Antisense oligonucleotides for inducing exon skipping and methods of use thereof

[0001] The invention relates to new compounds and antisense compositions suitable for carrying out exon skipping. It also provides methods for inducing "exon skipping" using new antisense compounds as well as therapeutic compositions adapted for use in the methods of the present invention.

Background Art [0002] Significant effort is currently being made in researching ways to inhibit or compensate for pathogenic mutations in genes. Using a number of different chemical strategies, antisense technologies have been developed to influence gene expression at many different levels (transcription, splicing, stability, translation). Much of this research has focused on the use of antisense compounds to repair or compensate for abnormal or pathogenic genes in many different disease states.

[0003] Antisense molecules are capable of inhibiting gene expression with excellent specificity. Therefore, numerous scientific studies on oligonucleotides as modulators of gene expression have focused on inhibiting the expression of target genes such as oncogenes or viral genes. Antisense oligonucleotides target both RNA (nothing sense) and DNA, with which they form triple helix structures that inhibit transcription catalyzed by RNA II polymerase. To achieve the desired effect in the event of down-regulation of a particular gene, oligonucleotides must either promote the breakdown of the target mRNA or block the translation of this mRNA, thereby effectively preventing de novo synthesis of the unwanted target protein.

[0004] These techniques are not useful in cases where the goal is to upregulate the native protein or compensate for mutations that cause premature translation termination, such as nonsense mutations or mutations leading to a change in reading frame.

[0005] In addition, it has been shown that in the event that the structure of a normally functional protein is prematurely disrupted due to a mutation occurring in the gene encoding this protein, it is possible to restore the production of functional protein to some extent using antisense oligonucleotide technology by intervention during splicing processes [Sierakowska H et al., (1996) Proc. Natl Acad Sci USA 93, 12840-12844 ;. Wilton SD, et al., (1999) Neuromusc Disorders 9, 330338; .. Deutekom JC van et al. (2001) Human Mol Genet 10, 1547/54]. In such cases, the transcript of the defective gene should not be subject to targeted degradation, which means that the chemical modifications of the antisense oligonucleotides should not promote the degradation of the target mRNA.

[0006] In many genetic diseases, the effect of mutations on the possible expression of a gene can be regulated in the process of targeted exon skipping during the splicing (transcript assembly) process. The splicing process is catalyzed by a complicated multi-molecular apparatus that pulls the exon-intron adjacent pre-mRNAs to each other and cleaves the phosphodiester bonds at the ends of the introns with their further transformation between the exons to be joined. This complex and very precise process is done through the sequence motifs present in pre-mRNA, which are relatively short semiconservative RNA segments to which various nuclear splicing factors bind and then participate in splicing reactions. By changing the way the splicing apparatus reads or

EP1766010B1PL recognizes the motifs involved in pre-mRNA processing, it is possible to obtain mRNA molecules in various ways. It is now known that most human genes undergo alternative splicing during normal gene expression, although no mechanisms have been identified to govern this process. Using antisense oligonucleotides, it has been shown that errors and deficiencies in the encoded mRNA can be bypassed or removed from mature gene transcripts.

[0007] The extent of the occurrence of a genetic deletion or "exon skipping" in a naturally occurring splicing process is not fully understood, although these processes have been shown to occur at many levels, although usually to a very low degree (Sherrat TG, et al., 1993 ) Hum Genet Am J 53, 1007/15). It is assumed that if exons associated with pathogenic mutations can be specifically removed from some genes, sometimes a shortened protein product with biological properties similar to the native protein or biological activity sufficient to improve the disease state caused by the mutation present in the target gene may be obtained [Lu QL et al. (2003) Nature Medicine 9, 1009/14; Aartsma-Rus et al. (2004) Hum Genet Am J 74: 83 92].

[0008] This process of targeted exon skipping can be particularly useful for long genes containing many exons and introns, where there is redundancy of exons in the gene, or when the protein encoded by such genes performs its functions despite the absence of one or more specific exons ( for example. for the dystrophin gene, which consists of 79 exons, or probably for some collagen genes that encode repetitive sequence blocks, or for the huge nebulin and thitin genes, which are composed of 80 and over 370 exons, respectively).

[0009] Attempts to redirect gene processing to treat genetic diseases associated with truncations caused by mutations in various genes have focused on the use of antisense oligonucleotides that: (1) completely or partially overlap the elements involved in the splicing process, or (2) bind to pre-mRNA at a location close enough to that element in a way that interferes with the binding and action of splicing factors that normally mediate specific splicing reactions occurring for this element (e.g., they bind to pre-mRNA at a site within the 3rd, 6th or 9th nucleotide of the element to be blocked).

[0010] For example, in vitro and in vivo modulation has been described using antisense oligorybonucleotides in the case of splicing pre-mRNA encoding a dystrophin mutant. In one type of dystrophin mutation, described in Japan, a 52 bp deletion mutation causes exon 19 and its adjacent introns to be spliced ​​in the process [Matsuo et al. (1991) J Clin Invest. 87: 21272131]. A minigen-based splicing system was used in an in vitro system to show that a 31mer 2'-O-methyl oligorybonucleotide complementary to half the sequence located at the 5 'end of the deletion sequence in exon 19 of the Kobe dystrophin gene, inhibited wild-type pre-mRNA splicing [Takeshima et al. (1995), J. Clin. Invest., 95, 515-520]. The same oligonucleotide was used to induce exon skipping from a native dystrophin gene transcript in human lymphoblastoid culture.

[0011] Dunckley et al. In Nucleosides & Nucleotides, (1997), 16, 1665-1668 described in vitro constructs for splicing analysis of exon 23 of mutated dystrophin in genetically modified mdx mice as a model of muscular dystrophy. There are discussed in vitro analysis plans for these constructs using 2 'modified oligonucleotides that target the splice sites located within the exon 23 of mouse dystrophin or sites adjacent to this exon. However, no target sites or sequences are given in this paper.

[0012] It was then announced that 2'-O-methyl-oligorybonucleotides have the ability to correct dystrophin defect in myoblasts derived from mdx mice. An antisense oligonucleotide directed to the 3 'splice site of intron 22 in the mouse dystrophin gene was found to omit the mutated exon as well as several flanking exons and create a new frame-compatible dystrophin transcript that contained a new internal deletion. This mutated dystrophin was expressed in 1-2% myotubes in mdx mice administered an antisense oligonucleotide. The use of other oligonucleotide modifications, such as 2'-O-methoxyethyl phosphodiester, has been described [Dunckley et al. (1998) Human Mol. Genetics, 5, 1083-90].

[0013] Thus, antisense molecules can be a tool for the treatment of genetic disorders such as Duchenne Muscular Dystrophy (DMD). However, attempts to induce exon skipping using antisense molecules have so far led to inconclusive results. Errington et al. [(2003) J Gen Med 5, 518-527] described studies on exon 19 dystrophin, in which it was possible to induce omission of this exon from dystrophin pre-mRNA using various antisense molecules directed to flanking splice sites or defining its positions motifs within this exon.

[0014] In contrast to the skipping of exon 19, which seems easy to perform, the first report of skipping exon 23 in mdx mice by Dunckley et al. (1998) is now considered a report on a rather only naturally occurring revertant transcript or an artifact, not a product of any real antisense oligonucleotide activity. In addition to the lack of constant generation of transcripts devoid of exon 23, Dunckley et al. (1998) did not show any course during induced omission of the exon or at least titration of antisense oligonucleotides in order to show the dependence of observed effects on dose, where exon skip levels would correlate with increasing or decreasing amounts of the antisense oligonucleotide. In addition, other researchers failed to repeat the experiments described in this paper.

[0015] The first example of specific and repetitive skipping of an exon on a mouse mdx model has been described by Wilton et al. [(1999) Neuromuscular Disorders 9, 330-338]. By directing antisense molecules to the donor splice site, during 6 hours of treatment of the cell culture, a constant and efficient skipping of exon 23 in dystrophin mRNA was induced. Wilton et al. (1999) also describe the use of longer antisense oligonucleotides directed to the mouse dystrophin pre-mRNA acceptor region. The authors of the paper failed to repeat the results published by Dunckley et al. (1998). Using the selected set of antisense oligonucleotides directed to the acceptor splice site of intron 22, it was not possible to repeatedly detect the skipping of both exon 23 itself and multiple removal of several flanking exons.

[0016] While the first antisense oligonucleotide directed to intron 23 donor splice induced constant exon skipping in primary myoblast culture, this compound was significantly less effective for immortalized cell cultures expressing higher dystrophin levels. However, when the selection of target sequences and the design of antisense oligonucleotides was improved, the efficiency of specific exon removal increased by almost an order of magnitude [see: Mann CJ et al. (2002) Gen J Med. 4, 644654].

[0017] Other disclosures regarding DMD therapy include CA2507125 and Matsuo M: "Duchenne And Becker Muscular Dystrophy: From Gene Diagnosis To Molecular Therapy" IUBMB LIFE,

EP1766010B1PL vol. 53, no. 3, 1 March 2002 (2002-03-01), pp. 147-152, as well as document WO 2004/083446, published on 30.09.2004.

[0018] Thus, there is still a need to provide antisense oligonucleotides capable of binding and modifying splicing of a target nucleotide sequence. Simply directing antisense oligonucleotides to motifs that are considered key to splicing does not guarantee the effectiveness of this compound in the treatment process.

Summary of the Invention [0019] The present invention provides an antisense oligonucleotide that binds to the human dystrophin gene by inducing omission of the exon in the dystrophin gene consisting of the SEQ ID sequence. ID NO. 181, wherein optionally the uracil (U) bases are replaced by thymine (T) bases.

[0020] In addition, the invention provides a composition comprising the antisense molecule of the present invention and one or more pharmaceutically acceptable carriers and / or diluents.

[0021] The invention further provides an antisense molecule or composition of the present invention for use in a method of treating muscular dystrophy in a patient.

[0022] More specifically, the present invention is defined in the appended claims.

[0023] The choice of destination is crucial for the efficiency of skipping an exon, and thus for its further use as a potential treatment method. The mere design of antisense molecules directed to pre-mRNA target regions in which they are assumed to be involved in splicing does not guarantee effective and specific skipping of the exon. The most obvious or easily defined targets for intervention at the splice level are donor and acceptor splice sites. However, there are less defined or less conserved motifs, including splicing enhancer sequences, silencing elements and branching regions.

[0024] The acceptor and donor splice sites have consensus sequences of 16 and 8 bases, respectively (see Figure 1, which is a diagram of the motifs and domains involved in exon recognition, intron removal, and splicing).

[0025] The invention also relates to the use of purified and single antisense oligonucleotides of the present invention in the manufacture of a medicament for the treatment of a genetic disease. [0026] The solution of the invention can also be used to treat a condition known as Duchenne muscular dystrophy, by administering to a patient that requires treatment an effective amount of an appropriately designed antisense oligonucleotide according to the present invention, appropriate to the specific genetic change occurring in that patient.

[0027] In addition, the invention can be used for the prophylactic treatment of a patient to prevent the development of Duchenne muscular dystrophy or at least to minimize its symptoms, which treatment comprises the step of: administering to the patient an effective amount of an antisense oligonucleotide or pharmaceutical composition containing one or more of these molecules biological.

[0028] Also described herein are kits for treating a genetic disease, which kits consist at least of the antisense oligonucleotide of the present invention, which is in a suitable container, and instructions for its use.

[0029] Other aspects and advantages of the present invention will become apparent to those skilled in the art based on the disclosure contained in the description, which includes references to the following Figures.

Short description of the drawings

EP1766010B1EN [0030]

Figure 1. Schematic representation of the motifs and domains involved in exon recognition, intron removal and splicing.

Figure 2. Schematic representation of the concept of skipping an exon induced by an antisense oligonucleotide to bypass pathogenic mutations (scale not kept). The hatched rectangle means an exon containing a mutation that prevents the translation of the distal portion of the mRNA resulting in a protein product. The black rectangle represents an antisense oligonucleotide that prevents this exon from being included in the mature mRNA.

[0031] Table 1 in the "Examples" section describes 2'-O-methylthiophosphate antisense oligonucleotides that were used to study induced exon skipping during dystrophin pre-mRNA processing. Because 2'-O-methyl-antisense oligonucleotides are more similar to RNA, U is uracil. For other chemical forms of antisense oligonucleotides, such as peptide nucleic acids or morpholine oligonucleotides, these U bases may exist as "T" bases.

Detailed description of the invention

General information [0032] It will be apparent to those skilled in the art that variations and modifications other than those specifically described may be allowed for the invention described herein. It should be understood that the invention includes all such changes and modifications. The invention also includes all steps, properties, compositions and compounds referred to or that have been demonstrated in the specification, on an individual or collective basis, and any combination of two or more stages or properties.

[0033] The invention cannot be limited by the scope of the specific embodiments described herein, which are merely examples. It is obvious that products, compositions and methods that are equivalent in terms of functionality fall within the scope of the invention described herein.

[0034] The sequence identification numbers (SEQ ID NO) contained in this specification, containing information regarding the nucleotide and amino acid sequence, have been collected at the end of the description and prepared using Patentin, version 3.0. Each nucleotide or amino acid sequence is marked on the sequence list by the numeric index <210> followed by the sequence identifier (e.g. <210> 1 <210> 2, etc.). The length, type of sequence and organism indicating the source of origin of each nucleotide or amino acid sequence are indicated by the information contained in the numeric indicator fields <211>, <212> and <213>, respectively. The nucleotide and amino acid sequences referred to in the specification are identified by the information contained in the numeric indicator field <400> followed by the sequence identifier (e.g. <400> 1, <400> 2, etc.).

[0035] The naming system for antisense molecules has been proposed and published to distinguish between different antisense molecules [see: Mann et al., (2002) Gen Med J 4, 644-654]. This nomenclature, which is described below, was particularly important when testing several slightly different antisense molecules directed at the same target region:

H # A / D (x: y) [0036] The first letter denotes a species (e.g. H: human, M: mouse, C: dog)

EP1766010B1EN "#" is the dystrophin exon target number.

[0037] "A / D" means the acceptor or donor splice site at the beginning and end of the exon, respectively.

[0038] (xy) represents the hybridization coordinates, where "-" or "+" indicates sequences within introns or exons, respectively. For example, A (-6 + 18) would indicate the last 6 bases of the intron preceding the target exon and the first 18 bases of the target exon. The nearest splice site would be the acceptor splice site, so these coordinates will be preceded by the letter "A". The D (+ 2-18) assay may describe the hybridization coordinates at the donor splice site, where the last 2 exon bases and the first 18 intron bases correspond to the hybridization site of the antisense molecule. Coordinates indicating the hybridization site only within the exon, represented by the expression A (+65 +85), would indicate a place between the 65th and 85th nucleotide from the beginning of this exon.

[0039] It has not been found that any of the references constitute prior art or is part of the knowledge generally available to persons in the field to which the present invention relates.

[0040] The term "originating" and "originating" as necessarily necessitated herein means that a specific unit can be obtained from a given source, albeit not directly from that source.

[0041] In this specification, unless the context indicates a different meaning, the word "include" or variants such as "includes" or "including" should be understood as an indication of the inclusion of a given entity or group of entities, and not as exclusion of another entity or group of units.

[0042] Other definitions of selected concepts used in this document can be found in the detailed description of the present invention and used throughout the document. Unless otherwise defined, all other scientific and technical terms used herein have the same meanings as those commonly understood by one of ordinary skill in the art to which this invention belongs.

Description of a Preferred Embodiment of the Invention [0043] When the antisense molecule / antisense molecules target nucleotide sequences involved in exon splicing in pre-mRNA sequences, normal exon splicing can be inhibited, which causes the splice apparatus to bypass all mutated exon in mature mRNA. The concept of skipping an antisense oligonucleotide induced exon is shown in Figure 2. For many genes, deletion of the entire exon would lead to the production of a non-functional protein due to loss of important functional domains or a change in reading frame. However, for some proteins, it is possible to shorten the protein by deleting one or more exons, without changing the reading frame and without significantly altering the biological activity of the protein. Such proteins usually act as structural proteins or have functional domains at their ends. The present invention describes antisense molecules capable of binding specific target sequences in dystrophin pre-mRNA and redirecting the processing of this gene.

Antisense Molecules [0044] According to a first aspect of the present invention, antisense molecules are provided, as defined in the claims, capable of binding to a selected target sequence to induce exon skipping. Designing antisense molecules to completely mask consensus splice sites may not necessarily generate exon skipping. Also,

The inventors have found that the size or length of the antisense oligonucleotide alone is not always a major factor in the design of antisense molecules. For some target sequences, such as exon 19, short 12 base long antisense oligonucleotides were able, though not as efficiently as longer oligonucleotides (20-31 bases), to induce exon skipping. For other target sequences, such as murine dystrophin exon 23, antisense oligonucleotides of only 17 bases induced a more efficient skipping of the exon compared to another 25-nucleotide complementary compound.

[0045] The inventors have also found that there is no standard motif that can be blocked or masked by antisense molecules to redirect splicing. In some exons, such as exon 23 of mouse dystrophin, the donor splice site was the most susceptible site for targeted redirection of skipping this exon. It should be noted that the design and testing of a series of exon 23 specific antisense molecules that have associated with overlapping regions of the donor splice site showed significant differences in the efficiency of induced exon skipping. In studies published by Mann et al. (2002) no significant changes in the efficiency of bypassing nonsense mutations that were dependent on hybridization of the antisense nucleotide ["Improved antisense oligonucleotide induced exigon skipping in the mdx mouse model of muscular dystrophy" were demonstrated. J Gen Med 4: 644-654]. Selecting an acceptor site in exon 23 or several internal domains as the target sequence did not lead to the induction of any constant skipping of exon 23.

[0046] For other exons selected for removal, masking of the donor splice site did not induce any skipping of the exon. However, by directing antisense molecules to acceptor splice sites (human exon 8), strong and constant skipping of the exon was induced. It should be noted that the removal of human exon 8 was closely related to the simultaneous removal of exon 9. Since the exon 8 antisense oligonucleotides and the corresponding regions in exon 9 do not show high homology, it does not appear that the simultaneous removal of exon 9 is the result of a cross-reaction. Rather splicing these two exons is inseparably connected with this situation. The described example is not an isolated case. The same effect is observed in dog-derived cells, where selecting for removal of exon 8 resulted in simultaneous skipping of exon 9. Choosing to remove exon 23 in mouse dystrophin pre-mRNA also results in frequent exon 22 removal. This effect is dose-dependent and also indicates the coordinated processing of two adjacent exons.

[0047] For other exons selected for removal, the antisense molecules directed to the donor or acceptor splice site did not induce exon skipping, while the best effect was seen when hybridizing the antisense molecules to regions inside the exon (e.g., exon splicing enhancer sequences present in exon 6 of human dystrophin). Some exons, e.g. both murine and human exon 19 are easily omitted by antisense molecules for which the target sequence is different motifs. This means that the skipping of the selected exon is induced after the use of antisense oligonucleotides that block the donor and acceptor splice sites or exon splicing enhancer sequences present in exon.

[0048] In order to identify and select antisense oligonucleotides suitable for use in exon skip modulation, the nucleic acid sequence whose function is to be modulated must first be determined. For example, it may be a gene (or mRNA obtained by transcription of that gene), the expression of which is associated with a specific disorder or condition, or

EP1766010B1EN nucleic acid molecule derived from an infectious agent. In the context of the present invention, the preferred target (s) are sites involved in mRNA splicing (i.e., donor splice sites, acceptor splice sites, or exon splicing enhancer sequence elements). Potential branching regions for mRNA splicing are also splice branching regions and exon recognition sequences or splicing enhancer sequences.

[0049] Preferably, the present invention aims to provide antisense molecules capable of binding to selected target sequences in dystrophin pre-mRNA to induce effective and constant skipping of the exon. Duchenne muscular dystrophy is the result of a mutation that prevents the synthesis of a functional gene product that encodes dystrophin. Gene defects identified in Duchenne muscular dystrophy usually take the form of nonsense mutations or rearrangements within the genome structure, such as deletions, repeats or microdeletions or insertions that interfere with the reading frame. Because the human dystrophin gene is a large and complex gene, containing 79 exons, which are linked together in splicing to generate mature mRNA with an open reading frame, about 11,000 bases long, so there are many places where these mutations can occur . As a result, treatment based on the use of antisense oligonucleotides, characterized by a wide range of action, i.e. directed at many different pathogenic mutations present in the dystrophin gene, will require the selection of many exons to be removed during the splicing process.

In the context of the present invention, the preferred target (s) are sites involved in mRNA splicing (i.e., donor splice sites, acceptor splice sites, or exon splicing enhancer sequence elements). Splicing branch sites and exon recognition sequences or splicing enhancers are also potential targets for modulation of mRNA splicing.

[0051] The oligonucleotide and DNA or RNA are complementary to each other when a sufficient number of appropriate positions in each of the molecules are occupied by nucleotides that are capable of forming hydrogen bonds with each other. Thus, "capable of specifically hybridizing" and "complementary" are terms that are used to describe such a sufficient degree of complementarity or pairing precision for which a stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of the antisense molecule need not be 100% complementary to its target sequence in order to be able to specifically hybridize. An antisense molecule is capable of specific hybridization when the binding of a compound to a target DNA or RNA molecule interferes with the normal functioning of the target DNA or RNA, resulting in a loss of function, and when the sequence is sufficiently complementary that there is no non-specific binding of the antisense compound to non-target sequences under conditions where specific binding is desired, that is, in physiological conditions in the case of in vivo tests or therapy and in the case of in vitro tests - in the conditions in which these tests are performed.

[0052] Although the above method can be used to select antisense molecules capable of removing any exon within a protein that can be truncated without losing its biological function, removal of the exon should not lead to a change in reading frame in the truncated mRNA transcript. So, if in a linear sequence of three exons, the end of the first exon encodes two of the three nucleotides in the codon and the next exon is deleted, then the third exon in this linear sequence

EP1766010B1PL must start with a single nucleotide that allows the three nucleotides to be completed in a codon. If the third exon does not start with a single nucleotide, the reading frame will change, which could lead to the production of a truncated or non-functional protein.

[0053] It should be noted that the codon structure at the end of exons in structural proteins may not always be broken at the codon end. Therefore, there may be a need to remove more than one exon from pre-mRNA to ensure reading of mRNA within the reading frame. In such a situation, most of the antisense oligonucleotides will have to be selected by applying the method of the present invention, in which each oligonucleotide is directed to a different region responsible for induction of splicing of exons to be removed.

[0054] The length of the antisense molecule can be different, provided that the molecule must be able to selectively bind to a selected site within the pre-mRNA molecule. The length of such sequences can be determined using selected methods described herein. Generally, the length of the antisense molecule will be from about 10 to 50 nucleotides. However, it should be noted that with the described method, a nucleotide of any length within this range can be used. Preferably, the length of the antisense molecule is from 17 to 30 nucleotides.

[0055] To determine which exons can be combined in the dystrophin coding gene, reference should be made to the map of exon regions. The connection of a given exon to the second exon occurs when the number on the 3 'border of the given exon is the same as the number on the 5' border of the second exon with which the exon is to be connected. Therefore, if exon 7 has been removed, to maintain the reading frame, exon 6 must be connected to either exon 12 or exon 18. Thus, antisense oligonucleotides should be selected that first redirect exon 7 splicing to exon 11 and then from exon 7 to exon 17. Another somewhat simplified approach to restore the reading frame within the exon 7 deletion would be to remove two flanking exons. The induction of skipping exons 6 and 8 should lead to a transcript consistent with the reading frame being the product of splitting exons 5 to 9. However, selecting exon 8 as the destination to be removed from pre-mRNA in practice results in the simultaneous removal of exon 9. Therefore, the resulting transcript would contain exon 5 connected to exon 10. Enabling or disabling exon 9 does not change the reading frame. When the antisense molecules to be tested are identified, they are produced using standard techniques known in the art. The most common method of producing antisense molecules is methylation of the hydroxyl group at the 2 'position of ribose and incorporation of the phosphorothioate skeleton. In this way, molecules are obtained that seemingly resemble RNA, but are much more resistant to degradation resulting from the action of nucleases.

[0056] To avoid degradation of pre-mRNA during duplex formation with antisense molecules, the antisense molecules used in this method can be adjusted to minimize or prevent degradation catalyzed by endogenous RNases H. This property is very advantageous because treatment of RNA with unmethylated oligonucleotides, carried out either intracellularly or in crude extracts containing RNase H, leads to degradation of pre-mRNA: antisense oligonucleotide duplexes. Any form of modified antisense molecule that is resistant to or will not induce degradation can be used in the present method. 2'-O-methyl- derivatives are an example of antisense molecules that do not undergo cellular RNase H catalyzed hydrolysis of their duplex with RNA. 2'9

EP1766010B1PL

O-methyl-oligorybonucleotides are very stable in the cell environment and in the tissues of animals, and their RNA duplexes are characterized by higher Tm values ​​than their ribo- or deoxyribo- equivalents. [0057] Antisense molecules that do not activate RNase H can be produced using known techniques (see, e.g., US Pat. No. 5,149,797). Such antisense molecules, which may be deoxyribonucleotide sequences or ribonucleotide sequences, simply contain any structural modification that is a steric obstacle hindering or preventing the binding of RNase H to the duplex molecule containing the given oligonucleotide as one of the components of this duplex. Such structural modification does not substantially impede the process of forming the duplex structure. Because the oligonucleotide regions involved in duplex formation are significantly different from the regions involved in binding RNase H to this nucleotide, many antisense molecules are available that do not activate RNase H. Such antisense molecules can be, for example, oligonucleotides in which at least one or all of the phosphate groups that form nucleotide bonds are modified phosphate groups, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholides, phosphoropiperazides and phosphorophosphates. For example, any one phosphate residue forming a linkage within a nucleotide can be modified as described above. In another non-limiting example, such antisense molecules are molecules in which at least one or all nucleotides contain in the 2 'position a low molecular weight alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl , propyl, 1-propenyl, 2-propenyl and isopropyl). For example, each of the nucleotides can be modified as described.

[0058] While the preferred form of antisense molecules are antisense oligonucleotides, the present invention also contemplates other oligomeric antisense molecules, including but not limited to oligonucleotide mimetics such as those described below.

[0059] Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or oligonucleotides containing unnatural bonds between nucleosides. As defined in this specification, modified backbone oligonucleotides include molecules that retain a phosphorus atom in the backbone and molecules that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as compounds described in the art, modified oligonucleotides that do not have a phosphorus atom in the nucleoside backbone are also considered such oligonucleosides.

[0060] For other preferred oligonucleotide mimetics, both the sugar residue and the nucleoside linkage, i.e. the skeleton, located in the nucleotide subunit are replaced with new groups. The bases are maintained to ensure hybridization with the appropriate target nucleic acid compound. As one of such oligomeric compounds, mention is made of peptide nucleic acid (PNA), which is an oligonucleotide mimetic. It has been shown to have excellent hybridization properties. The sugar backbone of the oligonucleotide in PNA compounds is replaced with a polyamide backbone, in particular a backbone made of N- (2-aminoethyl) glycine. Nitrogen bases are retained and bonded directly or indirectly to the nitrogen atoms in the azo group present in the skeletal amide region.

[0061] The modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides may also include nitrogen base modification or substitution (often referred to simply as "base" in the art). Some nitrogen bases are especially so

EP1766010B1EN useful when the goal is to increase the binding affinity of the oligomeric compounds of the present invention. These are 5-substituted pyrimidines, 6-aza-pyrimidines and purines substituted at the N-2, N-6 and O-6 positions, including 2-aminopropyl-adenine, 5-propynyl-uracil and 5-propynylcytosine. Substitution with 5-methylcytosine has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C. 5-methylcytosine is currently the preferred base replacement, which becomes even more beneficial when combined with modifications of the 2'-O-methoxyethyl sugar residue.

[0062] Another modification of the oligonucleotides of the present invention includes the chemical binding of an oligonucleotide to one or more moieties or conjugates that increase the activity, distribution in the cell, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol, cholic acid, thioether, e.g. hexyl-Triphenylmethanethiol (hexyl-S-tritylthiol), thiocholesterol, an aliphatic chain, e.g. dodecanediol or undecyl residues, phospholipid, e.g. 1,2-dihexadecyl-rac-glycerin or 1,2-dihexadecyl-rac-glyceryl-3-H-triethylammonium phosphonate, polyamine or polyethylene glycol chains or acetic acid adamantane, palmityl or octadecyl group or hexylamino carbonyl oxycholesterol.

[0063] Uniform modification of all positions in a given compound is not necessary. In fact, more than one of the modifications mentioned above may be introduced in one compound or even in one single nucleoside within an oligonucleotide. The invention also includes antisense compounds that are chimeric compounds. In the context of the present invention, "chimeric" antisense compounds or "chimeras" are antisense molecules, in particular oligonucleotides, which contain two or more regions differing in chemical structure, each of which consists of at least one monomer, i.e. a nucleotide - in case in an oligonucleotide compound. These oligonucleotides typically contain at least one region in which the oligonucleotide is modified to provide increased resistance to nuclease-catalyzed degradation, increased cellular uptake, and an additional region providing increased binding affinity for the target nucleic acid.

Methods for making antisense molecules [0064] Antisense molecules used in accordance with the present invention can be easily and routinely produced using the well-known solid phase synthesis technique. Apparatus for such synthesis is sold by several manufacturers, including for example the company Applied Biosystems (Foster City, CA). One method of synthesizing oligonucleotides on a modified solid support is described in US Patent 4,458,066.

[0065] Additionally or alternatively, any other methods of such synthesis known in the art can be used. The use of similar techniques for the preparation of oligonucleotides such as phosphorothioate and alkyl derivatives is well known . In one such automated embodiment, diethyl phosphate phosphates are used as starting materials, which can be synthesized as described by Beaucage et al. (1981) Tetrahedron Letters, 22: 1859-1862.

[0066] The antisense molecules of the present invention are synthesized in vitro and do not contain antisense compositions of biological origin or genetic vector constructs intended for direct synthesis of antisense molecules in vivo. To facilitate uptake, distribution and / or absorption, the molecules of the present invention may also be mixed, encapsulated, conjugated or otherwise bound to other molecules, molecular structures or

EP1766010B1EN mixtures of compounds, such as, for example, with liposomes, receptor-recognizing molecules, oral formulations, rectal formulations, topical formulations or other formulations.

Therapeutic Factors [0067] The present invention can also be used for prophylactic or therapeutic purposes, where it can be used to treat genetic diseases.

[0068] Accordingly, one embodiment of the present invention provides antisense molecules that bind to a selected target region in dystrophin pre-mRNA to induce the efficient and constant skipping of an exon described herein in an amount that provides therapeutic effect that is mixed with a pharmaceutically acceptable carrier, diluent or excipient.

[0069] The term "pharmaceutically acceptable" refers to the molecules themselves and the compositions themselves, which, when administered to a patient, are tolerated under physiological conditions and usually do not cause allergic reactions or unforeseen adverse reactions such as upset stomach, etc. The term "carrier" means a diluent , adjuvant, excipient or follicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including liquids made from petroleum, animal, vegetable or synthetic liquids, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably, in particular for injection solutions, water or saline solutions and aqueous dextrose and glycerol solutions are used as carriers. Suitable pharmaceutical carriers are described in: Martin, Remington's Pharmaceutical Sciences, 18 Ed. Mack Publishing Co, Easton, PA, (1990).

[0070] In a more specific embodiment, the present invention provides pharmaceutical compositions containing therapeutically effective amounts of an antisense molecule together with pharmaceutically acceptable diluents, preservatives, solubilizing agents, emulsifiers, adjuvants and / or carriers. Such compositions include diluents containing various buffers (e.g. Tris-HCl, acetate, phosphate) with different pH and ionic strength and additives such as detergents and solubilizing agents (e.g. Tween 80, Polysorbate 80), antioxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. Thimersol, benzyl alcohol) and bulking agents (e.g. lactose, mannitol). The substance can be incorporated into specific preparations composed of polymeric compounds, such as polylactic acid, polyglycolic acid, etc. or to liposomes. Hyaluronic acid may also be used. Such compositions may affect the physical state, stability, release rate in vivo and the rate of in vivo elimination of present proteins and derivatives. See, e.g., Martin, Remington's Pharmaceutical Sciences, 18 Ed. (1990, Mack Publishing Co, Easton, PA 18042, pages 1435-1712). The compositions may be made in liquid form or in the form of a dried powder, such as a lyophilized form.

[0071] It should be noted that the pharmaceutical compositions provided according to the present invention may be administered by methods known in the art. Preferably, the pharmaceutical compositions intended for administration are administered by injection, orally or into the lung, or intranasally. Preferably, the antisense molecules are delivered by intravenous, intraarterial, intraperitoneal, intramuscular or subcutaneous administration.

Antisense Molecule Therapy [0072] The present invention also describes the use of the antisense molecules of the present invention in the manufacture of a medicament modulating a genetic disease.

[0073] Delivery of a therapeutically useful amount of antisense molecules can be achieved using previously published methods. For example, delivery of an antisense molecule to the interior of a cell can be accomplished using a composition comprising an admixture of the antisense molecule and an effective amount of block copolymer. An example of this method is described in US patent application 20040248833.

[0074] Other methods of delivering antisense molecules to the cell nucleus are described in: CJ Mann et al., (2001) ["Antisense-induced exon skipping and the synthesis of dystrophin in the mdx mouse". Proc., Natl. Acad. Science, 98 (1) 42-47] and in: Gebski et al. [(2003). Human Molecular Genetics, 12 (15): 1801-1811].

[0075] A method of introducing a nucleic acid molecule into a cell, using an expression vector or in the form of naked DNA or a complex of DNA with lipid carriers, is described in US Patent 6,806,084.

[0076] It may be desirable to provide antisense molecules in colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, granules, lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, liposomes or liposome formulations.

[0077] Liposomes are artificial membrane vesicles that are useful as carriers in vitro and in vivo. These formulations have a net cationic, anionic or neutral charge and are useful in in vitro, in vivo and ex vivo delivery methods. Large single-layered vesicles (LUV) have been shown to be in the range of 0.2-4.0. PHI.m, can close a significant percentage of aqueous buffer containing large macromolecules. RNA and DNA can be enclosed in the aqueous environment of the interior of the liposome and delivered to cells in a biologically active form (Fraley, et al., Trendy Biochem. Sci., 6:77, 1981).

[0078] In order for liposomes to be an effective carrier for gene transfer, they should have the following properties:

(1) high closing efficiency of the antisense molecule of interest without reducing their biological activity; and (2) specific and significant binding to the target cell as compared to non-target cells; (3) delivering high efficiency water content of the vesicle to the cytoplasm of the target cell; and (4) accurate and effective expression of genetic information [Mannino, et al., Biotechniques, 6: 682, 1988].

[0079] The composition of liposomes is usually a mixture of phospholipids, especially phospholipids with a high phase transition temperature, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0080] Alternatively, for the preparation of a pharmaceutical composition, antisense constructs may be combined with other pharmaceutically acceptable carriers or diluents. Suitable carriers and diluents include isotonic salt solutions, e.g., phosphate buffered sodium chloride solution. The composition may be prepared as a formulation for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.

[0081] The routes of administration described are indicative only, as a skilled practitioner will be able to easily determine the optimal route of administration and any dosage for a particular animal

EP1766010B1EN specific conditions. Many ways have been tried to introduce new functional genetic material into cells, both in vitro and in vivo (Friedmann (1989) Nauka, 244: 12751280).

[0082] These methods include integrating the gene to be expressed into modified retroviruses [Friedmann, (1989), supra; Rosenberg (1991) Cancer Research 51 (18), suppl: 5074S-5079S], integration with non-retroviral vectors [Rosenfeld, et al. (1992) Cell, 68: 143-155; Rosenfeld, et al. (1991) Science, 252: 431-434] or delivery, via liposomes, of a transgene associated with the heterologous promoter-enhancer sequence element [Friedmann (1989), supra; Brigham, et al. (1989) Am. J. Med. Sci, 298: 278-281 ;. Nabel, et al. (1990) Science, 249: 1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec. Biol., 4: 206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. (USA), 84: 7851-7855], in combination with ligand-specific cationic transport systems [Wu and Wu (1988) J. Biol. Chem., 263: 14621-14624] or using naked DNA, expression vectors [Nabel et al. (1990), supra; Wolff et al. (1990) Science, 247: 1465-1468]. Direct injection of transgenes into tissue induces only local expression [Rosenfeld (1992) supra; Rosenfeld et al. (1991) supra; Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et al. (1991) supra].

Brigham et al. [Am. J. Med. Sci. (1989) 298: 278-281 and Clinical Research (1991) 39 (abstract)] described in vivo transfection only in the lungs of mice after intravenous or intratracheal administration of the liposomal DNA complex. An example of a review article on human gene therapy procedures is: Anderson, Science (1992) 256: 808-813.

[0083] The antisense molecules of the present invention include any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound that, when administered to an animal, including a human, is able to provide (directly or indirectly) a biologically active metabolite or product thereof. Thus, this disclosure, for example, contemplates prodrugs and pharmaceutically acceptable salts of the compounds of this invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0084] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present invention, i.e., salts that retain the desired biological activity of the parent substance and do not render it undesirable toxic.

[0085] In the case of oligonucleotides, preferred examples of pharmaceutically acceptable salts include, but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium ion, magnesium, calcium, polyamines such as spermine and spermidine, etc. , (b) addition salts formed with inorganic acids, e.g. with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc .; (c) salts formed with organic acids, such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid , palmitic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid etc., and (d) salts formed from basic anions, such as chlorine, bromine and iodine. The pharmaceutical compositions of the present invention may be administered in a variety of ways, depending on whether local or systemic treatment is desired, and depending on the area to be treated. These compositions may be administered topically (including ocular and mucosal administration, including rectal administration), into the lungs, e.g. by inhalation or insufflation of powders or aerosols (including the use of a nebulizer, intratracheal, intranasal, epidermal and transdermal administration), orally or parenterally. Pass

Parenteral includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, e.g. intrathecal or intraventricular. Oligonucleotides with at least one 2'-O-methoxyethyl modification are considered particularly useful for oral administration.

[0086] The pharmaceutical formulations of the present invention, which can easily be prepared in the form of a dosage unit, can be made using conventional techniques well known in the pharmaceutical industry. These techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier (s) or excipient (s). In general, formulations are prepared by preparing a homogeneous and accurate combination of active ingredients with liquid carriers or finely divided solid carriers, or with both carrier forms, and then, if necessary, forming the product.

Kits of the Invention [0087] The invention also provides kits for treating a patient having a genetic disease, wherein the kit comprises at least an antisense molecule placed in a suitable container together with instructions for its use.

[0088] In a preferred embodiment, the kits will contain at least one antisense molecule. Kits may also contain reagents less important compared to the main ingredient, such as buffers, stabilizers, etc.

[0089] Those skilled in the art should be aware that the uses of the method described above are widely used to identify antisense molecules suitable for use in the treatment of many other diseases.

EXAMPLES [0090] The following examples are intended to more fully describe how to use the invention described above, as well as to present the best methods considered for the various embodiments of this invention. It is understood that these examples are in no way intended to limit the true scope of this invention, but are provided for illustrative purposes.

[0091] Molecular cloning methods, methods used in immunology and protein chemistry that are not explicitly described in the examples below, are described in the literature and known to those skilled in the art. Review texts describing prior art conventional molecular biology, microbiology and recombinant DNA techniques include, for example, Sambrook et al., Molecular Cloning .: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd., Oxford, UK (1985); and Ausubel, F., Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K. Current Protocols In Molecular Biology. Greene Publishing Associates / Wiley Intersciences, New York (2002). Determination of induced exon skipping in human muscle cells [0092] Attempts by the inventors to develop a rational approach to the design of antisense molecules have not been fully successful. It turned out that no logical rule was observed that could be applied to all exons. Therefore, the identification of the most effective antisense molecules, and thus having the best therapeutic properties, was the result of empirical research.

[0093] These empirical studies involved the use of computer programs to identify motifs potentially involved in the splicing process. Other computer programs were used

EP1766010B1EN also for determining pre-mRNA regions that might not have extensive secondary structures and thus potential hybridization sites of antisense molecules. None of these strategies was characterized by full reliability in the design of antisense oligonucleotides for reliable and efficient induction of exon skipping.

[0094] Hybridization sites in human dystrophin pre-mRNA were selected for testing, initially based on known or predictable motifs or regions involved in the splicing process. Antisense oligonucleotides with 2'-O-methyl modification, complementary to the target sequences of the experiment, were designed and synthesized using the Expedite 8909 nucleic acid synthesizer. After the synthesis, the oligonucleotides were "cleaved" from the medium and the protective groups removed before the desalting step in ammonium hydroxide. The quality of oligonucleotide synthesis was monitored at each stage of deprotection during the synthesis, based on the intensity of the signals from the trityl groups of signals observed in the synthesis log. The concentration of antisense oligonucleotides was estimated by measuring absorbance at 260 nm, made for a diluted aliquot.

[0095] Subsequently, specific amounts of antisense molecules were tested for their ability to induce exon skipping in an in vitro assay as described below.

[0096] Briefly, primary normal myoblast cultures were obtained from human muscle biopsies with the consent of the donor. Cells were propagated and allowed to differentiate into myotubes using standard cell culture techniques. The cells were then transfected with antisense oligonucleotides, supplying the cells with oligonucleotides in the form of cationic lipoplexes, mixtures of antisense molecules or preparations of cationic liposomes.

[0097] Cells were then propagated for a further 24 hours, after which total RNA was extracted and molecular analysis started. To examine selected regions of dystrophin pre-mRNA or induced exon rearrangements, a PCR-coupled reverse transcription reaction (RT-PCR) was performed.

[0098] For example, when testing an antisense molecule designed to induce exon skipping 19, to detect inclusion of any adjacent exons, several exons were screened using RT-PCR. For example, by inducing omission of exon 19, RT-PCR was performed using primers that allow amplification within exons 17 and 21. In order to ensure that there is minimal non-specific amplification for shorter transcripts that are the product of the bypass splice process, amplification of much longer products of this region (i.e. exons 13-26) was also carried out. Truncated products or products resulting from exon skipping tend to be more efficiently amplified and may affect the estimation of normal and induced transcript. [0099] The sizes of the amplification reaction products were estimated on an agarose gel and compared with the appropriate standards. Final confirmation of the identity of these products was carried out by direct DNA sequencing to determine whether the correct or expected junctions between exons were maintained.

[0100] When efficient exon skipping is induced with one antisense molecule, subsequent overlapping antisense molecules can be synthesized, which will then be tested using the test described above. Our definition of an efficient antisense molecule refers to a molecule that causes a strong and constant skipping of an exon under conditions when used during transfection at concentrations of the order of 300 nM or less.

Antisense oligonucleotides directed at exon 51

[0101] Exon 51 antisense oligonucleotides were prepared and tested for their ability to induce exon skipping in human muscle cells using methods similar to those described above.

[0102] Antisense oligonucleotide H51A (+ 66 + 90) [SEQ. ID NO. 180] showed greater ability to induce exon 51 skipping. Table 1 below contains antisense molecules that were tested in the concentration range of 25, 50, 100, 300 and 600 nM. These antisense molecules showed different ability to induce 51 exon skipping. The strongest exon skip inducers were H51A (+ 61 + 90) antisense oligonucleotides [SEQ ID NO: 2]. ID NO. 179] and H51A (+ 66 + 95) [SEQ. ID NO. 181].

<td>Name antisense nucleotide</td><td>Sequence</td><td>Ability to induce skipping</td>

<td>H51A (-01 + 25)</td><td>ACC AGA GUA ACA GUC UGA GUA GGA GC</td><td>Weak skip</td>

<td>H51D (+ 16-07)</td><td>CUC AUA CCU UCU GCU UGA UGA UC</td><td>You will miss at 300 nM</td>

<td>H51A (+ 111 + 134)</td><td>UUC UGU CCA AGC CCG GUU GAA AUC</td><td>A necessary repetition test</td>

<td>H51A (+ 61 + 90)</td><td>ACA UCA AGG AAG AUG GCA UUU CUA GUU UGG</td><td>Very strong skip</td>

<td>H51A (+ 66 + 90)</td><td>ACA UCA AGG AAG AUG GCA UUU CUA G</td><td>Omission</td>

<td>H51A (+ 66 + 95)</td><td>CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG</td><td>Very strong skip</td>

<td>H51D (+ 08-17)</td><td>AUC AUU UUU UCU CAU ACC UUC UGC U</td><td>No skipping</td>

<td>H51A / D (+ 08-17) &amp; (15+?)</td><td>AUC AUU UUU UCU CAU ACC UUC UGC UAG GAG CUA AAA</td><td>No skipping</td>

<td>H51A (+ 175 + 195)</td><td>CAC CCA CCA UCA CCC UCY GUG.</td><td>No skipping</td>

<td>H51A (+ 199 + 220)</td><td>AUC AUC UCG UUG AUA UCC UCA A</td><td>No skipping</td>

SEQUENCE LIST [0103] &lt;110&gt; University of Western Australia &lt;120&gt; Antisense oligonucleotides for induction of exon skipping and methods of their use &lt;130&gt; 111819 &lt;160&gt; 211 &lt;170&gt; Patent In version3.3 &lt;210&gt; 1 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 1 gauagguggu aucaacaucu guaa 24

EP1766010B1EN &lt;210&gt; 2 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 2 gauagguggu aucaacaucu g 21 &lt;210&gt; 3 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 3 gauagguggu aucaacaucu guaag 25 &lt; 210&gt; 4 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 4 ggugguauca acaucuguaa 20 &lt;210&gt; 5 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 5 guaucaacau cuguaagcac 20 &lt;210&gt; 6 &lt; 211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 6 ugcauguucc agucguugug ugg 23 &lt;210&gt; 7

EP1766010B1EN &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 7 cacuauucca gucaaauagg ucugg 25 &lt;210&gt; 8 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 8 auuuaccaac cuucaggauc gagua 25 &lt;210&gt; 9 &lt; 211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 9 ggccuaaaac acauacacau a 21 &lt;210&gt; 10 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 10 cauuuuugac cuacaugugg 20 &lt;210&gt; 11 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 11 uuugaccuac auguggaaag 20 &lt;210&gt; 12 &lt;211&gt; 26 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 12 uacauuuuug accuacaugu ggaaag 26 &lt;210&gt; 13 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 13 auuuuugacc uacaugggaa ag 22 &lt;210&gt; 14 &lt;211&gt; 23 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 14 uacgaguuga uugucggacc cag 23 &lt;210&gt; 15 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 15 guggucuccu uaccuaugac ugugg 25 &lt;210&gt; 16 &lt;211&gt; 17 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 16 ggucuccuua ccuauga 17 &lt;210&gt; 17 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 17 ugucucagua aucuucuuac cuau 24 &lt;210&gt; 18 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 18 ucuuaccuau gacuauggau gaga 24 &lt;210&gt; 19 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 19 gcaugaacuc uuguggaucc 20 &lt;210&gt; 20 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 20 ccaggguacu acuuacauua 20 &lt;210&gt; 21 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 21 aucguguguc acagcaucca g 21 &lt;210&gt; 22 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 22 uguucagggc augaacucuu guggauccuu 30

EP1766010B1EN &lt;210&gt; 23 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 23 uaggaggcgc cucccauccu guaggucacu g 31 &lt;210&gt; 24 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 24 aggucuagga ggcgccuccc auccuguagg u 31 &lt;210&gt; 25.

&lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 25 gcgccuccca uccuguaggu cacug 25 &lt;210&gt; 26 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 26 cuucgaggag gucuaggagg cgccuc 26 &lt;210&gt; 27 &lt;211 &gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 27 cucccauccu guaggucacu g 21 &lt;210&gt; 28

EP1766010B1EN &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 28 uaccaguuuu ugcccuguca gg 22 &lt;210&gt; 29 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 29 ucaauaugcu gcuucccaaa cugaaa 26 &lt;210&gt; 30 &lt; 211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 30 cuaggaggcg ccucccaucc uguag 25 &lt;210&gt; 31 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 31 uuaugauuuc caucuacgau gucaguacuu c 31 &lt;210&gt; 32 &lt;211 &gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 32 cuuaccugcc aguggaggau uauauuccaa a 31 &lt;210&gt; 33 &lt;211&gt; 25 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 33 caucaggauu cuuaccugcc agugg 25 &lt;210&gt; 34 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 34 cgaugucagu acuuccaaua uucac 25 &lt;210&gt; 35 &lt;211&gt; 18 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 35 accauucauc aggauucu 18 &lt;210&gt; 36 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 36 accugccagu ggaggauu 18 &lt;210&gt; 37 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 37 ccaauauuca cuaaaucaac cuguuaa 27 &lt;210&gt; 38 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 38 caggauucuu accugccagu ggaggauuau 30 &lt;210&gt; 39 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 39 acgaugucag uacuuccaau auucacuaaa 31 31 &lt;210&gt; 40 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 40 auuuccaucu acgaugucag uacuuccaau a 31 &lt;210&gt; 41 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 41 caggagcuuc caaaugcugc a 21 &lt;210&gt; 42 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 42 cuugucuuca ggagcuucca aaugcugca 29 &lt;210&gt; 43 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 43 uccucagcag aaagaagcca cg 22

EP1766010B1EN &lt;210&gt; 44 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 44 uuagaaaucu cuccuugugc 20 &lt;210&gt; 45 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 45 5 uaaauugggu guuacacaau 20 &lt;210 &gt; 46 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 46 cccugaggca uucccaucuu gaau 24 &lt;210&gt; 47 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 47 aggacuuacu ugcuuuguuu 20 &lt;210&gt; 48 &lt; 211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 48 cuugaauuua ggagauucau cug 23 &lt;210&gt; 49

EP1766010B1EN &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 49 caucuucuga uaauuuuccu guu 23 &lt;210&gt; 50 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 50 ucuucuguuu uuguuagcca guca 24 &lt;210&gt; 51 &lt; 211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 51 ucuauguaaa cugaaaauuu 20 &lt;210&gt; 52 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 52 uucuggagau ccauuaaaac 20 &lt;210&gt; 53 &lt;211&gt; 24 &lt; 212&gt; DNA &lt;213&gt; human &lt;400&gt; 53 cagcaguugc gugaucucca cuag 24 &lt;210&gt; 54 &lt;211&gt; 21 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 54 uucaucaacu accaccacca in 21 &lt;210&gt; 55 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 55 cuaagcaaaa uaaucugacc uuaag 25 &lt;210&gt; 56 &lt;211&gt; 28 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 56 cuuguaaaag aacccagcgg ucuucugu 28 &lt;210&gt; 57 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 57 caucuacaga uguuugccca uc 22 &lt;210&gt; 58 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 58 gaaggauguc uuguaaaaga acc 23 &lt;210&gt; 59 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 59 accuguucuu caguaagacg 20 &lt;210&gt; 60 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 60 caugacacac cuguucuuca guaa 24 &lt;210&gt; 61 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400 &gt; 61 cauuugagaa ggaugucuug 20 &lt;210&gt; 62 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 62 aucucccaau accuggagaa gaga 24 &lt;210&gt; 63 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 63 gccaugcacu aaaaaggcac ugcaagacau u 31 &lt;210&gt; 64 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 64 ucuuuaaagc caguugugug aauc 24

EP1766010B1EN &lt;210&gt; 65 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 65 uuucugaaag ccaugcacua a 21 &lt;210&gt; 66 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 66 guacauacgg ccaguuuuug aagac 25 &lt; 210&gt; 67 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 67 cuagauccgc uuuuaaaacc uguuaaaaca a 31 &lt;210&gt; 68 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 68 ucuuuucuag auccgcuuuu aaaaccuguu a 31 &lt; 210&gt; 69 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 69 cuagauccgc uuuuaaaacc uguua 25 &lt;210&gt; 70

EP1766010B1EN &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 70 ccgucuucug ggucacugac uua 23 &lt;210&gt; 71 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 71 cuagauccgc uuuuaaaacc uguuaa 26 &lt;210&gt; 72 &lt; 211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 72 ccgcuuuuaa aaccuguuaa 20 &lt;210&gt; 73 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 73 uggauugcuu uuucuuuucu agaucc 26 &lt;210&gt; 74 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 74 caugcuuccg ucuucugggu cacug 25 &lt;210&gt; 75 &lt;211&gt; 23 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 75 gaucuuguuu gagugaauac agu 23 &lt;210&gt; 76 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 76 guuauccagc caugcuuccg uc 22 &lt;210&gt; 77 &lt;211&gt; 25 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 77 ugauaauugg uaucacuaac cugug 25 &lt;210&gt; 78 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 78 guaucacuaa ccugugcugu ac 22 &lt;210&gt; 79 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 79 cagcaguagu ugucaucugc 20 &lt;210&gt; 80 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 80 gccugagcug aucugcuggc aucuugcagu 31 31 &lt;210&gt; 81 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 81 cuggcagaau ucgauccacc ggcuguuc 28 &lt;210&gt; 82 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 82 cagcaguagu ugucaucugc uc 22 &lt;210&gt; 83 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 83 ugauggggug guggguugg 19 &lt;210&gt; 84 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 84 aucugcauua acacccucua gaaag 25 &lt;210&gt; 85 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 85 ccggcuguuc aguuguucug aggc 24

EP1766010B1EN &lt;210&gt; 86 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 86 aucugcauua acacccucua gaaagaaa 28 &lt;210&gt; 87 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 87 gaaggagaag agauucuuac cuuacaaa 28 &lt; 210&gt; 88 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 88 auucgaucca ccggcuguuc 20 &lt;210&gt; 89 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 89 cugcuggcau cuugcaguu 19 &lt;210&gt; 90 &lt; 211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 90 gccgguugac uucauccugu gc 22 &lt;210&gt; 91

EP1766010B1EN &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 91 cugcauccag gaacaugggu cc 22 &lt;210&gt; 92 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 92 gucugcaucc aggaacaugg guc 23 &lt;210&gt; 93 &lt; 211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 93 guugaagauc ugauagccgg uuga 24 &lt;210&gt; 94 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 94 uacuuacugu cuguagcucu uucu 24 &lt;210&gt; 95 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 95 cacucauggu cuccugauag cgca 24 &lt;210&gt; 96 &lt;211&gt; 22 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 96 cugcaauucc ccgagucucu gc 22 &lt;210&gt; 97 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 97 acugcuggac ccauguccug aug 23 &lt;210&gt; 98 &lt;211&gt; 21 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 98 cuaaguugag guauggagag 21 21 &lt;210&gt; 99 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 99 uauucacaga ccugcaauuc ccc 23 &lt;210&gt; 100 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 100 acaguggugc ugagauagua uaggcc 26 &lt;210&gt; 101 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 101 uaggccacuu uguugcucuu gc 22 &lt;210&gt; 102 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 102 uucagagggc gcuuucuuc 19 &lt;210&gt; 103 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400 &gt; 103 gggcaggcca uuccuccuuc aga 23 &lt;210&gt; 104 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 104 ucuucagggu uuguauguga uucu 24 &lt;210&gt; 105 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 105 cugggcugaa uugucugaau aucacug 27 &lt;210&gt; 106 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 106 cuguuggcac augugauccc acugag 26

EP1766010B1EN &lt;210&gt; 107 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 107 gucuauaccu guuggcacau guga 24 &lt;210&gt; 108 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 108 ugcuuucugu aauucaucug gaguu 25 &lt; 210&gt; 109 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 109 ccuccuuucu ggcauagacc uuccac 26 &lt;210&gt; 110 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 110 ugugucaucc auucgugcau cucug 25 &lt;210&gt; 111 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 111 uuaaggccuc uugugcuaca ggugg 25 &lt;210&gt; 112

EP1766010B1EN &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 112 gggccucuuc uuuagcucuc uga 23 &lt;210&gt; 113 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 113 gacuuccaaa gucuugcauu uc 22 &lt;210&gt; 114 &lt; 211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 114 gccaacaugc ccaaacuucc uaag 24 &lt;210&gt; 115 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 115 cagagauuuc cucagcuccg ccagga 26 &lt;210&gt; 116 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 116 cuuaraucua gcaccucaga g 21 &lt;210&gt; 117 &lt;211&gt; 25 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 117 uccgccaucu guuagggucu gugcc 25 &lt;210&gt; 118 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 118 auuuggguua uccucugaau gucgc 25 &lt;210&gt; 119 &lt;211&gt; 22 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 119 cauaccucuu cauguaguuc uc 22 &lt;210&gt; 120 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 120 cauuugagcu gcguccaccu ugucgucugu g 31 &lt;210&gt; 121 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213 &gt; human &lt;400&gt; 121 uccugggcag acuggaugcu cuguuc 26 &lt;210&gt; 122 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 122 uugccugggc uuccugaggc auu 23 &lt;210&gt; 123 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 123 uucugaaaua acauauaccu gugc 24 &lt;210&gt; 124 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 124 uaguuucuga aauaacauau accug 25 &lt;210&gt; 125 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 125 gacuugucaa aucagauugg a 21 &lt;210&gt; 126 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 126 guuucugaaa uaacauauac cugu 24 &lt;210&gt; 127 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 127 caccagaaau acauaccaca 20

EP1766010B1EN &lt;210&gt; 128 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 128 caaugauuua gcugugacug 20 &lt;210&gt; 129 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 129 cgaaacuuca uggagacauc uug 23 &lt;210 &gt; 130 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 130 cuuguagacg cugcucaaaa uuggc 25 &lt;210&gt; 131 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 131 20 caugcacaca ccuuugcucc 20 &lt;210&gt; 132 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 132 ucuguacaau cugacgucca gucu 24 &lt;210&gt; 133

EP1766010B1EN &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 133 gucuuuauca ccauuuccac uucagac 27 &lt;210&gt; 134 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 134 ccgucugcuu uuucuguaca aucug 25 &lt;210&gt; 135 &lt; 211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 135 uccauaucug uagcugccag cc 22 &lt;210&gt; 136 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 136 ccaggcaacu ucagaaucca aau 23 &lt;210&gt; 137 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 137 uuucuguuac cugaaaagaa uuauaaugaa 30 &lt;210&gt; 138 &lt;211&gt; 25 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 138 cauucauuuc cuuucgcauc uuacg 25 &lt;210&gt; 139 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 139 ugaucucuuu gucaauucca uaucug 26 &lt;210&gt; 140 &lt;211&gt; 27 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 140 uucagugaua uagguuuuac cuuuccc 27 &lt;210&gt; 141 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 141 cuguagcugc cagccauucu gucaag 26 &lt;210&gt; 142 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 142 ucuucugcuc gggaggugac a 21 &lt;210&gt; 143 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 143 20 ccaguuacua uucagaagac 20 &lt;210&gt; 144 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 144 ucuucaggug caccuucugu 20 &lt;210&gt; 145 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400 &gt; 145 ugugaugugg uccacauucu gguca 25 &lt;210&gt; 146 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 146 ccauguguuu cugguauucc 20 &lt;210&gt; 147 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 147 cguguagag ccaccuuugg gcgua 25 &lt;210&gt; 148 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 148 uacuaauuuc cugcaguggu cacc 24

EP1766010B1EN &lt;210&gt; 149 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 149 uucuguguga aauggcugca aauc 24 &lt;210&gt; 150 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 150 ccuucaaagg aauggaggcc 20 &lt;210 &gt; 151 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 151 ugcugaauuu cagccuccag ugguu 25 &lt;210&gt; 152 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 152 ugaagucuuc cucuuucaga uucac 25 &lt;210&gt; 153 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 153 cuggcuuucu cucaucugug auuc 24 &lt;210&gt; 154

EP1766010B1EN &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 154 guuguaaguu gucuccucuu 20 &lt;210&gt; 155 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 155 uugucuguaa cagcugcugu 20 &lt;210&gt; 156 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 156 gcucuaauac cuugagagca 20 &lt;210&gt; 157 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 157 cuuugagacc ucaaauccug uu 22 &lt;210&gt; 158 &lt;211&gt; 25 &lt;212 &gt; DNA &lt;213&gt; human &lt;400&gt; 158 cuuuauuuuc cuuucaucuc ugggc 25 &lt;210&gt; 159 &lt;211&gt; 27 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 159 aucguuucuu cacggacagu gugcugg 27 &lt;210&gt; 160 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 160 gggcuuguga gacaugagug auuu 24 &lt;210&gt; 161 &lt;211&gt; 22 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 161 accuucagag gacuccucuu gc 22 &lt;210&gt; 162 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 162 uauguguuac cuacccuugu cgguc 25.

&lt;210&gt; 163 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 163 ggagagagcu uccuguagcu 20 &lt;210&gt; 164 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 164 ucacccuuuc cacaggcguu gca 23 &lt;210&gt; 165 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 165 uuugugucuu ucugagaaac 20 &lt;210&gt; 166 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400 &gt; 166 aaagacuuac cuuaagauac 20 &lt;210&gt; 167 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 167 aucugucaaa ucgccugcag 20 &lt;210&gt; 168 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 168 uuaccuugac uugcucaagc 20 &lt;210&gt; 169 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 169 uccagguuca agugggauac 20

EP1766010B1EN &lt;210&gt; 170 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 170 gcucuucugg gcuuauggga gcacu 25 &lt;210&gt; 171 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 171 accuuuaucc acuggagauu ugucugc 27 &lt; 210&gt; 172 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 172 uuccaccagu aacugaaaca g 21 &lt;210&gt; 173 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 173 ccacucagag cucagaucuu cuaacuucc 29 &lt;210&gt; 174 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 174 cuucaacuca gagcucagau cuucuaa 27 &lt;210&gt; 175

EP1766010B1EN &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 175 gggauccagu auacuuacag gcucc 25 &lt;210&gt; 176 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 176 accagaguaa cagucugagu aggagc 26 &lt;210&gt; 177 &lt; 211&gt; 23 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 177 cucauaccuu cugcuugaug auc 23 &lt;210&gt; 178 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 178 uucuguccaa gcccgguuga aauc 24 &lt;210&gt; 179 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 179 acaucaagga ggauggcauu ucuag 25 &lt;210&gt; 180 &lt;211&gt; 25 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 180 acaucaagga agauggcauu ucuag 25 &lt;210&gt; 181 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 181 cuccaacauc aaggaagaug gcauuucuag 30 &lt;210&gt; 182 &lt;211&gt; 25 &lt;212&gt; DNA &lt; 213&gt; human &lt;400&gt; 182 aucauuuuuu cucauaccuu cugcu 25 &lt;210&gt; 183 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 183 aucauuuuuu cucauaccuu cugcuaggag cuaaaa 36 &lt;210&gt; 184 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213 &gt; human &lt;400&gt; 184 cacccaccau cacccucugu g 21 &lt;210&gt; 185 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 185 aucaucucgu ugauauccuc aa 22 &lt;210&gt; 186 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 186 uccugcauug uugccuguaa g 21 &lt;210&gt; 187 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 187 uccaacuggg gacgccucug uuccaaaucc 30 &lt;210&gt; 188 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 188 acuggggacg ccucuguucc a 21 &lt;210&gt; 189 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 189 ccguaaugau uguucuagcc 20 &lt;210&gt; 190 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 190 uguuaaaaaa cuuacuucga 20

EP1766010B1EN &lt;210&gt; 191 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 191 cauucaacug uugccuccgg uucugaaggu g 31 &lt;210&gt; 192 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 192 cuguugccuc cgguucugaa ggug 24 &lt;210&gt; 193 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 193 cauucaacug uugccuccgg uucug 25 &lt;210&gt; 194 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 194 21 uacuaaccuu gguuucugug a 21 &lt; 210&gt; 195 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 195 cugaaggugu ucuuguacuu caucc 25 &lt;210&gt; 196

EP1766010B1EN &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 196 uguauaggga cccuccuucc augacuc 27 &lt;210&gt; 197 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 197 cuaaccuugg uuucugugau uuucu 25 &lt;210&gt; 198 &lt; 211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 198 gguaucuuug auacuaaccu ugguuuc 27 &lt;210&gt; 199 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 199 auucuuucaa cuagaauaaa ag 22 &lt;210&gt; 200 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 200 gauucugaau ucuuucaacu agaau 25 &lt;210&gt; 201 &lt;211&gt; 20 &lt;212&gt; DNA

EP1766010B1EN &lt;213&gt; human &lt;400&gt; 201 aucccacuga uucugaauuc 20 &lt;210&gt; 202 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 202 uuggcucugg ccuguccuaa ga 22 &lt;210&gt; 203 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213 &gt; human &lt;400&gt; 203 cucuuuucca gguucaagug ggauacuagc 30 &lt;210&gt; 204 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 204 caagcuuuuc uuuuaguugc ugcucuuuuc c 31 &lt;210&gt; 205 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 205 uauucuuuug uucuucuagc cuggagaaag 30 &lt;210&gt; 206 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; human

EP1766010B1EN &lt;400&gt; 206 cugcuuccuc caaccauaaa acaaauuc 28 &lt;210&gt; 207 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 207 ccaaugccau ccuggaguuc cuguaa 26 &lt;210&gt; 208 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt; 400&gt; 208 uccuguagaa uacuggcauc 20 &lt;210&gt; 209 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 209 ugcagaccuc cugccaccgc agauuca 27 &lt;210&gt; 210 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 210 cuaccucuuu uuucugucug 20 &lt;210&gt; 211 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; human &lt;400&gt; 211 uguuuuugag gauugcugaa 20

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