned emotions, or memory contents .

The fact that neuroserpin is a potent inhibitor of tissue-type plasminogen activator (tPA) is particularly interesting, because tPA has been found to play a role in the pathogenesis of neuronal cell damage or neuronal cell death in the context of excitotoxin- induced epileptic seizures (see Tsirka et al . , Nature 377, pages 340-344, 1995) .

The gene expression pattern of the neuroserpins in the spinal cord and in the sensory ganglia is interesting, because these molecules are expressed in the adult nervous system in neurons of those brain regions that are thought to play a role in the processing of pain, as well as in the pathogenesis of pathological pain. The neuroserpins were found in connection with a study aimed at discovering proteins that are secreted from axons of neurons (see Stoeckli et al . , Eur . J. Biochem. 180, pages 249-259, 1989). Their preparation has now been described in several papers that are herein comprised by reference (see Osterwalder et al . , EMBO J. 15, pages 2944, 1996; S. P. Schrimpf et al . Human Neuroserpin (PI12): cDNA Cloning and Chromosomal Localization to 3q26, Genomics , Vol . 39, pages 1-8 (1997); St.R. Krueger et al . , Expression of Neuroserpin, an Inhibitor of Tissue Plasminogen Activator, in the Developing and Adult Nervous System of the Mouse, The Journal of Neuroscience, 17 (23 ) , 8984-8996, (1997)).

This procedure for the cloning can also be used for the isolation of homologous sequences of other species, such as rat, rabbit, guinea pig, cow, sheep, pig, primates, birds, zebra fish (Brachydanio rerio) , Drosophila melanogaster, Caenorhabditis elegans etc. Such sequences are preferred for the veterinary use in order to avoid incompatibility reactions. The coding nucleotide sequences obtained e.g. by the above described methods can be used for the production of proteins with the coded amino acid sequences as defined above.

The coding sequences of the sequences of the Tables I or II can also be used as starting sequences for the isolation of alleles and splice variants of the sequences shown in Tables I or II, or parts thereof can be used as probes for the isolation of the genes corresponding to the sequences shown in Tables I and II. Both the polymerase chain reaction and the nucleic acid hybridization can be used for this purpose. The coding sequences of the sequences of the

Tables I or II can be used as starting sequences for so- called "site-directed mutagenesis", in order to generate nucleotide sequences encoding proteins as defined above, in particular those shown in Tables I or II, or parts thereof, but whose nucleotide sequence is degenerated with respect to the sequences shown in Tables I or II due to use of alternative codons . Such mutagenesis can be desired dependent of the host cells used for the expression of the protein of interest. The coding sequences disclosed in Tables I or

II can be used as starting sequences for the production of sequence variants exhibiting altered function by means of so-called site-directed mutagenesis. Such altered functions can e.g. provide for proteins with longer lifetime, i.e. slower degradation, enhanced activity etc.

The coding sequences can be used for the production of vectors for use in gene therapy and cell engineering .

The coding sequences can be used for the generation of transgenic animals overexpressing the coding and the coded sequences of the sequences of the Tables I or II.

The coding sequences can be used for the diagnostics of disorders in the gene corresponding to the sequence of in particular Table I.

The amino acid sequences coded by the above described nucleic acid sequences can be used as active substances, as antigens for the production of antibodies, and as targets for drug development . The just outlined uses of nucleic acid sequences and amino acid sequences as defined above has been shown in the scope of the present invention to be very suitable for protease involving disorders, in particular tPA involving diseases, and especially suitable for the treatment of stroke. For example in stroke treatment the neuroserpins or the neuroserpin derived proteins are suitable pharmaceuticals in acute treatment as well as in long-time treatment.

In an acute state, i.e. within the first few hours after a stroke, a presently preferred mode of application is the direct application of a high amount of neuroserpin protein, preferably an intrathecal application, i.e. an injection directly into the cerebro- spinal fluids.

For the long term therapy of stroke, i.e. the restitution of damages, a preferred method is cell therapy.

For gene therapy and/or cell therapy a nucleic acid sequence coding for neuroserpin (the expression neuroserpin is considered as including alleles and mutants with protease inhibitor, at least tPA inhibitor activity) is introduced into a suitable vector allowing the expression of neuroserpin in the addressed nerve cells or specific therapy cells. Such a vector suitable for gene therapy and allowing expression of the neuroserpin comprises the neuroserpin encoding gene under the control of a nerve cell specific promotor.

For gene therapy suitable vectors are neurotrophic viruses that can be applied either directly or in transport cells.

Neuroserpin expressing cells can also be encapsulated so that they can be brought to the center of desired action by surgery treatment and with much reduced risk for incompatibility reactions. Such cells can be removed as soon as they are no longer needed or as soon as they have lost their activity and thus need replacement.

All the above described methods for the treatment of stroke are similarly applicable to other disorders induced by proteases, in particular tPA. Such disorders also comprise tumors such as those induced by tPA due to its effect on cell migration, but also tumors generally involving at least one protease in their growth, expansion, infiltration, metastasis and promotion of blood vessels or neoangiogenesis. Such proteases are preferably members of at least one of the following protease families:

- Serine Protease family such as urolinase, tissue-type plasminogen activator (tPA) , urokinase-type plasminogen activator (uPA) , plasmin, elastases, cathepsin G,

- Matrix Metalloproteinases family such as collagenases , gelatinases, stromelysins , matrylisins, - Cystein Proteases family such as cathepsin

B and cathepsin D.

- Glycosidases .

Besides of the above further described treatments, the present invention also provides for very useful diagnostic tools. By PCR and hybridization methods, as already mentioned above, genetic defects in the neuroserpin encoding protein that is localized in man to 3q26 (see S. P. Schrimpf et al . Human Neuroserpin (PI12): cDNA Cloning and Chromosomal Localization to 3q26, Genomics, Vol . 39, pages 1-8 (1997)) can be determined. Such determination helps for the diagnosis of disorders the symptoms of which are already noticeable as well as for the determination of persons or groups of persons, such as families, with enhanced risk to develop such a disorder.

It is of course also possible to produce by synthetic or chemical method proteins, peptides or nucleic acid sequences representing at least part of the sequences defined above and having the ability to mimic or to block, respectively, the biological activity of neuroserpin, in particular the protease inhibitory activity. Furthermore, the characterization and isolation of a deficient gene or a deficient protein encoded by such a gene provides efficient tools for screening possible drugs to improve the health of patients suffering from disorders due to such defects.

In particular for the search of further disorders and drugs also transgenic animals are of great value .

This and further aspects of the present invention are now further illustrated by the following examples. It has, however to be understood that they are not at all intended to reduce the scope of the present invention. They are of mere illustrative purpose.

Example 1 :

Production of the coded protein of the sequences of the Tables I and II in a procaryotic expression system

The production of the coded protein of the sequences of the Tables I and II can be carried out in procaryotic and eucaryotic expression systems . The protein coding sequences can be obtained as described in S. P. Schrimpf et al . Human Neuroserpin (PI12) : cDNA Cloning and Chromosomal Localization to 3q26, Genomics, Vol . 39, pages 1-8 (1997). In the following part, the production of the coded protein of the sequence of the Table I (human neuroserpin) in a procaryotic expression system is described.

The sequence described in Table I was cytoplasmically expressed in E. coli with a stretch of six histidines fused to the carboxyterminus of the protein. A fragment of the cDNA encoding amino acids 1 through 394 was amplified in a PCR using the oligodeoxynucleotide primers 5 ' -AAT TTC TAG AGA AAG GAG ATA CAT ATG ACA GGG GCC ACT TTC CCT-3 ' and 5 ' -GGG AAG CTT CTA GTG GTG ATG GTG GTG GTG AAG TTC TTC GAA ATC ATG GTC C-3 ' . The cDNA fragment was cloned into the vector pAK400 (see Krebber et al . , J. Immunol. Methods 201 , pages 35- 55, 1997) via the Xbal and Hindlll sites of the vector, allowing expression of the cDNA from the lac operator/promoter located immediately upstream. For expression, a colony of E. coli strain BL21DE3 harboring the expression plasmid was precultured overnight at 37 °C in 100 ml LB medium containing 30 μg/ml chloramphenicol . After inoculation of the same medium with the preculture, bacteria were grown at 25 °C and induced with 1 mM Isopropyl-1-thio-b-D-galactosidase (IPTG) at an OD₆₀o of 0.5. The bacteria were harvested by centrifugation 6 hrs after induction, resuspended in Ni-NTA-binding buffer (1 M NaCl, 50 mM TrisCl pH8.0), and disrupted in a French press . The soluble protein extract was incubated over night at 4 °C with 0.4 ml of Ni-NTA resin (Qiagen, Chatsworth, CA) . Following extensive washing with Ni-NTA- binding buffer, bound proteins were eluted with Ni-NTA- binding buffer containing 200 mM imidazole. The eluted protein was dialyzed against PBS and immediately frozen at -80 °C.

Example 2 :

Production of the coded protein of the sequence of the Table II in a eucaryotic expression system.

The protein of the sequence of the Table II (obtainable according to St.R. Krueger et al . , Expression of Neuroserpin, an Inhibitor of Tissue Plasminogen

Activator, in the Developing and Adult Nervous System of the Mouse, The Journal of Neuroscience, 17 (23 ) , 8984-8996 (1997)) was recombinantly expressed in human embryonic kidney cells (cell line 293) either in its unaltered form or, for single-step purification by metal chelate chromatography, fused carboxyterminally to a tag of six consecutive histidines. For heterologous expression of the unaltered form of neuroserpin, a Spe I-Ssp I fragment from the lambda phage cDNA clone mmns 4.1, containing the full length open reading frame of mouse neuroserpin, 111 bp of 5' untranslated region, and 100 bp of 3' untranslated region, was cloned into the expression vector pcDNA3.1 ( - )MycHisA (Invitrogen, Carlsbad, CA) . The construct was electroporated and the cells were subsequently tested for expression of neuroserpin by a dot blot assay. For heterologous expression of neuroserpin containing a carboxyterminal polyhistidine tag, the mouse neuroserpin cDNA was amplified in a polymerase chain reaction using the oligonucleotides 5' -GC TCT AGA CAT ATG ACA GGG GCA ACG TTC CCA-3 ' (5', sense) and 5 ' -GGG AAG CTT CTA GTG GTG ATG GTG GTG GTG AAG TTC CTC AAA GTC ATG GC-3 ' (3', antisense, encoding an additional segment of six consecutive histidines) . The entire amplification product was sequenced to exclude any polymerase chain reaction errors and a Sty I - Hind III fragment of the amplification product was used to replace a Sty I - Hind III fragment from the expression construct containing the unaltered form of mouse neuroserpin. Transfection of the construct into 293 cells and subsequent detection of the produced protein was done as described above .

The proteins produced according to Examples 1 and 2 were found to provide protease inhibitory activity (for the method see St.R. Krueger et al . , Expression of Neuroserpin, an Inhibitor of Tissue Plasminogen Activator, in the Developing and Adult Nervous System of the Mouse, The Journal of Neuroscience, 17 (23) , 8984-8996 (1997); T. Osterwalder et al . Neuroserpin, an axonally secreted serine protease inhibitor, The EMBO Journal , Vol . 15, No . 12 , pages 2944-2953 (1996).

Example 3 :

Neuroserpin expression is enhanced in neurons of the ipsilateral hemisphere after focal ischemic stroke

Recent observations obtained with tissue- type plasminogen activator (tPA) indicate that tPA contributes to the development of tissue-damage in an animal model of excitoxicity-induced neuronal cell death and stroke (see Tsirka et al . , Nature 377 , pages 340-344, 1995; Wang et al . , Nature Medicine 4, pages 148-150, 1998) . In brief, the damage of cerebral tissue after kainate-induced seizures and experimentally induced focal cerebral ischemia was significantly smaller in tPA^_/" mice, as compared with wild- ype mice. These observations indicate that tPA enhances excitotoxicity-induced degenerative processes.

For the analysis of the effect of neuroserpin as a tPA inhibitor and with respect to its potential role as neuroprotective agent attenuating the harmful effects of tPA derived from the tissue reaction in the injured brain, we started with a permanent occlusion model of the medial cerebral artery of the mouse. In order to produce a focal ischemic stroke, the mice were anaesthetized by intraperitoneal injection of Xylacine/Ketamme. Using a stereo microscope, a small incision was made between the eye and the ear on the right side. The temporal muscle was pushed forward after a small incision was made on the upper side of the anterior segment and one of the lower side of the midline of the muscle. The parotic gland and the surrounding tissue was pushed down. Through the half-transparent temporal scull the medial cerebral artery (MCA) becomes visible. Approximately 3 mm frontal of the MCA, a small hole was made using a 0.18 drill. The internal layer of the scull was removed with fine tweezers and the dura was opened carefully. Thereby the medial cerebral artery becomes visible and accessible. The MCA was coagulated by using an electro-surgical unit (MB-la, Asatom) . After electrocoagulation, the environment of the artery was humidified using 0.9 % NaCl. Subsequently the hole in the scull was closed with absorbable bone glue (Absele, Ethicon) . The temporal muscle and the parotic gland were moved back to the original place and the incision was closed. After surgery the animals were kept warm. They recovered quickly from surgery. The animals were sacrificed three days or seven days after the electrocoagulation of the MCA. The brains were directly frozen and subsequently cut with a Kryostat in order to investigate the lesion histologically using in situ- hybridization and immunocytochemistry .

Investigation of the infarct and the marginal zone of the infarct revealed a strong astrocytic reaction that was spread with diminishing intensity over almost the entire hemisphere ipsilateral to the infarct. In contrast, the microglial activation, as revealed by the specific marker for activated microglia, F4/80, was limited to the marginal zone of the infarct and was quite sharply delineated. In the same area as the activated microglia we also observed a very strong upregulation of tPA messenger RNA as indicated by in situ-hybridization with an antisense probe for tPA. Based on the analysis of consecutive sections, the expression of tPA is colocalized with the marker for activated microglia. Therefore we concluded that activated microglia accumulate in the reactive zone of a focal ischemic stroke and tPA is produced from the activated microglial cells of this area. In in-situ-hybridizations using an antisense probe for neuroserpin, we found that neuroserpin messenger RNA is significantly upregulated in many neurons spread over almost the entire hemisphere ipsilateral to the lesion. As neuroserpin is a potent and fast-acting inhibitor of tPA, its enhanced expression in the zone surrounding the infarct might indicate that neuroserpin is an element of a defensive process aimed at reducing the range of action of microglial tPA after ischemic stroke. Enhancing and prolonging the neurons' endogenous neuroserpin response might, therefore, be beneficial in the treatment of stroke. Both the enhancement of neuronal production and the administration of exogenous neuroserpin to the extracellular space of the cerebral tissue might be promising future therapies for reducing the range of tPA's action and, thus, limiting tissue damage following ischemic stroke.

Example 4 : Neuroserpin and tPA form complexes in the adult brain

In an other series of experiments it was investigated whether a direct interaction between neuroserpin and tPA could be observed in vivo. The brains from mice were quickly removed from killed animals and homogenized in a buffer (1 ml/100 mg of tissue) containing 140 mM NaCl, 10 mM Tris-Cl, pH 8.0, 0.1% (v/v) TritonX-100. Tissue extracts were cleared from insoluble material by ultracentrifugation and immunoprecipitated with the monoclonal antibody C7C10 coupled to cyanogen bromide activated Sepharose 4B (Pharmacia, Uppsala, Sweden) for 3 h at 4°C. The slurry was washed four times with a buffer containing 140 mM NaCl, 10 mM Tris-Cl, pH 8.0, 0.1% (v/v) TritonX-100. Neuroserpin was eluted by boiling the affinity resin for 5 min in 15% (v/v) glycerol, 3% (w/v) SDS, 60 mM Tris-Cl, pH 6.8, 0.01% (w/v) bromphenol blue, and 5% β-Mercaptoethanol . Samples were subjected to SDS-PAGE and blotted to nitrocellulose (Schleicher & Schuell, Dassel, Germany). Immunodetection was performed with the biotinylated monoclonal antibody C7C10 and the BM Chemilummescence Western blotting kit (Boehringer Mannheim, Germany) according to the supplier's recommendations. Using this approach, we could demonstrate on Western blots that extracts of mouse brain contained two additional bands that were immunoreactive for neuroserpin but exhibited a higher molecular weight than the native neuroserpin molecule. Based on the occurrence of two forms of tPA, namely the single chain and the two chain form, these two bands very likely reflect the occurrence of neuroserpin-tPA complexes in the normal adult brain.

Example 5 :

Overexpression of neuroserpin in CNS neurons using transgenic mice technology

Because tPA-deficient mice exhibit an enhanced resistance against neurotoxicity-induced neuronal cell death, we postulate that transgenic mice which overexpress neuroserpin (a potent inhibitor of tPA) in neurons, are less vulnerable after an ischemic stroke. To test this hypothesis, we have generated transgenic mice that overexpress neuroserpin. The overexpression of a gene in a transgenic mouse is a relatively direct way to study the function of a protein in vivo. For the first series of experiments chicken neuroserpin was expressed under the control of the promoter of the Thy-1 gene. The Thy-1 gene is expressed in the nervous system relatively late (postnatal day 4-10, depending on the location). The expression of neuroserpin under the control of the Thy-1 promoter (Gordon et al . , 1987) ensures that the earlier developmental stages are not affected. This point is essential. First, neuroserpin is expressed in some regions of the developing nervous system, such as the floor plate of the spinal cord, relatively early, and, thus, it could play a role in axonal pathfinding functions that are controlled by the floor plate. Second, it is known that protease nexin-1, a relative of neuroserpin with a partially overlapping inhibitory pattern, is involved in the regulation of axon growth during neurogenesis . By using a late onset promoter it was intended to prevent perturbations of early stages of neurogenesis in the transgenic animals .

For the first transgenic mice, we chose to overpress the neuroserpin of the chicken, because of its potential of being selectively detected with species- specific monoclonal antibodies. The chicken neuroserpin exhibits an amino acid sequence identity with its counterpart of the mouse of 75%. Thus, a highly conserved function can be assumed. To control this assumption the neuroserpin of the mouse was overexpressed as well. Further controls include the overexpression of inactive mutants of neuroserpin. A virtually inactive form of chicken neuroserpin has recently been generated in our laboratory by a triple amino acid mutation in the reactive site loop.

The construct of the transgene is based on an expression vector for Thy-1 in which the translated region of Thy-1 has been substituted by a Xho-I linker (Gordon et al . , 1987). The 1.25 kb long DNA fragment of chicken neuroserpin used for the overexpression is derived from the chicken cDNA digested with Afllll (3 bp upstream of the start ATG) and Cacδl (9 bp downstream of the TAA stop codon) . This fragment is inserted into the Thy-1 expression vector at the Xho-I linker site by a blunt-end ligation and the orientation controlled with a Hindlll digest. The plasmid is rescued and the fragment to be used for the injection into the pronucleus of fertilized mouse oocytes is cut out by digestion at the two flanking Pvul sites. The 7.5 kb long injection fragment is separated on a 1% agarose gel, the band purified with a QIAEXII-kit, and the DNA eluted from the QIAEX particles with injection buffer. The generation of transgenic mice was achieved by pronuclear injection following standard protocol. The litters were screened for the presence of the transgene by PCR and Southern blotting.

By this procedure, three mouse lines overexpressing the chicken neuroserpin and two lines overexpressing the mouse neuroserpin were raised. The expression of the transgene was verified at the mRNA level by Norhern blotting and in-situ-hybridization and at the proteine level by Western blotting. A typical overexpression was in the order of 6 to 12 fold.

Example 6:

Overexpression of neuroserpin in CNS neurons results in reduced tPA activity in the brain

In the scope of the present invention an in vivo effect could be demonstrated making neuroserpin a highly promising candidate for pharmaceutical compound for the treatment of excitotoxicity-mediated cell loss. To test for a potential beneficial effect of neuroserpin in focal ischemic stroke, transgenic mice overexpressing neuroserpin in neurons under the control of the Thy-1 promoter were used. These mice produce approximately 6 - 8 times more neuroserpin in the cerebral cortex and the hippocampus than wild-type mice. It was then investigated whether the overexpression of neuroserpin in transgenic mice under the control of the Thy-1 promoter indeed resulted in a diminished activity of tPA in these brains. The wildtype and transgenic mice overexpressing neuroserpin were anesthetized with pentobarbital and transcardially perfused with 10 ml PBS^" to completely remove the blood from the brain. The brains were homogenized with 10 volumes of 1% SDS, 50 mM Tris, pH 6.8 , in a Dounce homogenizer, followed by several passages through a 25- gauge needle to shear the DNA. The protein concentration was quantified by the BCA method (Pierce) . 0.25 -0.5 :g total brain homogenate were electrophoretically processed through a 0.1% SDS-10% polyacrylamide gel with a 4% stacking gel. Plasminogen (0.04 units/ml: Sigma) and casein (1 mg/ml: Merck) were copolymerized in the polyacrylamide. Following electrophoresis, the gel was incubated in 2.5% Triton X-100, washed with water and then sandwiched between two pieces of Whatmann 3M chromatography paper, which had been saturated with 0.1 M Tris-HCl, pH 8.1, and incubated at 37°C for 12-16 h. The gel was stained with 0.1% Coomassie brilliant blue. The cleared bands were quantified on a Molecular Dynamics Densitometer .

Using this zymographic approach we could demonstrate that transgenic mice overexpressing neuroserpin approximately 6-8 fold in the brain had a markedly diminished activity of tPA in the order of approximately 45% (of remaining activity) . Therefore we concluded that neuroserpin locally produced by neurons in the CNS reduces ^"the activity of tPA in the brain.

Example 7

Overexpression of neuroserpin in CNS neurons results in an attenuated microglial activation in the reactive zone of a focal ischemic stroke The potential role of neuroserpin as a protective agent against the harmful effects of the tPA was analyzed with the same stroke model using transgenic mice overexpressing neuroserpin approximately 6-8 fold. So far two series of experiments have been carried out using the stroke model. In each experiment 4 transgenic animals overexpressing neuroserpin approximately 6-8 fold in neurons under the control of the Thy-1 promoter were analyzed in comparison with their non-transgenic litter mates. Analyses included immunohistochemical staining of microglial cells using F4/80, a marker for activated microglial cells and in-situ-hybridisation with an antisense probe for tPA.

Using this experimental approach, we found that neuroserpin overexpressing mice exhibited a marked decrease in the microglial activation in the marginal zone of the infarct and this decrease of microglial activation was accompanied by markedly decreased in-situ- hybridization signals for tPA in the same area, indicative of a reduced expression of tPA. Therefore, neuroserpin is likely to attenuate both the microglial activation and the microglial production of tPA in the reactive zone of the infarct after a focal ischemic stroke. From the attenuation of the microglial activation and the reduction of the microglial tPA production, a neuroprotective effect is expected.

In the following part statements concerning the sequences of the Tables I or II are given: SEQUENCE LISTING

<110> Sonderegger, Peter <120> Neuroserpin

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<150> US 09/023129 <151> 1998-02-13 <160> 4

<170> Patentln Ver . 2.1

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<213> Homo sapiens

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<210> 2

<211> 410 <212> PRT <213> Homo sapiens <400> 2

Met Ala Phe Leu Gly Leu Phe Ser Leu Leu Val Leu Gin Ser Met Ala 1 5 10 15 Thr Gly Ala Thr Phe Pro Glu Glu Ala lie Ala Asp Leu Ser Val Asn 20 25 30

Met Tyr Asn Arg Leu Arg Ala Thr Gly Glu Asp Glu Asn lie Leu Phe 35 40 45

Ser Pro Leu Ser lie Ala Leu Ala Met Gly Met Met Glu Leu Gly Ala 50 55 60 Gin Gly Ser Thr Gin Lys Glu lie Arg His Ser Met Gly Tyr Asp Ser 65 70 75 80

Leu Lys Asn Gly Glu Glu Phe Ser Phe Leu Lys Glu Phe Ser Asn Met 85 90 95

Val Thr Ala Lys Glu Ser Gin Tyr Val Met Lys lie Ala Asn Ser Leu 100 105 110

Phe Val Gin Asn Gly Phe His Val Asn Glu Glu Phe Leu Gin Met Met 115 120 125

Lys Lys Tyr Phe Asn Ala Ala Val Asn His Val Asp Phe Ser Gin Asn 130 135 140 Val Ala Val Ala Asn Tyr lie Asn Lys Trp Val Glu Asn Asn Thr Asn 145 150 155 160

Asn Leu Val Lys Asp Leu Val Ser Pro Arg Asp Phe Asp Ala Ala Thr 165 170 175

Tyr Leu Ala Leu lie Asn Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser 180 185 190

Gin Phe Arg Pro Glu Asn Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp 195 200 205

Glu Ser Glu Val Gin lie Pro Met Met Tyr Gin Gin Gly Glu Phe Tyr 210 215 220 Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu Ala Gly Gly lie Tyr Gin 225 230 235 240

Val Leu Glu lie Pro Tyr Glu Gly Asp Glu lie Ser Met Met Leu Val 245 250 255

Leu Ser Arg Gin Glu Val Pro Leu Ala Thr Leu Glu Pro Leu Val Lys 260 265 270

Ala Gin Leu Val Glu Glu Trp Ala Asn Ser Val Lys Lys Gin Lys Val 275 280 285

Glu Val Tyr Leu Pro Arg Phe Thr Val Glu Gin Glu lie Asp Leu Lys 290 295 300 Asp Val Leu Lys Ala Leu Gly lie Thr Glu lie Phe He Lys Asp Ala 305 310 315 320

Asn Leu Thr Gly Leu Ser Asp Asn Lys Glu He Phe Leu Ser Lys Ala 325 330 335 He His Lys Ser Phe Leu Glu Val Asn Glu Glu Gly Ser Glu Ala Ala 340 345 350

Ala Val Ser Gly Met He Ala He Ser Arg Met Ala Val Leu Tyr Pro 355 360 365

Gin Val He Val Asp His Pro Phe Phe Phe Leu He Arg Asn Arg Arg 370 375 380 Thr Gly Thr He Leu Phe Met Gly Arg Val Met His Pro Glu Thr Met 385 390 395 400

Asn Thr Ser Gly His Asp Phe Glu Glu Leu 405 410

<210> 3 <211> 2944 <212> DNA

<213> Mus musculus

<400> 3 cggcacgaga tccggagcag tctcagcctg cccagcatcc tctccagcat cccgagcggg 60 gattgcaggt gtgaaggaga cttgaaacca tcccatcatg acttaccttg aactgcttgc 120 tttgctggcc ttgcaaagtg tggtgacagg ggcaacgttc ccagatgaaa ccataactga 180 gtggtcagtg aacatgtata accaccttcg aggcaccggg gaagatgaaa acattctctt 240 ctctccacta agcattgccc ttgcgatggg aatgatggag cttggggctc aaggatctac 300 taggaaagaa atccgccatt caatgggata tgagggtctg aaaggtggtg aagaattttc 360 tttcctgagg gatttttcta atatggcctc tgccgaagaa aaccaatatg tgatgaaact 420 tgccaattcg ctctttgtac aaaatggatt tcatgtcaat gaggaattct tgcaaatgct 480 gaaaatgtac tttaatgcag aagtcaacca tgtggacttc agtcaaaatg tggctgtggc 540 taactccatc aataaatggg tggagaatta tacaaacagt ctgttgaaag atctggtgtc 600 tccggaggac tttgatggtg tcactaattt ggccctcatc aatgctgtat atttcaaagg 660 aaactggaag tctcagttta gacctgaaaa taccagaact ttctccttca cgaaagatga 720 tgaaagtgaa gtgcagattc caatgatgta tcaacaagga gaattttatt atggtgaatt 780 tagtgatgga tccaatgagg ctggtggtat ctaccaagtc cttgagatac cctatgaggg 840 agatgagatc agcatgatgc tggcactgtc cagacaggaa gtcccactgg ccacactgga 900 gcctctgctc aaagcacagc tgatcgaaga atgggcaaac tctgtgaaga aacaaaaggt 960 ggaagtgtac ttgcccaggt tcactgtgga acaggaaatt gatttaaaag acatcttgaa 1020 agcccttggg gtcactgaaa ttttcatcaa agatgcaaat ttgactgcca tgtcagataa 1080 gaaagagctg ttcctctcca aagctgttca caagtcctgc attgaggtta atgaagaagg 1140 gtcagaagcc gctgcagcct ccggaatgat tgcgattagt aggatggctg tgctgtaccc 1200 tcaggttatt gtcgaccatc catttctcta tcttatcagg aacaggaaat ctggcataat 1260 cttattcatg ggacgagtca tgaaccctga aacaatgaat acaagtggcc atgactttga 1320 ggaactttaa atgacgacgt ttgagtacaa agaaagcagg aacaaagcac attatgtttg 1380 caagtggtat atatttagga tttctgtttt atagtgttac ttagggaaat atttaaatag 1440 ttctggatag tagtaatcca tgtgacctat aagttagcct gtcaaaagct gttatcagta 1500 taaagagtat ggtcccattg tgtcattgtg tctggtgtgc tgctgtttaa aataaaagta 1560 catattgaaa ctgtgaacca ctttttttca ttttgaaagt agttgtagtc tatacaatac 1620 tatgtctgag atttgaaacc tatgctgttt ctttaggaat tgtagtaaaa tgatcctaca 1680 aggcaaaatg tagaaactgt tgtttctgag tttcttcata atcatgcaga atcaaacacc 1740 aaagtaagca acatacatat atatatataa taagcaatac tgtgaagggg aggccaaaag 1800 gcagagaaat tgagattgtt atttagtgtg gcattccatg acaaaagatt taggaggaaa 1860 tgtgggatat gtaagaccca tagatgtata ttttgtatat ctgtagtatt atacttttaa 1920 tttattaaag tataactctt ttatttattt ttaaaagttt cctgtgaacc aatatgccac 1980 atgactctac tagcaagttc agatatctca ttagctattc tggatgacat caagaggcct 2040 catggaggga atcccgtgta ccatttacgt tttagtgatt ttttgtgatg ttcacacaaa 2100 gatgaaatca cattgttgca cactctctag actatatcca agaaaggcat caagtggtac 2160 attggtgtgc caggaaaata gatgtaatta ctttattaaa aaagttcctg gtattgtgca 2220 tcatatggaa tcagtgctgc ttaaacttag tacgtcctgc tgacacctgg tcacttatta 2280 caaatatagg ttcttatcca ggatgtctaa ggtagagtgg gaaccacagc tttctatcat 2340 tactgacatc caaatgatgc cgcagatatc tgaccatagc ctttgctgag agtcccttgg 2400 gttgcaatgt cgtacttgaa gtcagcctca cattttcaca gactgagatt ggagagatga 2460 gggtgcaggg aggagataat ctacactagt gatacgatgc ctttgtcaag cactggtgtg 2520 atctcgaagt attctagtac acactctaga taaattcttc tgtacattac aacacttgaa 2580 atgcagtcgt taaaaatatg gagacattta taggcaatac ccatgaaaga atttatgact 2640 atccgaggac acagtactta acaatgaatc ttttacagct tatattttca gaggacttgt 2700 agtttattca taaatcttca tgttattgta caatagtgct cttgttttca tttataattt 2760 atgaagctga gatgctggtg ttaattcagt gttcacattc tctgctaaga acagtcttta 2820 tctctgtatc cttcttgtta atatgacatc tatagctata tctatatgtt cattagttaa 2880 acaaatgtat ggcctgtaag gaagaataaa cattattatg caatcatgta aaaaaaaaaa 2940 aaaa 2944

<210> 4

<211> 410 <212> PRT

<213> Mus musculus

<400> 4

Met Thr Tyr Leu Glu Leu Leu Ala Leu Leu Ala Leu Gin Ser Val Val 1 5 10 15

Thr Gly Ala Thr Phe Pro Asp Glu Thr He Thr Glu Trp Ser Val Asn 20 25 30 Met Tyr Asn His Leu Arg Gly Thr Gly Glu Asp Glu Asn He Leu Phe 35 40 45

Ser Pro Leu Ser He Ala Leu Ala Met Gly Met Met Glu Leu Gly Ala 50 55 60

Gin Gly Ser Thr Arg Lys Glu He Arg His Ser Met Gly Tyr Glu Gly 65 70 75 80

Leu Lys Gly Gly Glu Glu Phe Ser Phe Leu Arg Asp Phe Ser Asn Met 85 90 95

Ala Ser Ala Glu Glu Asn Gin Tyr Val Met Lys Leu Ala Asn Ser Leu 100 105 110 Phe Val Gin Asn Gly Phe His Val Asn Glu Glu Phe Leu Gin Met Leu 115 120 125

Lys Met Tyr Phe Asn Ala Glu Val Asn His Val Asp Phe Ser Gin Asn 130 135 140

Val Ala Val Ala Asn Ser He Asn Lys Trp Val Glu Asn Tyr Thr Asn 145 150 155 160

Ser Leu Leu Lys Asp Leu Val Ser Pro Glu Asp Phe Asp Gly Val Thr 165 170 175

Asn Leu Ala Leu He Asn Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser 180 185 190 Gin Phe Arg Pro Glu Asn Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp 195 200 205

Glu Ser Glu Val Gin He Pro Met Met Tyr Gin Gin Gly Glu Phe Tyr 210 215 220 Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu Ala Gly Gly He Tyr Gin 225 230 235 240 Val Leu Glu He Pro Tyr Glu Gly Asp Glu He Ser Met Met Leu Ala

245 250 255

Leu Ser Arg Gin Glu Val Pro Leu Ala Thr Leu Glu Pro Leu Leu Lys 260 265 270

Ala Gin Leu He Glu Glu Trp Ala Asn Ser Val Lys Lys Gin Lys Val 275 280 285

Glu Val Tyr Leu Pro Arg Phe Thr Val Glu Gin Glu He Asp Leu Lys 290 295 300

Asp He Leu Lys Ala Leu Gly Val Thr Glu He Phe He Lys Asp Ala 305 310 315 320 Asn Leu Thr Ala Met Ser Asp Lys Lys Glu Leu Phe Leu Ser Lys Ala

325 330 335

Val His Lys Ser Cys He Glu Val Asn Glu Glu Gly Ser Glu Ala Ala 340 345 350

Ala Ala Ser Gly Met He Ala He Ser Arg Met Ala Val Leu Tyr Pro 355 360 365

Gin Val He Val Asp His Pro Phe Leu Tyr Leu He Arg Asn Arg Lys 370 375 380

Ser Gly He He Leu Phe Met Gly Arg Val Met Asn Pro Glu Thr Met 385 390 395 400 Asn Thr Ser Gly His Asp Phe Glu Glu Leu

405 410

Further information to the sequences;

Sequences of Table I, SEQ. ID. NOS. 1 (nucleic acid sequence) and 2 (amino acid sequence) :

LENGTH , STRANDEDNESS and TOPOLOGY of nucleic acid: 1577 base pairs, single stranded, linear

LENGTH and LOCATION of amino acid: see below MOLECULE TYPE: cDNA to mRNA

IMMEDIATE SOURCE (2 sources) :

LIBRARY: oligo (dT) -primed human fetal retina cDNA library in the lambda Uni-ZAP XR vector, catalog Nr . 937202, Stratagene, La Jolla, CA, USA LIBRARY: oligo (dT)- and random-primed human fetal brain (17-18 weeks of gestation) cDNA library in the lambda Uni-ZAP XR vector, catalog Nr . 936206, Stratagene, La Jolla, CA, USA

CLONE: IVb POSITION IN GENOME: Chromosome 3 / Segment q26

FEATURES (location of specific parts of the amino acid)

signal peptide: 82 .. 129 mature peptide: 130 .. 1311 coding sequence: 82 .. 1311 reactive site loop: 1108 .. 1209 amino acid PI: 1165 .. 1167 amino acid PI': 1168 .. 1170 inhibiting segment: 1108 .. 1209 amino acid PI: 1165 .. 1167 amino acid PI': 1168 .. 1170 polyA signal: 1535 .. 1540 polyA segment: 1560 .. 1577

3'UTR: 1312 .. 15 5'UTR: 1 .. 81 Sequences of Table II, SEQ. ID. NOS. 3 (nucleic acid sequence) and 4 (amino acid sequence) :

LENGTH STRANDEDNESS and TOPOLOGY of nucleic acid: 2944 base pairs, single stranded, linear

LENGTH and LOCATION of amino acid: see below MOLECULE TYPE: cDNA to mRNA ORIGINAL SOURCE, ORGANISM: Mus musculus

IMMEDIATE SOURCE (2 sources) :

LIBRARY: oligo (dT) -primed mouse brain cDNA library in the lambda Uni-ZAP-XR vector, from Balb c mice, postnatal day 20, catalog Nr . 937319, Stratagene, La Jolla, CA, USA

CLONE : mmns 4.1 FEATURES (location of specific parts of the amino acid) : signal peptide: 98 .. 145 mature peptide: 146 .. 1327 coding sequence: 98 .. 1327 reactive site loop: 1124 .. 1225 amino acid Pi: 1181 .. 1183 amino acid Pi': 1184 .. 1186 inhibiting segment: 1124 .. 1225 amino acid Pi: 1181 .. 1183 amino acid Pi': 1184 .. 1186 polyA signal: 1551 .. 1556 and 2905 .. 2910 polyA segment: 2930 .. 2944 3'UTR: 1328 .. 2929

5'UTR: 1 .. 97

Claims

PATENT CLAIMS

1. DNA sequence encoding a polypeptide product having at least part of the primary structural conformation of that of neuroserpin, said DNA sequence being selected from a) the DNA sequences of Tables I and II, b) DNA sequences which hybridize under stringent conditions to the protein coding regions of the DNA sequences of Tables I or II or respective genomic sequences, c) sequences that but for the degeneracy of the genetic code would hybridize to the DNA sequences defined under a) and b) , d) DNA sequences that encode alleles and/or mutants of the neuroserpins of figures I and II, e) parts of the sequences defined under a) to d) that encode a protein with protease inhibitory activity, and f) the complementary strands of the sequences defined above, for the use as pharmaceutical or diagnostic agent .

2. The DNA of claim 1 which is the nucleotide sequence of Table I .

3. A protein with the amino acid sequence as encoded by the DNA sequences of claim 1 or 2 for the use as pharmaceutical or diagnostic agent.

4. The protein of claim 3 which has protease inhibitory activity.

5. Pharmaceutical composition, characterized in that it comprises a DNA sequence according to anyone of claims 1 or 2 or a protein according to anyone of claims 3 or 4.

6. Use of a DNA sequence or a protein as defined in one of claims 1 to 4 for the preparation of a medicament for the treatment of disorders of the nervous system, in particular the central nervous system, most preferably the brain.

7. Use according to claim 6, characterized in that said disorders of the nervous system, in particular brain, are disorders due to at least one protease.

8. Use according to claim7, characterized in that the protease is tissue-type plasminogen activator, abbreviated as tPA, urokinase-type plasminogen activator, abbreviated as uPA, or plasmin.

9. Use according to claim 7 or 8, characterized in that the protease is tPA.

10. Use according to one of claims 7 to 9 , characterized in that the medicament is a medicament for the minimization of the tissue destruction during and/or after stroke.

11. Use according to anyone of claims 7 to 9, characterized in that it prevents the cell death of cells of the nervous system.

12. Use of a DNA sequence or a protein as defined in one of claims 1 to 4 for the preparation of a medicament for the treatment of tumors, including prevention or reduction of the growth, the expansion, the infiltration and the metastasis of primary and metastatic tumors, in particular brain tumors or tumors of the retina.

13. Use according to claim 12, characterized in that said tumors involve at least one protease in their growth, expansion, infiltration, metastasis and promotion of blood vessels or neoangiogenesis.

14. Use according to claim 13, characterized in that the protease is a member of at least one of the following protease families:

- Serine Protease family such as urolinase, tissue-type plasminogen activator (tPA) , urokinase-type plasminogen activator (uPA) , plasmin, elastases, cathepsin G, - Matrix Metalloproteinases family such as collagenases, gelatinases, stromelysins , matrylisins,

- Cystein Proteases family such as cathepsin B and cathepsin D. - Glycosidases .

15. A method for the production of proteins as defined in claims 2 or 3 , characterized in that suitable host cells are transfected with a DNA sequence as defined in one of claims 1 or 2 in a vector ensuring the expression of said DNA sequence in said host cell and in that said transfected cells are cultured under suitable conditions allowing said expression.

16. A synthetic or chemical method for the production of proteins, peptides or nucleic acid sequences representing at least part of the sequences defined in one of claims 1 to 4 and having the ability to mimic or to block, respectively, the biological activity of neuroserpin, in particular the protease inhibitory activity.

17. Use of the DNA sequences and/or the proteins as defined in one of claims 1 to 4 as tools in the screening of pharmaceutical drugs .

18. Use of a sequence as defined in one of claims 1 or 2 as a means to produce antigens or as antigen for the production of antibodies.

19. Transgenic animal, characterized in that it comprises an exogenous DNA sequence as defined in claim 1 or 2 in an environment allowing protein expression.

20. Use of a DNA sequence or as defined in one of claims 1 or 2 or parts thereof for the preparation of a diagnostic preparation for the diagnostic of disorders due to defects in the genomic sequence comprising a DNA sequence according to one of claims 1 or 2.

21. A vector or artificial chromosome comprising a DNA sequence as defined in one of claims 1 or 2 for the use in gene therapeutical applications in humans and in animals .