WO2016188729A1

WO2016188729A1 - Methods for the therapy of protein-misfolding-diseases

Info

Publication number: WO2016188729A1
Application number: PCT/EP2016/060339
Authority: WO
Inventors: Detlev Riesner; Julian VICTOR; Gerhard Steger; Volker Alfred ERDMANN
Original assignee: Heinrich-Heine-Universität Düsseldorf; Erdmann Technologies Gmbh
Priority date: 2015-05-22
Filing date: 2016-05-09
Publication date: 2016-12-01

Abstract

The present invention relates to a method for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins in a subject suffering from such a disease or disorder by lowering the concentration of the corresponding proteins in said subject by applying DNAzymes or/and RNA-DNAzymes in form of a pharmaceutical composition. The invention further relates to DNAzymes or/and RNA-DNAzymes for use in said methods.

Description

Methodi for the Therapy of Protein-Misfolding-Diseases

The present invention is in the area of medical therapy. In particular, a method is provided for the therapy of protein-misfolding diseases by lowering the concentration of the corresponding proteins in a subject suffering from such a disease by applying DNAzymes or RNA- DNAzymes, or combinations thereof, ia form of a pharmaceutical composition. The invention, further relates to DNAzymes or RNA-DNAzymes, or combinations thereof, for use in said methods.

It is known that specific RNAs (ribozymes) (Kruger et al. 1982, Guerrier-Takada et al. 1983) as well as DNAs (DNAzymes) (Santoro and Joyce, 1997) exist which hydrolyze RNA at specific sequences of nucleotides (Nts); these are 3 Nts (e.g. GUC) for ribozymes or 2 Nts (e.g. GU) for DNAzymes, respectively. Upstream and downstream of the cleavage-site short double-strands are formed between the target-R A and ribozyme or DNAzyme, respectively, by which only the GUC- or GU- sequences, respectively, with the corresponding flanking Nt- sequences are cleaved. Enzymatically active RNAs (ribozymes) exist in nature (Kruger et al. 1982, Guerrier-Takada et al. 1983, Haumann et al. 2012), which is not known for enzymatically active DNAs (DNAzymes).

In various embodiments of the present invention DNAzymes and/or RNA-DNAzymes (cf. below) are applied for use in the treatment of protein-misfolding diseases. DNAzymes and RNA-DNAzymes exert a superior haemolytic stability as compared to, for example, ribozymes.

Ribozymes cleave the target RNA with high activity 3 'of GUC and AUC but with lower activity also 3 'of UUC and CUC (Zoumadakis and Tabler 1995). The third nucleotide, i.e. the C, can be replaced by A or U but will lower the activity. Since the 5 nucleotide of the triplet is base paired to the 3 'nucleotide of the catalytically active core of the ribozyme (cf. seqOl), the CA at the 3 'end of seqOl has to be replaced by UA for the AUC triplet, by GA for the CUC triplet and by AA for the UUC triplet, respectively. DNAzymes and RNA-DNAzymes cleave the target MNA between a purine and a pyrrolidine nucleotide, particularly between G and U or between A and U or between G and C or between A and C, respectively. Cleavage occurs with highest activity between G and U and A and U, respectively, and with less activity between G and C and between A and C, respectively. Thus, for cleaving the target RNA between GU and AU, respectively, the 5 'nucleotide of the core sequence of the DNAzyme or the RNA-DNAzyme is an A, which forms a basepair to the U of the target sequence.

Correspondingly, if the target RNA is to be cleaved between GC and AC, respectively, the 5 ^'nucleotide of the core sequence of the DNAzyme or the RNA-DNAzyme has to be a G, or the modified nucleotide Inosine (I) (Caims et al. 2003). Use of Inosine leads to a higher cleaving activity of the DNAzyme or RNA-DNAzyme as compared to G.

The present invention shows a beneficial effect for therapies of protein-misfblding diseases (cf. below) when the concentration of proteins prone for misfolding is lowered. This can be achieved by taking advantage of the hydrolyzing activity of DNAzymes and/or RNA- DNAzymes, which can be used to lower the concentration/amount of corresponding messenger RNAs (mR As). The DNAzymes and/or RNA-DNAzymes align with the corresponding sequences of the mRNA to produce an intermediate, which is used to modulate the concentration of the protein of therapeutic interest.

Of particular interest within the scope of the present invention are proteins, which are prone for being misfolded and which can be knocked out by genetic ablation with little or no phenotypic consequences in the corresponding organism. Accordingly, adverse side effects are not to be expected, which makes these proteins particularly interesting for use in therapy. Diseases such as, for example, Huntington disease or some forms of ALS, where lowering the corresponding proteins huntingtin and TPD43, respectively, leads to serious or even lethal effects, are thus not comprised within the scope of the present invention.

The DNAzyme technology has never been used before to modulate the pathogenic action of misfolded pathogenic proteins through the herein suggested approach.

Misfolded proteins lead to serious even lethal diseases (Eisenberg and Jucker, 2012; Prusiner, 2012), like Alzheimer disease (AD, Masters et al. 1985), Parkinson disease (Luk et al. 2012), Creutzfeldt- Jakob disease (CJD, Hornlimann et al. 2007), Amyotrophic lateral sclerosis (ALS, Grad et al. 2011), Frontal temporal dementia (FID, Radcmakers and Hutton 2007), Tauopathy (Sydow and Mandelkow 2010), Down syndrome (Masters et al. 2011), Glaucoma, Diabetes and Fibrosis, all in humans. In animals, Scrapie in sheep and goat, bovine spongiforme encephalopathy (BSE) and Chronic Wasting disease (CWD) in deer are known (Homlimann et al. 2007). In all of these diseases, one or several specific host-encoded proteins are expressed in a native or a cellular, i.e. non-pathological isoform. However, these proteins have the tendency to switch their native conformation into a so-called misfolded isoform, which isoform in most cases shows a tendency for oligo- or multimerization and represents the highly pathological isoform. This switch in conformation can be induced either by infection, i.e. invading of an already misfolded protein from outside as is the case with prion diseases (scrapie, CWD, CJD), or sporadically, i.e. without known influence from outside but with increasing probability with age (e.g. AD), or by a genetic predisposition in the gene of the protein increasing the tendency to misfold (e.g. familial forms of CJD and AD). The term "protein-mi sfolding disease" is used in the literature not in a unique way. Since the concept of protein misfolding as the cause for diseases originated from the prion diseases (scrapie, BSE, CJD), also the terms "prion-like diseases" or "prion-diseases" in general are used. "Prion-diseases" in the narrow sense are used for diseases where the specific Prion Protein is misfolded, but the term "Prion-diseases" is also applied in the wider sense to diseases, where the concept of a host encoded protein switching from a cellular isoform into a pathological isoform can be applied. Prion-diseases in the original sense stand for infectious particles (Prusiner 1 82), in the wider sense for diseases induced by misfolded proteins.

For the Prion Protein it has been shown that it is presented via a glycolipid anchor on the cell surface and acts as a receptor for the misfolded Prion Protein (A. Salwiertz, 2009) and most probably also for other pathogenic misfolded proteins (Lauren, J., et al., 2009). Therefore, downregulation of the Prion Protein not only should prevent the pathogenic effect of misfolded Prion Protein but also of other misfolded pathogenic proteins, which bind to or associate with the Prion Protein receptor on the cell surface.

In most cases the cellular functions of those misfolded proteins are under debate or even not known, and as mentioned above transgenic animals with one or several of those proteins knocked out do show little or no phenotypic changes (Biieler et al. 1993). Most of the proteins have no enzymatic activity, but possibly might have regulatory functions which, however, are not essential for the organism. Since the pathological misfolding is in all known cases connected with oligomerization or multimerization of the protein, it is proposed herein, without however being bound thereto, that the misfolding and in consequence the disease can be prevented by lowering the concentration of the protein having the tendency to misfold. Consequently, by using the herein described approach, regulating down the concentration of misfolding proteins has the potential of preventing the disease without serious side effects. The therapeutic effects could be shown by gene therapy in mice, when the protein expression was regulated down by a tetracycline-depcndent promoter (Tremblay et al. 1998). This method, however, can as a matter of principle not be applied to humans.

Within the scope of the present invention the use of DNAzymes and/or RNA-DNAzymes are described for lowering the concentration of proteins, which have a tendency for misfblding but show substantially no phenotypic effects after gene ablation (cf. above) and thus substantially no negative side effects. Diseases associated with these mis folding or misfolded proteins can therefore be prevented or cured by the methods disclosed herein.

Known prior art approaches focus on the use for overexpressed, misregulated or mutated proteins. Misfolding is different from overexpressing and misregulation, because in case of protein-misfolding diseases, the diseased organisms have the same or very similar amounts of expression of the misfolding proteins as healthy organisms. Misfolding is a consequence of infection, deficiency of clearance or just age and sporadic events. In some cases mutations in the gene of a protein are involved, which lead to a tendency to misfoid, whereas mutations in proteins described in other diseases lead to dysfunction or loss of function of the mutated protein.

Art-kno applications focus on proteins with catalytic activity (enzymes), carrier proteins (e.g. Hemoglobin for oxygen transport) or structure proteins (e.g. actin, collagen), which are caused by or induced by overexpressing, or underexpressing of a gene or by expressing a mutated protein.. In contrast, the application to misfolding proteins according to the present invention is independent of over- or underexpression.

Nearly all therapeutic approaches are directed against the protein target. Several attempts against the mRNA have been reported, but they have been carried out with chemically modified nucleic acids and work in the mode of antisense-, small interfering- or micro-nucleic acids. Because of the chemical modifications they often have unfavorable features that can lead to serious side-effects. The cleavage activity of ribozymes and DNAzymes can also be applied to inactivate cellular RNA and foreign RNA, i.e. those from infectious agents. In a series of recent articles (Watt and Corey 2012; Burnett and Rossi 2012; Hausseckcr 2012; Lightfoot and Hall 2012; Sharma et al. 2014; Fokina et al. 2015) the application of nucleic acids as therapeutic agents is reviewed, mostly to treat cancer and infectious diseases.

It was surprisingly found with the present invention that DNAzyme-techniques and RNA™ DNAzyme techniques can be applied to misfolded proteins as disclosed herein for prevention or treatment of diseases associated with misfolding and/or misfolded proteins.

The performance of the DNAzymes or the RNA-DNAzymes according to the present invention for use in animal or human therapy may be further improved by (a) stabilizing the DNAzymcs or the RNA-DNAzymes, for example by chemically modifying one or more of the nucleotides of the DNAzymcs or the RNA-DNAzymes using art-known, techniques, to obtain, for example, thiophosphate nucleotides, morpholinonucleotides, linked nucleotides, etc. or by (b) enhancing the penetration of the cell membrane and the blood-brain-barri er, for example by using the catalytic nucleic acids in combination with membrane- transferring oligonucleotides, peptides or vesicle based systems. Said systems or technologies have been described in the literature (cf. Watt and Corey 2012; Burnett and Rossi 2012; Haussecker 2012; Lightfoot and Hall 2012; Sharma et al. 2014; Fokina et al. 2015).

A therapeutic intervention for targeting a misfolded protein and/or a protein having a tendency to misfold may be approached in different ways within the scope of the present invention:

1) Prophylactic approach: The concentration of a protein with the tendency to misfold, but which is not yet misfolded, is kept low to prevent its misfolding, and prevent occurrence o a disease.

2) Halting approach: A protein with the tendency to misfold has already started to misfold, but the concentration of the misfolded protein is still low. The concentration of misfolded protein is kept low to stop the occurrence of a disease associated with the pathological misfolded protein.

3) Treatment approach: High concentration of a misfolded protein is lowered by clearance effects when the production of new protein is blocked.

The present invention provides methods for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolding and/or misfolded pathogenic proteins in a subject suffering from such a disease or disorder by lowering the concentration of a protein, which can switch its native conformation to a misfolded pathogenic isoform (misfolding protein) or of a protein, which is already present in a misfolded pathogenic isoform (misfolded protein) in the subject to be treated. The present invention further provides catalytic polynucleotides, which are capable of cleaving a target mRNA of such a misfolding protein or misfolded protein, and a pharmaceutical compositions comprising said catalytic polynucleotides for use in such methods.

The catalytic polynucleotide may be a ribozyme and/or a DNAzyme and/or a RNA- DNAzymc, or a combination thereof, but in particular a DNAzyme or a RNA-DNAzyme, or a combination thereof.

The ribozyme may comprise a hammerhead motif, or a hairpin, hepatitis delta virus, group 1 intron, Neurospora VS RNA or RNaseP RNA motif. In particular, the present invention relates to a catalytic polynucleotide selected from the group of RNA-DNAzyme and DNAzyme capable of cleaving a target mR A of a protein, which can switch its native conformation to a misfolded pathogenic isoform (misfolding protein) or is present in a misfolded pathogenic isoform (misfolded protein), for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded or misfolding pathogenic proteins (protein-misfolding diseases) in a subject suffering from such a disease or disorder to lower the concentration of said protein in the subject.

The subject to be treated with the catalytic polynucleotides in the method according to the invention may be an animal or a human.

The misfolding protein or misfolded protein may be a protein, which is prone for being misfolded and show substantially no changes or alterations of the phenotype in the corresponding organism when modified by the catalytic polynucleotide according to the present invention, including, without being limited thereto, changes in the day- night-rhythm, changes in the action potential, etc. Accordingly, adverse side effects are not to be expected, which makes these proteins particularly interesting for use in therapy

In particular, the misfolding protein or misfolded protein may be a protein selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, Tau protein, superoxide dismutase 1 (SOD) protein, and insulin.

The disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins than can be treated by the method according to the present invention is, in various embodiments of the invention, a disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins binding to the prion receptor on the cell surface.

In a specific embodiment of the invention, the disease, condition or disorder is selected from the group consisting of Creutzfeldt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

The catalytic polynucleotide for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded and/or misfolding pathogenic proteins in a subject suffering from such a disease or disorder is, in a specific embodiment of the invention, a DNAzyme comprising a catalytic core region comprising the catalytic domain, flanked 3^* and/or 5' by a flanking region comprising antiseese deoxyribonucleotide sequencers) which is (are) complementary to the target-mRNA sequence flanking the cleaving site, particularly the dinucleotide cleavage site, on the target mRNA and bind to the target RNA molecule.

In a specific embodiment of the invention, at least one of the deoxyribonucleotides of the core region, particularly the 5' deoxyribonucleotide of the core region, binds to the pyrimidine nucleotide Y of the dinucleotide cleavage site XY on the target RNA, whereas the purine nucleotide X remains unbound.

Accordingly, in one embodiment the dinucleotide cleavage site on the target RNA is XY, wherein X=G or A and Y=U or C, and the at least one deoxyribonucleotide of the core region, particularly the 5' deoxyribonucleotide of the core region binding to the pyrimidine nucleotide Y of the dinucleotide cleavage site on the target RNA, is A or G.

In a specific embodiment of the invention, the cleavage site is characterized by a trinucleotide selected from the group consisting of CGU-, AGU-, UGU.

The 3^* and 5^! flanking region comprises deoxyribonucleotides complementary to the nucleotides 5' and 3' of the dinucleotide cleavage site on the target RNA as defined above. In various embodiments of the invention, the catalytic polynucleotide is a DNAzyme, wherein the catalytic core region of the DNAzyme has between 2 and 100 deoxyribonucleotides, particularly between 8 and 50 deoxyribonucleotides, particularly between 10 and 20 deoxyribonucleotides, particularly between 13 and 19 deoxyribonucleotides, particularly 15- 17 deoxyribonucleotides, particularly 16 deoxyribonucleotides.

Each of the flanking regions 3^* and/or 5^* of the catalytic core region has an anti sense sequence comprising between 2 and 100 deoxyribonucleotides, particularly between 2 and 24 deoxyribonucleotides, particularly between 2 and 12 deoxyribonucleotides, particularly between 8 and 12 deoxyribonucleotides complementary to the target-mRNA.

In a specific embodiment of the invention, the catalytic core region of the DNAzyme of the present invention has 16 deoxyribonucleotides with the 5 ' deoxyribonucleotide binding to the pyrimidine Y of the XY dinucleotide cleavage site on the target RNA, and each flanking region 3' and/or 5^* of the catalytic core region has between 8 and 12 deoxyribonucleotides, which are complementary to the nucleotides 5' and 3' of the cleavage site, particularly the dinucleotide cleavage site, on the target mRNA, and bind to the target RNA molecule..

In another specific embodiment, the invention provides a RNA-DNAzyme, particularly a RNA-DNAzyme for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded and/or misfblding pathogenic proteins in a subject suffering from such a disease or disorder, wherein said RNA-DNAzyme comprises a core region of d coxyribonucleotides comprising the catalytic domain flanked 3' and/or 5' by a ribonucleotide anti sense sequence(s), which is (are) complementary to the target-mKNA sequence flanking the cleavage site, particularly the dinucleotide cleavage site, on the target mRNA, and bind to the target RNA molecule.

The RNA-DNAzyme comprises the core region of the DNAzymes as disclosed herein above, flanked 3' and/or 5^* by anti sense ribonucleotide sequences) which are complementary to the target-mRNA sequence flanking the cleavage site, particularly the dinucleotide cleavage site, on the target mRNA, and bind to the target RNA molecule. Because RNA-DNAzymes form RNA-RNA double strands with the target mRNA they have the potential of competing more effectively with the internal structure of the mRNA, i.e forming RNA-RNA double strands, whereas DNAzymes form less stable DNA-RNA double strands with the target mRNA.

In a specific embodiment of the invention, at least one of the deoxynbonucleotides of the core region, particularly the 5' deoxyribonucleotide of the core region, binds to the pyrimidine nucleotide Y of the dinucleotide cleavage site XY on the target RNA, whereas the purine nucleotide X remains unbound.

Accordingly, in one embodiment the cleavage site on the target RNA is XY, wherein X=G or A and Y=U or C, and the at least one deoxyribonucleotide of the core region, particularly the

5" deoxyribonucleotide of the core region binds to the pyrimidine nucleotide Y of the dinucleotide cleavage site on the target RNA is A or G.

In particular, the pyrimidine nucleotide Y of the dinucleotide cleavage site is U or C, and the purine nucleotide X is G or A, wherein a RNA-DNAzyme targeting the purine/pyrimidine pairs GU and AU is preferred.

In various embodiments of the invention, the catalytic core region of the RNA-DNAzyme of the present invention has between 2 and 100 deoxyribonucleo ides, particularly between 8 and 50 deoxyribonucleotides, particularly between 10 and 20 deoxyribonucleotides, particularly between 13 and 19 deoxyribonucleotides, particularly 15-17 deoxyribonucleotides , particularly 16 deoxyribonucleotides.

Each of the flanking regions 3' and/or 5^* of the catalytic core region has an anti sense sequence comprising between 2 and 100 ribonucleotides, particularly between 2 and 24 ribonucleotides, particularly between 2 and 12 ribonucleotides, particularly between 8 and 10 ribonucleotides, particularly between 6 and 8 ribonucleotides, complementary to the corresponding sequences 5' and 3' of the cleavage site, particularly the dinucleotide cleavage site, on the target-mR A, and bind to the target R A molecule.

In a specific embodiment of the invention, the catalytic core region of the RNA-DNAzyme of the present invention has 16 deoxyribonucleoudes and each flanking region 3' and/or 5' of the catalytic core region has between 6 and 8 ribonucleotides.

The 3 ^s and 5' flanking region comprises ribonucleotides complementary to the nucleotides 5' and 3^* of the cleavage site, particularly the dinucleotide clea age site, on the target-mRNA as defined above, and bind to the target RNA molecule..

At least one of the nucleotides of the flanking region, particularly at least the nucleotide of the 3' flanking region, which is 3' of the catalytic core region may be a deoxyribonucleotide, which is variable and binds to the corresponding nucleotide 5' of the dinucleotide XY cleavage site on the target RNA.

Accordingly, the target mRNA may be cleaved by the DNAzyme and/or the RNA-DNAzyme of the invention between nucleotides X (purine) and Y (pyrimidine), wherein X=G or A and

Y=U or C,

In a specific embodiment of the invention, the target mRNA is cleaved between nucleotides G and U.

In another specific embodiment of the invention, the target mRNA is cleaved between nucleotides A and U.

In another specific embodiment of the invention, the target mRNA is cleaved between nucleotides G and C.

In another specific embodiment of the invention, the target mRNA is cleaved between nucleotides A and C.

In another specific embodiment of the invention, the cleavage site is characterized by a trinucleotide selected from the group consisting of CGU-, AGU-, UGU, and the target mRNA is cleaved between nucleotides CG and U, AG and U, or UG and U.

in a specific embodiment, the invention provides a a catalytic polynucleotide, particularly a DNAzyme and/or a RNA-DNAzyme or a combination thereof, for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded and/or misfolding pathogenic proteins in a subject suffering from such a disease or disorder, wherein said catalytic polynucleotide cleaves one or all of the above dinucleotide sequences. In one embodiment, the invention provides a DNAzyme as disclosed herein in the various embodiments, wherein the catalytic core region has

i) the sequence shown ia SEQ ID NO: 2; and/or

ii) the sequence shown in SEQ ID NO: 29 and/or

iii) the sequence shown, in SEQ ID NO: 30;

iv) a sequence having between 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 2, 29 and 30;

v) which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 2, 29 and 30..

In another specific embodiment, the invention provides a RNA-DNAzyme, particularly a RNA-DNAzyme for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded and/or misfolding pathogenic proteins in a subject suffering from such a disease or disorder, wherein said RNA-DNAzyme cleaves one or all of the above dinucleotide sequences.

In one embodiment, the invention provides a RNA-DNAzyme as disclosed herein in the various embodiments, wherein the catalytic core region has the sequence shown in

i) SEQ ID NO: 2; and/or

ii) SEQ ID NO: 29 and/or

iii) SEQ ID NO: 30,

iv) a sequence having between 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 2, 29 and 30

v) which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 2, 29 and 30.

particularly for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded and/or misfolding pathogenic proteins in a subject suffering from such a disease or disorder.

In another embodiment, the invention provides a catalytic polynucleotide, particularly a DNAzyme and/or a RNA-DNAzyme or a combination thereof, particularly for use in the method according to the present invention as disclosed herein in the various embodiments for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfoided and/or misfolding pathogenic proteins in a subject suffering from such a disease or disorder, wherein said catalytic polynucleotide recognizes the CGU, AGU, and UGU trinucleotide cleavage sites and cleaves the target RNA between nucleotides CG and U, AG and U, or IJG and U and wherein the flanking regions 3' and/or 5' of the catalytic core region of the catalytic polynucleotide comprises deoxyribonucleotides or ribonucleotides, which are in antisense orientation to the sequences 5' and 3 ' to the CGU. AGU. and UGU cleavage sites on the following sequences:

2144-CUCAUAAUUGUCAAAAACC 2162

1 138-GGUCUUCCUGUUUUCACCA 1 156

2682-AAAAAAAUUGUAAAUGUUU-2700

1364 CCAGUAAAAGUAUAACAGC 1382

1 O-AUUUUUACAGUCAAUGAGC-28

1289- AAACAUAGAGUAGACCUGA 1307

1893 - AUUUUAG AUGUUU AAAGG A- 1911

1761 -GUAAAUAUUGUC AC AAC AC- 1779

2094 - AAAUGGUCAGUGUGC AAAG-21 12

2264-UGAUlJUGAAGUGGAAAAAG— 2282

2297-UUAAUUAAAGUAAAAUUAU— 2315

1957-AGCUGAAAAGUAAAUUGCC-l 975

2327 UUUGAUAUUGUCACCUAGC -2345

1752 UCAACAAGAGUAAAUAUUG- 1770

2688 -AUUGUAAAUG U UU AAUAUC 2706

2133 - UUUAUUUCUGUCUCAUAAU- -2151

1995-AUCUCCUUUGUCCAUUUAC--2013

829 ACAUGCACCGUUACCCCAA 847

1234-UCUCUCUUUGUCCCGGAUA- 1252

2395 UAUUCUAUGUAAAAAUAU- 2413

1080-UCUCCACCUGUGAUCaJCC-l 098

2386-ACUUUGUGAGUAUUCUAUG-2 04

2666-UCUUGUUUUGUUAUAUAAA-2684

670 ACAAGCCGAGUAAGCCAAA 688 2287--UCIJGUUAAUGUUAAIJUAAA-2305 2621 -AAUAUGCAUGUACUUUAUA 2639

88-L UCAACUCGUbTJUUUCCG-l 06 13 S 8- OCC AGGCC AG I AAA AGU AU- 1376 2661-CAUGUUCUUGUUUUGUUAU-2679 2047-GCUGCAGUUGUGAAAGCAC-2065 2167-UTJAGGUC AAGUUC AU AGUU-2185 656 CC AC AG U C AGUG G AAC A AG - 674 2317--CCCUGAAUUGUUUGALJALiU-2335

66- CLO AUGC AAGJLJGliUC AAGC - 84 100 - UG AGC AG AUGUGU AUC ACQ 1019 1502-UAGAGCUC AGUAUACUAAU- 1520 2461 -UUUAGAGCAGUU AACAUCU- 2479 2643 -UCmUAUUUGUAACUUUGC-2661 1812-AACAUAACUGUAACAUAUA- 1830 1326- UUUGAUUGAGUUCAUCAUG -l 344 1413-UIJGG ACU JAGUGC AAC AGG- 1431 2281 - AGAAAUUCUG JUAAUGUUA- 2299 96» GAGACCGACGUUAAGAUGA-987 1911 ACCCU AU AUGUGGC AUUCC- 1 29 2382HU JGCACUUUGUGAGUAUUC-2400 2518-ACUUAAUAUGUGGGAAACC-2536 2574 - UCGUXJUC AUGU AAG AAUCC-2592 412- CC ACAIJGG AGUG ACCUGGG -430 814-GUU ACUAUCGUGAAAAC AU-832 866-CAUGGAUGAGUACAGCAAC-884 1721 - UUUUCACAGUAUGGGCUA 1739 2474- AC AUCUGAAGUGUCU AAUG-2492 2535 CCCUUUUGCGUGGUCCUUA-2553 805 -AUG AGG ACCGUU ACU AUCG-823 2174-AAGlJUC AUAGIJIJ UCUGUAA ^~21 2 2567 CACUGAAUCGUUUCAUGUA-2585 843 -CCCAACC AAGUGUACUAC A-861 79O-AUUUCGGCAGUGACUAUGA-808

2495-UUAACUUUUGUAAGGUACU-2513

1706- UGGGGUGAUGLIJUUACIJUU 1724

180O-AUAU JCAC AGUGAAC AUAA- 1818

888- AACAACUUUGUGCACGACU- -906

1013-UAUCACCCAGUACGAGAGG- 1031

900-C ACG ACUGCGUC AAUAUC A-918

2044- AGAGCUGCAGUUGUGAAAG-2062

2555-GCUIJAC AAUGUGC ACUGAA-2573

1682-AUGAGCUCUGUGUGUACCG-l 700

1686 GCUCUGUGUGUACCGAGAA 1704

2343- AGCAGAUAUGUAUUACUUU-2361

3 9-GUUCUCUUUGUGGCCACAU-417

In a specific embodiment of the invention, the nucleotide of the 3* flanking region, which is 3' of the catalytic core region of the DNAzyme or the RNA-DNAzyme is a deoxynocleotide, which is complementary to and hinds to the 5' ribonucleotide of the trinucleotide cleaving site on the target RNA.

In another embodiment, the invention provides a catalytic polynucleotide, particularly a DNAzyme, for use in the method according to the invention as disclosed herein in the various embodiments, wherein the

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 5;

(ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 8;

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 11 ;

(iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 15;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 17;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 20 or SEQ ID NO: 22;

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 24;

(viii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 32;

(ix) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 34;

(x) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 36;

(xi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 38; (xii) catalytic polynucleotide has a sequence, which shows between 90%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to my one of the sequences of SEQ ID NOs: 5, 8, 1 1, 13, 15, 17, 20, 22_» 24_» 32, 34, 36, 38;

(xiii) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 5, 8, 11, 13, 15, 17, 20, 22, 24, 32, 34, 36, 38.

hi another anbodiment, the invention provides a catalytic polynucleotide, particularly a DNAzyme, for use in the method according to the invention as disclosed herein in the various embodiments, wherein the

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 5 and the target sequence is the sequence shown in SEQ ID NO: 3;

(ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 8 and the target sequence is the sequence shown in SEQ ID NO: 6;

(iii) catalytic polyEUcleoti.de has the sequence as shown in SEQ ID NO: 11 and the target sequence is the sequence shown in SEQ ID NO: 9;

(iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 15 and the target sequence is the sequence shown in SEQ ID NO: 12;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 17 and the target sequence is the sequence shown in SEQ ID NO: 16;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 20 or SEQ ID NO: 22 and the target sequence is the sequence shown in SEQ ID NO: 19;

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 24 and the target sequence is the sequence shown in SEQ ID NO: 23;

(viii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 32 and the target sequence is the sequence shown in SEQ ID NO: 31 ;

(ix) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 34 and the target sequence is the sequence shown in SEQ ID NO: 33;

(x) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 36 and the target sequence is the sequence shown in SEQ ID NO: 35;

(xi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 38 and the target sequence is the sequence shown in SEQ ID NO: 37;

(xii) catalytic polynucleotide has a sequence, which shows between 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 5, 8, 11, 13, 15, 17, 20, 22, 24, 32, 34, 36, 38 and/or the corresponding target sequence has a sequence, which shows between 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 3, 6, 9, 12, 16, 19, 23, 31, 33, 35, 37;

(xiii) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 5, 8, 11, 13, 15, 17, 20, 22, 24, 32, 34, 36, 38 and/or the corresponding target sequence has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 3, 6, 9, 12, 16, 19, 23, 31, 33, 35, 37

In another embodiment, the invention provides a catalytic polynucleotide, particularly a RNA-DNAzyme, particularly for use in the method according to the invention as disclosed herein in the various embodiments, wherein the

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 39;

(ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 40;

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 42;

(iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 43;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 44;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 45

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 46;

(viii) catalytic polynucleotide has a sequence, which shows between 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46;

(ix) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 39, 0, 42, 43, 44, 45, 46.

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 39 and the target sequence is the sequence shown in SEQ ID NO: 16; (ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 40 and the target sequence is the sequence shown in SEQ ID NO; 16;

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 42 and the target sequence is the sequence shown in SEQ ID NO: 12;

(iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 43 and the target sequence is the sequence shown in SEQ ID NO: 12;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 44 and the target sequence is the sequence shown in SEQ ID NO: 19;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 45 and the target sequence is the sequence shown in SEQ ID NO: 19;

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 46 and the target sequence is the sequence shown in SEQ ID NO: 23;

(viii) catalytic polynucleotide has a sequence, which shows between 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46 and/or the corresponding target sequence has a sequence, which shows between 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 16, 12, 19, 23;

(ix) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46 and/or the corresponding target sequence has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 16, 12, 19, 23.

Further disclosed herein is a ribozyme for use in the method disclosed herein in the various embodiments, wherein said ribozyme. comprising a catalytic core region flanked 5' and/or 3' by antiseese sequence(s) which are complementary to the target-mRNA sequence flanking the cleaving site on the target mRNA.

In particular, the anti sense sequence 5^* and/or 3^* of the catalytic core region comprises between 2 and 100 ribonucleotides, particularly between 2 and 24 ribonucleotides, particularly between 2 and 12 ribonucleotides complementary to the target-mRNA.

The target mRNA is cleaved 3' ofthe triplet XUY, with X= G, A U or C and Y=C, A or U.

In particular, the target mRNA is cleaved 3' of the triplet GUC, GUA or GUU.

In particular, the target mRNA is cleaved 3' of the triplet (i) AUC, AUA or AUU and have at its 3 'site a U to match the target triplets AUC, AUA and AUU; or

(ii) CUC_? CUA or CUU and have at its 3 'site a G to match the target triplets CUC, CUA and CUU; or

(iii) UUC, UUA or UUU and have at its 3 'site an A to match the target triplets UUC- UUA and UUU.

In a specific aspect, a ribozyme is disclosed for use in the method as disclosed herein in the various embodiments, wherein said ribozyme cleaves one or ail of the above triplet sequences.

The ribozyme for use in the method as disclosed herein in the various embodiments may have the catalytic core region with lie sequence shown in

SEQ ID NO: 1; and/or

SEQ ID NO: 26; and/or

SEQ ID NO: 27; and/or

SEQ ID NO: 28.

In particular disclosed is a catalytic polynucleotide, which has the sequence as shown in SEQ ID NO: 4, SEQ ID NO: 7_» and SEQ ID NO: 10.

The present invention further provides a pharmaceutical composition comprising one or more of the catalytic polynucleotides defined herein in any one of the embodiment disclosed herein for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolding and/or misfolded pathogenic proteins in a subject suffering from such a disease or disorder to lower the concentration of said protein in the subject.

In one embodiment, the invention provides a pharmaceutical composition for use in the method according to the invention, comprisin one or more DNAzymes or one or more RNA- DNAzymes as defined in any one of the embodiments disclosed herein or a combination thereof.

In one embodiment, the invention provides a pharmaceutical composition for use in the method according to the invention as described herein in the various embodiments for the treatment of an animal.

In another embodiment, the invention provides a pharmaceutical composition for use in the method according to the invention as described herein in the various embodiments for the treatment of a human. The pharmaceutical composition as defined herein in the various embodiments may be used for lowering the concentration, of a protein which can switch its native conformation to a misfolded pathogenic isoform (misfolding protein) or a protein which is present in a misfolded pathogenic isoform (misfolded protein), in a subject suffering from a disease, condition or disorder associated with misfolded or misfolding pathogenic proteins.

In particular, the pharmaceutical composition may be used for lowering the concentration of a protein, which is prone for being misfolded (misfolding protein) or a protein which is present in a misfolded pathogenic isoform (misfolded protein) and shows little or no changes or alterations of the phenotype in the subject when treated with the catalytic polynucleotide according to the present invention, including, without being limited thereto, changes in the day- night-rhythm, changes in the action potential, etc.

In particular, said protein is, in a specific embodiment, selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, tau protein, superoxide dismutase 1 (SOD) protein, and insulin.

The disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins may be a disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins binding to the prion receptor on the cell surface.

In particular, the disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins may be selected from the group consisting of Creutzfeldt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FID), Tenopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

In a specific embodiment, the pharmaceutical composition for use in the method according to the invention as disclosed herein in the various embodiment comprises the catalytic polynucleotide, particularly a catalytic DNAzyme or a catalytic RNA-DNAzyme, or a combination thereof, in a therapeutically effective amount, particularly together with a pharmaceutically acceptable carrier and/or excipient.

I one embodiment, the catalytic polynucleotide, particularly the catalytic DNAzyme or the catalytic RNA-DNAzyme, or a combination thereof, or the pharmaceutical composition comprising said catalytic polynucleotide's) as described herein may be used in the method according to the invention as disclosed herein in the various embodiments for cleaving the mRNA of i) prion protein;

ii) tan protein;

iii) alpha-synuclein protein;

iv) amyloid precursor protein (APP);

v) superoxide dismutase 1 (SOD) protein; and/or

vi) a misfolding or misfolded protein binding to the prion receptor on the eel! surface.

The present invention further relates to a method for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded or misfolding pathogenic proteins comprising administering a catalytic polynucleotide or the pharmaceutical composition as defined herein in any of the various embodiments to a subject suffering from such a disease or disorder to lower the concentration of said protein in the subject.

The catalytic polynucleotide may be a ribozyme and/or a DNAzyme and/or a RNA- DNAzyme, but particularly a DNAzyme and/or a RNA-DNAzyme, and the pharmaceutical composition may comprise said ribozyme and/or a DNAzyme and/or RNA-DNAzyme, or a combination thereof, but particularly a DNAzyme and/or a RNA-DNAzyme or a combination thereof.

The subject to be treated with the catalytic polypeptides in the method according to the invention may be an animal or a human.

The misfolding protein or misfolded protein may be a protein which shows little or no changes or alterations of the phenotype in the corresponding organism when modified by the catalytic polynucleotide according to the present invention, including, without being limited thereto, changes in the day- night-rhythm, changes in the action potential, etc.

The disease, condition or disorder than can be treated by the method according to the present invention is any disease, condition or disorder that is associated with misfolding pathogenic proteins or misfolded pathogenic proteins, particularly with misfolding pathogenic proteins or misfolded pathogenic proteins binding to the prion receptor on the cell surface. In particular, the disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins may be, in a specific embodiment of the invention, a disease, condition or disorder selected from the group consisting of Creutzfeldt- J akob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

Further disclosed herein is a method for producing of a pharmaceutical composition which comprises ribozymes which are capable of cleaving a given sequence of a cellular RNA, in particular containing the triplet GUC and others with U in the middle position (AUG, UUC, CUC, GUA, AUA, UUA, CUA, GUU, AUU, UUU, CUU) with otherwise any sequences attached upstream and downstream of the triplet, and wherein the ribozyme is intended for administration in a pharmacologically effective dose. Typically, the ribozyme is mixed with pharmaceutical excipients and/or carriers.

in various embodiments, the invention relates to a method for producing of a pharmaceutical composition which comprises DNAzymes and/or RNA-DNAzymes or a combination thereof, which are capable of cleaving a given sequence of a cellular RNA in particular containing the duplet GU or AU or with less activity GC and AC with C, A or U at the 5 'site of the said duplets and otherwise any sequences attached upstream and downstream of the duplet, and wherein the DNAzyme and/or the RNA-DNAzyme or a combination thereof is intended for administration in a pharmacologically effective dose. Typically, the DNAzyme and/or the RNA-DNAzyme or a combination thereof is mixed with pharmaceutical excipients and/or carriers.

The invention relates to a use of (or method of using) catalytic polynucleotides as described herein, particularly DNAzymes and/or the RNA-DNAzymes or a combination thereof to lower the concentration of proteins which have a tendency for misfolding or to lower the concentration of already misfolded proteins.

In a specific embodiment, the invention, relates to a method for selection of GU-, AU-, GC-, AC- sequences, particularly U, CGU, AGU, UGU, respectively, by applying Sequential- Folding-Algorithms (Bernhart et al. 2006; Tafer et a). 2008) in order to predict the accessibility for DNAzymes and/or the RNA-DNAzymes, respectively, during the synthesis of the corresponding mRNA, for the use to lower the concentration of proteins which have a tendency for misfolding or to lower the concentration of already misfolded proteins. Further disclosed herein is a method for selection of GUC-, AUC-, UUC-, CUC-, GUA-, AUA-, UUA-, CUA-, GUU-, AUU-, UUU-, CUU triplets.

The pharmaceutical composition according to the invention can be further optimized ^' for example to stabilize the catalytic polynucleotides as described herein, particularly DNAzymes and/or the RN A-DN Azymes by chemically modified nucleotides and to enhance the penetration of the cell membrane and the blood-brain-barrier by using catalytic polynucleotides as described herein, particularly DNAzymes and or the RNA-DNAzymes in combination with membrane-transfcmng oligonucleotides, peptides or vesicle based systems. Vesicle based systems can be for example lipid vesicles or virus-like protein capsids.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES FIGURES

Figure 1 :

Gel electrophoretic analysis of the cleavage activity of three Ribozymes Rzl, Rz2 and Rz3, respectively, cleaving target segments of the mRNA of the murine Prion Protein. The target fragments are fluorescence labeled at their 5 'end. Slots 1 , 4, 7 are controls containing the target fragments only; slots 2, 5, 8, contain 10-fold molar excess of the corresponding ribozymes arid slots 3,6,9 a 100-fold molar excess of the ribozymes.

The Ribozymes consist of the core sequence (24 ribonucleotides):

5 CUGAUGAGGCCG AAAGGCCGAAAC3 ' (SEQ ID NO: 1) and the 5 'and 3 'flanking sequences specific for targets Tl, T2 and T3, respectively.

Targetl : 5 TLG AAGGAGUCCCAGG3 ' (SEQ ID NO: 3)

Ribozymel (Rzl): 5TCUGGCUGAUGAGGCCGAAAGGCCGAAACUCCU3 (SEQ ID NO: 4)

Target2: 5 'FIXUGUUUGUCCCCCA3 ^'(SEQ ID NO: 6)

Ribozyme2 (Rz2): 5 'UGGGGCUGAUGAGGCCGAAAGGCCGAAACAAAC3 ' (SEQ ID NO: 7)

Target3: 5 FLCACACAGUCACCAC3 (SEQ ID NO: 9)

RibozymeS (Rz3): 5 GUCGUCUGAUGAGGCCGAAAGGCCGAAACUGUC3 ^' (SEQ ID NO: 10) Bfi_ffeJ:

Gel electrophoretic analysis of the cleavage activity of the DNAzymes Dzl, Dz2, Dz3, respectively, cleaving the identical target segments as in Fig, 1 , The target fragments are fluorescence labeled at their 5 'ends. Slots 1, 4, 7 are controls containing the target fragments only; slots 2, 5, 8, contain a 10-fold molar excess of the DNAzymes and slots 3, 6, a 100- fold excess of the DNAzymes.

The DNAzymes consist of the core-sequence (15/16 deoxyribonucleotides):

5 ' AGGCTAGCTACAACGA3 ' (SEQ ID NO: 2) and the 5 'and 3 'flanking sequences specific for targets Tl, T2 and T3, respectively.

Targetl : 5 'FLGAAGGAGUCCCAGG3 ^'(SEQ ID NO: 3)

DNAzyme! (Dzl): 5 'CTGGGAGGCTAGCTACAACGATCCTTC3 '(SEQ ID NO: 5)

Target2: 5 'FLCUGUUUGUCCCCCA3 (SEQ ID NO: 6)

DNAzyme2 (Dz2): 5 'GGGGGAGGCTAGCTAC AACG AAAACAG3 '(SEQ ID NO: 8)

Targets: 5 ' FLC AC AC AGUC ACC AC3 (SEQ ID NO: 9)

DNAzyme3 (Dz3): 5 TGGTGAGGCTAGCTACAACG ATGTGTG3 '(SEQ ID NO: 11) Figure 3:

Visual representation of the target fragments of the mRNA of the human Prion Protein in complexes with their corresponding DNAzymes. The cleavage sites are designated by black arrows, the numbers designate the cleavage site in the numbering of the mRNA., the pairs of numbers, i.e. 9/9, designate the numbers of base pairs in the double stranded region. Target T 736 was also combined with DNAzyme 733 and vice versa because of the close neighborhood of the two cleavage sites. The sequence numbers are allocated to the target fragments and the DNAzymes: T736 (SEQ ID NO: 12), T839 (SEQ ID NO: 16), T824: (SEQ ID NO: 19), T733 (SEQ ID NO: 23), Dz736 (SEQ ID NO: 13), Dz736 inv (SEQ ID NO: 14), Dz839 (SEQ ID NO: 17), Dz839 inv (SEQ ID NO: 18), Dz824 (SEQ ID NO: 20), Dz824 inv (SEQ ID NO: 21), Dz733 (SEQ ID NO: 24) and Dz733 inv (SEQ ID NO: 25).

Figure 4:

Gel electrophoretic analysis of the cleavage activity of the DNAzymes Dz736, Dz839, Dz824, Dz773 cleaving the corresponding target fragments of the mRNA of the human Prion Protein. The complexes of the DNAzymes with their corresponding target fragments are presented in Fig.3. DNAzymes designated as„inv" contain the catalytic core in inverted direction of the nucleotide sequence. They serve as controls but show in some cases activity. Figure 5;

Gel electrophoretic analysis of the cleavage activity of D Azyme Dz736 applied to all four target fragments of the mRNA of the human Prion Protein, The specific combinations, i.e. Dz736 with T736 (slot 2), Dz839 with T839 (slot 5), Dz824 with T824 (slot 9), and Dz733 with T733 (slot 13), are shown as positive controls. No cleavage activity can be detected for non-corresponding combinations of Dz736, i.e. with T839 (slot 6), T824 {slot 19); cleavage of Dz736 with T733 as expected (Fig. 3). Incubation of target molecules in the absence of

DNAzymes (slots 1 , 4, 8, 12) serve as negative controls. DNAzymes designated as "inv" (slots 3, 7, 11 , 15) contain the catalytic core in inverted direction of the nucleotide sequence. They serve as additional controls.

Figure 6:

Gel electrophoretic analysis of the cleavage activity of DNAzyme Dz839 applied to all four target fragments of the mRNA of the human Prion Protein. The specific combinations, i.e. Dz736 and T736 (slot 2), Dz839 and T839 (slot 6), Dz824 and T824 (slot 9), and Dz733 and

T733 (slot 13) are shown as positive controls. No cleavage activity can be detected for non- corresponding combinations of Dz839, i.e. with T736 (slot 3), T824 (slot 10), T733 (slot 14). Incubation of target molecules in the absence of DNAzymes (slots 1 , 5, 8, 12) serve as negative controls. DNAzymes designated as "inv" (slots 4, 7, 1 1 , 15) contain the catalytic core in inverted direction of the nucleotide sequence. They serve as additional controls but show in the case of Dz839inv activity.

Figure 7:

Gel electrophoretic analysis of the cleavage activity of DNAzyme Dz824 applied to all four target fragments of the mRNA of the human Prion Protein. The specific combinations, i.e. Dz736 and T736 (slot 2), Dz839 and T839 (slot 6), Dz824 and T824 (slot 10) and Dz733 and

T733 (slot 13) are shown as positive controls. No cleavage activity could be detected for non- corresponding combinations of Dz824, i.e. with T736 (slot 3), T839 (slot 7), T733 (slot 14). Incubation of target molecules in the absence of DNAzymes (slots 1 _» 5, 9, 12) serve as negative controls. DNAzymes designated as "inv" (slots 4, 8, 1 1 , 15) contain the catalytic core in inverted direction of the nucleotide sequence. They serve as additional controls.

Figure 8:

Gel electrophoretic analysis of the cleavage activity of DNAzyme Dz733 applied to all four target fragments of the mRNA of the human Prion Protein. The specific combinations Dz733 and T736 (slot 3), Dz839 and T839 (slot 6), Dz824 and T824 (slot 10), and Dz733 and T733 (slot 14) are shown as positive controls. No cleavage activity could be detected for non- corresponding combinations ofDz733, i.e. with T839 (slot 7), T824 (slotl 1). The cleavage activity of Dz733 with. T736 is expected because of the close neighbourhood of both cleavage sites (of Fig. 3), Incubation of target molecules in the absence of DNAzymes (slots 1, 5, 9, 13) serve as negative controls. DNAzymes designated as "inv" (slots 4, 8, 12, 15) contain the catalytic core in inverted direction of the nucleotide sequence. They serve as additional controls.

Figure 9:

Gel electrophoretic analysis of the cleavage activities ofD Azyme Dz736, Dz839, Dz824, and Dz733 incubated with their corresponding target fragments T736, T839, T824 and T733, respectively, with 10-fold molar excess of target fragment over DNAzymes. Only the combination Dz839-T839 shows 65-75% extent of cleavage (slot 5), the other combinations only about 10% extent of cleavage (slots 2, 8, 1 1). Incubation of the target fragments in the absence of DNAzymes (slots 1 , 4, 7, 10) and with DNAzymes designated as "inv" with the catalytic core in inverted direction of the nucleotide sequence (slots 3, 6, 9, 12) serve as negative controls. All sequences may be seen from Fig. 3 and the list of sequences.

Figure 10:

Visual representation of the target fragments T736 and T824 of mRNA of the human Prion Protein in complexes with their corresponding DNAzymes. The complexes as in Fig. 3 with 9 base pairs (designated 9/9) in both double stranded regions and with shortened double stranded regions of T736 7/6 and with extended double stranded regions of T824 1 1/10 are shown.

F

Gel electrophoretic analysis of the cleavage activities of Dz839, Dz736, and Dz824 cleaving the corresponding target fragments of the mRN A of the human Prion Protein. The ratio Dz839:T839 with double stranded regions 9/9 was 0.1 (slot 2), the ratio Dz736:T736 with double stranded regions 9/9 was 10 (slot 4) and 0.1 (slot 5) and with shortened double stranded regions 6/7 was 10 (slot 6) and 0.1 (slot 7), the ratio Dz724:t824 with double stranded regions 9/9 was 10 (slot 9) and 0.1 (slot 10) and with extended double stranded regions 10/11 was 10 (slot 11) and 0.1 (slot 12). All sequences may be seen from Fig. 3 and in the list of sequences, respectively. It can be seen, that shortening the flanking double-stranded regions of T736 from 9/9 to 6/7 raises the cleaving activity slightly (slot 7 compared to slot 5), extending the flanking double-stranded regions of T824 from 9/9 to 10/11 raises the cleaving activity to full activity (slot 10 compared to slot 12). Incubation of the target fragments in the absence of DNAzyme (slots 1, 3, 8) serve as negative controls.

Single-turnover cleavage kinetics of DNAzymes at XGU cleavage triplets with the same flanking segments. The RNA fragments Tl 186 (SEQ ID NO : 31, 33, 35, 37) represent a segment of the mRNA for the murine Prion Protein. Different variants were designed that contain the cleavage sites AGU (SEQ ID NO : 31), UGU (SEQ ID NO : 33), CGU (SEQ ID NO :35), and GGU {SEQ ID NO : 37) instead of the wildtype sequence AAU and the

DNAzymes contain the corresponding deoxynucleotide complementary to X (SEQ ID NO :32,34,36,38). The target RNAs were incubated with a 10-fold molar excess of the corresponding DNAzyme. With the DNAzyme in excess, hydrolysis of the phosphodiester is the rate-limiting step of the KNA cleavage reaction, whereas association of the DNAzyme to the KNA target is much faster. CGU(e) and AGU (A) target sites are cleaved at similar rates, whereas cleavage of UGU (♦) is sligthly slower and GGU (·) at least 10-fold slower. Plateau regions might be similar but are not reached in this experiment. The samples were analyzed on an 18 % PAA gel with 7 M urea arid images were acquired using the ChemiDoc™ MP System (Bio-Rad). The signal intensities were analyzed using the Image Lab 5.0 volume tool (Bio-Rad).

Figure 13:

Visual representation of R A-DNAzymes in complexes with the target fragment T839 of the mRNA of the human Prion Protein. The cleavage sites are designated by black arrows, the number designates the cleavage site in the numbering of the mRNA, the pairs of numbers,i.e. 9/9, designate the numbers of base pairs in the double stranded region. Boxed in letters designate ribonucleotides, free letters deo yribonucleotides . The direct neighbour basepairs of the cleavage site are DNA-RNA base pairs, all other flanking pairs are RNA-RNA pairs. The sequence numbers are allocated to target fragment and the R A-DNAzymes : T839 (SEQ ID NO: 16), RDz839 9/9 (SEQ ID NO: 39), RDz839 7/7 (SEQ ID NO: 40).

Figure 14:

Multiple- turnover cleavage kinetics or two RNA-DNAzymes (RDz) and DNAzyme 839. A concentration of 0.4μΜ target RNA T839 was incubated with 0.04μΜ of RNA-DNAzymes and DNAzymes, respectively. The RNA-DNAzymes RDz839_7/7 (SEQ ID NO: 39) and RDz839_ 9/9 (SEQ ID NO: 40) comprise the catalytic centre of DNAzymes but the substrate recognition arms are composed of RNA. The RNA-DNAzyme RDz839_7/7 binds to its RNA target via 7 ribonucleotides on each side of the catalytic centre whereas RDz839_9/9 binds via 9 ribonucleotides on each side (cf Fig. 13). While RDz839 7/7 (■) cleaves the RNA target at a slightly higher rate than RDz839 9/9 (·), whereas Dz839 (♦) shows significantly higher activity. However, the extents of cleavage after 22h, i.e. in the plateau, are similar for DZ and RDz. Cleavage kinetics is derived from gelelectrophoretic analysis as described in Fig. 12.

Figure I S:

Gel electrophoretic analysis of the DNAzyme-mediated hydrolysis of full-length RNA transcripts. The activity of the DNAzymes Dz736_9/9, Dz736 6/7, Dz839 9/9, Dz824 9/9, Dz824_l 1/10, and Dz733 9/9 respectively (cf Fig. 3), cleaving the human PrP coding sequence (CDS) mRNA during in vitro transcription is shown. The symbol (→) designates full-length transcripts, (>) cleavage products, respectively. The Azide Cy5 RNA T7

Transcription Kit (Jena Bioscience),

http://www.jenabioscience.aim cm

ling_kits.html) was used to produce in vitro transcripts of the human PrP CDS in which a portion of the uridines (U) are randomly switched for 5-azido-propyl-uridine (N3-U), The original RNA sequence is preserved. An Azadibenzocyclooctyne (DBCO)-functionalized Cy5 dye is later used to fluorescence label the transcripts at each N3-U in a reaction known as Strain-Promoted Azide- Alkyne Click Chemistry (SPAAC). Slot 1 is a control without DNAzymes; in slots 2-7, the respective DNAzymes are added to a final concentration of 10 μΜ. Effective cleavage during transcription is shown for Dz736_9/9 (slot 2), Dz839 9/9 (slot 4), and Dz824_l 1/10 (slot 6), whereas significant cleavage was not observed for Dz736_6/7 (slot 3, shortened flanking regions), Dz824_9/9 (slot 5, shorter flanking regions compared to Dz824_l 1/10 (slot 6)), and Dz733 9/9 (slot 7). The samples were analyzed on a 5 % PAA gel with 7 M urea.

Quantitative PGR (qPCR) analysis of DNAzyme-mediated knockdown of the human Prion Protein mRNA in cell culture. WAC II neuroblastoma cells were transfected with different doses of polynucleotides using lipofectamine® 2000. The level of PrP mRNA expression is normalized on β2 microglobulin mRNA levels which are not affected by the polynucleotides. The relative PrP mRNA levels are shown as a fraction of the level in cells treated with

Lipofectamine® 2000 alone (1st bar). Treating the cells for 24 h with a DNAzyme without substrate specificity for PrP but with the catalytic core (scramble) reduces PrP mRNA expression to 68 % (2nd bar). Transfecting an anti sense deoxyoligonucleotide specific for the Dz824 _l 1/10 binding site but without catalytic activity (ASO) at a final dose of 50, 100, and 200 nM does reduce PrP raR A levels only slightly at the highest concentration (3rd to 5th bar). Treatment with increasing doses of Dz824_l 1/10 reduces PrP mRNA expression up to 58 % (6th to 8th bar). Adding two thymidines in L-ctmfiguration to the 3' end of

Dz824 11/10 (Dz824_LL) protects the DNAzyme from 3'->5' exonuclease activity. When using the Dz824 LL variant, PrP mRNA expression levels are reduced to up to 37 % at the concentration of 200μΜ. Each bar represents an average of qPCR performed in triplicates using the same cDNA as a template. Error bars show the standard deviation. The total RNA of each sample was isolated using the NucleoSpin RNA Plus Kit (Macherey&Nagel, Dflren) according to the manufacturer's recommendations. 2 total RNA were used for reverse transcription using the M-MLV reverse transcriptase cDNA synthesis kit (Promega GmbH, Mannheim) and random hexamer primers according to the manufacturer's protocol.

Assuming a 1 :1 reaction, the cDNA was diluted to a 10 ng/μΐ concentration.

Quantitative PGR was carried out using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories GmbH, M nchen).

SEQUENCES

SEQ ID NO: 1 :

Hammerhead Ribozyme core sequence: 5 ' CUG AUGAGGCCGAAAGGCCGAAAC3 ' (24 nt) Type: RNA

SEQ ID NO: 2:

DNAzyme/RNA-DNAzyme core sequence: 5 ^* AGGCTAGCTAC AACG A3 ' (16 nt)

Type: DNA

SEQ ID NO: 3:

Target! : 5 'FLGAAGGAGUCCCAGG3 ' (14 nt)

Type: RNA

SEQ ID NO: 4:

Rzl: 5 'CCUGGCUGAUGAGGCCG AAAGGCCGAAACUCCU3 ' (33 nt)

Type: RNA

SEQ ID NO: 5:

Dzl : 5⁴ CTGGG AGGCTAGCTAC AACG ATCCITC3⁴ (27 nt)

Type: DNA

SEQ ID NO: 6:

Target2: 5 LCUGUUUGUCCCCCA3 ' (14 nt)

Type: RNA SEQ ID NO: 7:

Rz2: 5 ' UGGGGCUGAUGAGGCCGAAAGGCCG AAAC AAAC3 ' (33 nt)

Type; RNA

SEQ ID NO: 8:

Dz2: 5 'GGGGGAGGCTAGCTAC AACGAAAACAG3 ' (27 nt)

Type: DNA

SEQ ID NO: 9;

Target3: 5'FLCACACAGUCACCAC3 ' (14 nt)

Type: RNA

SEQ ID NO: 10:

Rz3: 5⁴ GUCGUCUGAUGAGGCCG AAAGGCCGAAACUGUC3 ^* (33 nt)

Type: RNA

SEQ ID NO: 11:

Dz3: 5 ' TGGTG AGGCT AGCT AC AACG ATGTGTG3 ' (27 nt)

Type: DNA

SEQ ID NO: 12;

T736: 5 'FLGCUGGGGC AGUGGUGGGGGGCCUUG3 ' (25 nt)

Type: RNA

SIQ ID NO: 13:

Dz736_ 9/9: 5'GGCCCCCCAGGCTAGCTACAACGACACTGCCCC3' (33 nt)

Type: DNA

SEQ ID NO: 14:

Dz736_9/9_inv: 5 'GGCCCCCCAAGC AAC ATCGATCGGCACTGCCCC3⁴ (33 nt)

Type: DNA

SEQ ID NO: 15:

Dz736 6/7: 5'CCCCCAGGCTAGCTAC ACGACACTGCC3' (28 nt)

Type: DNA

SEQ ID NO: 16:

T839: 5 'FL AAAAC AUGC ACCGUUACCCCAACC A3 ' (25 nt)

Type: RNA

SEQ ID NO: 17;

Dz839 9/9: 5 'TTG 3GGTAAGGCTAGCTAC AACGAGGTGC ATGT3⁴ (33 nt)

Type: DNA

SEQ IB NO: IS: Dz839_9/9_inv: 5 'TTGGGGTAA AGC AAC ATCG ATCGGGGTGC ATGT3 ' (33 nt)

Type: DMA

SEQ ID NO: If:

T824: 5TI^CCGUUACUAUCGUGAAAACAUGCA3' (25 nt)

Type: RNA

SEQ ID NO: 20:

Dz824__9/9: 5 'ATGT ITC AGGCTAGCTAC AACGAGATAGTAAC3 ^* (33 nt)

Type: DNA

SEQ ID NO: 21:

Dz824 9/9 inv: 5 ' ATG ITITCAAGC AAC ATCG ATCG K5ATAGTAAC3 ' (33 nt)

Type: DNA

SEQ ID NO: 22:

Dz824_ 10/11 : 5 ' C ATGTITTC AGGCTAGCTAC AACG AG ATAGTAACGG ' (36 nt) Type: DNA

SEQ ID NO: 23:

T733: 5 TLX3C AGCUGGGGCAGUGGUGGGGGGCC3⁴ (25 nt)

Type: RNA

SEQ ID NO: 24:

Dz733 9/9: 5 'CCCCCACC AGGCTAGCTAC A ACGATGCCCCAGC3' (33 nt)

Type: DNA

SEQ ID NO: 25:

Dz733_9/9_inv: 5 'CCCCCACC AAGCAACATCGATCGGTGCCCCAGC3 ' (33 nt)

Type: DNA

SEQ ID NO: 26:

Hammerhead Ribozyme core sequence: 5 'CU G AU G AGG CCG AAAGGCCG AAAU3 ' (24 nt)

SEQ ID NO: 27:

Hammerhead Ribozyme core sequence: 5 'CUGAUGAGGCCG AAAGGCCG AAAG3 ' (24 nt)

SEQ ID NO: 28:

Hammerhead Ribozyme core sequence: 5'CUGAUGAGGCCGAA AGGCCGAAAA3 ' (24 nt)

SEQ ID NO: 29:

DNAzyme/RNA-DNAzyme core sequence: 5 ' GGGCTAGCT AC AACG A3 ' (16 nt) SEQ ID NO: 30:

DNAzyme RNA-DNAzyme core sequence: 5 'IGGCTAGCTAC AACGA3 ' (16 nt)

SEQ ID NO: 311

Ti l 86 AGU: 5 'FLGGGCCA AUAAGAGUAUA AC ACCAAA3 ' (25 nt)

Type: R A

SEQ ID NO: 32:

Dzl 186 AGU: 5 TGTGTTATAGGCI AGCTAC AACG1 CTTATTGG3 ' (33 nt) Type: DNA

SEQ ID NO: 33:

Ti l 86 UGU: 5 'FLGGGCC AAU AAGUGUAUAACACCAAA3⁴ (25 nt)

Type: RNA

SEQ ID NO: 34:

Dzl 186 UGU: 5 'GGTGTTATAGGCn AGCTAC A ACGTACITATTGG3 ' (33 nt) Type: DNA

SEQ ID NO: 35:

T 186_CGU: 5 'FLGGGCC AAUAAGCGUAUAACACC A AA3 ' (25 nt)

Type: RNA

SEQ ID NO: 36:

Dzl 186 CGU: 5 'GGTGTTATAGGCTAGCTACAACGTGCTTATTGG3 ' (33 nt) Type: DNA

SEQ ID NO: 37:

T1186_GGU: 5⁵ FLGGGCC AAU AAGGGUAUAACACCAAA3 ^* (25 nt)

Type: RNA

SEQ m NO: 38:

Dzl l86_ GGU: ' GGTGTTATAGGCTAGCTAC AACGTCCITATTGG3 ^* (33 nt)

Type: DNA

SEQ ID NO: 39:

RDz839_7/7: 5'rG rG rG rG rU rA dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dG rG rU rG rC rA rU3' (29 nt)

Type: RNA-DNA-RNA

SEQ ID NO: 40:

RDz839_9/9: 5 'rU rU rG rG rG rG rU rA dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dG rG rU rG rC rA rU rG rU3' (33 nt)

Type: RNA-DNA-RNA SEQ ID NO: 41:

Dz824 10/11_LL: S'CATGTTTTCAGGCTAGCTACAACGAGATAGTAACGG-p-L-dT-P- L-dT3'(38 nt)

Type: DNA stabilized

SEQ ID NO: 42:

RDz736_9/9: 5 'rG rG rC rC rC rC rC iC dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dC rA rC rtJ rGrCrCrC rC3' (33 nt)

Type: RNA-DNA-RNA

SEQ ID NO: 43:

RDz736_6/7: 5'rC rC rC rC rC dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dC rArCrlirGrCrC3' {28 et)

Type: RNA-DNA-RNA

SEQ ID NO: 44:

RDz824_9/9: 5'rA rU rG rU rU rUrUrCdA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dG rA rU rA rG rU rA rA rC3 ' (33 nt)

Type: RNA-DNA-RNA

SEQ ID NO: 45:

RDz824 10/11: 5'rC rArUrG rU rU rU rU rC dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dG rA rU rA rG rU rArArCrGrG3' (36 nt)

Type: RNA-DNA-RNA

SEQ ID NO: 46:

RDz733_9/9: S'rC rC rC rC rC rA rC rC dA dG dG dC dT dA dG dC dT dA dC dA dA dC dG dA dT rG rC rC rC rC rA rGrC3^* (33 nt)

Type: RNA-DNA-RNA

SEQ ID NO: 47:

2144 - CUC AUA AUUGUCAAAAACC-2162

SEQ ID NO: 48:

1138- GGUCUUCCUGUUUUCACCA-1156

SEQ ID NO: 49:

2682- AAAAAAAUUGUAAAUGUUU 2700

SEQ ID NO: 50:

1364- CCAGUA AAA GUAUAACAGC 1382

SEQ ID NO: 51:

10 AUUUUUACAGUCAAUGAGC -28 SEQ ID NO: 52:

1289-AAAC AUAG AGUAGACCUGA-1307

SEQ ID MO; 53:

1893 AUUUU AG AUGUUUAAAGG A- 1911

SEQ ID NO: 54:

1761 -GUAAAUAUUGUCACAA.CAC 1779 SEQ ID NO: 55:

2094 -AAAUGGUCAGUGUGCAAAG 21 12 SEQ ID NO: 56:

2264 UGAULJUGAAGUGGAAAAAG-2282 SEQ ID NO: 57:

2297-UUAAUUAAAGUAAAAUUAU-2315 SEQ ID NO: 58:

1957-AGCUGAAAAGUAAAUUGCC-1975 SEQ ID NO: 59:

2327-UUUGAU AUUGUC ACCUAGC-2345 SEQ ID NO: 60:

1752-UCAACAAGAGUAAAUAUUG-l 770 SEQ ID NO: 61:

2688-AUUGUAAAUGLJUUAAUAUC-2706 SEQ ID NO: 62:

2133-UUUAUUUCUGUClJCAU AAU-2151 SEQ ID NO: 63:

1995 AUCUCCUUUGUCCALTJUAC-2013 SEQ ID NO: 64:

829 ACAUGCACCGUUACCCCA 847 SEQ ID NO: 65:

1234-UCUCUCTJUUGUCCCGGAUA-l 252 SEQ ID NO: 66:

2395 GUAUUCUAUGUAAAAAUAU-2413 SEQ ID NO: 67:

1080 UCUCCACCUGlJGAUCCUCC-1098 SEQ ID NO: 68: 2386-ACUUUGUGAGUAUUCUAUG--2404 SEQ ID NO: 69:

2666-UCXJUGUUUUGUUAUAUAAA-2684

SEQ IP NO: 70:

670-ACAAGCCGAGUAAGCCAAA-688

SEQ ID NO: 71:

2287 UCUGUUAAUGUUAAUU AAA- 2305

SEQ ID NO: 72:

2621 - AAU AUGCAUGU ACUUU AU A -2639 SEQ ID NO: 73:

88-UCUCAACUCGUUUUUUCCG-106

SEQ ID NO: 74:

1358-GCC AGGCC AG U A AA AGUAU- 1376 SEQ ID NO: 75:

2661 -CAUGUUCUUG U U UUGUUAU-2679 SEQ ID NO: 76:

2047-GCUGCAGUIJGUGAAAGCAC-2065 SEQ ID NO: 77:

2167-UUAGGUCAAGUUCAUAGUU-2185 SEQ ID NO: 78:

656-CCACAGUCAGUGGAACAAG 674 SEQ ID NO: 79:

2317 CCCUG AAUUGUIJ UG AU AUU 2335 SEQ ID NO: 80:

66-CUGAUGCAAGUGIJUCAAGC-84 SEQ ID NO: 81:

1001 - UG AGC AG AUGUGU AUG ACC- 1019 SEQ ID NO: 82:

1502-UAGAGCUCAGUAUACUAAU-l 520 SEQ ID NO: 83:

2461 -UUUAGAGCAGUUAACAUCU 2479 SEQ ID NO: 84:

2643 UCUAUAUlJUGUAACJUUUGC-2661 SEQ ID NO: 85:

1812- A AC AUA ACUGU AAC AU AU A - 1830

SEQ ID NO: 86:

1326 UUUGAUUGAGUUCAUCAUG - 1344

SEQ ID NO: 87:

1413-UUGG ACUUAGUGCAACAGG- 1431 SEQ ID NO: 88:

2281 -AGAAAUUCUG UUAAUGUUA-2299

SEQ ID NO: 89:

969-GAGACCGACGUUAAGAUGA-987

SEQ ID NO: 90:

191 1 -ACCCUAUAUGUGGCAUUCC- l 929 SEQ ID NO: 91:

2382-UUGC ACUUUGUGAG U AUUC-2400 SEQ ID NO: 92:

2518 ACUUAAUAUGUGGGAAACC 2536 SEQ ID NO: 93:

2574— UCGUUUC AUGUAAG AAUCC— 2592 SEQ ID NO: 94:

412-CCACAUGGAGUG ACCUGGG-430 SEQ ID NO: 95:

814-GUUACUAUCGUGAAAAC Αϋ-832 SEQ ID NO: 96:

866-CAUGGAUGAGUACAGCAAC-884 SEQ ID NO: 97:

1721 -CUUUUCACAGUAUGGGCUA-1739 SEQ ID NO: 98:

2474-ACAUCUGAAGUGUCUAAUG— 2492 SEQ ID NO: 99:

2535-CCCUUUUGCGUGGUCCUUA-2553 SEQ ID NO: 100:

805-AUGAGGACCGUUACUAUCG-823 SEQ ID NO: 101: 2174-AAGUUC AU AGUUUCUGUA A-21 2

SEQ ID NO: 102:

2567-CAC JGAAUCGUUUCAUGUA 2585

SEQ ID MO: 103:

843 - CCC A ACC AAGUGU AC U AC A - 861

SEQ ID NO: 104:

790 -AUUUCGGCAGUGACIJAUGA -808

SEQ ID NO: 105:

2495 "UUAACUUUUGUAAGGUACU 2513

SEQ ID NO: 106:

1706-UGGGGUGAUGUUUUAOJUU-1724

SEQ ID NO: 107:

1800-AUAUUCACAGUGAACAUAA— 1818 SEQ ID NO: 108:

888 -AACAAClJUUGUGCACGACU 906 SEQ ID NO: 109:

1013-UAUCACCCAGUACG AGAGG- 1031 SEQ ID NO: 110:

900 CACGACUGCGUCAAUALJCA-918 SEQ ID NO: 111:

2044 - AGAGCUGCAGUUGUGAAAG 2062 SEQ ID NO: 112:

2555 GCUUACAAUGUGCACUGAA- 2573 SEQ ID NO: 113:

1682 AUGAGCUCUGUGUGUACCG 1700 SEQ ID NO: 114:

1686- GCUCUGUGUGUACCGAGAA- 1704 SEQ ID NO: 115:

2343- AGCAGAUAUGUAUUACUUU - 2361 SEQ ID NO: 116:

399 GUUCU JUUGUGGCC ACAU 17 DEFINITIONS

The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art if not otherwise indicated herein below.

As used in this specification and the appended embodiments, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes one or more compounds.

The term "Ribozymes" as used herein refers to catalytically active ribonucleic acids which hydrolyze KNA at the 3 'side of specific sequences of 3 nucleotides (GUC and others with U in the middle position). Ribozymes consist of the catalytic core-sequence of 24 ribonucleotides and of two flanking regions which form on both sides of GUC base-paired double strands with the target RNA of 2 or more base pairs to select a specific GUC-cleavage site.

The term "DNAzymes" as used herein refers to single-stranded catalytically active desoxyribonucleic acids which hydrolyze RNA between G and U or A and U or with less activity between G and C or A and C, respectively. They consist of a catalytic core region comprising the catalytic domain and a 3'- and 5 '-flanking region, which is complementary to the 5'- and 3' region on the target RNA flanking the cleavage site. The nucleotide sequence of the flanking region is in antisense orientation to the corresponding region on the target RNA and binds to the target RNA through complementary base pairing.

The term„RNA-DNAzyme" as used herein refers to single- stranded catalytically active hybrid nucleic acid molecules comprising RNA and DNA sequences, which hydrolyzes RNA between G and U or A and U or with less activity between G and C or A and C, respectively. The RNA-DNAzyme consists of a catalytic core region comprising the catalytic domain and a 3'- and 5 '-flanking region, which is complementary to the 5'- and 3' region on the target RNA flanking the cleavage site. The flanking region consists of ribonucleotides and the sequence of the flanking region is in antisense orientation to the corresponding region on the target RNA and binds to the target RNA through complementary base pairing.

The ratio of RNA to DNA in the„RNA-DNAzyme" is about 1.0 : 1.2; 1 ,0 : 1.1; 1.0 : 1.0; 0.95 : 1.0; 0.90 : 1.0; 0.85 : 1.0; 0.80 : 1.0; 0.75 : 1.0; 0.70 : 1.0; 0.65 : 1.0; 0.60 : 1.0.

In particular, the ratio of RNA to DNA in the„RNA-DNAzyme" is in a range of between 1 ,0 ; 1 ,2 and 0.6 : 1.0, particularly in a range of 0.95 : 1,0 and 0.6 ; 1 ,0. The DNA sequence may be located in the center of the„RN A-D Azyme' ' forming the catalytically active "core region", which is flanked by DNA/RNA flanking sequences. The

„RN A-DN Azyme" may be constructed symmetrically, with the same number of ribonucleotides on each side of the DNA core sequence, or asymmetrically, with varying numbers of ribonucleotides on each side of the DNA core sequence.

The DNAzymes and/or RNA-DNAzymes may be modified to protect against degradation in vivo. Methods for protecting DNAzymes against degradation are known in the art. Examples include incorporating a 3'-3'inversioii at one or more terminae of the DN Azyme. This entails modifying the 3 "terminal nucleotide so that covalent phosphate bonding occurs between the 3 'carbons of the terminal nucleotide and its adjacent nucleotide. Alternatively, or in addition, the DNAzymes may contain modified nucleotides or nucleotide linkages, for example N3'- P5'phosphoramidate linkages, 2'-0-methyl substitutions and peptide-nucleic acid linkages. An alternative strategy for stabilising the DNAzymes is to employ a stem-loop structure at one or each terminus, as described in Gavin and Gupta, J. Biol. Chem., 1 97; 272: 1451 -1472.

The term "complementary" as used herein refers to a nucleotide sequence that base-pairs by non-covalent bonds to a target nucleic acid of interest. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and vice versa. Typically, "complementary" refers to a nucleotide sequence that is at least partially complementary. The term "complementary" may also encompass duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotide is complementary to every nucleotide in the target nucleic acid in all the corresponding positions.

The term "label", as used herein, in the context of a labeled polynucleotide (e.g., a labeled target sequence) refers to moiety via which an oligonucleotide can be detected or purified. Examples of such labels are mass tags, fluorescent tags, chemiluminescent tags and radio tags such as nucleosides harboring radioactive phosphorous, sulfur or hydrogen.

The term "nucleotide" as used herein is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrirnidines, acylated purines or pyrimidines, alkylated purines or pyrirnidines, halogenated purines or pyrirnidines, deaza- purines or pyrimidines or other heterocycles. In addition, the term "nucleotide" includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugare as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxy! groups are replaced with halogen atoms or aliphatic groups, are functionalized as ethers, amines, such as but not limited to MOE, LNA or the likes

The term "Misfolding proteins" as used herein refers to proteins which are expressed in the cell in a cellular or non-pathogenic conformation but have the tendency for misfolding in a pathogenic conformation. Some examples of misfolding protein are, amongst others: Prion Protein, amyloid precursor protein (APP), alpha-synuclein, Tau protein, superoxide dismutase 1 (SOD) protein, insulin and others.

The term "Misfolded proteins" as used herein refers to proteins, which can exist in a cellular, i.e. non-pathological conformation and in a misfiled, i.e. in most cases pathological and disease-related conformation and which are present in a misfolded pathogenic isoform. . Aminoacid sequence and chemical modifications, if present are identical in both conformations.

The term "hybridize™ as used herein refers to conventional hybridization conditions, preferably to hybridization conditions at which 5xSSPE, 1 % SDS, IxDenhardts solution is used as a solution and/or hybridization temperatures are between 35°C and 70°C, preferably 65°C. After hybridization, washing is preferably carried out first with 2xSSC, 1% SDS and subsequently with 0.2xSSC at temperatures between 35°C and 70°C, preferably at 65°C (regarding the definition of SSPE, SSC and Denhardts solution see Sambrook et al. loc. tit). Stringent hybridization conditions as for instance described in Sambrook et al, supra, are particularly preferred. Particularly preferred stringent hybridization conditions are for instance present if hybridization and washing occur at 65°C as indicated above. Nonstringent hybridization conditions, for instance with hybridization and washing carried out at 45 °C are less preferred and at 35°C even less

The term "Sequence Identity" as used herein can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Generics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to deteraiiee whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. The deviations appearing hi the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program "fasta20u66" (version 2.0u66, September 1 98 by William R. Pearson and the University of Virginia; see also W.R. Pearson (1990), Methods in Enzyrnology 183, 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the "default" parameter settings may be used.

The term "Protein misfolding diseases" as used herein refers to diseases which are induced by particular misfolded proteins. Few examples are, amongst others: Creutzfeld Jakob disease (CJD), Alzheimer^'s disease (AD), Parki son's disease (PD), Down^'s Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FID), Tenopathy, Glaucoma, Diabetes, Fibrosis, all in humans and Chronic wasting disease (CWD), Bovine spongiform encephalopathy (BSE) and Scrapie in animals. Protein-misfolding diseases are called also "Prion-like diseases" or even simpler "Prion-diseases".

The term "Sequential-Folding-Algorithms" (Bernhart et al. 2006; Tafer et al. 2008) as used herein refers to an algorithm that calculate the formation of the secondary structure of a target-RNA during the synthesis of the RNA. From that the accessibility of a target RNA for base pairing with the corresponding segment in the sequence in a second nucleic acid (anti ense, cleaving or other nucleic acids) can be derived.

In contrast to other algorithms described in the literature the algorithm used for the present inventions determines the structure or accessibility to binding of other nucleic acids of the 40 nucleotides at the 3' site during synthesis which form the fastest structure which might be different from the most stable structure formed later.

The term "pharmaceutically acceptable" is meant to encompass any carrier, excipient, diluent or vehicle, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.

A "therapeutically effective amount" refers to that amount which provides a therapeutic effect for a given condition and administration regimen. In particular, "therapeutically effective amount" means an amount that is effective to prevent, reverse, alleviate or ameliorate symptoms of the disease or prolong the survival of the subject being treated, which may be a human or non-human animal. Determination of a therapeutically effective amount is within the skill of the person skilled in the art. hi particular, in the present case a "therapeutically or prophylactically effective amount" refers to the amount of the compound or pharmaceutical composition which, when administered to a human or animal, leads to a therapeutic or prophylactic effect in said human or animal. The effective amount is readily determined by one of skill in the art following routine procedures. The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a maimer known in the relevant art. The dosage can vary within wide limits and will, of course, have to be adjusted to the individual requirements in each particular case.

The expressions "pharmaceutical composition" is used herein in the widest sense. It is meant to refer, for the purposes of the present invention, to a therapeutically effective amount of the active ingredient of the invention, and, optionally, a pharmaceutically acceptable carrier or diluent.

It embraces compositions that are suitable for the curative treatment, the control, the amelioration, an improvement of the condition or the prevention of a disease or disorder in a human being or a non-human animal. Thus, it embraces pharmaceutical compositions for the use in the area of human or veterinary medicine. Such a "therapeutic composition" is characterized in that it embraces at least one compound of the invention or a physiologically acceptable salt thereof, and optionally a carrier or excipient whereby the salt and the carrier and excipient are tolerated by the target organism that is treated therewith.

Suitable pharmaceutical carriers, diluents and/or excipients are well known in the art and include, for example, phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, etc.

Formulation of the pharmaceutical composition according to the invention can be accomplished according to standard methodology know to those skilled in the art.

The compositions of the present invention may be administered to a subject in the form of a solid, liquid or aerosol at a suitable, pharmaceutically effective dose. Examples of solid compositions include pills, creams, and implantable dosage units. Pills may be administered orally. Therapeutic creams may be administered topically. Implantable dosage units may be administered locally, or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously. Examples of liquid compositions include formulations adapted for injection intramuscularly, subcutaneously, intravenously, intra- arterially, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhaler formulations for administration to the lungs.

The compositions may be administered by standard routes of administration. In general, the composition may be administered by topical, oral, rectal, nasal, interdermal, intraperitoneal, or parenteral (for example, intravenous, subcutaneous, or intramuscular) routes.

It is well know to those skilled in the pertinent art that the dosage of the composition will depend on various factors such as, for example, the condition of being treated, the particular composition used, and other clinical factors such as weight, size, sex and general health condition of the patient, body surface area, the particular compound or composition to be administered, other drugs being administered concurrently, and the route of administration.

A "patient" or "subject" for the purposes of the present invention is used interchangeably and meant to include both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therap and veterinary applications. In the preferred embodiment the patient or subject is a mammal, and in the most preferred embodiment the patient or subject is a human.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may he therapeutic in terms of partially or completely curing a disease and/or adverse effects attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing a disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease (d) reversing the disease symptoms,

EXAMPLES

Example 1 : Proof of Ribozyme (Rz) activity to be compared with DNAzvme ( z) activity for cleaving target segments of the mRNA of the murine Prion Protein mRNA

Gel electrophoretic analysis was performed of the cleavage activity of three Ribozymes Rzl, Rz2 and Rz3, respectively, cleaving target segments of the mRNA of the murine Prion Protein (see Figure 1). The target fragments are fluorescence labelled at their 5 ^'end. Slots 1 , 4, 7 are controls containing tie target fragments only; slots 2, 5, 8, contain 10-fold molar excess of the corresponding ribozymes and slots 3, 6_» 9 a 100-fold molar excess of the ribozym.es. The cleavage reaction of 0,4 μΜ 5 '-6-FAM-labclled target RNA molecules in. the presence of 4 μΜ or 40 μΜ ribozyme, respectively, was carried out in 50 mM Tris-HCl pH 7,5 in the presence of 10 mM MgC12 for 3 h at 37°C after a pre-incubation step at 73 °C and a slow cooling down to RT, The analysis was done via 18% PAA gel electrophoresis with 7 M urea. The Ribozymes consist of the core sequence (24 ribonucleotides):

5 ^'CUGAUGAGGCCGAAAGGCCGAAAC3 ^' (SEQ ID NO: 1) and the 5 'and 3 'flanking sequences specific for targets Tl , T2 and T3, respectively.

Target!; 5 TLGAAGGAGUCCC AGG3 ^' (SEQ ID NO: 3)

Ribozyme! (Rzl): 5 CCUGGCLJGAUGAGGCCGAAAGGCCGAAACUCCU3 (SEQ ID NO: 4)

Target2: 5 TXCUGUUUGUCCCCCA3 ' (SEQ ID NO: 6)

Ribozyme2 (Rz2): 5 UGGGGCUG AUGAGGCCGAAAGGCCGAAACAAAC3 ' (SEQ ID NO: 7)

Target3: 5 'FLCACACAGUC ACCAC3 '(SEQ ID NO: 9)

Ribozyme3 (Rz3): 5 'GUCGUCUGAUGAGGCCGAAAGGCCGAAACUGUC3 ^' (SEQ ID NO: 10)

In Fig. 1 the cleavage is shown with three target segments and the three corresponding ribozymes (Rzl , Rz2, and Rz3). In slots 1 , 4, 7 the target sequences 1, 2, 3, respectively, in the absence of ribozymes are incubated and analyzed showing the full length target segments as controls. In slots 2, 5, 8, a 10-fold molar excess of ribozymes was added achieving complete cleavage; only the 5 labelled part of the cleaved target segment can be seen. The same result was obtained with 100-fold excess of ribozyme over target segment (slots 3, 6, 9). This example is not part of the invention but serves for comparison with DNAzymes.

Example 2: Proof of concept of D Azvme (Dz) activity for cleaving target segments of the mRNA of the murine prion protein mRNA

Gel electrophoretic analysis was performed of the cleavage activity of the three DNAzymes Dzl , Dz2, Dz3, respectively, cleaving the identical target segments as in Fig. 1 (see Example 1). The target fragments were fluorescence labelled at their 5 ^'ends. Slots 1 , 4, 7 are controls containing the target fragments only; slots 2, 5, 8, contain a 10-fold molar excess of the corresponding DNAzymes and slots 3, 6, 9 a 100-fold excess of the DNAzymes. The cleaving activity of 0.4 μΜ 5 -6-FAM-labeled target RNA molecules in the presence of 4 μΜ or 40 μΜ DNAzyme was earned out in 50 ml Tris-HCl pH 7.5 in the presence of 10 mM MgC12 for 3 h at 37°C after a pre-incubation step at 73°C and a slow cooling down to RT. Analysis was done via 18% PAA gel electrophoresis with 7M urea.

The DNAzymes consist of the Core -Sequence (15/16 deoxyribonucleotides): 5 ' AGGCTAGCTACAACGA3 ^' (SEQ ID NO: 2) and the 5 ^'and 3 ^'flanking sequences specific for targets Tl, T2 and T3, respectively.

Targetl : 5 'FLGAAGGAGUCCCAGG3 '(SEQ ID NO: 3)

DNAzymel (Dzl): 5 'CTGGGAGGCTAGCTACAACGATCCTTC3 '(SEQ ID NO: 5)

Target2: 5 'FLCUGUUUGUCCCCCA3 '(SEQ ID NO: 6)

DNAzyme2 (Dz2): 5 GGGGGAGGCTAGCTACAACGAAAACAG3'(SEQ ID NO: 8) Targets: 5 'FLCACACAGUCACCAC3 (SEQ ID NO: 9)

DNAzyme3 (Dz3): 5 GGTGAGGCTAGCTAC AACGATGTGTG3 ^'(SEQ ID NO: 1 1) It is seen from the gel electrophoresis analysis in Fig. 2 that the DNAzymes cleave the target segments in an identical manner as compared to the ribozymes. The same result was obtained with 100-fold excess of DNAzyme over target segment (slots 3, 6, 9). Target segments 1 , 2, 3 are identical to those of Example 1 (see above and list of sequences). Sequences of Dzl, Dz2, Dz3, are shown above and in the list of sequences.

Example 3: Proof of ..concept of DNAzyme (Dz) activity for cleaving target segments of the P';U A of tin liuman Prion Protein mRNA

In Fig. 3 all combinations of target segments (upper strands) and their corresponding DNAzymes (lower strands) are depicted. The cleavage sites are designated by black arrows. The numbers of target segments are the nucleotide numbers in the mRNA of the human Prion Protein where the cleavage at its 3 ^'side occurs. The numbers of the DNAzymes are also the cleaving sites and the number pairs 9/9 represent the double stranded flanking regions at both sides. Because of the close neighborhood of cleavage site 733 and 736 target segments T733 and T736 and DNAzymes Dz733 and Dz736, respectively, were combined counterwise.

Gel electrophoretic analysis was performed of the cleavage activity of the DNAzymes Dz736, Dz739, Dz824, Dz773 cleaving the corresponding target fragments of the mRNA of the human Prion Protein (see Figure 4). The complexes of the DNAzymes with their corresponding target fragments are presented in Fig.3. DNAzymes designated as „inv" contain the catalytic core in inverted direction of the nucleotide sequence. They serve as controls. All target RNA molecules were labeled with 6-FAM at their 5 ^'end, allowing the visualization of uncleaved molecules and the 5 'cleavage products. The cleaving activity of 0.4M T736, T839, T824, md T733 in the presence of 4 μΜ Dz736, Dz839, Dz824, and Dz733, respectively, was carried out in 50mM Tris-HCl, pH 7.4, lOmM MgC12 for 3 h at 37°C after a 2 min denaturation step at 73°C and a slow cooling down to RT. Control reactions include the incubation of target molecules in the absence of DNAzymes (slots 1 , 4, 7_» and 10) and in the presence of DNAzymes "inv" in which the sequence of the catalytic motif is inverted. The reaction products were separated via 18% polyacrylamide gel electrophoresis (PAGE) with 7 M urea.

In Fig. 4 it is shown that complete cleavage was achieved with all four combinations of target segments and corresponding DNAzymes (slots 2, 5, 8, 11). The bands of the fluorescence labelled target segment in the absence of DNAzyme (slots 1 , 4, 7, 10) are controls; they are shifted by applying 10-fold molar excess of DNAzyme to the position of the 5 'labelled cleavage product. As an additional control the DNAzymes were applied with the catalytic core segment inverted in respect to the 5 'to 3 'directio (cf. Dz "inv", Fig. 4). No activity was seen for Dz 736 inv (slot 3) and Dz 733 inv (slot 12), but significant activity for Dz 839 inv (slot 6) and slight activity for Dz 824 inv (slot 9, Fig. 4)). As found out later, some of the Dz "inv" contain impurities from the synthesis of the corresponding Dz.

Example rf rf sBetifi^

m¾ _. jofthe human Prion Protein-

The cleaving activity of 0.4μΜ T736, T839, T824, and T733 in the presence of 4μΜ Dz736, Dz839, Dz824 and Dz733, respectively, was carried out in SOmM Tris-HCl, pH 7.4, 10 mM MgC12 for 3 h at 37°C after a 2 min denaturation step at 73 °C and a slow cooling down to RT. Control reactions include the incubatio of target molecules in the absence of DNAzymes (slots 1, 4, 7, and 10) and in the presence of DNAzymes Dz inv in which the sequence of the catalytic motif is inverted. The reaction products were separated via 18% polyacrylamide gel electrophoresis (PAGE) with 7 M urea. Gel electrophoretic analysis of the cleavage activity was performed in all cases applying one DNAzyme in combination with the four different target segments, with the corresponding combinations in all cases as positive controls. DNAzymes designated as "inv" contain the catalytic core in inverted direction of the nucleotide sequence. They serve as controls but show in some cases activity (cf Example 3). All target RNA molecules were labelled with 6-FAM at their 5 'end, allowing the visualisation of uncleaved molecules and the 5 'cleavage products.

In Fig. 5, 6, 7 and 8 the cleaving activity of Dz736, Dz839, Dz824 and Dz733 with the four different target RNAs is shown, respectively. In Figs. 5, 6, 7 and 8, corresponding combinations, e.g. Dz736 with target T736, are compared in respect to cleavage activity with non-corresponding, e.g. Dz839 with target T736 etc. The results presented in Figures 5, 6, 7 and 8 show that the cleavage activity is completely specific for the given Dz-target complex. Although 10-fold excess of DNAzyme over target RNA was applied, no unspecific cleavage was detected. There was, however, some cross activity between cleavage sites 733 and 736 which can be explained by the close neighbourhood of these two cleavage sites and can be seen from the presentation of double stranded Dz-target complexes in Fig. 3.

Example 5: Proof of the catalytic mechanism of the activity of DNAzymes for cleaving target segments of the mRNA of the human Prs tein. In the Examples 1 to 4 a molar excess of DNAzyme over the target segments was applied in order to prove the principles of the cleavage reactions. It is known from the literature and as the name "DNAzymes" suggests, DNAzymes should act in much lower molar concentration as compared to the concentration of the target segment, in the sense that a catalytic action runs through several binding/ cleavage/ dissociation cycles and cleave an excess of the target fragment. In Fig, 9 instead of a 10-fold excess, i.e. 4 μΜ DNAzyme, a concentration of 0,04 μΜ DNAzyme is applied for the cleavage of 0.4 μΜ target segment (T). Not only the ratio Dz:T (1:10) but also the absolute concentration of DNAzyme is lowered by a factor of 100. The four specific combinations of DNAzymes and target segments are analysed in Fig. 9. The double stranded structure of the complexes can be seen from Fig. 3. The cleaving activity of 0.4 μΜ T736, T839, T824, and T733 in the presence of 0.04 μΜ Dz736, Dz839, Dz824, and Dz733, respectively, was carried out i 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2 for 3 h at 37°C after a 2 min denaturation step at 73 °C and a slow cooling down to RT. Control reactions include the incubation of target molecules in the absence of DNAzymes (slots 1, 4, 7, 10) and in the presence of DNAzymes Dz inv in which the sequence of the catalytic motiv is inverted. The reaction products were separated via 18% polyacrylamide gel electrophoresis (PAGE) with 7M urea.

In Fig. 9 it is shown that only Dz839 acts catalytically; 65-75% of the target segment are cleaved but only 10% DNAzyme of the target segment are applied (slot 5, Dz:T=T :10). The other three specific combinations (slots 2, 8, 1 1) show only around 10% cleavage extent. All target RNA molecules were labelled with 6-FAM at their 5 'end, allowing the visualisation of uncleaved molecules and the 5 'cleavage products. Example 6; Optimization of the double stranded regions between the DNAzymcs and the target fragments of the imRNA of the human Prion Protein to achieve catalytic cleavage activity.

The absence of catalytic, i.e. multiple cycle cleavage activity of the combinations Dz736 and T736 or Dz824 and T824, respectively, could originate from too unstable binding of the target RNA to the DNAzyme, or from too strong binding of DNAzyme to the cleaved products preventing release of the products. It was estimated from the thermodynamic stability of the Dz-T complexes, that the first possibility, i.e. too unstable binding, is expected for the combination Dz824-T824. Therefore, two base pairs were added on the 5 'site of T824 and one on the 3 'site, respectively; the double stranded structures of the Dz824-T824 with arm lengths 9/9 and the elongated arm lengths 10/1 1 are shown in Fig. 10. It was estimated from the thermodynamic stability of the DZ-T complexes, that the second possibility, i.e. too strong binding of the cleavage products, is expected for the combination Dz736-T736. Therefore, three basepairs were left out at the 5 'site of T736 and two at the 3 'site, respectively; the double stranded structures of Dz736-T736 with the arm lengths 9/9 and the shortened arm lengths 6/7 are shown in Fig. 10. The cleaving activity of 0.4μΜ T839, T736, and T824 in the presence of 4μΜ Dz736 (slots 4, 6) and of Dz824 (slots 9, 11) and 0.04μΜΟζ839 (slot 2), Dz736 (slots 5, 7) and Dz824 (slots 10, 12), respectively, was carried out in 50mM Tris-HCl, pH 7.4, lOmM MgC12 for 3 h at 37°C after a 2 min denaturation step at 73°C and a slow cooling down to RT. Control reactions include the incubation of target molecules in the absence of DNAzymes (slots t , 3, 8). The reaction products were separated via 18% polyacrylamide gel electrophoresis (PAGE) with 7M urea.

The gelel ectrophoretic analysis in Fig. 11 shows, that the combination Dz824-T824 with the extended double structures 10/11 excerts clearly catalytic cleavage; the extent of cleavage with a Dz:T ratio of 0.1 (1 ;10) (slot 12) is similar to that with the ratio Dz:T of 10 (10: 1) (slot 11). In the case of the double stranded structure 9/9 the ratio Dz:T of 10 only shows high extend of cleavage (slot 9), while the ratio of 0.1 shows very low extent (slot 10). In summary, the results shown in slots 8-12 in Figure 11 confirm that the absence of catalytic, i.e. multiple cycle cleavage activity of the Dz824 and T824 originate from too unstable binding of the target RNA to the DNAzyme.

In the case of Dz736-T736, the shortening of double stranded regions of 9/9 (slots 4, 5) to 6/7, respectively, excerts a minor increase in cleavage extent with a ratio Dz:T of 0.1 (1 :10) by comparing 9/9 (slot 5) and 6/7 (slot 7). Even though after shortening of double stranded regions the observed increase in cleavage is minor, still it showed the right tendency of improving catalytic cleavage at Dz736-T736~! : 10 ratio, and therefore partially confirming the hypothesis of too strong binding of DNAzyme to the cleaved products preventing release.

compHi » i ^ / 11 ' i juence cleavage site on the RNA target and influence... of the S^? ribonucleotide next to the cleavage site.

Hie catalytic activity of Dz's at the cleavage sites (triplets) AAU, GAU, UAU, CAU and AGU, GGU, UGU, CGU were determined applying 10 fold excess of Dz over target RNA in order to compare catalytic activity after saturation of binding of Dz and target. The Dz's contained at the 3* site of the catalytic core the deoyxnucleotide complementary to the 5^* ribonucleotide of the cleavage triplet (cf. secondary structures in Fig.3). Otherwise the sequences were identical of that of RNA substrate T 839 and Dz 839 9/9 as specified in Fig. 3. All target RNA molecules were labeled with 6-FAM at their 5' end. The cleaving activity of 4μΜ Dz on 0.4μΜ target RNA. was carried out in 50 mM Tris-HCl, pH 7.4, lOmM MgCi₂ for incubation times between 1 min and 22 h at 37°C after a 2 min denaturation step at 73°C and slow cooling down to RT. The reaction products were separated via 18% polyacrylamide gel electrophoresis (PAGE) with 7M urea. Comparing the 4 triplets with cleavage site GU with those with AU after lb and 4h incubation times demonstrated that cleavage at GU is at least six times more active as compared with cleavage at AU (not shown in a Fig.). Comparison of the four cleavage triplets with GU cleavage site is shown in Fig.12 in form of cleavage kinetics from zero to 10 min. Within the limits of experimental error cleavage activities of CGU, AGU and UGU are similar, but activity of GGU is surprisingly lower.

Example 8: Proof c af RNA-DNAzvme (RDz) activity for cleaving target segments of the mRN'A of the human Prion Protein mRNA

DNAzymes as described in the literature consist of a catalytic core and antisense sequences flanking the catalytic core which binds to the target RNA; both parts consis of deoxyribonucl eotides . However, the antisense binding can be accomplished also by RNA- DNAzyme consisting of DNA catalytic core and RNA antisense flanking regions. The hydrolytic stability of the catalytic core is conserved, and the RNA-RNA double strands have higher thermodynamic stability. The target RNA molecules were labeled with 6-FAM at their 5 'end. The cleaving activity of 0.4pM target RNA in the presence of 0.04μΜ RDz was carried out in 50mM Tris-HCl, pH 7.4, 10 mM MgCl₂ for incubation times between 1 min and 22 h at 37° after a 2 min denaturation step at 73 °C and slow cooling down, to RT. The reaction products were separated via 18% polyacryl amide gel electrophoresis (PAGE) with 7 M urea. Fig 13 show the sequences and secondary structures of DRz839 7/7 and DRz839 9/9. From Fig. 14 it is obvious that the RNA-DNAzymes act catalytically although less effectively as compared with tie DNAzyme but to the same plateau region. Because the RNA-RNA double strands are stronger than the RNA-DNA double strands it can be expected that in vivo RNA- DNAzymes might have preference over the intramolecular structure of the mRNA.

Example 9: DNAzyme mediated cleavage of mRNA of the human Prion Protein during transcription. The mRNA of the coding sequence of the human Prion Protein was transcribed by T7 polymerase in vitro from a plasmid containing the coding sequence (CDS) of the human Prion Protein downstream of the T7 promoter. Each transcription was carried out using the Azide Cy5 RNA T7 Transcription Kit (Jena Bioscience). A pETI la vector with huprP23-230 insert downstream of the T7 promoter was linearized with Bamffl which cleaves the plasmid downstream of the insert. In a total volume of 10 μΐ, 200 ng linearized plasmid, ImM each of ATP, GTP, and CTP, 0.75 niM UTP and 0.25 mM 5-Azidc-propyl-UTP, 1 unit/μΐ RNase inhibitors, 10 units/μΐ T7 RNA polymerase, lx 17 reaction buffer (Jena Bioscience) and 4 μΜ DNAzyme (specified in Fig. 15 and Fig. 3) were incubated in the dark for 1 h at 37°C, and water was added to a total volume of 25 μΐ. The samples were then purified by gel filtration using illustra MicroSpin G-25 Columns (GE Healthcare). The columns were first centrifuged without sample for 1 min at 700 rcf. The samples were applied to the column and then centrifuged for 2 min at 700 rcf. Afterwards, 5nmol of DBCO-Sulfo- Cy5 were added and the samples were incubated in the dark for 1 h at 37°C. The samples were diluted 1:1 in 2x RNA loading buffer, denaturated at 96°C for 10 min and analyzed via 5% PAA gel electrophoresis with 7 M urea. From the gel electrophoretic analysis in Fig.15 it can be seen that DNAzyme (Dz) 736 9/9 (slot 2) is active in the cleavage reaction wherreas Dz 6/7 (slot 3) with the shorter Dz-target RNA (T) double strands is not, Dz 839 9/9 (slot 4) is active, Dz 824 9/9 (slot 5) is not active, but Dz 10/11, i.e. with longer Dz-T-double strands (slot 6) is active, and Dz 733 9/9 (slot 7) is not active. These results show: i) Sites which are cleaved in short segments (cf. Fig.3) are also cleaved, except site 733, during transcription of the full length coding sequence, and the dependence of the cleavage activity upon the length of the Dz-T double-stranded segments i.e. 9/9, 6/7, 10/11 is very similar to that in the fragments. ii) The results are in agreement with the predictions of the algorithm calculating the accessibility of cleavage sites including their binding sites to the DNAzymes. The algorithm predicted good accessibility of sites 736, 839 and 824 but very low accessibility of site 733, as found experimentally in slot 7.

Example 10: Knockdown of the mRNA of the human, prion Protein in neuroblastoma WAC II cells, mediated by antisense deoxyribonucleotides (ASO) and two DNAzymes, Neuroblastoma cells, containing the endogeneous gene of the human Prion Protein, were grown in culture vessels and transferred into solution. They were transfected with lipofectamine alone as control or additionally with catalytically active DNAzyme without substrate specificity for PrP mRNA (scrambled), antisense-deoxyoligonucleotide of the same Dz-target double strand sequence as DNAzyme 824 10/11 (ASO), DNAzyme (Dz) 824 10/11 (Dz824) and DNAzyme 824 10/11 IX (Dz824LL) containing two L-isomeric nucleotides at the 3'site for stabilisation against cellular 3' exonucleases. The extent of knockdown of the mRNA was determined by qPCR with fluorescence labelling of the PCR-products. (cf. Fig. legend of Fig.16). The results in Fig.16 show that within the limits of experimental error Dz scramble 200 nM, ASO 50 and 100 nM, Dz 824 50 and 100 nM and 824LL 50 and 100 nM knock down the mRNA compared to lipofectamine alone to 60-70%, which effect might be unspecific or only antisense inhibition. Solely 200nM Dz 824 and much more expressed 824LL knock down significantly mRNA to 58% or 37%, respectively.

Summary of disclosed Aspects

1. A catalytic polynucleotide,

particularly a ribozyme and/or a DNAzyme,

capable of cleaving a target m NA of a protein, which can switch its native structure to a misfolded pathogenic isoform (misfolding protein) or is present in a misfolded, pathogenic isoform (misfolded protein),

particularly a protein selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, huntingtin, Tau protein, superoxide distnutase 1 (SOD) protein, and insulin,

for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins (protein-misfolding diseases), particularly a disease, condition or disorder selected from the group consisting of Creutzfel dt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie,

in a subject,

particularly an animal, particularly a human,

suffering from such a disease or disorder to lower the concentration of said protein in the subject.

2. The catalytic polynucleotide for use according to aspect 1, wherein the catalytic polynucleotide is a ribozyme comprising a catalytic core region flanked 5' and/or 3' by anti sense sequence(s) wh ch are complementary to the target-mRNA sequence flanking the cleaving site on the target mRNA,

particularly an antlsem.se sequence comprising between 2 and 100 ribonucleotides, particularly between 2 and 24 ribonucleotides, particularly between 2 and 12 ribonucleotides complementary to the target-mRNA.

3. The catalytic polynucleotide for use according to aspect 2, wherein the target mRNA is cleaved 3' of the triplet XUY, with X= G, A U or C and Y=C, A or U, particularly

a. 3' of the triplet GUC, GUA or GUU; or b. 3' of the triplet AUC, AUA or AUU, wherein the catalytic polynucleotide has at its 3 'site a U to match the target triplets AUC, AUA and AUU; or c. 3* of tie triplet CUC, CUA or CUU, wherein the catalytic polynucleotide has at its 3 'site a G to match, the target triplets CUC, CUA and CUU, or

3' of the triplet UUC, UUA or UUU, wherein the catalytic polynucleotide has at its 3 'site an A to match the target triplets UUC, UUA and UUU.

The catalytic polynucleotide for use according to aspect 3, wherein the catalytic core region of the ribozyme has the sequence shown in

a. SEQ ID NO: l ; or

b. SEQ ID NO: 26; or

c. SEQ ID NO: 27; or

d. SEQ ID NO: 28

The catalytic polynucleotide for use according to aspect 1, wherein the catalytic polynucleotide is a DNAzyme comprising a catalytic core region flanked 5' and/or 3' by antisense sequence(s) which are complementary to the target-mRNA sequence flanking the cleaving site on the target mRNA,

particularly an antisense sequence comprising between 2 and 100 ribonucleotides, particularly between 2 and 24 ribonucleotides, particularly between 2 and 12 ribonucleotides complementary to the target-mRNA.

The catalytic polynucleotide for use according to aspect 5, wherein the target mRNA is cleaved between nucleotides X and Y, wherein X=G or A and Y=U or C, particularly

a. between nucleotides G and U; or

b. between nucleotides A and U; or

c. between nucleotides G and C; or

d. between nucleotides A and C

The catalytic polynucleotide for use according to aspect 6, wherein the catalytic core region of the DNAzyme has the sequence shown in

a. SEQ ID NO: 2; or

b. SEQ ID NO: 29; or

c. SEQ ID NO: 30.

The catalytic polynucleotide for use according to aspect 1 , wherein the

i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 5 and the target sequence is the sequence shown, in SEQ ID NO: 3; or (ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 7 or SEQ ID NO: 8 and the target sequence is the sequence shown in SEQ ID NO: 6; or

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 11 and the target sequence is the sequence shown in SEQ ID NO: 9; or

(iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 and the target sequence is the sequence shown in SEQ ID NO: 12; or

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18 and the target sequence is the sequence shown in SEQ ID NO: 16; or

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 20 or SEQ ID NO: 21, or SEQ ID NO: 22 and the target sequence is the sequence shown in SEQ ID NO: 19; or

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 24 or SEQ ID NO: 25 and the target sequence is the sequence shown in SEQ ID NO: 23.0. A pharmaceutical composition comprising one or more of the catalytic polynucleotides defined in any one of aspects 1-8, particularly

a. one or more ribozymes as defined in any one of aspects 1 to 4 and 8; and/or b. one or more DNAzymes as defined in any one of aspects 5 to 8,

which catalytic polynucleotides are capable of cleaving a target mRNA of a protein, which can switch its native structure to a misfolded pathogenic isofom (misfolding protein) or is present in a misfolded, pathogenic isoform (misfolded protein),

particularly a protein selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, huntingtin, Tau protein, superoxide dismutase 1 (SOD) protein, and insulin,

for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins (protein-misfolding disease); particularly a disease, condition or disorder selected fro the group consisting of Creutzfeldt-Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie,

in a subject,

particularly an animal; particularly a human;

1. The pharmaceutical composition for use according to aspect 9 comprising the catalytic polynucleotide in a therapeutically effective amount, particularly together with a pharmaceutically acceptable carrier and/or excipient.

2. The catalytic polynucleotide for use according to any one of aspects 1 to 8 or the pharmaceutical composition for use according to any one of aspects 9 to 10, comprising

a. cleaving one of all triplet sequences as defined in any one of aspects 3 to 4; and/or

b. cleaving one of all duplet sequences as defined in aspect 6.

3. The catalytic polynucleotide for use according to any one of aspects 1 to 8 or the pharmaceutical composition for use according to any one of aspects 9 to 10, comprising cleaving the mKNA of

a. prion protein; and/or

b. tau protein; and/or

c. alpha-synuclein protein; and/or

d. huntingtin protein; and/or

e. amyloid precursor protein (APP); and/or

f. superoxide dismutase 1 (SOD) protein. 4. A method for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins (protein-misfolding diseases),

particularly a disease, condition or disorder selected from the group consisting of Creutzfeldt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie,

comprising administering the catalytic polynucleotide as defined in any one of aspects 1 to 8 and 11 to 12, or the pharmaceutical composition as defined in any one of aspects 9 to 12, to a subject particularly an animal;

particularly a human;

5. Use of the catalytic polynucleotide as defined in any one of aspects 1 to 8 and 11 to 12, or the pharmaceutical composition as defined in any one of aspects 9 to 12, for cleaving the mRNA of a target protein, which can switch its native structure to a misfolded pathogenic isoform (misfolding protein) or is present in a misfolded, pathogenic isoform (misfolded protein),

in vitro or in vivo, particularly by bringing the catalytic polynucleotide into contact with the mRNA of the target protein.

6. The method of aspect 13 or the use of aspect 14 comprising selecting of GUC-, AUC-, UUC-, CUC-, GUA-, AUA-, UUA-, CUA-, GUU-, AUU-, UUIJ-, CUU- or GU-, AU - , GC-, AC- sequences, respectively, by applying Sequential-Folding- Algorithras to predict the accessibility for ribozymes or DNAzymes, respectively, during the synthesis of the corresponding mRNA.

REFERENCE LIST

Bemhart SHI, Hofacker JL, Stadler PF. Local RNA base pairing probabilities in large sequences. Bioinformatics 2006; 22: 614-615

Bfleier Agozzi A, Sailer A et.al. Mice devoid of PrP are resistant to sorapie. Cell 1993; 73; 1393-1347

Biieler H, Fischer M, Lang Y, et.al. Normal development and behavior of mice lacking the neuronal cell-surface PrP protein. Nature 1992; 356: 577-582

Burnett JC, Rossi JJ. RNA-based therapeutics: current Progress and future prospects. Chem. Biol. 2012; 19: 60-71

Cairns MJ, King A, Sun L-Q. Optimization of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purinee-cytosine target sites. Nucleic Acids Res. 2003; 31 : 2883-2889

Eisenberg D, Jucker M. The amyloid state of proteins in human diseases. Cell 2012; 148: 1 188-1203

Fokina AA, Stetsenko DA, Francois JC. DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes. Expert opinion on Biological Therapy. Informa healthcare 2015; 15: 689-711

Gavin and Gupta, J. Biol. Chem., 1997; 272: 1451-1472

Grad LI, Guest WC, Yanai A, Pokrishevsky E, O'Neill MA, Gibbs E, Semenchenko V, Yousefi M, Wishart DS, Plotkin SS, Cashman NR. Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc. Natl. Acad. Sci. USA 201 1 , 108: 16398-16403 Guerricr-Takada C, Gardiner K, Marsh T, Pace N, Altman S. The RNA moiety of

ribonuclease P is the catalytic sub unit of the enzyme. Cell 1983; 35: 849-857

Haumann C, Luptak A, Perreault J, de la Pena M. The ubiquitous hammerhead ribozyme. RNA 2012; 18: 871-885

Haussecker D. The Business ofRNAi Therapeutics in 2012. Mol. Ther. Nucleic Acids. 2012;

2, eg

Hornlimann B, Riesner D, Kretzschmar H (eds.) Prions in Humans and Animals. De Gruyter, Berlin, New York; 2007

Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequences of

Tetrahymena. Cell 1982; 31 :147-157

Lauren, J, Gimbel, D.A., Nygaard, H.B., Gilbert, J.W. and Shittmatter, S.M. (2009) Cellular prion protein mediales impairment of synaptic plasticity by amyloid-β oligomers. Nature 457, 1128-1132.

Lightfoot HL, Hall J. Target mRNA inhibition by oligonucleotide drugs in man. Nucleic Acids Res. 2012; 40: 10585-10595

Luk KC, Kehm VM, Zhang B, O'Brien P, Trojanowski JQ, Lee VM. Intracerebral inoculation of pathological a-synuclein initiates a rapidly progressive neurodegenerative a- synucleinopathy in mice. J. Exp. Med 201 ; 209: 975-986

Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. Amyolid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. USA 1985. 82, 4245-4249

Pearson, W.R. (1990), Methods in Enzymology 183, 63-98 Pmsiner SB, Novel proteinaceous infections particles cause scrapie. Science 1982; 216: 136- 144

Pmsiner SB, Shattuck Lecture- Neurodegenerative diseases and prions. Cell. 2012; 148, 1188-1203

Rademakers R, Hutton M. The genetics of frontotemporal lobar degeneration. Curr. Neurol.

Neurosci. Rep. 2007; 7: 434-442

Salwierz A. Studies on the interaction of the cellular and the pathogenic isofoms of the prion protein in the presence of the lipid membrane, Thesis 2009, Htinrich-Htine-University Diisseldorf

Santoro SW, Joyce GF. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA. 1997; 94; 4262-4266

Sharma VK, Rungta P, Prasad Ak. Nucleic acid therapeutics: basic concepts and recent developments. RSC Adv. 2014; 4: 16618-16631

Sydow A, Mandelkow EM. 'Proin-like' propagation of mouse and human tau aggregates in an inducible mouse model of tauopathy. Neurodegener. Dis. 2010; 7: 28-31

Tafer H, Arneres SI, Obemosterer G, Gebeshuber CA, Schroeder R, Martinez J, Hofacker XL. The impact of target site accessibility on the design of effective siRNAs. Nat. Biotech. 2008, 26: 578-583

The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 1993; 72: 971-983

Tremblay P, Meiner Z, Galon M, et.al. Deoxycycline control of prion protein transgene expression modulates prion disease in mice. Proc. Natl. Acad. Sci. USA 1998; 95: 12580- Watts IK, Corey DR. SilenciBg disease genes in the laboratory and the clinic. J. Pathol. 2012; 226; 365-379

Zoumadakis M, Tabler M.Comparative analysis of cleaYage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymcs. Nucleic Acids Res. 1995; 23: 1192-1 196

Claims

1. A catalytic polynucleotide selected from the group of RNA-DNAzyme and DNAzyme capable of cleaving a target mR A of a protein, which can switch its native conformation to a misfolded pathogenic isoform (misfolding protein) or is present in a misfolded pathogenic isoform (misfolded protein), for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded or misfolding pathogenic proteins (protem-misfolding diseases) in a subject suffering from such a disease or disorder to lower the concentration of said protein in the subject.

2. The catalytic polynucleotide for use according to claim 1 which is a RNA-DNAzyme.

3. The catalytic polynucleotide for use according to claim 1 which is a DNAzyme.

4. The catalytic polynucleotide for use according to any one of claims 1-3, wherein the subject is an animal.

5. The catalytic polynucleotide for use according to any one of claims 1-3, wherein the subject is a human.

6. The catalytic polynucleotide for use according to any one of claims 1-5, wherein the misfolding protein or the misfolded protein is a protein which shows substantially no changes or alterations of the phenotype in the subj ct when treated with the catalytic polynucleotide.

7. The catalytic polynucleotide for use according to any one of claims 1-6, wherein the misfolding protein or the misfolded protein is a protein selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, Tau protein, superoxide dismutase 1 (SOD) protein, and insulin.

8. The catalytic polynucleotide for use according to any one of claims 1-7, wherein the disease, condition or disorder is associated with misfolding pathogenic proteins or misfolded pathogenic proteins, which bind to the prion receptor on the cell surface.

9. The catalytic polynucleotide for use according to any one of claims 1-8, wherein the disease, condition or disorder associated with misfolding pathogenic proteins or misfolded pathogenic proteins is selected from the group consisting of Creutzfeldt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

10. The catalytic polynucleotide for use according to any one of claims 2-9, wherein said catalytic polynucleotide is a R A-DNAzyme comprising a core region of deoxyribonucleotides comprising the catalytic domain, flanked 3' and 5' by antisense sequences consisting of ribonucleotides, which are complementary to the target mRNA sequences flanking the cleavage site on the target mRNA.

1 1. The catalytic polynucleotide for use according to claim 10, wherein the core region comprising the catalytic domain has between 2 and 100 deoxyribonucleotides, particularly between 8 and 50 deoxyribonucleotides, particularly betwee 10 and 20 deoxyribonucleotides, particularly between 13 and 19 deoxyribonucleotides, particularly 15-17 deoxyribonucleotides, particularly 16 deoxyribonucleotides. .

12. The catalytic polynucleotide for use according to claim 10, wherein each of the flanking regions 3' and/or 5' of the core region has a antisense sequence comprising between 2 and 100 ribonucleotides, particularly between 2 and 24 ribonucleotides, particularly between 2 and 12 ribonucleotides, particularly between 8 and 10 ribonucleotides, particularly between 6 and 8 ribonucleotides, complementary to the corresponding sequences 5' and 3^* of the cleavage site on the target-mRNA.

13. The catalytic polynucleotide for use according to claim 12 wherein at least the nucleotide of the 3' flanking region, which is 3^* of the catalytic core region is a deoxyribonucleotide, which binds to the corresponding nucleotide 5' of the dinucleotide cleavage site on the target RNA.

14. The catalytic polynucleotide according to claims 10, wherein the catalytic core region of the RNA-DNAzyme has 16 deoxyribonucleotides and each flanking region 3' and/or 5' of the catalytic core region has between 6 and 8 ribonucleotides, which are complementary to the nucleotides 5' and 3' of the dinucleotide cleavage site on the target RNA. .

15. The catalytic polynucleotide according to any one of claims 2-14, wherein at least one of the deoxyribonucleotides of the core region, particularly the 5' deoxyribonucleotide of the core region, binds to the pyrimidine nucleotide of the dinucleotide cleavage site on the target RNA, whereas the purine nucleotide remains unbound.

16. The catalytic polynucleotide for use according to any one of claims 1 and 3-9, wherein the catalytic polynucleotide is a DNAzyme comprising a core region of deoxyribonucleotides comprising the catalytic domain flanked 3' and 5' by antisense sequences consisting of deoxyribonucleotides which are complementary to the target mRNA sequences flanking the cleavage site on the target mRNA.

17. The catalytic polynucleotide for use according to claim 16, wherein the core region of the DNAzyme has between 2 and 100 deoxyribonucleotides, particularly between 8 and 50 deoxyribonucleotides, particularly between 10 and 20 deoxyribonucleotides, particularly between 13 and 19 deoxyribonucleotides, particularly 15-17 deoxyribonucleotides, particularly 16 deoxyribonucleotides. ,

18. The catalytic polynucleotide for use according to claim 17, wherein the catalytic core region of the DNAzyme has 16 deoxyribonucleotides with the 5' deoxyriboriucleoti.de binding to the pyrimidine of the dinucleotide cleavage site on the target RNA, and each flanking region 3' and/or 5' of the catalytic core region has between 8 and 12 deoxyribonucleotides, which are complementary to the nucleotides 5* and 3^* of the dinucleotide cleavage site on the target RNA..

19. The catalytic polynucleotide for use according to any one of claims 1, 3-9, and 16-18, at least one of the deoxyribonucleotides of the core region, particularly the 5^* deoxyribonucleotide of the core region, bind to the pyrimidine nucleotide of the dinucleotide cleavage site on the target RNA, whereas the purine nucleotide remains unbound.

20. The catalytic polynucleotide for use according to any one of claims 1-1 , wherein the target mRNA is cleaved between nucleotides X and Y, wherein X=G or A and Y=U or C.

21. The catalytic polynucleotide for use according to claim 19, wherein the target mRNA is cleaved

a) between nucleotides G and U;

b) .between nucleotides A and U;

c) .between nucleotides G and C;

d) between nucleotides A and C; or

e) between CG and U, AG and U, and UG and U.

22. The catalytic polynucleotide for use according to any one of claims 1-21, wherein the catalytic core region has the sequence shown in

(i) SEQ ID NO: 2;

(ii) SEQ ID NO: 29;

(iii) SEQ ID NO: 30.

23. The catalytic polynucleotide for use according to any one of claims 1-21, wherein said catalytic polynucleotide recognizes the CGU, AGU, and UGU cleavage sites and cleaves the target RNA between nucleotides CG and U, AG and U, or UG and U and wherein the flanking regions 3' and/or 5' of the catalytic core region comprises deoxyribonucleotides or riboaucleotides, which are in anti sense orientation to one of the following sequences 5' and 3' to the CGU, AGU, and UGU cleavage sites:

1 21 AU AAUUGUCAAA AACC-2 Ϊ 62

2 1 38 GGUCUUCCUGUUL^JCACCA-l 156

3 2682-AAAAAAAUUGUAAAUGUUU-2700

4 1364 CCAGU AA AAGUAUAACAGC - 1382

5 1 O-AUUUUUACAGUCAAUGAGC-28

6 1289-AAACAU AG AGU AG ACCUGA 1307

7 1893-AUUUUAGAUGUUUAAAGGA-l 911

8 1761 -GUAA AU AIJUGUC AC AAC AC- 1779

9 2094 AAAUGGUCAG UGUGC AAAG-2112

10 2264 -UGAUUUGAAGUGGAAAAAG -2282

11 2297 -UUAAUUAAAGUAAAAUUAU-2315

12 1957 AGCUGAA AAGU AAAUUGCC- 1975

13 2327-UUUGAUAUUGUCACCUAGC-2345

14 1752 -UCAACAAGAGUAAAUAUUG- 1770

15 2688-AUUGUAAAUGUUUAAUAUC-2706

16 2133-UUUAUUUCUGUCUCAUAAU-2151

17 1995-AUCUCCUUUGUCCAUIJUAC-2013

18 829- ACAUGCACCGUUACCCCAA-847

19 1234-UCUCUOJUUGUCCCGGAUA-12S2

20 2395-GUAUUCUAUGUAAAAAUAU-2413

21 108fr-UCUCCACCUGUGAUCCUCC- 1 98

22 2386-ACUUUGUGAGUAUUCUAUG- 2404

23 2666-UCUUGUUUUGUUAUAUAAA-2684

24 67Q-ACAAGCCGAGUAAGCCAAA 688

25 2287 - UCUGUUAAUGUUAAUU AAA 2305

26 2621 -AAUAUGCAUGUACUUUAUA-2639

27 88-UCUCAACUCGUUUUUUCCG-l 06

28 358 GCC AGGCCAGUAAAAGUAU-1376

29 2661 -CAUGUUCUUGUUUUGUU AU 2679

30 2047H3CUGCAGUUGUGAAAGCAC-2065 2167-UUAGGUCAAGUUCAU AGUU-2185 656 CCACAGUCAGUGGAACAAG-674 2317 CCCUGAAUUGUIJUGAUAUU -2335 66-CUGAUGCAAGUGUUCAAGC-84 1001 -UG AGC AG AUGUGU AUG ACQ 1019 1502-UAGAGCUCAGUAUAOJAAU-1520 2461 -UUUAG AGC AGUUAAC AUCU-2479 2643 UCUAUAUUUGUAACUUUGC-2661 1812-AAC AUAACUGU AACAU AUA-- 1830 1326-UUUG AUUGAGUUC AUCAUCJ- 1344 1413-UUGG ACUUAGUGC AACAGG- 1431 2281 -AGAAAUUCUGUUAAUGUUA-2299 969- GAGACCGACGUUAAGAUGA-987 1 11-ACCCUAUAUGUGGCAUUCC-l 929 2382 -UUGCACUUUGJJGAGUAUUC-2400 2518- ACUUAAUAUGUGGGAAACC-2536 2574 -U CG U UUCAUGUAAG AAUCC 2592 412-CC ACAUGGAGUG ACCUGGG 430 8 4--GUUACU AUCGUG A A AAC AU-832 866-C AUGG AUGAGUAC AGC AAC-884 1721 -GUUUUCACAGUAUGGGCUA- 1739 2474-ACAUCUGAAGUGUCUAAUG 2492 2535 -CCCUUUUGCGUGGUCCUUA-2553 805- AUGAGGACCG JUACUAUCG 823 2174 AAGUUCAUAGUU UCUGU AA-2192 2567-CACUGAAUCGUUUCAUGUA-2585 843-CCCAACCAAGUGU ACUACA-861 790-AUUUCGGCAGUGACUAUGA-808 2495 U U AACU J JUGUA AGG U ACU-- 2513 170fr UGGGGUG AUGUliTOA TJU- 1724 1800-AU AUUCACAGUGAAC AUAA- 1818 888— AACAACUUUGUGCACGACU-906 1013-UAUC ACCC AGUACGAGAGG- 1031 64 9QQ-CACGACUGCGUCAAUAUCA-918

65 2044-AGAGCUGCAGUUGUGAAAG-2062

66 2555 JCUUACAAUGUGCACUGAA"2573

67 1682-AUGAGCUCUGUGUGUACCG- 700

68 1 86-GCUCUGUGUGUACCGAGAA- 1704

69 2343-AGCAGAUAUGUAUUACUUU-2361

70 399-GUUCUCULTUGUGGCCACAU-417

24. The catalytic polynucleotide for use according to any one of claims 1, 3-9, and 16-23, wherein the

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 5;

(ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 8;

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 11 ;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 17;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 20 or SEQ

ID NO: 22;

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 24;

(viii) catalytic polynucleotide has the sequence as shown in SEQ ED NO: 32;

(ix) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 34;

(x) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 36;

(xi) catalytic polynucleotide has the sequence as shown in SEQ ED NO: 38;

(xii) catalytic polynucleotide has a sequence, which shows between 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 5, 8, 11, 13, 15, 17, 20, 22, 24, 32, 34, 36, 38;

(xiii) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 5, 8, 11 , 13, 15, 17, 20, 22, 24, 32, 34, 36, 38.

25. The catalytic polynucleotide for use according to claims 1, 2-15, and 20-23, wherein the

(i) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 39;

(ii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 40;

(iii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 42; (iv) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 43;

(v) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 44;

(vi) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 45

(vii) catalytic polynucleotide has the sequence as shown in SEQ ID NO: 46;

(viii) catalytic polynucleotide has a sequence, which shows between 90%, 91%, 92%, 93%, 94%, 95%, 36%, 97%, 98%, 99% sequence identity to any one of the sequences of SEQ ID NOs: 39, 40, 42, 43, 44, 45, 46;

(ix) catalytic polynucleotide has a sequence, which hybridizes, particularly under stringent hybridization conditions as defined herein, to any one of the sequences complementary to SEQ ID NOs: 39, 40_» 42, 3_» 44, 45, 46.

26. The catalytic polynucleotide according to any one of claims 1-25 for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded or misfolding pathogenic proteins (protein-misfolding diseases) in a subject suffering from such a disease or disorder, particularly an animal, particularly a human, to lower the concentration of said protein in the subject.

27 A pharmaceutical composition comprising one or more of the catalytic polynucleotides defined in any one of claims 1-25 for use in the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins (protein-misfolding disease) in a subject suffering from such a disease or disorder, particularly an animal, particularly a human, to lower the concentration of said protein in the subject.

28. The pharmaceutical composition for use according to claim 27, comprising one or more RN A-DN Azymes as defined in any one of claims 1, 2-15, 20-23, 25 and 26.

29. The pharmaceutical composition for use according to claim 28, comprising one or more DN Azymes as defined in any one of claims 1 , 3-9, and 16-24.

30. The pharmaceutical composition for use according to claim 25, comprising a combination of one or more DNAzymes as defined in any one of claims 1, 3-9, and 16-24 and/or one or more RNA-D Azymes as defined in any one of claims 1, 2-15, 20-23, 25 and 26.

31. The pharmaceutical composition for use according to any one of claims 27-30, wherein the subject is an animal.

32. The pharmaceutical composition for use according to any one of claims 27-30, wherein the subject is a human.

33. The pharmaceutical composition for use according to any one of claims 27-32, wherein the misfolding protein or the misfolded protein is a protein which shows substantially no changes or alterations of the phenotype in the subject when treated with the catalytic polynucleotide.

34. The pharmaceutical composition for use according to any one of claims 27-33, wherein the misfolding protein or the misfolded protein, is a protein selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, Tau protein, superoxide dismutase 1 (SOD) protein, and insulin.

35. The pharmaceutical composition for use according to any one of claims 27-34, wherein the disease, condition or disorder is associated with misfolding pathogenic proteins or misfolded pathogenic proteins, which bind to the prion receptor on the cell surface.

36. The pharmaceutical composition for use according to any one of claims 27-35, wherein the disease, condition or disorder associated with misfolded pathogenic proteins is selected from the group consisting of Creutzfeldt-Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tenopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

37. The pharmaceutical composition for use according to any one of claims 27-36 comprising the catalytic polynucleotide in a therapeutically effective amount.

38. The pharmaceutical composition for use according to any one of claims 27-37 comprising a pharmaceutically acceptable carrier and/or excipient.

39. The catalytic polynucleotide for use according to any one of claims 1-26 or the pharmaceutical compositio for use according to any one of claims 27-38, comprising cleaving one or all duplet sequences as defined in any one of claims 20-21.

40. The catalytic polynucleotide for use according to any one of claims 1-26 or the pharmaceutical composition for use according to any one of claims 27-38, comprising cleaving the mRNA of

(i) the prion protein;

(ii) the tan protein; and/or

(iii) alpha-synuclein protein; and/or

(iv) the amyloid precursor protein (APP);

(v) superoxide dismutase 1 (SOD) protein; and/or (vi) a misfoiding or misfolded protein binding to the prion receptor on the cell surface.

41. A method for the treatment, prevention and/or alleviation of a disease, condition or disorder associated with misfolded pathogenic proteins (protein-misfolding diseases) comprising administering a catalytic polynucleotide as defined in any one of claims 1- 26 or the pharmaceutical composition according to any one of claims 27-38 to a subject suffering from such a disease or disorder to lower the concentration of said protein, in. the subject.

42. The method of claim 41 wherein the subject is an animal.

43. The method of claim 41 , wherein the subject is a human.

44. The method of any one of claims 41 -43, wherein the misfoiding protein or the misfolded pathogenic protein is selected from the group of prion protein, amyloid precursor protein (APP), alpha-synuclein, tau protein, superoxide dismutase 1 (SOD) protein, and insulin.

45. The method of any one of claims 41 -44, wherein the disease, condition or disorder associated with misfolded pathogenic proteins is selected from the group consisting of Creutzfel dt- Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD), Down's Syndrome, Amyotrophic lateral sclerosis (ALS), Frontal temporal dementia (FTD), Tauopathy, Glaucoma, Diabetes, Fibrosis or eradication of animal Chronic Wasting disease (CWD), and scrapie.

46. Use of the catalytic polynucleotide as defined in any one of claims 1-26 or the pharmaceutical composition according to any one of claims 27-38, for cleaving the mRNA of a target protein, which can switch its native conformation to a misfolded pathogenic isoform (misfolding protein) or is present in a misfolded, pathogenic isoform (misfolded protein),

particularly a protein selected from the group of prion protein, beta amyloid precursor protein (APP), alpha-synuclein, Tau protein, superoxide dismutase 1 (SOD) protein, and insulin,

47 The method of any one of claims 41 -44 or the use of claim 46 comprising selecting or GU-, AU-, GC-, AC-, or CGU-, AGU-, UGU-sequences, respectively, by applying Sequential-Folding- Algorithms to predict the accessibility for DNAzymes or RNA- DNAzymes, respectively, during the synthesis of the corresponding mRNA. , Use of the catalytic polynucleotide as defined in any one of claims 1-26, wherein said catalytic polynucleotide recognizes the CGU, AGU, and UGU cleavage sites and wherein the flanking regions 3' aad/or 5' of the catalytic core region comprises deoxyribonucleotides or ribonucleotides, which are in antisense orientation to one of the following sequences 5^* and 3⁵ to the CGU, AGU. and UGU cleavage sites:

1 2144-CUC AUAAUUGUCAAAAACC 2162

2 1 138-GGUCUUCCUGUUUUC ACCA- 1156

3 2682-AAAAAAAUUGUAAAUGUUU-2700

4 1364 -CCAGUAAAAGUAUAACAGC-1382

5 10- AUUUUUACAGUC AAUGAGC -28

6 1289-AAAC AUAG AGU AGACCUG A- 1307

7 893-AUUUUAGAUGUUUAAAGGA-l 911

8 1761 -GUAAAUAUUGUC AC AACAC-1779

9 2094 AAAUGGUCAGUGUGCAAAG-21 12

10 2264-UGAUUUGAAGUGGAAAAAG-2282

11 2297 -UUAAUUAAAGUAAAAUUAU-2315

12 1957- AGCUGAAAAGUAAAUUGCC 197S

13 2327-UUUGAUAUUGUCACCUAGC-2345

14 1752 - UCAAC AAGAGUAAAUAUUG- 1770

15 2688- AUUGUAAAUGUUUAAUAUC - 2706

16 2133 -UUUAUUUCUGUCUCAUAAU-2151

17 1995-AUCUCCUUUGUCCAUUUAC-2013

18 829-ACAUGCACCGUUACCCCAA-847

19 1234-UCUCUCUUUGUCCCGGAUA- 1252

20 2395-GUAUUCUAUGU AAAAAUAU-2413

21 1080-UCUCCACCUGUGAUCCUCC-l 098

22 2386 ACUUUGUGAGUAUUCUAUG-2404

23 2666-UOJUGUUUUGUUAUAUAAA-2684

24 670-AC AAGCCGAGU AAGCCAAA-688

25 2287 UCUGUUAAUGUUAAUUAAA-2305

26 2621 -AAUAUGCAUGUACUUUAU A - 2639

27 88 - UCUCAACUCGUUUUUUCCG-106

28 1358-GCCAGGCCAGUAAAAGUAU-l 376 2661 -CAUGUUCUUGUUUUGUUAU-2679 2047--GCUGCAGUUGUGAAAGCAC-2065 2167-UU AGGUCAAGUUC AUAGUU-2185 656 -CCACAGUCAGUGGAACAAG -674 2317-CCCUGAAUUGUUUGAUAUU-2335 66 CUGAUGCAAGjUGUUCAAGC-84 1001 -UGAGCAGAUGUGUAUCACC-1019 1502 -UAGAGCUCAGUAUACtJAAU-1520 2461-UUUAGAGCAGUUAACAUCU-2479 2643-UCUAUAUUUGUAACUUUGC-2661 1812-AAC AU AACUGUAAC AUAU A- 1830 1326-UUUGAUUGAGUUCAUCAUG-13 4 1413-UUGGACUUAGUGCAACAGG 1431 2281 - AG AAAUUCUGUU AAUGUUA-2299 969-GAGACCGACGlJUAAGAUGA 987 1911 ACCCU AU AUGUGGC AL JCC-- 1929 2382-UUGCACUUUGUGAGUAUUC--2400 2518- ACUUAAU AUGUGGG AAACC-2536 2574 "UCGUtJUCAUGUAAGAAUCC- -2592 412-CC AC AUGGAGUG ACCUGGG-430 814--GUUACUAUCGUGAAAACAU -832 866-CAUGGAUGAGUACAGCAAC- 884 1721 -CUUUUCACAGUAUGGGCUA-1739 2474-ACAUCUGAAGUGUCUAAUG -2492 2535-CCCUUUUGCGUGGUCCUUA-2553 805^~AUGAGGACCGUUACUAUCG--823 2174— AAGUUC AUAGUUUCUGUAA-21 2 2567-CACUGAAUCGUUUCAUGUA -2585 843-CCC AACC AAGUGUACUAC A-861 790-AUUUCGGCAGUGACUAUGA-808 2495-UUAACUUTOJGUAAGGUACU-2513 1706 UGGGGUGAUGUUUU ACUUU- 1724 1800- AUAUUC AC AGUG AAC AUAA- 1818 62 888-AACAACUUUGUGCACGACU-906

63 1013 UAUCACCCAGUACGAGAGG- 1031

64 900-CACGACUGCGUCAAUAUCA-918

65 2044-AGAGCUGCAGUUGUGAAAG-2062

66 2555 -GCUU AC AAUGUGC ACUG AA 2573

67 1682-AUGAGCUCUGUGUGUACCG-l 700

68 1686-GCUCUGUGUGUACCGAGAA-l 704

69 2343 AGCAGAUAUGUAUUACUUU-2361

70 399-GUUCUCUUUGUGGCCACAU--417;

for cleaving in vivo or in vitro one of more of the above target RNA sequences between nucleotides CG and U, AG and U, or UG and U

49. A MNA-DNAzyme capable of cleaving a target mBNA comprising a core region of deoxyribonucleo tides comprising the catalytic domain, which core region is flanked 5* and 3' by antisense sequences consisting of ribonucleotides which are complementary to the target mRNA sequences flanking the cleavage site on the target mRNA.

50. The RNA-DNAzyme of claim 49, wherein the core region contains a deoxyribonucl eotide which is complementary to and binds to the pyrimidine nucleotide of the dinucleotide cleavage site on the target RNA.

51. The RNA-DNAzyme of claim 50, wherein the deoxynbonucleotide binding to the pyrimidine nucleotide of the dinucleotide cleavage site on the target RNA is the 5^* deoxynbonucleotide of the core sequence.

52. The RNA-DNAzyme as defined in any one of claims 1, 2-15, 20-23, 25 and 26.

53. The RNA-DNAzyme according to any one of claims 49-52 for use in therapy.