HK1167433B

HK1167433B - Oligonucleotide comprising an inosine for treating dmd

Info

Publication number: HK1167433B
Application number: HK12108169.4A
Authority: HK
Inventors: Christina Theodora Van Deutekom Judith; Johannes De Kimpe Josephus; Johannes Platenburg Gerardus
Original assignee: Biomarin Technologies B.V.
Priority date: 2009-04-24
Filing date: 2010-04-26
Publication date: 2017-03-10

Description

Field of the invention

The invention relates to the fields of molecular biology and medicine.

Background of the invention

A muscle disorder is a disease that usually has a significant impact on the life of an individual. A muscle disorder can have either a genetic cause or a non-genetic cause. An important group of muscle diseases with a genetic cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD). These disorders are caused by defects in a gene for a muscle protein.

Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are genetic muscular dystrophies with a relatively high incidence. In both Duchenne and Becker muscular dystrophy the muscle protein dystrophin is affected. In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and/or the dystrophin present is abnormally formed. Both diseases are associated with recessive X-linked inheritance. DMD results from a frameshift mutation in the DMD gene. The frameshift in the DMD gene's transcript (mRNA) results in the production of a truncated non-functional dystrophin protein, resulting in progressive muscle wasting and weakness. BMD occurs as a mutation does not cause a frame-shift in the DMD transcript (mRNA). As in BMD some partly to largely functional dystrophin is present in contrast to DMD where dystrophin is absent, BMD has generally less severe symptoms then DMD. The onset of DMD is earlier than BMD. DMD usually manifests itself in early childhood, BMD in the teens or in early adulthood. The progression of BMD is slower and less predictable than DMD. Patients with BMD can survive into mid to late adulthood. Patients with DMD rarely survive beyond their thirties.

Dystrophin plays an important structural role in the muscle fiber, connecting the extracellular matrix and the cytoskeleton. The N-terminal region binds actin, whereas the C-terminal end is part of the dystrophin glycoprotein complex (DGC), which spans the sarcolemma. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis.

For most genetic muscular dystrophies no clinically applicable and effective therapies are currently available. Exon skipping techniques are nowadays explored in order to combat genetic muscular dystrophies. Promising results have recently been reported by us and others on a genetic therapy aimed at restoring the reading frame of the dystrophin pre-mRNA in cells from the mdx mouse, the GRMD dog (reference 59) and DMD patients^1-11. WO 2006/000057 describes a list of antisense oligonucleotides targeting different exons of the DMD gene with varying degree of exon skipping efficiency. By the targeted skipping of a specific exon, a DMD phenotype (lacking dystrophin) is converted into a milder BMD phenotype (partly to largely functional dystrophin). The skipping of an exon is preferably induced by the binding of antisense oligoribonucleotides (AONs) targeting either one or both of the splice sites, or exon-internal sequences. Since an exon will only be included in the mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been considered obvious targets for AONs. More preferably, one or more AONs are used which are specific for at least part of one or more exonic sequences involved in correct splicing of the exon. Using exon-internal AONs specific for an exon 46 sequence, we were previously able to modulate the splicing pattern in cultured myotubes from two different DMD patients with an exon 45 deletion¹¹. Following AON treatment, exon 46 was skipped, which resulted in a restored reading frame and the induction of dystrophin synthesis in at least 75% of the cells. We have recently shown that exon skipping can also efficiently be induced in human control and patient muscle cells for 39 different DMD exons using exon-internal AONs^{1, 2, 11-15}.

Hence, exon skipping techniques applied on the dystrophin gene result in the generation of at least partially functional - albeit shorter - dystrophin protein in DMD patients. Since DMD is caused by a dysfunctional dystrophin protein, it would be expected that the symptoms of DMD are sufficiently alleviated once a DMD patient has been provided with functional dystrophin protein. However, the present invention provides the insight that, even though exon skipping techniques are capable of inducing dystrophin synthesis, the oligonucleotide used for exon skipping technique can be improved any further by incorporating an inosine and/or a nucleotide containing a base able to form a wobble base pair in said oligonucleotide. WO 2001/083503 describes oligonucleotides having inosine for guanine substitutions and relates to a method for labelling oligonucleotides which could be used in diagnostic arrays. WO 2005/115479 discloses inosine for guanine substitutions to reduce aggregation of G-rich PMO-peptide conjugates.

Description of the invention Oligonucleotide

In a first aspect of the invention, there is provided an antisense oligonucleotide comprising at least one inosine, wherein the oligonucleotide comprises a sequence which targets at least part of a dystrophin pre-mRNA exon part being a contiguous stretch comprising at least 13 nucleotides and wherein the antisense oligonucleotide is capable of exon-skipping. In a further aspect, there is provided an oligonucleotide according to the disclosure comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. The use of an inosine and/or a nucleotide containing a base able to form a wobble base pair in an oligonucleotide of the invention is very attractive as explained below. Inosine for example is a known modified base which can pair with three bases: uracil, adenine, and cytosine. Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a ß-N9-glycosidic bond. Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. A wobble base pair is a G-U and I-U / I-A / I-C pair fundamental in RNA secondary structure. Its thermodynamic stability is comparable to that of the Watson-Crick base pair. Wobble base pairs are critical for the proper translation of the genetic code. The genetic code makes up for disparities in the number of amino acids (20) for triplet codons (64), by using modified base pairs in the first base of the anti-codon. Similarly, when designing primers for polymerase chain reaction, inosine is useful in that it will indiscriminately pair with adenine, thymine, or cytosine. A first advantage of using such a base allows one to design a primer that spans a single nucleotide polymorphism (SNP), without worry that the polymorphism will disrupt the primer's annealing efficiency. Therefore in the invention, the use of such a base allows to design an oligonucleotide that may be used for an individual having a SNP within the dystrophin pre-mRNA stretch which is targeted by an oligonucleotide of the invention. A second advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is when said oligonucleotide would normally contain a CpG if one would have designed it as being complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 13 nucleotides. The presence of a CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (reference 60). This increased immunogenicity is undesired since it may induce the breakdown of muscle fibers. Replacing one, two or more CpG by the corresponding inosine and/or a base able to form a wobble base pair in said oligonucleotide is expected to provide an oligonucleotide with a decreased and/or acceptable level of immunogenicity. Immunogenicity may be assessed in an animal model by assessing the presence of CD4⁺ and/or CD8⁺ cells and/or inflammatory mononucleocyte infiltration in muscle biopsy of said animal. Immunogenicity may also be assessed in blood of an animal or of a human being treated with an oligonucleotide of the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said oligonucleotide using a standard immunoassay known to the skilled person. An increase in immunogenicity preferably corresponds to a detectable increase of at least one of these cell types by comparison to the amount of each cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having at least one inosine and/or a base able to form a wobble base pair. Alternatively, an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said oligonucleotide using a standard immunoassay. A decrease in immunogenicity preferably corresponds to a detectable decrease of at least one of these cell types by comparison to the amount of corresponding cell type in a corresponding muscle biopsy of an animal before treatment or treated with a corresponding oligonucleotide having no inosine and/or a base able to form a wobble base pair. Alternatively a decrease in immunogenicity may be assessed by the absence of or a decreasing amount of said compound and/or neutralizing antibodies using a standard immunoassay.

A third advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to avoid or decrease a potential multimerisation or aggregation of oligonucleotides. It is for example known that an oligonucleotide comprising a G-quartet motif has the tendency to form a quadruplex, a multimer or aggregate formed by the Hoogsteen base-pairing of four single-stranded oligonucleotides (reference 61), which is of course not desired: as a result the efficiency of the oligonucleotide is expected to be decreased. Multimerisation or aggregation is preferably assessed by standard polyacrylamid non-denaturing gel electrophoresis techniques known to the skilled person. In a preferred embodiment, less than 20% or 15%, 10%, 7%, 5% or less of a total amount of an oligonucleotide of the invention has the capacity to multimerise or aggregate assessed using the assay mentioned above.

A fourth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is thus also to avoid quadruplex structures which have been associated with antithrombotic activity (reference 62) as well as with the binding to, and inhibition of, the macrophage scavenger receptor (reference 63 .).

A fifth advantage of using an inosine and/or a base able to form a wobble base pair in an oligonucleotide of the invention is to allow to design an oligonucleotide with improved RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (http://www.unc.edu/∼cail/biotool/oligo/index.html) for single stranded RNA using the basic Tm and the nearest neighbour model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). If a Tm is too high, the oligonucleotide is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 65°C and an acceptable free energy may be ranged between 15 and 45 kcal/mol.

The skilled person may therefore first choose an oligonucleotide as a potential therapeutic compound. In a second step, he may use the invention to further optimise said oligonucleotide by decreasing its immunogenicity and/or avoiding aggregation and/or quadruplex formation and/or by optimizing its Tm and/or free energy of the AON-target complex. He may try to introduce at least one inosine and/or a base able to form a wobble base pair in said oligonucleotide at a suitable position and assess how the immunogenicity and/or aggregation and/or quadruplex formation and/or Tm and/or free energy of the AON-target complex have been altered by the presence of said inosine and/or a base able to form a wobble base pair. If the alteration does not provide the desired alteration or decrease of immunogenicity and/or aggregation and/or quadruplex formation and/or its Tm and/or free energy of the AON-target complex he may choose to introduce a further inosine and/or a base able to form a wobble base pair in said oligonucleotide and/or to introduce a given inosine and/or a base able to form a wobble base pair at a distinct suitable position within said oligonucleotide.

An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair may be defined as an oligonucleotide wherein at least one nucleotide has been substituted with an inosine and/or a base able to form a wobble base pair. The skilled person knows how to test whether a nucleotide contains a base able to form a wobble base pair. Since for example inosine can form a base pair with uracil, adenine, and/or cytosine, it means that at least one nucleotide able to form a base pair with uracil, adenine and/or cytosine has been substituted with inosine. However, in order to safeguard specificity, the inosine containing oligonucleotide preferably comprises the substitution of at least one, two, three, four nucleotide(s) able to form a base pair with uracil or adenine or cytosine as long as an acceptable level of a functional activity of said oligonucleotide is retained as defined later herein. An oligonucleotide comprising an inosine and/or a base able to form a wobble base pair is preferably an olignucleotide, which is still able to exhibit an acceptable level of a functional activity of a corresponding oligonucleotide not comprising an inosine and/or a base able to form a wobble base pair. A functional activity of said oligonucleotide is preferably to provide an individual with a functional dystrophin protein and/or mRNA and/or at least in part decreasing the production of an aberrant dystrophin protein and/or mRNA. Each of these features are later defined herein. An acceptable level of such a functional activity is preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the functional activity of the corresponding oligonucleotide which does not comprise an inosine and/or a base able to form a wobble base pair. Such functional activity may be as measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to the functional activity of a corresponding oligonucleotides not comprising an inosine and/or a base able to form a wobble base pair. The assessment of the functionality may be carried out at the mRNA level, preferably using RT-PCR. The assessment of the functionality may be carried out at the protein level, preferably using western blot analysis or immunofluorescence analysis of cross-sections.

Within the context of the disclosure, an inosine and/or a base able to form a wobble base pair as present in an oligonucleotide is/are present in a part of said oligonucleotide which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides. Therefore, in a preferred embodiment, an oligonucleotide comprising an inosine and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-mRNA exon or at least part of a non-exon region of a dystrophin pre-mRNA said part being a contiguous stretch comprising at least 8 nucleotides and wherein said inosine and/or a nucleotide containing a base able is/are present within the oligonucleotide sequence which is complementary to at least part of a dystrophin pre-mRNA as defined in previous sentence.

However, as later defined herein such inosine and/or a base able to form a wobble base pair may also be present in a linking moiety present in an oligonucleotide of the disclosure. Preferred linking moieties are later defined herein. In a preferred embodiment, In a preferred embodiment, an oligonucleotide according to the invention is preferably a medicament. More preferably, said medicament is for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or a patient. As defined herein a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD or BMD patient. A patient is preferably intended to mean a patient having DMD or BMD or a patient susceptible to develop DMD or BMD due to his or her genetic background. In the case of a DMD patient, an oligonucleotide used will preferably correct at least one of the DMD mutations as present in the DMD gene of said patient and therefore will preferably create a dystrophin that will look like a BMD dystrophin: said dystropin will preferably be a functional dystrophin as later defined herein. In the case of a BMD patient, an oligonucleotide as used will preferably correct at least one of the BMD mutations as present in the DMD gene of said patient and therefore will preferably create a, or more of a, dystrophin, which will be more functional than the dystrophin which was originally present in said BMD patient. Even more preferably, said medicament provides an individual with a functional or more (of a) functional dystrophin protein and/or mRNA and/or at least in part decreases the production of an aberrant dystrophin protein and/or mRNA. Preferably, a method of the invention by inducing and/or promoting skipping of at least one exon of the DMD pre-mRNA as identified herein in one or more cells, preferably muscle cells of a patient, provides said patient with an increased production of a more (of a)functional dystrophin protein and/or mRNA and/or decreases the production of an aberrant or less functional dystrophin protein and/or mRNA in said patient. Providing a patient with a more functional dystrophin protein and/or mRNA and/or decreasing the production of an aberrant dystrophin protein and/or mRNA in said patient is typically applied in a DMD patient. Increasing the production of a more functional or functional dystrophin and/or mRNA is typically applied in a BMD patient. Therefore a preferred method is a method, wherein a patient or one or more cells of said patient is provided with an increased production of a more functional or functional dystrophin protein and/or mRNA and/or wherein the production of an aberrant dystrophin protein and/or mRNA in said patient is decreased, wherein the level of said aberrant or more functional dystrophin protein and/or mRNA is assessed by comparison to the level of said dystrophin and/or mRNA in said patient at the onset of the method.

As defined herein, a functional dystrophin is preferably a wild type dystrophin corresponding to a protein having the amino acid sequence as identified in SEQ ID NO: 1. A functional dystrophin is preferably a dystrophin, which has an actin binding domain in its N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) each of these domains being present in a wild type dystrophin as known to the skilled person. The amino acids indicated herein correspond to amino acids of the wild type dystrophin being represented by SEQ ID NO: 1. In another embodiment, a functional dystrophin is a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. "At least to some extent" preferably means at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a corresponding activity of a wild type functional dystrophin. In this context, an activity of a wild type dystrophin is preferably binding to actin and to the dystrophin-associated glycoprotein complex (DGC)⁵⁶. Binding of dystrophin to actin and to the DGC complex may be visualized by either co-immunoprecipitation using total protein extracts or immunofluorescence analysis of cross-sections, from a biopsy of a muscle suspected to be dystrophic, as known to the skilled person.

Individuals suffering from Duchenne muscular dystrophy typically have a mutation in the gene encoding dystrophin that prevents synthesis of the complete protein, i.e a premature stop prevents the synthesis of the C-terminus of the protein. In Becker muscular dystrophy the dystrophin gene also comprises a mutation compared to the wild type but the mutation does typically not include a premature stop and the C-terminus of the protein is typically synthesized. As a result a functional dystrophin protein is synthesized that has at least the same activity in kind as a wild type protein, although not necessarily the same amount of activity. In a preferred embodiment, a functional dystrophin protein means an in frame dystrophin gene. The genome of a BMD individual typically encodes a dystrophin protein comprising the N terminal part (first 240 amino acids at the N terminus), a cystein-rich domain (amino acid 3361 till 3685) and a C terminal domain (last 325 amino acids at the C terminus) but its central rod shaped domain may be shorter than the one of a wild type dystrophin⁵⁶. Exon -skipping for the treatment of DMD is preferably but not exclusively directed to overcome a premature stop in the pre-mRNA by skipping an exon in the rod-domain shaped domain to correct the reading frame and allow synthesis of remainder of the dystrophin protein including the C-terminus, albeit that the protein is somewhat smaller as a result of a smaller rod domain. In a preferred embodiment, an individual having DMD and being treated using an oligonucleotide as defined herein will be provided a dystrophin, which exhibits at least to some extent an activity of a wild type dystrophin. More preferably, if said individual is a Duchenne patient or is suspected to be a Duchenne patient, a functional dystrophin is a dystrophin of an individual having BMD: preferably said dystrophin is able to interact with both actin and the DGC, but its central rod shaped domain may be shorter than the one of a wild type dystrophin (Aartsma-Rus et al (2006, ref 56). The central rod domain of wild type dystrophin comprises 24 spectrin-like repeats⁵⁶. For example, a central rod shaped domain of a dystrophin as provided herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like repeats as long as it can bind to actin and to DGC. Decreasing the production of an aberrant dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of aberrant dystrophin mRNA, is still detectable by RT PCR. An aberrant dystrophin mRNA or protein is also referred to herein as a non-functional or less to non-functional or semi-functional dystrophin mRNA or protein. A non-functional pre-mRNA dystrophin is preferably leads to an out of frame dystrophin protein, which means that no dystrophin protein will be produced and/or detected. A non functional dystrophin protein is preferably a dystrophin protein which is not able to bind actin and/or members of the DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA does typically not have, or does not encode a dystrophin protein with an intact C-terminus of the protein.

Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the mRNA level (by RT-PCR analysis) and preferably means that a detectable amount of a functional or in frame dystrophin mRNA is detectable by RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a functional or in frame dystrophin mRNA. Increasing the production of a functional dystrophin in said patient or in a cell of said patient may be assessed at the protein level (by immunofluorescence and western blot analyses) and preferably means that a detectable amount of a functional dystrophin protein is detectable by immunofluorescence or western blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin protein is a functional dystrophin protein.

An increase or a decrease is preferably assessed in a muscular tissue or in a muscular cell of an individual or a patient by comparison to the amount present in said individual or patient before treatment with said molecule or composition of the invention. Alternatively, the comparison can be made with a muscular tissue or cell of said individual or patient, which has not yet been treated with said oligonucleotide or composition in case the treatment is local.

In a preferred method, one or more symptom(s) from a DMD or a BMD patient is/are alleviated and/or one or more characteristic(s) of a muscle cell or tissue from a DMD or a BMD patient is/are alleviated using a molecule or a composition of the invention. Such symptoms may be assessed on the patient self. Such characteristics may be assessed at the cellular, tissue level of a given patient. An alleviation of one or more characteristics may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, decreased oxidative stress, and/or improved muscle fiber function, integrity, and/or survival. These parameters are usually assessed using immunofluorescence and/or histochemical analyses of cross sections of muscle biopsies.

Alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual using a molecule or a composition of the invention may be assessed by any of the following assays: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life. Each of these assays is known to the skilled person. As an example, the publication of Manzur at al (2008, ref 58) gives an extensive explanation of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of a parameter measured in an assay has been found, it will preferably mean that one or more symptoms of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy has been alleviated in an individual using a molecule or composition of the invention. Detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al (2006, ref 57). Alternatively, the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.

An oligonucleotide as used herein preferably comprises an antisense oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied. Exon skipping interferes with the natural splicing processes occurring within a eukaryotic cell. In higher eukaryotes the genetic information for proteins in the DNA of the cell is encoded in exons which are separated from each other by intronic sequences. These introns are in some cases very long. The transcription machinery of eukaryotes generates a pre-mRNA which contains both exons and introns, while the splicing machinery, often already during the production of the pre-mRNA, generates the actual coding region for the protein by splicing together the exons present in the pre-mRNA.

Exon-skipping results in mature mRNA that lacks at least one skipped exon. Thus, when said exon codes for amino acids, exon skipping leads to the expression of an altered product. Technology for exon-skipping is currently directed towards the use of antisense oligonucleotides (AONs). Much of this work is done in the mdx mouse model for Duchenne muscular dystrophy. The mdx mouse carries a nonsense mutation in exon 23. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin positive fibers have been observed in mdx muscle tissue. These dystrophin-positive fibers are thought to have arisen from an apparently naturally occurring exon-skipping mechanism, either due to somatic mutations or through alternative splicing. AONs directed to, respectively, the 3' and/or 5' splice sites of introns 22 and 23 in dystrophin pre-mRNA, have been shown to interfere with factors normally involved in removal of intron 23 so that also exon 23 was removed from the mRNA^{3, 5, 6, 39, 40}.

By the targeted skipping of a specific exon, a DMD phenotype is converted into a milder BMD phenotype. In the disclosure, the skipping of an exon is preferably induced by the binding of AONs targeting either one or both of the splice sites, or exon-internal sequences. An oligonucleotide of the invention directed toward an exon internal sequence typically exhibits no overlap with non-exon sequences. It preferably does not overlap with the splice sites at least not insofar, as these are present in the intron. An oligonucleotide directed toward an exon internal sequence preferably does not contain a sequence complementary to an adjacent intron. Further provided is thus an oligonucleotide according to the invention, wherein said oligonucleotide, or a functional equivalent thereof, is for inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA produced from splicing of said pre-mRNA. An exon skipping technique is preferably applied such that the absence of an exon from mRNA produced from dystrophin pre-mRNA generates a coding region for a more functional - albeit shorter - dystrophin protein. In this context, inhibiting inclusion of an exon preferably means that the detection of the original, aberrant dystrophin mRNA and/or protein is decreased as earlier defined herein.

Since an exon of a dystrophin pre-mRNA will only be included into the resulting mRNA when both the splice sites are recognised by the spliceosome complex, splice sites have been obvious targets for AONs. One embodiment of the disclosure therefore provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a non-exon region of a dystrophin pre mRNA. In one embodiment of the disclosure an AON is used which is solely complementary to a non-exon region of a dystrophin pre mRNA. This is however not necessary: it is also possible to use an AON which comprises an intron-specific sequence as well as exon-specific sequence. Such AON of the disclosure comprises a sequence which is complementary to a non-exon region of a dystrophin pre mRNA, as well as a sequence which is complementary to an exon region of a dystrophin pre mRNA. Of course, an AON is not necessarily complementary to the entire sequence of a dystrophin exon or intron. AONs of the disclosure, which are complementary to a part of such exon or intron are preferred. An AON of the disclosure is preferably complementary to at least part of a dystrohin exon and/or intron, said part having at least 8, 10, 13, 15, 20 nucleotides.

Splicing of a dystrophin pre-mRNA occurs via two sequential transesterification reactions. First, the 2'OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat. The branch point and splice sites of an intron are thus involved in a splicing event. Hence, an oligonucleotide of the disclosure comprising a sequence, which is complementary to such branch point and/or splice site is preferably used for exon skipping. Further provided is therefore an oligonucleotide of the disclosure, or a functional equivalent thereof, which comprises a sequence which is complementary to a splice site and/or branch point of a dystrophin pre mRNA.

Since splice sites contain consensus sequences, the use of an oligonucleotide or a functional equivalent thereof of the disclosure (herein also called an AON) comprising a sequence which is complementary of a splice site involves the risk of promiscuous hybridization. Hybridization of AONs to other splice sites than the sites of the exon to be skipped could easily interfere with the accuracy of the splicing process. To overcome these and other potential problems related to the use of AONs which are complementary to an intron sequence, one preferred embodiment of the disclosure provides an oligonucleotide, or a functional equivalent thereof, comprising a sequence which is complementary to a dystrophin pre-mRNA exon. Preferably, said AON is capable of specifically inhibiting an exon inclusion signal of at least one exon in said dystrophin pre-mRNA. Interfering with an exon inclusion signal (EIS) has the advantage that such elements are located within the exon. By providing an AON for the interior of the exon to be skipped, it is possible to interfere with the exon inclusion signal thereby effectively masking the exon from the splicing apparatus. The failure of the splicing apparatus to recognize the exon to be skipped thus leads to exclusion of the exon from the final mRNA. This embodiment does not interfere directly with the enzymatic process of the splicing machinery (the joining of the exons). It is thought that this allows the method to be more specific and/or reliable. It is thought that an EIS is a particular structure of an exon that allows splice acceptor and donor to assume a particular spatial conformation. In this concept, it is the particular spatial conformation that enables the splicing machinery to recognize the exon. However, the invention is certainly not limited to this model. In a preferred embodiment, use is made of an oligonucleotide, which is capable of binding to an exon and is capable of inhibiting an EIS. An AON may specifically contact said exon at any point and still be able to specifically inhibit said EIS.

Within the context of the invention, a functional equivalent of an oligonucleotide preferably means an oligonucleotide as defined herein wherein one or more nucleotides have been substituted and wherein an activity of said functional equivalent is retained to at least some extent. Preferably, an activity of said functional equivalent is providing a functional dystrophin protein. Said activity of said functional equivalent is therefore preferably assessed by quantifying the amount of a functional dystrophin protein or by quantifying the amount of a functional dystrophin mRNA. A functional dystrophin protein (or a functional dystrophin mRNA) is herein preferably defined as being a dystrophin protein (or a dystrophin protein encoded by said mRNA) able to bind actin and members of the DGC protein. The assessment of said activity of an oligonucleotide is preferably done by RT-PCR (m-RNA) or by immunofluorescence or Western blot analyses (protein). Said activity is preferably retained to at least some extent when it represents at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% or at least 95% or more of corresponding activity of said oligonucleotide the functional equivalent derives from. Such activity may be measured in a muscular tissue or in a muscular cell of an individual or in vitro in a cell by comparison to an activity of a corresponding oligonucleotide of said oligonucleotide the functional equivalent derives from. Throughout this application, when the word oligonucleotide is used it may be replaced by a functional equivalent thereof as defined herein.

Hence, an oligonucleotide, or a functional equivalent thereof, comprising or consisting of a sequence which is complementary to a dystrophin pre-mRNA exon provides good DMD therapeutic results. In one preferred embodiment an oligonucleotide, or a functional equivalent thereof, is used which comprises or consists of a sequence which is complementary to at least part of either dystrophin pre-mRNA exons 2 to 75 said part having or comprising at least 13 nucleotides. However, said part may also have at least 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Within the disclosure, said part may also have at least 8, 9, 10, 11, 12 nucleotides. A part of dystrophin pre-mRNA to which an oligonucleotide is complementary may also be called a contiguous stretch of dystrophin pre-mRNA.

Most preferably an AON is used which comprises or consists of a sequence which is complementary to at least part of dystrophin pre-mRNA exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, 55, 2, 11, 17, 19, 21, 57, 59, 62, 63, 65, 66, 69, and/or 75 said part having or comprising at least 13 nucleotides. However, said part may also have at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Within the disclosure, said part may also have at least 8, 9, 10, 11, 12 nucleotides. More preferred oligonucleotides are represented by a sequence that comprises or consists of each of the following sequences SEQ ID NO: 2 to SEQ ID NO:539 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil. Accordingly, an even more preferred oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:2 to SEQ ID NO:486 or SEQ ID NO:539, even more preferably SEQ ID NO:2 to NO 237 or SEQ ID NO:539, most preferably SEQ ID NO:76 wherein at least one inosine and/or a base able to form a wobble base pair is present in said sequence. Preferably, an inosine has been introduced in one of these sequences to replace a guanosine, adenine, or a uracil.

Accordingly, in another preferred embodiment, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:540 to SEQ ID NO:576. More preferably, an oligonucleotide as used herein is represented by a sequence that comprises or consists of SEQ ID NO:557.

Said exons are listed in decreasing order of patient population applicability. Hence, the use of an AON comprising a sequence, which is complementary to at least part of dystrophin pre-mRNA exon 51 is suitable for use in a larger part of the DMD patient population as compared to an AON comprising a sequence which is complementary to dystrophin pre-mRNA exon 44, et cetera.

In a preferred embodiment, an oligonucleotide of the invention, which comprises a sequence that is complementary to part of dystrophin pre-mRNA is such that the complementary part is at least 50% of the length of the oligonucleotide of the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99%, or even more preferably 100%. In a most preferred embodiment, the oligonucleotide of the invention consists of a sequence that is complementary to part of dystrophin pre-mRNA as defined herein. As an example, an oligonucleotide may comprise a sequence that is complementary to part of dystrophin pre-mRNA as defined herein and additional flanking sequences. In a more preferred embodiment, the length of said complementary part of said oligonucleotide is of at least 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Within the disclosure, said part may also have at least 8, 9, 10, 11, 12 nucleotides. Preferably, additional flanking sequences are used to modify the binding of a protein to the oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, more preferably to modify target RNA binding affinity.

One preferred embodiment provides an oligonucleotide, or a functional equivalent thereof which comprises:

a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is hybridized to another part of a dystrophin pre-mRNA exon (closed structure), and
a sequence which is complementary to a region of a dystrophin pre-mRNA exon that is not hybridized in said dystrophin pre-mRNA (open structure).

For this embodiment, reference is made to WO 2004/083432 . RNA molecules exhibit strong secondary structures, mostly due to base pairing of complementary or partly complementary stretches within the same RNA. It has long since been thought that structures in the RNA play a role in the function of the RNA. Without being bound by theory, it is believed that the secondary structure of the RNA of an exon plays a role in structuring the splicing process. The structure of an exon is one parameter which is believed to direct its inclusion into the mRNA. However, other parameters may also play a role therein. Herein this signalling function is referred to as an exon inclusion signal. A complementary oligonucleotide of this embodiment is capable of interfering with the structure of the exon and thereby capable of interfering with the exon inclusion signal of the exon. It has been found that many complementary oligonucleotides indeed comprise this capacity, some more efficient than others. Oligonucleotides of this preferred embodiment, i.e. those with the said overlap directed towards open and closed structures in the native exon RNA, are a selection from all possible oligonucleotides. The selection encompasses oligonucleotides that can efficiently interfere with an exon inclusion signal. Without being bound by theory it is thought that the overlap with an open structure improves the invasion efficiency of the oligonucleotide and prevents the binding of splicing factors (i.e. increases the efficiency with which the oligonucleotide can enter the structure), whereas the overlap with the closed structure subsequently increases the efficiency of interfering with the secondary structure of the RNA of the exon, and thereby interfere with the exon inclusion signal. It is found that the length of the partial complementarity to both the closed and the open structure is not extremely restricted. We have observed high efficiencies with oligonucleotides with variable lengths of complementarity in either structure. The term complementarity is used herein to refer to a stretch of nucleic acids that can hybridise to another stretch of nucleic acids under physiological conditions. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the oligonucleotide one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may, to some extent, be allowed, if under the circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridising to the complementary part. In this context, "sufficiently" preferably means that using a gel mobility shift assay as described in example 1 of EP 1619 249 , binding of an oligonucleotide is detectable. Optionally, said oligonucleotide may further be tested by transfection into muscle cells of patients. Skipping of the targeted exon may be assessed by RT-PCR (as described in EP 1 619 249 ). The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-)mRNA in the system. The risk that also one or more other pre-mRNA will be able to hybridise to the oligonucleotide decreases with increasing size of the oligonucleotide. It is clear that oligonucleotides comprising mismatches in the region of complementarity but that retain the capacity to hybridise to the targeted region(s) in the pre-mRNA, can be used in the present invention. However, preferably at least the complementary parts do not comprise such mismatches as these typically have a higher efficiency and a higher specificity, than oligonucleotides having such mismatches in one or more complementary regions. It is thought, that higher hybridisation strengths, (i.e. increasing number of interactions with the opposing strand) are favourable in increasing the efficiency of the process of interfering with the splicing machinery of the system. Preferably, the complementarity is between 90 and 100%. In general this allows for approximately 1 or 2 mismatch(es) in an oligonucleotide of around 20 nucleotides

The secondary structure is best analysed in the context of the pre-mRNA wherein the exon resides. Such structure may be analysed in the actual RNA. However, it is currently possible to predict the secondary structure of an RNA molecule (at lowest energy costs) quite well using structure-modelling programs. A non-limiting example of a suitable program is RNA mfold version 3.1 server⁴¹. A person skilled in the art will be able to predict, with suitable reproducibility, a likely structure of the exon, given the nucleotide sequence. Best predictions are obtained when providing such modelling programs with both the exon and flanking intron sequences. It is typically not necessary to model the structure of the entire pre-mRNA.

The open and closed structure to which the oligonucleotide is directed, are preferably adjacent to one another. It is thought, that in this way the annealing of the oligonucleotide to the open structure induces opening of the closed structure whereupon annealing progresses into this closed structure. Through this action the previously closed structure assumes a different conformation. The different conformation results in the disruption of the exon inclusion signal. However, when potential (cryptic) splice acceptor and/or donor sequences are present within the targeted exon, occasionally a new exon inclusion signal is generated defining a different (neo) exon, i.e. with a different 5' end, a different 3' end, or both. This type of activity is within the scope of the present invention as the targeted exon is excluded from the mRNA. The presence of a new exon, containing part of the targeted exon, in the mRNA does not alter the fact that the targeted exon, as such, is excluded. The inclusion of a neo-exon can be seen as a side effect, which occurs only occasionally. There are two possibilities when exon skipping is used to restore (part of) an open reading frame of dystrophin that is disrupted as a result of a mutation. One is that the neo-exon is functional in the restoration of the reading frame, whereas in the other case the reading frame is not restored. When selecting oligonucleotides for restoring dystrophin reading frames by means of exon-skipping it is of course clear that under these conditions only those oligonucleotides are selected that indeed result in exon-skipping that restores the dystrophin open reading frame, with or without a neo-exon.

Further provided is an oligonucleotide, or a functional equivalent thereof, comprising a sequence that is complementary to a binding site for a serine-arginine (SR) protein in RNA of an exon of a dystrophin pre-mRNA. In WO 2006/112705 we have disclosed the presence of a correlation between the effectivity of an exon-internal antisense oligonucleotide (AON) in inducing exon skipping and the presence of a (for example by ESE finder) predicted SR binding site in the target pre-mRNA site of said AON. Therefore, in one embodiment an oligonucleotide is generated comprising determining a (putative) binding site for an SR (Ser-Arg) protein in RNA of a dystrophin exon and producing an oligonucleotide that is complementary to said RNA and that at least partly overlaps said (putative) binding site. The term "at least partly overlaps" is defined herein as to comprise an overlap of only a single nucleotide of an SR binding site as well as multiple nucleotides of said binding site as well as a complete overlap of said binding site. This embodiment preferably further comprises determining from a secondary structure of said RNA, a region that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide that at least partly overlaps said (putative) binding site and that overlaps at least part of said closed structure and overlaps at least part of said open structure. In this way we increase the chance of obtaining an oligonucleotide that is capable of interfering with the exon inclusion from the pre-mRNA into mRNA. It is possible that a first selected SR-binding region does not have the requested open-closed structure in which case another (second) SR protein binding site is selected which is then subsequently tested for the presence of an open-closed structure. This process is continued until a sequence is identified which contains an SR protein binding site as well as a(n) (partly overlapping) open-closed structure. This sequence is then used to design an oligonucleotide which is complementary to said sequence.

Such a method, for generating an oligonucleotide, is also performed by reversing the described order, i.e. first generating an oligonucleotide comprising determining, from a secondary structure of RNA from a dystrophin exon, a region that assumes a structure that is hybridised to another part of said RNA (closed structure) and a region that is not hybridised in said structure (open structure), and subsequently generating an oligonucleotide, of which at least a part of said oligonucleotide is complementary to said closed structure and of which at least another part of said oligonucleotide is complementary to said open structure. This is then followed by determining whether an SR protein binding site at least overlaps with said open/closed structure. In this way the method of WO 2004/083432 is improved. In yet another embodiment the selections are performed simultaneously.

Without wishing to be bound by any theory it is currently thought that use of an oligonucleotide directed to an SR protein binding site results in (at least partly) impairing the binding of an SR protein to the binding site of an SR protein which results in disrupted or impaired splicing.

Preferably, an open/closed structure and an SR protein binding site partly overlap and even more preferred an open/closed structure completely overlaps an SR protein binding site or an SR protein binding site completely overlaps an open/closed structure. This allows for an improved disruption of exon inclusion.

Besides consensus splice sites sequences, many (if not all) exons contain splicing regulatory sequences such as exonic splicing enhancer (ESE) sequences to facilitate the recognition of genuine splice sites by the spliceosome^{42, 43}. A subgroup of splicing factors, called the SR proteins, can bind to these ESEs and recruit other splicing factors, such as U1 and U2AF to (weakly defined) splice sites. The binding sites of the four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55) have been analyzed in detail and these results are implemented in ESE finder, a web source that predicts potential binding sites for these SR proteins^{42, 43}. There is a correlation between the effectiveness of an AON and the presence/absence of an SF2/ASF, SC35 and SRp40 binding site. In a preferred embodiment, the invention thus provides a combination as described above, wherein said SR protein is SF2/ASF or SC35 or SRp40.

In one embodiment an oligonucleotide, or a functional equivalent thereof is capable of specifically binding a regulatory RNA sequence which is required for the correct splicing of a dystrophin exon in a transcript. Several cis-acting RNA sequences are required for the correct splicing of exons in a transcript. In particular within the disclosure, supplementary elements such as intronic or exonic splicing enhancers (ISEs and ESEs) or silencers (ISSs and ESEs) are identified to regulate specific and efficient splicing of constitutive and alternative exons. Using sequence-specific antisense oligonucleotides (AONs) that bind to the elements, their regulatory function is disturbed so that the exon is skipped, as shown for DMD. Hence, in one preferred embodiment of the disclosure an oligonucleotide or functional equivalent thereof is used which is complementary to an intronic splicing enhancer (ISE), an exonic splicing enhancer (ESE), an intronic splicing silencer (ISS) and/or an exonic splicing silencer (ESS). As already described herein before, a dystrophin exon is in one preferred embodiment skipped by an agent capable of specifically inhibiting an exon inclusion signal of said exon, so that said exon is not recognized by the splicing machinery as a part that needs to be included in the mRNA. As a result, a mRNA without said exon is formed.

An AON used in the disclosure is preferably complementary to a consecutive part or a contiguous stretch of between 8 and 50 nucleotides of dystrophin exon RNA or dystrophin intron RNA. In one embodiment an AON used herein is complementary to a consecutive part or a contiguous stretch of between 14 and 50 nucleotides of a dystrophin exon RNA or dystrophin intron RNA. Preferably, said AON is complementary to a consecutive part or contiguous stretch of between 14 and 25 nucleotides of said exon RNA. More preferably, an AON is used which comprises a sequence which is complementary to a consecutive part or a contiguous stretch of between 20 and 25 nucleotides of a dystrophin exon RNA or a dystrophin intron RNA.

Different types of nucleic acid may be used to generate an oligonucleotide according to the invention. Preferably, said oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable. Since one of the aims of the exon skipping technique is to direct splicing in subjects it is preferred that the oligonucleotide RNA comprises a modification providing the RNA with an additional property, for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc. Preferably, said modification comprises a 2'-O-methyl-phosphorothioate oligoribonucleotide modification. Preferably, said modification comprises a 2'-O-methyl-phosphorothioate oligodeoxyribonucleotide modification. One embodiment thus provides an oligonucleotide is used which comprises RNA which contains a modification, preferably a 2'-O-methyl modified ribose (RNA) or deoxyribose (DNA) modification.

In one embodiment the invention provides a hybrid oligonucleotide comprising an oligonucleotide comprising a 2'-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification and locked nucleic acid. This particular oligonucleotide comprises better sequence specificity compared to an equivalent consisting of locked nucleic acid, and comprises improved effectivity when compared with an oligonucleotide consisting of 2'- O-methyl-phosphorothioate oligo(deoxy)ribonucleotide modification.

With the advent of nucleic acid mimicking technology it has become possible to generate molecules that have a similar, preferably the same hybridisation characteristics in kind not necessarily in amount as nucleic acid itself. Such functional equivalents are of course also suitable for use in the invention. Preferred examples of functional equivalents of an oligonucleotide are peptide nucleic acid and/or locked nucleic acid. Most preferably, a morpholino phosphorodiamidate is used. Suitable but non-limiting examples of equivalents of oligonucleotides of the invention can be found in^44-50. Hybrids between one or more of the equivalents among each other and/or together with nucleic acid are of course also suitable. In a preferred embodiment locked nucleic acid is used as a functional equivalent of an oligonucleotide, as locked nucleic acid displays a higher target affinity and reduced toxicity and therefore shows a higher efficiency of exon skipping.

In one embodiment an oligonucleotide, or a functional equivalent thereof, which is capable of inhibiting inclusion of a dystrophin exon into dystrophin mRNA is combined with at least one other oligonucleotide, or functional equivalent thereof, that is capable of inhibiting inclusion of another dystrophin exon into dystrophin mRNA. This way, inclusion of two or more exons of a dystrophin pre-mRNA in mRNA produced from this pre-mRNA is prevented. This embodiment is further referred to as double- or multi-exon skipping^{2, 15}. In most cases double-exon skipping results in the exclusion of only the two targeted exons from the dystrophin pre-mRNA. However, in other cases it was found that the targeted exons and the entire region in between said exons in said pre-mRNA were not present in the produced mRNA even when other exons (intervening exons) were present in such region. This multi-exon skipping was notably so for the combination of oligonucleotides derived from the DMD gene, wherein one oligonucleotide for exon 45 and one oligonucleotide for exon 51 was added to a cell transcribing the DMD gene. Such a set-up resulted in mRNA being produced that did not contain exons 45 to 51. Apparently, the structure of the pre-mRNA in the presence of the mentioned oligonucleotides was such that the splicing machinery was stimulated to connect exons 44 and 52 to each other. Other preferred examples of multi-exon skipping are:

the use of an oligonucleotide targeting exon 17, and a second one exon 48 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 48.
the use of an oligonucleotide targeting exon 17, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 17 and exon 51.
the use of an oligonucleotide targeting exon 42, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 42 and exon 55.
the use of an oligonucleotide targeting exon 43, and a second one exon 51 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 51.
the use of an oligonucleotide targeting exon 43, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 43 and exon 55.
the use of an oligonucleotide targeting exon 45, and a second one exon 55 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 55.
the use of an oligonucleotide targeting exon 45, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 45 and exon 59.
the use of an oligonucleotide targeting exon 48, and a second one exon 59 which may result in the skipping of said both exons or of the entire region between exon 48 and exon 59.
the use of an oligonucleotide targeting exon 50, and a second one exon 51 which may result in the skipping of said both exons.
the use of an oligonucleotide targeting exon 51, and a second one exon 52 which may result in the skipping of said both exons.

Further provided in the disclosure is therefore an oligonucleotide which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first exon of a dystrophin pre-mRNA and wherein a nucleotide sequence is used which comprises at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon of said dystrophin pre-mRNA. Said first and said second exon may be the same.

In one preferred embodiment said first and said second exon are separated in said dystrophin pre-mRNA by at least one exon to which said oligonucleotide is not complementary. Alternatively, said first and said second exon are adjacent.

It is possible to specifically promote the skipping of also the intervening exons by providing a linkage between the two complementary oligonucleotides. Hence, in one embodiment stretches of nucleotides complementary to at least two dystrophin exons are separated by a linking moiety. The at least two stretches of nucleotides are thus linked in this embodiment so as to form a single molecule. Further provided is therefore an oligonucleotide, or functional equivalent thereof of the disclosure which is complementary to at least two exons in a dystrophin pre-mRNA, said oligonucleotide or functional equivalent comprising at least two parts wherein a first part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a first of said at least two exons and wherein a second part comprises an oligonucleotide having at least 8, preferably between 16 to 80, consecutive nucleotides that are complementary to a second exon in said dystrophin pre-mRNA. The linkage may be through any means, but is preferably accomplished through a nucleotide linkage. In the latter case, the number of nucleotides that do not contain an overlap between one or the other complementary exon can be zero, but is preferably between 4 to 40 nucleotides. The linking moiety can be any type of moiety capable of linking oligonucleotides. Preferably, said linking moiety comprises at least 4 uracil nucleotides. Currently, many different compounds are available that mimic hybridisation characteristics of oligonucleotides. Such a compound, called herein a functional equivalent of an oligonucleotide, is also suitable for the present invention if such equivalent comprises similar hybridisation characteristics in kind not necessarily in amount. Suitable functional equivalents are mentioned earlier in this description. As mentioned, oligonucleotides of the invention do not have to consist of only oligonucleotides that contribute to hybridisation to the targeted exon. There may be additional material and/or nucleotides added.

The DMD gene is a large gene, with many different exons. Considering that the gene is located on the X-chromosome, it is mostly boys that are affected, although girls can also be affected by the disease, as they may receive a bad copy of the gene from both parents, or are suffering from a particularly biased inactivation of the functional allele due to a particularly biased X chromosome inactivation in their muscle cells. The protein is encoded by a plurality of exons (79) over a range of at least 2.4 Mb. Defects may occur in any part of the DMD gene. Skipping of a particular exon or particular exons can, very often, result in a restructured mRNA that encodes a shorter than normal but at least partially functional dystrophin protein. A practical problem in the development of a medicament based on exon-skipping technology is the plurality of mutations that may result in a deficiency in functional dystrophin protein in the cell. Despite the fact that already multiple different mutations can be corrected for by the skipping of a single exon, this plurality of mutations, requires the generation of a series of different pharmaceuticals as for different mutations different exons need to be skipped. An advantage of an oligonucleotide or of a composition comprising at least two distinct oligonucleotide as later defined herein capable of inducing skipping of two or more exons, is that more than one exon can be skipped with a single pharmaceutical. This property is not only practically very useful in that only a limited number of pharmaceuticals need to be generated for treating many different DMD or particular, severe BMD mutations. Another option now open to the person skilled in the art is to select particularly functional restructured dystrophin proteins and produce compounds capable of generating these preferred dystrophin proteins. Such preferred end results are further referred to as mild phenotype dystrophins.

Dose ranges of oligonucleotide according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. A molecule or an oligonucleotide as defined herein may be used at a dose which is ranged between 0.1 and 20 mg/kg, preferably 0.5 and 10 mg/kg. In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged between 0.1 nM and 1 µM is used. Preferably, this range is for in vitro use in a cellular model such as muscular cells or muscular tissue. More preferably, the concentration used is ranged between 0.3 to 400 nM, even more preferably between 1 to 200 nM. If several oligonucleotides are used, this concentration or dose may refer to the total concentration or dose of oligonucleotides or the concentration or dose of each oligonucleotide added.

The ranges of concentration or dose of oligonucleotide(s) as given above are preferred concentrations or doses for in vitro or ex vivo uses. The skilled person will understand that depending on the oligonucleotide(s) used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of oligonucleotide(s) used may further vary and may need to be optimised any further.

An oligonucleotide as defined herein for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing DMD or BMD, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Duchenne and Becker muscular dystrophy have a pronounced phenotype in muscle cells, it is preferred that said cells are muscle cells, it is further preferred that said tissue is a muscular tissue and/or it is further preferred that said organ comprises or consists of a muscular tissue. A preferred organ is the heart. Preferably, said cells comprise a gene encoding a mutant dystrophin protein. Preferably, said cells are cells of an individual suffering from DMD or BMD.

An oligonucleotide of the invention may be indirectly administrated using suitable means known in the art. An oligonucleotide may for example be provided to an individual or a cell, tissue or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of a molecule as identified herein. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector^{4, 51, 52} and the like. Also, plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from PolIII promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are PolIII driven transcripts. Preferably, in the form of a fusion transcript with an Ulor U7 transcript^{4, 51, 52}. Such fusions may be generated as described^{53, 54}. The oligonucleotide may be delivered as is. However, the oligonucleotide may also be encoded by the viral vector. Typically, this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.

Improvements in means for providing an individual or a cell, tissue, organ of said individual with an oligonucleotide and/or an equivalent thereof, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. An oligonucleotide and/or an equivalent thereof can be delivered as is to an individual, a cell, tissue or organ of said individual. When administering an oligonucleotide and/or an equivalent thereof, it is preferred that an oligonucleotide and/or an equivalent thereof is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a muscle cell. Preferred are excipients capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine (PEI), or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell, preferably a muscle cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as antisense nucleic acids to a wide variety of cultured cells, including muscle cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.

Polycations such like diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an oligonucleotide across cell membranes into cells.

In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an oligonucleotide for use in the current invention to deliver it for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in humans.

In addition, an oligonucleotide could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake in to the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognising cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, an oligonucleotide is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery. Accordingly, the invention also encompasses a pharmaceutically acceptable composition comprising an oligonucleotide and further comprising at least one excipient and/or a targeting ligand for delivery and/or a delivery device of said oligonucleotide to a cell and/or enhancing its intracellular delivery. It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an oligonucleotide and a further adjunct compound as later defined herein.

A preferred oligonucleotide is for preventing or treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in an individual. An individual, which may be treated using an oligonucleotide of the invention may already have been diagnosed as having a DMD or a BMD. Alternatively, an individual which may be treated using an oligonucleotide of the invention may not have yet been diagnosed as having a DMD or a BMD but may be an individual having an increased risk of developing a DMD or a BMD in the future given his or her genetic background. A preferred individual is a human being.

Composition

In a further aspect, there is provided a composition comprising an oligonucleotide as defined herein. Preferably, said composition comprises at least two distinct oligonucleotide as defined herein. More preferably, these two distinct oligonucleotides are designed to skip distinct two or more exons as earlier defined herein for multi-exon skipping.

In a preferred embodiment, said composition being preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Such a pharmaceutical composition may comprise any pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. Each feature of said composition has earlier been defined herein.

If several oligonucleotides are used, concentration or dose already defined herein may refer to the total concentration or dose of all oligonucleotides used or the concentration or dose of each oligonucleotideused or added. Therefore in one embodiment, there is provided a composition wherein each or the total amount of oligonucleotide used is dosed in an amount ranged between 0.5 mg/kg and 10 mg/kg.

A preferred composition additionally comprises:

a) an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or
b) an adjunct compound for improving muscle fiber function, integrity and/or survival and/or
c) a compound exhibiting readthrough activity.

It has surprisingly been found that the skipping frequency of a dystrophin exon from a pre-MRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon, is enhanced if cells expressing said pre-mRNA are also provided with an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation, and/or an adjunct compound for improving muscle fiber function, integrity and/or survival. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

According to the present disclosure, even when a dystrophin protein deficiency has been restored in a DMD patient by administering an oligonucleotide of the invention, the presence of tissue inflammation and damaged muscle cells still continues to contribute to the symptoms of DMD. Hence, even though the cause of DMD - i.e. a dysfunctional dystrophin protein - is alleviated, treatment of DMD is still further improved by additionally using an adjunct therapy according to the present disclosure. Furthermore, the present disclosure provides the insight that a reduction of inflammation does not result in significant reduction of AON uptake by muscle cells. This is surprising because, in general, inflammation enhances the trafficking of cells, blood and other compounds. As a result, AON uptake/delivery is also enhanced during inflammation. Hence, before the present disclosure it would be expected that an adjunct therapy counteracting inflammation involves the risk of negatively influencing AON therapy. This, however, appears not to be the case.

An adjunct compound for reducing inflammation comprises any therapy which is capable of at least in part reducing inflammation, preferably inflammation caused by damaged muscle cells. Said adjunct compound is most preferably capable of reducing muscle tissue inflammation. Inflammation is preferably assessed by detecting an increase in the number of infiltrating immune cells such as neutrophils and/or mast cells and/or dendritic cells and/or lymphocytes in muscle tissue suspected to be dystrophic. This assessment is preferably carried out in cross-sections of a biopsy⁵⁷ of muscle tissue suspected to be dystrophic after having specifically stained immune cells as identified above. The quantification is preferably carried out under the microscope. Reducing inflammation is therefore preferably assessed by detecting a decrease in the number of immune cells in a cross-section of muscle tissue suspected to be dystrophic. Detecting a decrease preferably means that the number of at least one sort of immune cells as identified above is decreased of at least 1%, 2%, 3%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the number of a corresponding immune cell in a same individual before treatment. Most preferably, no infiltrating immune cells are detected in cross-sections of said biopsy.

An adjunct compound of the disclosure for improving muscle fiber function, integrity and/or survival comprises any therapy, which is capable of measurably enhancing muscle fiber function, integrity and/or survival as compared to an otherwise similar situation wherein said adjunct compound is not present. The improvement of muscle fiber function, integrity and/or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and/or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.

Creatine kinase may be detected in blood as described in 57. A detectable decrease in creatine kinase may mean a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same individual before treatment.

A detectable decrease of necrosis of muscle fibers is preferably assessed in a muscle biopsy, more preferably as described in 57 using biopsy cross-sections. A detectable decrease of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same individual before treatment.

A detectable increase of the homogeneity of the diameter of a muscle fiber is preferably assessed in a muscle biopsy cross-section, more preferably as described in 57.

In one embodiment of the disclosure, an adjunct compound for increasing turnover of damaged muscle cells is used. An adjunct compound for increasing turnover of damaged muscle cells comprises any therapy, which is capable of at least in part inducing and/or increasing turnover of damaged muscle cells. Damaged muscle cells are muscle cells, which have significantly less clinically measurable functionality than a healthy, intact muscle cell. In the absence of dystrophin, mechanical stress leads to sarcolemmal ruptures, causing an uncontrolled influx of calcium into the muscle fiber interior, thereby triggering calcium-activated proteases and fiber necrosis, resulting in damaged muscle cells. Increasing turnover of damaged muscle cells means that damaged muscle cells are more quickly broken down and/or removed as compared to a situation wherein turnover of damaged muscle cells is not increased. Turnover of damaged muscle cells is preferably assessed in a muscle biopsy, more preferably as described in 57 using a cross-section of a biopsy. A detectable increase of turnover may be an increase of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein turnover has been identified using a biopsy cross-section. The increase is measured by comparison to the turnover as assessed in a same individual before treatment.

Without wishing to be bound to theory, it is believed that increasing turnover of muscle cells is preferred because this reduces inflammatory responses.

According to the present disclosure, a composition of the invention further comprising an adjunct therapy for reducing inflammation, preferably for reducing muscle tissue inflammation in an individual, is particularly suitable for use as a medicament. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy as compared to a combination not comprising said adjunct compound. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

Further provided is therefore a composition further comprising an adjunct compound for reducing inflammation, preferably for reducing muscle tissue inflammation in said individual, for use as a medicament, preferably for treating or preventing counteracting DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein or altered or truncated dystrophin mRNA or protein is formed which is not sufficiently functional.

Preferred adjunct compound of the disclosure for reducing inflammation include a steroid, a TNFα inhibitor, a source of mIGF-1 and/or an antioxidant. However, any other compound able to reduce inflammation as defined herein is also encompassed within the present disclosure. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for improving muscle fiber function, integrity and/or survival.

Furthermore in the disclosure, a composition comprising an adjunct therapy for improving muscle fiber function, integrity and/or survival in an individual is particularly suitable for use as a medicament, preferably for treating or preventing DMD. Such composition is even better capable of alleviating one or more symptom(s) of Duchenne Muscular Dystrophy as compared to a composition not comprising said adjunct compound.

Preferred adjunct compounds for improving muscle fiber function, integrity and/or survival include an ion channel inhibitor, a protease inhibitor, L-arginine and/or an angiotensin II type I receptor blocker. However, any other compound able to improving muscle fiber function, integrity and/or survival as defined herein is also encompassed within the present disclosure. Each of these compounds is later on extensively presented. Each of the compounds extensively presented may be used separately or in combination with each other and/or in combination with one or more of the adjunct compounds used for reducing inflammation.

In a particularly preferred embodiment of the disclosure, a composition further comprises a steroid. Such composition results in significant alleviation of DMD symptoms. This embodiment also enhances the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

In one embodiment of the disclosure, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

A steroid is a terpenoid lipid characterized by a carbon skeleton with four fused rings, generally arranged in a 6-6-6-5 fashion. Steroids vary by the functional groups attached to these rings and the oxidation state of the rings. Steroids include hormones and drugs, which are usually used to relieve swelling and inflammation, such as for instance prednisone, dexamethasone and vitamin D.

According to the present disclosure, supplemental effects of adjunct steroid therapy in DMD patients include reduction of tissue inflammation, suppression of cytotoxic cells, and improved calcium homeostasis. Most positive results are obtained in younger boys. Preferably, the steroid is a corticosteroid, more preferably, a glucocorticosteroid. Preferably, prednisone steroids such as prednisone, prednizolone or deflazacort are used in a combination according to the invention²¹. Dose ranges of steroid or of a glucocorticosteroid to be used in the therapeutic applications as described herein are designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist. The usual doses are 0.5 - 1.0 mg/kg/day, preferably 0.75 mg/kg/day for prednisone and prednisolone, and 0.4 - 1.4 mg/kg/day, preferably 0.9 mg/kg/day for deflazacort.

In one embodiment of the disclosure, a steroid is administered to said individual prior to administering a composition as earlier defined herein. In this embodiment, it is preferred that said steroid is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering said composition.

In another preferred embodiment of the disclosure, a combination further comprises a tumour necrosis factor-alpha (TNFα) inhibitor. Tumour necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that stimulates the inflammatory response. Pharmacological blockade of TNFα activity with the neutralising antibody infliximab (Remicade) is highly effective clinically at reducing symptoms of inflammatory diseases. In mdx mice, both infliximab and etanercept delay and reduce the necrosis of dystrophic muscle^{24, 25}, with additional physiological benefits on muscle strength, chloride channel function and reduced CK levels being demonstrated in chronically treated exercised adult mdx mice²⁶. Such highly specific antiinflammatory drugs designed for use in other clinical conditions, are attractive alternatives to the use of steroids for DMD. In one embodiment, the use of a TNFα inhibitor is limited to periods of intensive muscle growth in boys when muscle damage and deterioration are especially pronounced.

A composition further comprising a TNFα inhibitor for use as a medicament is also provided. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. A preferred TNFα inhibitor is a dimeric fusion protein consisting of the extracellular ligand-binding domain of the human p75 receptor of TNFα linked to the Fc portion of human IgG1. A more preferred TNFα inhibitor is ethanercept (Amgen, America)²⁶. The usual doses of ethanercept is about 0.2 mg/kg, preferably about 0.5 mg/kg twice a week. The administration is preferably subcutaneous.

In another preferred embodiment of the disclosure, a composition of the invention further comprises a source of mIGF-1. As defined herein, a source of IGF-1 preferably encompasses mIGF-1 itself, a compound able of enhancing mIGF-1 expression and/or activity. Enhancing is herein synonymous with increasing. Expression of mIGF-1 is synonymous with amount of mIGF-1. mIGF-1 promotes regeneration of muscles through increase in satellite cell activity, and reduces inflammation and fibrosis²⁷. Local injury of muscle results in increased mIGF-1 expression. In transgenic mice with extra IGF-1 genes, muscle hypertrophy and enlarged muscle fibers are observed²⁷. Similarly, transgenic mdx mice show reduced muscle fiber degeneration²⁸. Upregulation of the mIGF-1 gene and/or administration of extra amounts of mIGF-1 protein or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]) thus promotes the effect of other, preferably genetic, therapies for DMD, including antisense-induced exon skipping. The additional mIGF-1 levels in the above mentioned transgenic mice do not induce cardiac problems nor promote cancer, and have no pathological side effects. As stated before, the amount of mIGF-1 is for instance increased by enhancing expression of the mIGF-1 gene and/or by administration of mIGF-1 protein and/or a functional equivalent thereof (especially the mIGF-1 Ea isoform [as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]). A composition of the invention further preferably comprises mIGF-1, a compound capable of enhancing mIGF-1 expression and/or an mIGF-1 activity, for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, such composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

Within the context of the disclosure, an increased amount or activity of mIGF-1 may be reached by increasing the gene expression level of an IGF-1 gene, by increasing the amount of a corresponding IGF-1 protein and/or by increasing an activity of an IGF1-protein. A preferred mIGF-1 protein has been earlier defined herein. An increase of an activity of said protein is herein understood to mean any detectable change in a biological activity exerted by said protein or in the steady state level of said protein as compared to said activity or steady-state in a individual who has not been treated. Increased amount or activity of mIGF-1 is preferably assessed by detection of increased expression of muscle hypertrophy biomarker GATA-2 (as described in 27).

Gene expression level is preferably assessed using classical molecular biology techniques such as (real time) PCR, arrays or Northern analysis. A steady state level of a protein is determined directly by quantifying the amount of a protein. Quantifying a protein amount may be carried out by any known technique such as Western blotting or immunoassay using an antibody raised against a protein. The skilled person will understand that alternatively or in combination with the quantification of a gene expression level and/or a corresponding protein, the quantification of a substrate of a corresponding protein or of any compound known to be associated with a function or activity of a corresponding protein or the quantification of said function or activity of a corresponding protein using a specific assay may be used to assess the alteration of an activity or steady state level of a protein.

In the invention, an activity or steady-state level of a said protein may be altered at the level of the protein itself, e.g. by providing a protein to a cell from an exogenous source.

Preferably, an increase or an up-regulation of the expression level of a said gene means an increase of at least 5% of the expression level of said gene using arrays. More preferably, an increase of the expression level of said gene means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. In another preferred embodiment, an increase of the expression level of said protein means an increase of at least 5% of the expression level of said protein using Western blotting and/or using ELISA or a suitable assay. More preferably, an increase of the expression level of a protein means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

In another preferred embodiment, an increase of a polypeptide activity means an increase of at least 5% of a polypeptide activity using a suitable assay. More preferably, an increase of a polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. The increase is preferably assessed by comparison to corresponding activity in the individual before treatment.

A preferred way of providing a source of mIGF1 is to introduce a transgene encoding mIGF1, preferably an mIGF-1 Ea isoform (as described in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577), more preferably in an AAV vector as later defined herein. Such source of mIGF1 is specifically expressed in muscle tissue as described in mice in 27.

In another preferred embodiment of the disclosure, a composition further comprises an antioxidant. Oxidative stress is an important factor in the progression of DMD and promotes chronic inflammation and fibrosis²⁹. The most prevalent products of oxidative stress, the peroxidized lipids, are increased by an average of 35% in Duchenne boys. Increased levels of the enzymes superoxide dismutase and catalase reduce the excessive amount of free radicals causing these effects. In fact, a dietary supplement Protandim® (LifeVantage) was clinically tested and found to increase levels of superoxide dismutase (up to 30%) and catalase (up to 54%), which indeed significantly inhibited the peroxidation of lipids in 29 healthy persons³⁰. Such effective management of oxidative stress thus preserves muscle quality and so promotes the positive effect of DMD therapy. Idebenone is another potent antioxidant with a chemical structure derived from natural coenzyme Q10. It protects mitochondria where adenosine triphosphate, ATP, is generated by oxidative phosphorylation. The absence of dystrophin in DMD negatively affects this process in the heart, and probably also in skeletal muscle. Idebenone was recently applied in clinical trials in the US and Europe demonstrating efficacy on neurological aspects of Friedreich's Ataxia³¹. A phase-IIa double-blind, placebo-controlled randomized clinical trial with Idebenone has recently been started in Belgium, including 21 Duchenne boys at 8 to 16 years of age. The primary objective of this study is to determine the effect of Idebenone on heart muscle function. In addition, several different tests will be performed to detect the possible functional benefit on muscle strength in the patients. When effective, Idebenone is a preferred adjunct compound for use in a combination according to the present disclosure in order to enhance the therapeutic effect of DMD therapy, especially in the heart. A composition further comprising an antioxidant for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the antioxidant, the skilled person will know which quantities are preferably used. An antioxidant may include bacoside, silymarin, curcumin and/or a polyphenol. Preferably, a polyphenol is or comprises epigallocatechin-3-gallate (EGCG). Preferably, an antioxidant is a mixture of antioxidants as the dietary supplement Protandim® (LifeVantage). A daily capsule of 675mg of Protandim® comprises 150 mg of B. monniera (45% bacosides), 225mg of S. marianum (70-80% silymarin), 150 mg of W. somnifera powder, 75mg green tea (98% polyphenols wherein 45% EGCG) and 75mg turmeric (95% curcumin).

In another preferred embodiment of the disclosure, a composition further comprises an ion channel inhibitor. The presence of damaged muscle membranes in DMD disturbs the passage of calcium ions into the myofibers, and the consequently disrupted calcium homeostasis activates many enzymes, e.g. proteases, that cause additional damage and muscle necrosis. Ion channels that directly contribute to the pathological accumulation of calcium in dystrophic muscle are potential targets for adjunct compounds to treat DMD. There is evidence that some drugs, such as pentoxifylline, block exercise-sensitive calcium channels³² and antibiotics that block stretch activated channels reduce myofibre necrosis in mdx mice and CK levels in DMD boys³³. A composition further comprising an ion channel inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

Preferably, an ion channel inhibitor of the class of xanthines is used. More preferably, said xanthines are derivatives of methylxanthines, and most preferably, said methylxanthine derivates are chosen from the group consisting of pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof. Most preferred is the use of pentoxifylline. Ion channel inhibitors of the class of xanthines enhance the skipping frequency of a dystrophin exon from a pre-mRNA comprising said exon, when using an oligonucleotide directed toward the exon or to one or both splice sites of said exon. The enhanced skipping frequency also increases the level of functional dystrophin protein produced in a muscle cell of a DMD or BMD individual.

Depending on the identity of the ion channel inhibitor, the skilled person will know which quantities are preferably used. Suitable dosages of pentoxifylline are between 1 mg/kg/day to 100 mg/kg/day, preferred dosages are between 10 mg/kg/day to 50 mg/kg/day. Typical dosages used in humans are 20 mg/kg/day.

In one embodiment of the disclosure, an ion channel inhibitor is administered to said individual prior to administering a composition comprising an oligonucleotide. In this embodiment, it is preferred that said ion channel inhibitor is administered at least one day, more preferred at least one week, more preferred at least two weeks, more preferred at least three weeks prior to administering a composition comprising an oligonucleotide.

In another preferred embodiment of the disclosure, a composition further comprises a protease inhibitor. Calpains are calcium-activated proteases that are increased in dystrophic muscle and account for myofiber degeneration. Calpain inhibitors such as calpastatin, leupeptin³⁴, calpeptin, calpain inhibitor III, or PD150606 are therefore applied to reduce the degeneration process. A new compound, BN 82270 (Ipsen) that has dual action as both a calpain inhibitor and an antioxidant increased muscle strength, decreased serum CK and reduced fibrosis of the mdx diaphragm, indicating a therapeutic effect with this new compound³⁵. Another compound of Leupeptin/Carnitine (Myodur) has recently been proposed for clinical trials in DMD patients.

MG132 is another proteasomal inhibitor that has shown to reduce muscle membrane damage, and to ameliorate the histopathological signs of muscular dystrophy³⁶. MG-132 (CBZ-leucyl-leucyl-leucinal) is a cell-permeable, proteasomal inhibitor (Ki=4nM), which inhibits NFkappaB activation by preventing IkappaB degradation (IC50 = 3 µM). In addition, it is a peptide aldehyde that inhibits ubiquitin-mediated proteolysis by binding to and inactivating 20S and 26S proteasomes. MG-132 has shown to inhibit the proteasomal degradation of dystrophin-associated proteins in the dystrophic mdx mouse model³⁶. This compound is thus also suitable for use as an adjunct pharmacological compound for DMD. A composition further comprising a protease inhibitor for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said combination is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the protease inhibitor, the skilled person will know which quantities are preferably used.

In another preferred embodiment of the disclosure, a composition further comprises L-arginine. Dystrophin-deficiency is associated with the loss of the DGC-complex at the fiber membranes, including neuronal nitric oxide synthase (nNOS). Expression of a nNOS transgene in mdx mice greatly reduced muscle membrane damage. Similarly, administration of L-arginine (the substrate for nitric oxide synthase) increased NO production and upregulated utrophin expression in mdx mice. Six weeks of L-arginine treatment improved muscle pathology and decreased serum CK in mdx mice³⁷. The use of L-arginine as a further constituent in a composition of the invention has not been disclosed.

A composition further comprising L-arginine for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional.

In another preferred embodiment of the disclosure, a composition further comprises angiotensin II type 1 receptor blocker Losartan, which normalizes muscle architecture, repair and function, as shown in the dystrophin-deficient mdx mouse model²³. A composition further comprising angiotensin II type 1 receptor blocker Losartan for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the angiotensin II type 1 receptor blocker, the skilled person will know which quantities are preferably used.

In another preferred embodiment of the disclosure, a composition further comprises an angiotensin-converting enzyme (ACE) inhibitor, preferably perindopril. ACE inhibitors are capable of lowering blood pressure. Early initiation of treatment with perindopril is associated with a lower mortality in DMD patients²². A composition further comprising an ACE inhibitor, preferably perindopril for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of an ACE inhibitor, preferably perindopril are about 2 to 4 mg/day²². In a more preferred embodiment of the disclosure, an ACE inhibitor is combined with at least one of the previously identified adjunct compounds.

In another preferred embodiment of the disclosure, a composition further comprises a compound exhibiting a readthrough activity. A compound exhibiting a readthrough activity may be any compound, which is able to suppress a stop codon. For 20% of DMD patients, the mutation in the dystrophin gene is comprising a point mutation, of which 13% is a nonsense mutation. A compound exhibiting a readthrough activity or which is able to suppress a stop codon is a compound which is able to provide an increased amount of a functional dystrophin mRNA or protein and/or a decreased amount of an aberrant or truncated dystrophin mRNA or protein. Increased preferably means increased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. Decreased preferably means decreased of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. An increase or a decrease of said protein is preferably assessed in a muscular tissue or in a muscular cell of an individual by comparison to the amount present in said individual before treatment with said compound exhibiting a readthrough activity. Alternatively, the comparison can be made with a muscular tissue or cell of said individual, which has not yet been treated with said compound in case the treatment is local. The assessment of an amount at the protein level is preferably carried out using western blot analysis.

Preferred compounds exhibiting a readthrough activity comprise or consist of aminoglycosides, including, but not limited to, geneticin (G418), paromomycin, gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), and derivatives thereof (references 64, 65).

A more preferred compound exhibiting a readthrough activity comprises or consists of PTC124™, and/or a functional equivalent thereof. PTC124™ is a registered trademark of PTC Therapeutics, Inc. South Plainfield, New Jersey. 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124™ (references 16, 17) belongs to a new class of small molecules that mimics at lower concentrations the readthrough activity of gentamicin (reference 55). A functional equivalent of 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin is a compound which is able to exhibit a readthrough activity as earlier defined herein. Most preferably, a compound exhibiting a readthrough activity comprises or consists of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known as PTC124™. A composition further comprising a compound exhibiting a readthrough activity, preferably comprising or consisting of gentamycin and/or 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. The usual doses of a compound exhibiting a readthrough activity, preferably 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of gentamicin are ranged between 3 mg/kg/day to 200 mg/kg/day, preferred dosages are between 10 mg/kg to 50 mg/kg per day or twice a day. In a more preferred embodiment, a compound exhibiting a readthrough activity is combined with at least one of the previously identified adjunct compounds.

In another preferred embodiment of the disclosure, a composition further comprises a compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing. Small chemical compounds, such as for instance specific indole derivatives, have been shown to selectively inhibit spliceosome assembly and splicing³⁸, for instance by interfering with the binding of serine- and arginine-rich (SR) proteins to their cognate splicing enhancers (ISEs or ESEs) and/or by interfering with the binding of splicing repressors to silencer sequences (ESSs or ISSs). These compounds are therefore suitable for applying as adjunct compounds that enhance exon skipping. A composition further comprising a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing for use as a medicament is also provided. Said medicament is preferably for alleviating one or more symptom(s) of DMD. In one embodiment, said composition is used in order to alleviate one or more symptom(s) of a severe form of BMD wherein a very short dystrophin protein is formed which is not sufficiently functional. Depending on the identity of the compound, which is capable of enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing, the skilled person will know which quantities are preferably used. In a more preferred embodiment, a compound for enhancing exon skipping and/or inhibiting spliceosome assembly and/or splicing is combined with a ACE inhibitor and/or with any adjunct compounds as identified earlier herein.

The disclosure thus provides a composition further comprising an adjunct compound, wherein said adjunct compound comprises a steroid, an ACE inhibitor (preferably perindopril), angiotensin II type 1 receptor blocker Losartan, a tumour necrosis factor-alpha (TNFα) inhibitor, a source of mIGF-1, preferably mIGF-1, a compound for enhancing mIGF-1 expression, a compound for enhancing mIGF-1 activity, an antioxidant, an ion channel inhibitor, a protease inhibitor, L-arginine, a compound exhibiting a readthrough activity and/or inhibiting spliceosome assembly and/or splicing.

In one embodiment of the disclosure an individual is further provided with a functional dystrophin protein using a vector, preferably a viral vector, comprising a micro-mini-dystrophin gene. Most preferably, a recombinant adeno-associated viral (rAAV) vector is used. AAV is a single-stranded DNA parvovirus that is non-pathogenic and shows a helper-dependent life cycle. In contrast to other viruses (adenovirus, retrovirus, and herpes simplex virus), rAAV vectors have demonstrated to be very efficient in transducing mature skeletal muscle. Application of rAAV in classical DMD "gene addition" studies has been hindered by its restricted packaging limits (< 5 kb). Therefore, rAAV is preferably applied for the efficient delivery of a much smaller micro- or mini-dystrophin gene. Administration of such micro- or mini-dystrophin gene results in the presence of an at least partially functional dystrophin protein. Reference is made to^18-20.

Each constituent of a composition can be administered to an individual in any order. In one embodiment, each constituent is administered simultaneously (meaning that each constituent is administered within 10 hours, preferably within one hour). This is however not necessary. In one embodiment at least one adjunct compound is administered to an individual in need thereof before administration of an oligonucleotide. Alternatively, an oligonucleotide is administered to an individual in need thereof before administration of at least one adjunct compound.

Use

In a further aspect, there is provided the use of a oligoucleotide or of a composition as defined herein for the manufacture of a medicament for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual. Each feature of said use has earlier been defined herein. A treatment in a use or in a method according to the invention is at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more. Each molecule or oligonucleotide or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing DMD or BMD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an oligonucleotide, composition, compound or adjunct compound of the invention may depend on several parameters such as the age of the patient, the mutation of the patient, the number of molecules (i.e. dose), the formulation of said molecule. The frequency may be ranged between at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.

Method

In a further aspect of the invention, there is provided an in vitro method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual or alleviate one or more characteristic(s)of a myogenic or muscle cell of said individual, the method comprising administering to said individual an oligonucleotide or a composition as defined herein. There is further provided a method for enhancing, inducing or promoting skipping of an exon from a dystrophin pre-mRNA in a cell expressing said pre-mRNA in an individual suffering from Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the method comprising administering to said individual an oligonucleotide or a composition as defined herein. Further provided is a method for increasing the production of a functional dystrophin protein and/or decreasing the production of an aberrant dystrophin protein in a cell, said cell comprising a pre-mRNA of a dystrophin gene encoding an aberrant dystrophin protein, the method comprising providing said cell with an oligonucleotide or composition of the invention and allowing translation of mRNA produced from splicing of said pre-mRNA. In one embodiment, said method is performed in vitro, for instance using a cell culture. Preferably within the disclosure, said method is in vivo.

In this context, increasing the production of a functional dystrophin protein has been earlier defined herein.

Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of" meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

Each embodiment as identified herein may be combined together unless otherwise indicated.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Brief description of the drawings

Figure 1. In human control myotubes, PS220 and PS305 both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies. PS220 reproducibly induced highest levels of exon 45 skipping (up to 73%), whereas with PS305 maximum exon 45 skipping levels of up to 46% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells)
Figure 2. Graph showing relative exon 45 skipping levels of inosine-containing AONs as assessed by RT-PCR analysis. In human control myotubes, a series of new AONs, all targeting exon 45 and containing one inosine for guanosine substitution were tested for relative exon 45 skipping efficiencies when compared with PS220 and PS305 (see Figure 1). All new inosine-containing AONs were effective, albeit at variable levels (between 4% and 25%). PS220 induced highest levels of exon 45 skipping (up to 72%), whereas with PS305 maximum exon 45 skipping levels of up to 63% were obtained. No exon 45 skipping was observed in non-treated cells. (M: DNA size marker; NT: non-treated cells).

Examples Example 1 Materials and methods

AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESEfinder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2'-O-methyl RNA and full-length phosphorothioate (PS) backbones.

Tissue culturing, transfection and RT-PCR analysis

Myotube cultures derived from a healthy individual ("human control") (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200nM for each AON (PS220 and PS305). Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 µl UNIFectylin per µg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results DMD exon 45 skipping.

Two AONs, PS220 (SEQ ID NO: 76; 5'-UUUGCC G CUGCCCAAUGCCAUCCUG-3') and PS305 (SEQ ID NO: 557; 5'-UUUGCC I CUGCCCAAUGCCAUCCUG-3') both targeting an identical sequence within exon 45, were directly compared for relative skipping efficiencies in healthy control myotube cultures. Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that both AONs were indeed capable of inducing exon 45 skipping. PS220, consisting a GCCGC stretch, reproducibly induced highest levels of exon 45 skipping (up to 73%), as shown in Figure 1. However, PS305, which is identical to PS220 but containing an inosine for a G substitution at position 4 within that stretch is also effective and leading to exon 45 skipping levels of up to 46%. No exon 45 skipping was observed in non-treated cells (NT).

Example 2 Materials and methods

AON design was based on (partly) overlapping open secondary structures of the target exon 45 RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESEfinder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2'-O-methyl RNA, full-length phosphorothioate (PS) backbones and one inosine for guanosine substitution.

Tissue culturing, transfection and RT-PCR analysis

Myotube cultures derived from a healthy individual ("human control") were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200nM for each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 µl UNIFectylin per µg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis using primers in the exons flanking the targeted exon 45. PCR fragments were isolated from agarose gels for sequence verification. For quantification, the PCR products were analyzed using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer (Agilent Technologies, USA).

Results DMD exon 45 skipping.

An additional series of AONs targeting exon 45 and containing one inosine-substitution were tested in healthy control myotube cultures for exon 45 skipping efficiencies, and directly compared to PS220 (without inosine; SEQ ID NO: 76)) and PS305 (identical sequence as PS220 but with inosine substitution; SEQ ID NO: 557). Subsequent RT-PCR and sequence analysis of isolated RNA demonstrated that all new AONs (PS309 to PS316) were capable of inducing exon 45 skipping between 4% (PS311) and 25% (PS310) as shown in Figure 2. When compared to PS220 and PS305, PS220 induced highest levels of exon 45 skipping (up to 72%). Of the new inosine-containing AONs PS305 was most effective, showing exon 45 skipping levels of up to 63%. No exon 45 skipping was observed in non-treated cells (NT).

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Sequence listing

DMD gene amino acid sequence

SEQ ID NO 2	GUACCUCCAACAUCAAGGAAGAUGG	SEQ ID NO 39	GAGAUGGCAGUUUCCUUAGUAACCA
SEQ ID NO 3	UACCUCCAACAUCAAGGAAGAUGGC	SEQ ID NO 40	AGAUGGCAGUUUCCUUAGUAACCAC
SEQ ID NO 4	ACCUCCAACAUCAAGGAAGAUGGCA	SEQ ID NO 41	GAUGGCAGUUUCCUUAGUAACCACA
SEQ ID NO 5	CCUCCAACAUCAAGGAAGAUGGCAU	SEQ ID NO 42	AUGGCAGUUUCCUUAGUAACCACAG
SEQ ID NO 6	CUCCAACAUCAAGGAAGAUGGCAUU	SEQ ID NO 43	UGGCAGUUUCCUUAGUAACCACAGG
SEQ ID NO 7	UCCAACAUCAAGGAAGAUGGCAUUU	SEQ ID NO 44	GGCAGUUUCCUUAGUAACCACAGGU
SEQID NO 8	CCAACAUCAAGGAAGAUGGCAUUUC	SEQ ID NO 45	GCAGUUUCCUUAGUAACCACAGGUU
SEQID NO 9	CAACAUCAAGGAAGAUGGCAUUUCU	SEQ ID NO 46	CAGUUUCCUUAGUAACCACAGGUUG
SEQ ID NO 10	AACAUCAAGGAAGAUGGCAUUUCUA	SEQ ID NO 47	AGUUUCCUUAGUAACCACAGGUUGU
SEQ ID NO 11	ACAUCAAGGAAGAUGGCAUUUCUAG	SEQ ID NO 48	GUUUCCUUAGUAACCACAGGUUGUG
SEQ ID NO 12	CAUCAAGGAAGAUGGCAUUUCUAGU	SEQ ID NO 49	UUUCCUUAGUAACCACAGGUUGUGU
SEQ ID NO 13	AUCAAGGAAGAUGGCAUUUCUAGUU	SEQ ID NO 50	UUCCUUAGUAACCACAGGUUGUGUC
SEQ ID NO 14	UCAAGGAAGAUGGCAUUUCUAGUUU	SEQ ID NO 51	UCCUUAGUAACCACAGGUUGUGUCA
SEQ ID NO 15	CAAGGAAGAUGGCAUUUCUAGUUUG	SEQ ID NO 52	CCUUAGUAACCACAGGUUGUGUCAC
SEQ ID NO 16	AAGGAAGAUCxGCAUUUCUAGUUUGG	SEQ ID NO 53	CUUAGUAACCACAGGUUGUGUCACC
SEQ ID NO 17	AGGAAGAUGGCAUUUCUAGUUUGGA	SEQ ID NO 54	UUAGUAACCACAGGUUGUGUCACCA
SEQ ID NO 18	GGAAGAUGGCAUUUCUAGUUUGGAG	SEQ ID NO 55	UAGUAACCACAGGUUGUGUCACCAG
SEQ ID NO 19	GAAGAUGGCAUUUCUAGUUUGGAGA	SEQ ID NO 56	AGUAACCACAGGUUGUGUCACCAGA
SEQ ID NO 20	AAGAUGGCAUUUCUAGUUUGGAGAU	SEQ ID NO 57	GUAACCACAGGUUGUGUCACCAGAG
SEQ ID NO 21	AGAUGGCAUUUCUAGUUUGGAGAUG	SEQ ID NO 58	UAACCACAGGUUGUGUCACCAGAGU
SEQ ID NO 22	GAUGGCAUUUCUAGUUUGGAGAUGG	SEQ ID NO 59	AACCACAGGUUGUGUCACCAGAGUA
SEQ ID NO 23	AUGGCAUUUCUAGUUUGGAGAUGGC	SEQ ID NO 60	ACCACAGGUUGUGUCACCAGAGUAA
SEQ ID NO 24	UGGCAUUUCUAGUUUGGAGAUGGCA	SEQ ID NO 61	CCACAGGUUGUGUCACCAGAGUAAC
SEQ ID NO 25	GGCAUUUCUAGUUUGGAGAUGGCAG	SEQ ID NO 62	CACAGGUUGUGUCACCAGAGUAACA
SEQ ID NO 26	GCAUUUCUAGUUUGGAGAUGGCAGU	SEQ ID NO 63	ACAGGUUGUGUCACCAGAGUAACAG
SEQ ID NO 27	CAUUUCUAGUUUGGAGAUGGCAGUU	SEQ ID NO 64	CAGGUUGUGUCACCAGAGUAACAGU
SEQ ID NO 28	AUUUCUAGUUUGGAGAUGGCAGUUU	SEQ ID NO 65	AGGUUGUGUCACCAGAGUAACAGUC
SEQ ID NO 29	UUUCUAGUUUGGAGAUGGCAGUUUC	SEQ ID NO 66	GGUUGUGUCACCAGAGUAACAGUCU
SEQ ID NO 30	UUCUAGUUUGGAGAUGGCAGUUUCC	SEQ ID NO 67	GUUGUGUCACCAGAGUAACAGUCUG
SEQ ID NO 31	UCUAGUUUGGAGAUGGCAGUUUCCU	SEQ ID NO 68	UUGUGUCACCAGAGUAACAGUCUGA
SEQ ID NO 32	CUAGUUUGGAGAUGGCAGUUUCCUU	SEQ ID NO 69	UGUGUCACCAGAGUAACAGUCUGAG
SEQ ID NO 33	UAGUUUGGAGAUGGCAGUUUCCUUA	SEQ ID NO 70	GUGUCACCAGAGUAACAGUCUGAGU
SEQ ID NO 34	AGUUUGGAGAUGGCAGUUUCCUUAG	SEQ ID NO 71	UGUCACCAGAGUAACAGUCUGAGUA
SEQ ID NO 35	GUUUGGAGAUGGCAGUUUCCUUAGU	SEQ ID NO 72	GUCACCAGAGUAACAGUCUGAGUAG
SEQ ID NO 36	UUUGGAGAUGGCAGUUUCCUUAGUA	SEQ ID NO 73	UCACCAGAGUAACAGUCUGAGUAGG
SEQ ID NO 37	UUGGAGAUGGCAGUUUCCUUAGUAA	SEQ ID NO 74	CACCAGAGUAACAGUCUGAGUAGGA
SEQ ID NO 38	UGGAGAUGGCAGUUUCCUUAGUAAC	SEQ ID NO 75	ACCAGAGUAACAGUCUGAGUAGGAG
SEQ ID NO 539	UCAAGGAAGAUGGCAUUUCU	SEQ ID NO 548	UCAAGGAAGAUGGCAUIUCU
SEQ ID NO 540	UCAAIGAAGAUGGCAUUUCU	SEQ ID NO 549	UCAAGGAAGAUGGCAUUICU
SEQ ID NO 541	UCAAGIAAGAUGGCAUUUCU	SEQ ID NO 550	UCAAGGAAGAUGGCAUUUCI
SEQ ID NO 542	UCAAGGAAIAUGGCAUUUCU	SEQ ID NO 551	UCIAGGAAGAUGGCAUUUCU
SEQ ID NO 543	UCAAGGAAGAUIGCAUUUCU	SEQ ID NO 552	UCAIGGAAGAUGGCAUUUCU
SEQ ID NO 544	UCAAGGAAGAUGICAUUUCU	SEQ ID NO 553	UCAAGGIAGAUGGCAUUUCU
SEQ ID NO 545	ICAAGGAAGAUGGCAUUUCU	SEQ ID NO 554	UCAAGGAIGAUGGCAUUUCU
SEQ ID NO 546	UCAAGGAAGAIGGCAUUUCU	SEQ ID NO 555	UCAAGGAAGIUGGCAUUUCU
SEQ ID NO 547	UCAAGGAAGAUGGCAIUUCU	SEQ ID NO 556	UCAAGGAAGAUGGCIUUUCU

		SEQ ID NO 109	GUUGCAUUCAAUGUUCUGACAACAG
SEQ ID NO 77	AUUCAAUGUUCUGACAACAGUUUGC	SEQ ID NO 110	UUGCAUUCAAUGUUCUGACAACAGU
SEQ ID NO 78	CCAGUUGCAUUCAAUGUUCUGACAA	SEQ ID NO 111	UGCAUUCAAUGUUCUGACAACAGUU
SEQ ID NO 79	CAGUUGCAUUCAAUGUUCUGAC	SEQ ID NO 112	GCAUUCAAUGUUCUGACAACAGUUU
SEQ ID NO 80	AGUUGCAUUCAAUGUUCUGA	SEQ ID NO 113	CAUUCAAUGUUCUGACAACAGUUUG
SEQ ID NO 81	GAUUGCUGAAUUAUUUCUUCC	SEQ ID NO 114	AUUCAAUGUUCUGACAACAGUUUGC
SEQ ID NO 82	GAUUGCUGAAUUAUUUCUUCCCCAG	SEQ ID NO 115	UCAAUGUUCUGACAACAGUUUGCCG
SEQ ID NO 83	AUUGCUGAAUUAUUUCUUCCCCAGU	SEQ ID NO 116	CAAUGUUCUGACAACAGUUUGCCGC
SEQ ID NO 84	UUGCUGAAUUAUUUCUUCCCCAGUU	SEQ ID NO 117	AAUGUUCUGACAACAGUUUGCCGCU
SEQ ID NO 85	UGCUGAAUUAUUUCUUCCCCAGUUG	SEQ ID NO 118	AUGUUCUGACAACAGUUUGCCGCUG
SEQ ID NO 86	GCUGAAUUAUUUCUUCCCCAGUUGC	SEQ ID NO 119	UGUUCUGACAACAGUUUGCCGCUGC
NO ID NO 87	CUGAAUUAUUUCUUCCCCAGUUGCA	SEQ ID NO 120	GUUCUGACAACAGUUUGCCGCUGCC
SEQ ID NO 88	UGAAUUAUUUCUUCCCCAGUUGCAU	SEQ ID NO 121	UUCUGACAACAGUUUGCCGCUGCCC
SEQ ID NO 89	GAAUUAUUUCUUCCCCAGUUGCAUU	SEQ ID NO 122	UCUGACAACAGUUUGCCGCUGCCCA
SEQ ID NO 90	AAUUAUUUCUUCCCCAGUUGCAUUC	SEQ ID NO 123	CUGACAACAGUUUGCCGCUGCCCAA
SEQ ID NO 91	AUUAUUUCUUCCCCAGUUGCAUUCA	SEQ ID NO 124	UGACAACAGUUUGCCGCUGCCCAAU
SEQ ID NO 92	UUAUUUCUUCCCCAGUUGCAUUCAA	SEQ ID NO 125	GACAACAGUUUGCCGCUGCCCAAUG
SEQ ID NO 93	UAUUUCUUCCCCAGUUGCAUUCAAU	SEQ ID NO 126	ACAACAGUUUGCCGCUGCCCAAUGC
SEQ ID NO 94	AUUUCUUCCCCAGUUGCAUUCAAUG	SEQ ID NO 127	CAACAGUUUGCCGCUGCCCAAUGCC
SEQ ID NO 95	UUUCUUCCCCAGUUGCAUUCAAUGU	SEQ ID NO 128	AACAGUUUGCCGCUGCCCAAUGCCA
SEQ ID NO 96	UUCUUCCCCAGUUGCAUUCAAUGUU	SEQ ID NO 129	ACAGUUUGCCGCUGCCCAAUGCCAU
SEQ ID NO 97	UCUUCCCCAGUUGCAUUCAAUGUUC	SEQ ID NO 130	CAGUUUGCCGCUGCCCAAUGCCAUC
SEQ ID NO 98	CUUCCCCAGUUGCAUUCAAUGUUCU	SEQ ID NO 131	AGUUUGCCGCUGCCCAAUGCCAUCC
SEQ ID NO 99	UUCCCCAGUUGCAUUCAAUGUUCUG	SEQ ID NO 132	GUUUGCCGCUGCCCAAUGCCAUCCU
SEQ ID NO 100	UCCCCAGUUGCAUUCAAUGUUCUGA	SEQ ID NO 133	UUUGCCGCUGCCCAAUGCCAUCCUG
SEQ ID NO 101	CCCCAGUUGCAUUCAAUGUUCUGAC	SEQ ID NO 134	UUGCCGCUGCCCAAUGCCAUCCUGG
SEQ ID NO 102	CCCAGUUGCAUUCAAUGUUCUGACA	SEQ ID NO 135	UGCCGCUGCCCAAUGCCAUCCUGGA
SEQ ID NO 103	CCAGUUGCAUUCAAUGUUCUGACAA	SEQ ID NO 136	GCCGCUGCCCAAUGCCAUCCUGGAG
SEQ ID NO 104	CAGUUGCAUUCAAUGUUCUGACAAC	SEQ ID NO 137	CCGCUGCCCAAUGCCAUCCUGGAGU
SEQ ID NO 105	AGUUGCAUUCAAUGUUCUGACAACA	SEQ ID NO 138	CGCUGCCCAAUGCCAUCCUGGAGUU
SEQ ID NO 106	UCC UGU AGA AUA CUG GCA UC	SEQ ID NO 139	UGUUUUUGAGGAUUGCUGAA
SEQ ID NO 107		SEQ ID NO 140
SEQ ID NO 108			UUUGCCICUGCCCAAUGCCAUCCUG
SEQ ID NO 558	UUUGCCGCUICCCAAUGCCAUCCUG	SEQ ID NO 566	UUUGCCGCUGCCCAIUGCCAUCCUG
SEQ ID NO 559	UUUGCCGCUGCCCAAUICCAUCCUG	SEQ ID NO 567	UUUGCCGCUGCCCAAUGCCIUCCUG
SEQ ID NO 560	UUUICCGCUGCCCAAUGCCAUCCUG	SEQ ID NO 568	UUUICCICUGCCCAAUGCCAUCCUG
SEQ ID NO 561	UUUGCCGCUGCCCAAUGCCAUCCUI	SEQ ID NO 569	UUUGCCGCUGCCCAAIGCCAUCCUG
SEQ ID NO 562	IUUGCCGCUGCCCAAUGCCAUCCUG	SEQ ID NO 570	UUUGCCGCUGCCCAAUGCCAICCUG
SEQ ID NO 563	UIUGCCGCUGCCCAAUGCCAUCCUG	SEQ ID NO 571	UUUGCCGCUGCCCAAUGCCAUCCIG
SEQ ID NO 564	UUIGCCGCUGCCCAAUGCCAUCCUG	SEQ ID NO 572	UUUGCCGCUGCCCIAUGCCAUCCUG
SEQ ID NO 565	UUUGCCGCIGCCCAAUGCCAUCCUG

SEQ ID NO 141	CUCUGGCCUGUCCUAAGACCUGCUC	SEQ ID NO 165	CAGCUUCUUCCUUAGCUUCCAGCCA
SEQ ID NO 142	UCUGGCCUGUCCUAAGACCUGCUCA	SEQ ID NO 166	AGCUUCUUCCUUAGCUUCCAGCCAU
SEQ ID NO 143	CUGGCCUGUCCUAAGACCUGCUCAG	SEQ ID NO 167	GCUUCUUCCUUAGCUUCCAGCCAUU
SEQ ID NO 144	UGGCCUGUCCUAAGACCUGCUCAGC	SEQ ID NO 168	CUUCUUCCUUAGCUUCCAGCCAUUG
SEQ ID NO 145	GGCCUGUCCUAAGACCUGCUCAGCU	SEQ ID NO 169	UUCUUCCUUAGCUUCCAGCCAUUGU
SEQ ID NO 146	GCCUGUCCUAAGACCUGCUCAGCUU	SEQ ID NO 170	UCUUCCUUAGCUUCCAGCCAUUGUG
SEQ ID NO 147	CCUGUCCUAAGACCUGCUCAGCUUC	SEQ ID NO 171	CUUCCUUAGCUUCCAGCCAUUGUGU
SEQ ID NO 148	CUGUCCUAAGACCUGCUCAGCUUCU	SEQ ID NO 172	UUCCUUAGCUUCCAGCCAUUGUGUU
SEQ ID NO 149	UGUCCUAAGACCUGCUCAGCUUCUU	SEQ ID NO 173	UCCUUAGCUUCCAGCCAUUGUGUUG
SEQ ID NO 150	GUCCUAAGACCUGCUCAGCUUCUUC	SEQ ID NO 174	CCUUAGCUUCCAGCCAUUGUGUUGA
SEQ ID NO 151	UCCUAAGACCUGCUCAGCUUCUUCC	SEQ ID NO 175	CUUAGCUUCCAGCCAUUGUGUUGAA
SEQ ID NO 152	CCUAAGACCUGCUCAGCUUCUUCCU	SEQ ID NO 176	UUAGCUUCCAGCCAUUGUGUUGAAU
SEQ ID NO 153	CUAAGACCUGCUCAGCUUCUUCCUU	SEQ ID NO 177	UAGCUUCCAGCCAUUGUGUUGAAUC
SEQ ID NO 154	UAAGACCUGCUCAGCUUCUUCCUUA	SEQ ID NO 178	AGCUUCCAGCCAUUGUGUUGAAUCC
SEQ ID NO 155	AAGACCUGCUCAGCUUCUUCCUUAG	SEQ ID NO 179	GCUUCCAGCCAUUGUGUUGAAUCCU
SEQ ID NO 156	AGACCUGCUCAGCUUCUUCCUUAGC	SEQ ID NO 180	CUUCCAGCCAUUGUGUUGAAUCCUU
SEQ ID NO 157	GACCUGCUCAGCUUCUUCCUUAGCU	SEQ ID NO 181	UUCCAGCCAUUGUGUUGAAUCCUUU
SEQ ID NO 158	ACCUGCUCAGCUUCUUCCUUAGCUU	SEQ ID NO 182	UCCAGCCAUUGUGUUGAAUCCUUUA
SEQ ID NO 159	CCUGCUCAGCUUCUUCCUUAGCUUC	SEQ ID NO 183	CCAGCCAUUGUGUUGAAUCCUUUAA
SEQ ID NO 160	CUGCUCAGCUUCUUCCUUAGCUUCC	SEQ ID NO 184	CAGCCAUUGUGUUGAAUCCUUUAAC
SEQ ID NO 161	UGCUCAGCUUCUUCCUUAGCUUCCA	SEQ ID NO 185	AGCCAUUGUGUUGAAUCCUUUAACA
SEQ ID NO 162	GCUCAGCUUCUUCCUUAGCUUCCAG	SEQ ID NO 186	GCCAUUGUGUUGAAUCCUUUAACAU
SEQ ID NO 163	CUCAGCUUCUUCCUUAGCUUCCAGC	SEQ ID NO 187	CCAUUGUGUUGAAUCCUUUAACAUU
SEQ ID NO 164	UCAGCUUCUUCCUUAGCUUCCAGCC	SEQ ID NO 188	CAUUGUGUUGAAUCCUUUAACAUUU

SEQ ID NO 189	UCAGCUUCUGUUAGCCACUG	SEQ ID NO 214	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 190	UUCAGCUUCUGUUAGCCACU	SEQ ID NO 215
SEQ ID NO 191	UUCAGCUUCUGUUAGCCACUG	SEQ ID NO 216	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 192	UCAGCUUCUGUUAGCCACUGA	SEQ ID NO 217	AGCUUCUGUUAGCCACUGAU
SEQ ID NO 193	UUCAGCUUCUGUUAGCCACUGA	SEQ ID NO 218	GCUUCUGUUAGCCACUGAUU
SEQ ID NO 194	UCAGCUUCUGUUAGCCACUGA	SEQ ID NO 219	AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 195	UUCAGCUUCUGUUAGCCACUGA	SEQ ID NO 220	GCUUCUGUUAGCCACUGAUUA
SEQ ID NO 196	UCAGCUUCUGUUAGCCACUGAU	SEQ ID NO 221	AGCUUCUGUUAGCCACUGAUUA
SEQ ID NO 197	UUCAGCUUCUGUUAGCCACUGAU	SEQ ID NO 222	GCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 198	UCAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 223	AGCUUCUGUUAGCCACUGAUUAA
SEQ ID NO 199	UUCAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 224	GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 200	UCAGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 225	AGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 201	UUCAGCUUCUGUUAGCCACUGAUA	SEQ ID NO 226	GCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 202	UCAGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 227	CCAUUUGUAUUUAGCAUGUUCCC
SEQ ID NO 203	UUCAGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 228	AGAUACCAUUUGUAUUUAGC
SEQ ID NO 204	UCAGCUUCUGUUAGCCACUGAUUAAA	SEQ ID NO 229	GCCAUUUCUCAACAGAUCU
SEQ ID NO 205	UUCAGCUUCUGUUAGCCACUGAUUAAA	SEQ ID NO 230	GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 206	CAGCUUCUGUUAGCCACUG	SEQ ID NO 231	AUUCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 207	CAGCUUCUGUUAGCCACUGAU	SEQ ID NO 232	UCUCAGGAAUUUGUGUCUUUC
SEQ ID NO 208	AGCUUCUGUUAGCCACUGAUU	SEQ ID NO 233	GUUCAGCUUCUGUUAGCC
SEQ ID NO 209	CAGCUUCUGUUAGCCACUGAUU	SEQ ID NO 234	CUGAUUAAAUAUCUUUAUAU C
SEQ ID NO 210	AGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 235	GCCGCCAUUUCUCAACAG
SEQ ID NO 211	CAGCUUCUGUUAGCCACUGAUUA	SEQ ID NO 236	GUAUUUAGCAUGUUCCCA
SEQ ID NO 212	AGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 237	CAGGAAUUUGUGUCUUUC
SEQ ID NO 213	CAGCUUCUGUUAGCCACUGAUUAA	SEQ ID NO 575	UCAICUUCUGUUAGCCACUG
SEQ ID NO 573	UCAGCUUCUIUUAGCCACUG	SEQ ID NO 576	UCAGCUUCUGUUAGCCACUI
SEQID NO 574	UCAGCUUCUGUUAICCACUG

SEQ ID NO 238	GCUUUUCUUUUAGUUGCUGCUCUUU	SEQ ID NO 265	CCAGGUUCAAGUGGGAUACUAGCAA
SEQ ID NO 239	CUUUUCUUUUAGUUGCUGCUCUUUU	SEQ ID NO 266	CAGGUUCAAGUGGGAUACUAGCAAU
SEQ ID NO 240	UUUUCUUUUAGUUGCUGCUCUUUUC	SEQ ID NO 267	AGGUUCAAGUGGGAUACUAGCAAUG
SEQ ID NO 241	UUUCUUUUAGUUGCUGCUCUUUUCC	SEQ ID NO 268	GGUUCAAGUGGGAUACUAGCAAUGU
SEQ ID NO 242	UUCUUUUAGUUGCUGCUCUUUUCCA	SEQ ID NO 269	GUUCAAGUGGGAUACUAGCAAUGUU
SEQ ID NO 243	UCUUUUAGUUGCUGCUCUUUUCCAG	SEQ ID NO 270	UUCAAGUGGGAUACUAGCAAUGUUA
SEQ ID NO 244	CUUUUAGUUGCUGCUCUUUUCCAGG	SEQ ID NO 271	UCAAGUGGGAUACUAGCAAUGUUAU
SEQ ID NO 245	UUUUAGUUGCUGCUCUUUUCCAGGU	SEQ ID NO 272	CAAGUGGGAUACUAGCAAUGUUAUC
SEQ ID NO 246	UUUAGUUGCUGCUCUUUUCCAGGUU	SEQ ID NO 273	AAGUGGGAUACUAGCAAUGUUAUCU
SEQ ID NO 247	UUAGUUGCUGCUCUUUUCCAGGUUC	SEQ ID NO 274	AGUGGGAUACUAGCAAUGUUAUCUG
SEQ ID NO 248	UAGUUGCUGCUCUUUUCCAGGUUCA	SEQ ID NO 275	GUGGGAUACUAGCAAUGUUAUCUGC
SEQ ID NO 249	AGUUGCUGCUCUUUUCCAGGUUCAA	SEQ ID NO 276	UGGGAUACUAGCAAUGUUAUCUGCU
SEQ ID NO 250	GUUGCUGCUCUUUUCCAGGUUCAAG	SEQ ID NO 277	GGGAUACUAGCAAUGUUAUCUGCUU
SEQ ID NO 251	UUGCUGCUCUUUUCCAGGUUCAAGU	SEQ ID NO 278	GGAUACUAGCAAUGUUAUCUGCUUC
SEQ ID NO 252	UGCUGCUCUUUUCCAGGUUCAAGUG	SEQ ID NO 279	GAUACUAGCAAUGUUAUCUGCUUCC
SEQ ID NO 253	GCUGCUCUUUUCCAGGUUCAAGUGG	SEQ ID NO 280	AUACUAGCAAUGUUAUCUGCUUCCU
SEQ ID NO 254	CUGCUCUUUUCCAGGUUCAAGUGGG	SEQ ID NO 281	UACUAGCAAUGUUAUCUGCUUCCUC
SEQ ID NO 255	UGCUCUUUUCCAGGUUCAAGUGGGA	SEQ ID NO 282	ACUAGCAAUGUUAUCUGCUUCCUCC
SEQ ID NO 256	GCUCUUUUCCAGGUUCAAGUGGGAC	SEQ ID NO 283	CUAGCAAUGUUAUCUGCUUCCUCCA
SEQ ID NO 257	CUCUUUUCCAGGUUCAAGUGGGAUA	SEQ ID NO 284	UAGCAAUGUUAUCUGCUUCCUCCAA
SEQ ID NO 258	UCUUUUCCAGGUUCAACGUGGGAUAC	SEQ ID NO 285	AGCAAUGUUAUCUGCUUCCUCCAAC
SEQ ID NO 259	UCUUUUCCAGGUUCAAGUGG	SEQ ID NO 286	GCAAUGUUAUCUGCUUCCUCCAACC
SEQ ID NO 260	CUUUUCCAGGUUCAAGUGGGAUACU	SEQ ID NO 287	CAAUGUUAUCUGCUUCCUCCAACCA
SEQ ID NO 261	UUUUCCAGGUUCAAGUGGGAUACUA	SEQ ID NO 288	AAUGUUAUCUGCUUCCUCCAACCAU
SEQ ID NO 262	UUUCCAGGUUCAAGUGGGAUACUAG	SEQ ID NO 289	AUGUUAUCUGCUUCCUCCAACCAUA
SEQ ID NO 263	UUCCAGGUUCAAGUGGGAUACUAGC	SEQ ID NO 290	UGUUAUCUGCUUCCUCCAACCAUAA
SEQ ID NO 264	UCCAGGUUCAAGUGGGAUACUAGCA

SEQ ID NO 291	AGCCUCUUGAUUGCUGGUCUUGUUU	SEQ ID NO 326	UUGGGCAGCGGUAAUGAGUUCUUCC
SEQ ID NO 292	GCCUCUUGAUUGCUGGUCUUGUUUU	SEQ ID NO 327	UGGGCAGCGUAAUGAGUUCUUCCA
SEQ ID NO 293	CCUCUUGAUUGCUGGUCUUGUUUUU	SEQ ID NO 328	GGGCAGCGGUAAUGAGUUCUUCCAA
SEQ ID NO 294	CCUCUUGAUUGCUGGUCUUG	SEQ ID NO 329	GGCAGCGGUAAUGAGUUCUUCCAAC
SEQ ID NO 295	CUCUUGAUUGCUGGUCUUGUUUUUC	SEQ ID NO 330	GCAGCGGUAAUGAGUUCUUCCAACU
SEQ ID NO 296	UCUUGAUUGCUGGUCUUGUUUUUCA	SEQ ID NO 331	CAGCGGUAAUGAGUUCUUCCAACUG
SEQ ID NO 297	CUUGAUUGCUGGUCUUGUUUUUCAA	SEQ ID NO 332	AGCGGUAAUGAGUUCUUCCAACUGG
SEQ ID NO 298	UUGAUUGCUGGUCUUGUUUUUCAAA	SEQ ID NO 333	GCGGUAAUGAGUUCUUCCAACUGGG
SEQ ID NO 299	UGAUUGCUGGUCUUGUUUUUCAAAU	SEQ ID NO 334	CGGUAAUGAGUUCUUCCAACUGGGG
SEQ ID NO 300	GAUUGCUGGUCUUGUUUUUCAAAUU	SEQ ID NO 335	GGUAAUGAGUUCUUCCAACUGGGGA
SEQ ID NO 301	GAUUGCUGGUCUUGUUUUUC	SEQ ID NO 336	GGUAAUGAGUUCUUCCAACUGG
SEQ ID NO 302	AUUGCUGGUCUUGUUUUUCAAAUUU	NO 337	GUAAUGAGUUCUUCCAACUGGGGAC
SEQ ID NO 303	UUGCUGGUCUUGUUUUUCAAAUUUU	SEQ ID NO 338	UAAUGAGUUCUUCCAACUGGGGACG
SEQ ID NO 304	UGCUGGUCUUGUUUUUCAAAUUUUG	SEQ ID NO 339	AAUGAGUUCUUCCAACUGGGGACGC
SEQ ID NO 305	GCUGGUCUUGUUUUUCAAAUUUUGG	SEQ ID NO 340	AUGAGUUCUUCCAACUGGGGACGCC
SEQ ID NO 306	CUGGUCUUGUUUUUCAAAUUUUGGG	SEQ ID NO 341	UGAGUUCUUCCAACUGGGGACGCCU
SEQ ID NO 307	UGGUCUUGUUUUUCAAAUUUUGGGC	SEQ ID NO 342	GAGUUCUUCCAACUGGGGACGCCUC
SEQ ID NO 308	GGUCUUGUUUUUCAAAUUUUGGGCA	SEQ ID NO 343	AGUUCUUCCAACUGGGGACGCCUCU
SEQ ID NO 309	GUCUUGUUUUUCAAAUUUUGGGCAG	SEQ ID NO 344	GUUCUUCCAACUGGGGACGCCUCUG
SEQ ID NO 310	UCUUGUUUUUCAAAUUUUGGGCAGC	SEQ ID NO 345	UUCUUCCAACUGGGGACGCCUCUGU
SEQ ID NO 311	CUUGUUUUUCAAAUUUUGGGCAGCG	SEQ ID NO 346	UCUUCCAACUGGGGACGCCUCUGUU
SEQ ID NO 312	UUGUUUUUCAAAUUUUGGGCAGCGG	SEQ ID NO 347	CUUCCAACUGGGGACGCCUCUGUUC
SEQ ID NO 313	UGUUUUUCAAAUUUUGGGCAGCGGU	SEQ ID NO 348	UUCCAACUGGGGACGCCUCUGUUCC
SEQ ID NO 314	GUUUUUCAAAUUUUGGGCAGCGGUA	SEQ ID NO 349	UCCAACUGGGGACGCCUCUGUUCCA
SEQ ID NO 315	UUUUUCAAAUUUUGGGCAGCGGUAA	SEQ ID NO 350	CCAACUGGGGACGCCUCUGUUCCAA
SEQ ID NO 316	UUUUCAAAUUUUGGGCAGCGGUAAU	SEQ ID NO 351	CAACUGGGGACGCCUCUGUUCCAAA
SEQ ID NO 317	UUUCAAAUUUUGGGCAGCGGUAAUCG	SEQ ID NO 352	AACUGGGGACGCCUCUGUUCCAAAU
SEQ ID NO 318	UUCAAAUUUUGGGCAGCGGUAAUGA	SEQ ID NO 353	ACUGGGGACGCCUCUCGUUCCAAAUC
SEQ ID NO 319	UCAAAUUUUGGGCAGCGGUAAUGAG	SEQ ID NO 354	CUGGGGACGCCUCUGUUCCAAAUCC
SEQ ID NO 320	CAAAUUUUGGGCAGCGGUAAUGAGU	SEQ ID NO 355	UGGGGACGCCUCUGUUCCAAAUCCU
SEQ ID NO 321	AAAUUUUGGGCAGCGGUAAUGAGUU	SEQ ID NO 356	GGGGACGCCUCUGUUCCAAAUCCUG
SEQ ID NO 322	AAUUUUGGGCAGCGGUAAUGAGUUC	SEQ ID NO 357	GGGACGCCUCUGUUCCAAAUCCUGC
SEQ ID NO 323	AUUUUGGGCAGCGGUAAUGAGUUCU	SEQ ID NO 358	GGACGCCUCUGUUCCAAAUCCUGCA
SEQ ID NO 324	UUUUGGGCAGCGGUAAUGAGUUCUU	SEQ ID NO 359	GACGCCUCUGUUCCAAAUCCUGCAU
SEQ ID NO 325	UUUGGGCAGCGGUAAUGAGUUCUUC

SEQ ID NO 360	CCAAUAGUGGUCAGUCCAGGAGCUA	SEQ ID NO 386	CUAGGUCAGGCUGCUUUGCCCUCAG
SEQ ID NO 361	CAAUAGUGGUCAGUCCAGGAGCUAG	SEQ ID NO 387	UAGGUCAGGCUGCUUUGCCCUCAGC
SEQ ID NO 362	AAUAGUGGUCAGUCCAGGAGCUAGG	SEQ ID NO 388	AGGUCAGGCUGCUUUGCCCUCAGCU
SEQ ID NO 363	AUAGUGGUCAGUCCAGGAGCUAGGU	SEQ ID NO 389	GGUCAGGCUGCUUUGCCCUCAGCUC
SEQ ID NO 364	AUAGUGGUCAGUCCAGGAGCU	SEQ ID NO 390	GUCAGGCUGCUUUGCCCUCAGCUCU
SEQ ID NO 365	UAGUGGUCAGUCCAGGAGCUAGGUC	SEQ ID NO 391	UCAGGCUGCUUUGCCCUCAGCUCUU
SEQ ID NO 366	AGUGGUCAGUCCAGGAGCUAGGUCA	SEQ ID NO 392	CAGGCUGCUUUGCCCUCAGCUCUUG
SEQ ID NO 367	GUGGUCAGUCCAGGAGCUAGGUCAG	SEQ ID NO 393	AGGCUGCUUUGCCCUCAGCUCUUGA
SEQ ID NO 368	UGGUCAGUCCAGGAGCUAGGUCAGG	SEQ ID NO 394	GGCUGCUUUGCCCUCAGCUCUUGAA
SEQ ID NO 369	GGUCAGUCCAGGAGCUAGGUCAGGC	SEQ ID NO 395	GCUGCUUUGCCCUCAGCUCUUGAAG
SEQ ID NO 370	GUCAGUCCAGGAGCUAGGUCAGGCU	SEQ ID NO 396	CUGCUUUGCCCUCAGCUCUUGAAGU
SEQ ID NO 371	UCAGUCCAGGAGCUAGGUCAGGCUG	SEQ ID NO 397	UGCUUUGCCCUCAGCUCUUGAAGUA
SEQ ID NO 372	CAGUCCAGGAGCUAGGUCAGGCUGC	SEQ ID NO 398	GCUUUGCCCUCAGCUCUUGAAGUAA
SEQ ID NO 373	AGUCCAGGAGCUAGGUCAGGCUGCU	SEQ ID NO 399	CUUUGCCCUCAGCUCUUGAAGUAAA
SEQ ID NO 374	GUCCAGGAGCUAGGUCAGGCUGCUU	SEQ ID NO 400	UUUGCCCUCAGCUCUUGAAGUAAAC
SEQ ID NO 375	UCCAGGAGCUAGGUCAGGCUGCUUU	SEQ ID NO 401	UUGCCCUCAGCUCUUGAAGUAAACG
NO 376	CCAGGAGCUAGGUCAGGCUGCUUUG	SEQ ID NO 402	UGCCCUCAGCUCUUGAAGUAAACGG
SEQ ID NO 377	CAGGAGCUAGGUCAGGCUGCUUUGC	SEQ ID NO 403	GCCCUCAGCUCUUGAAGUAAACGGU
SEQ ID NO 378	AGGAGCUAGGUCAGGCUGCUUUGCC	SEQ ID NO 404	CCCUCAGCUCUUGAAGUAAACGGUU
SEQ ID NO 379	GGAGCUAGGUCAGGCUGCUUUGCCC	SEQ ID NO 405	CCUCAGCUCUUGAAGUAAAC
SEQ ID NO 380	GAGCUAGGUCAGGCUGCUUUGCCCU	SEQ ID NO 406	CCUCAGCUCUUGAAGUAAACG
SEQ ID NO 381	AGCUAGGUCAGGCUGCUUUGCCCUC	SEQ ID NO 407	CUCAGCUCUUGAAGUAAACG
SEQ ID NO 382	GCUAGGUCAGGCUGCUUUGCCCUCA	SES ID NO 408	CCUCAGCUCUUGAAGUAAACGGUUU
SEQ ID NO 383	CUCAGCUCUUGAAGUAAACGGUUUA	SEQ ID NO 409	UCAGCUCUUGAAGUAAACGGUUUAC
SEQ ID NO 384	CAGCUCUUGAAGUAAACGGUUUACC	SEQ ID NO 410	AGCUCUUGAAGUAAACGGUUUACCG
SEQ ID NO 385	GCUCUUGAAGUAAACGGUUUACCGC	SEQ ID NO 411	CUCUUGAAGUAAACGGUUUACCGCC

SEQ ID NO 412	CCACAGGCGUUGCACUUUGCAAUGC	SEQ ID NO 443	UCUUCUUGCUAUGAAUAAUGUCAAU
SEQ ID NO 413	CACAGGCGUUGCACUUUGCAAUGCU	SEQ ID NO 444	CUUCUUGCUAUGAAUAAUGUCAAUC
SEQ ID NO 414	ACAGGCGUUGCACUUUGCAAUGCUG	SEQ ID NO 445	UUCUUGCUAUGAAUAAUGUCAAUCC
SEQ ID NO 415	CAGGCGUUGCACUUUGCAAUGCUGC	SEQ ID NO 446	UCUUGCUAUGAAUAAUGUCAAUCCG
SEQ ID NO 416	AGGCGUUGCACUUUGCAAUGCUGCU	SEQ ID NO 447	CUUGCUAUGAAUAAUGUCAAUCCGA
SEQ ID NO 417	GGCGUUGCACUUUGCAAUGCUGCUG	SEQ ID NO 448	UUGCUAUGAAUAAUGUCAAUCCGAC
SEQ ID NO 418	GCGUUGCACUUUGCAAUGCUGCUGU	SEQ ID NO 449	UGCUAUGAAUAAUGUCAAUCCGACC
SEQ ID NO 419	CGUUGCACUUUGCAAUGCUGCUGUC	SEQ ID NO 450	GCUAUGAAUAAUGUCAAUCCGACCU
SEQ ID NO 420	CGUUGCACUUUGCAAUGCUGCUG	SEQ ID NO 451	CUAUGAAUAAUGUCAAUCCGACCUG
SEQ ID NO 421	GUUGCACUUUGCAAUGCUGCUGUCU	SEQ ID NO 452	UAUGAAUAAUGUCAAUCCGACCUGA
SEQ ID NO 422	UUGCACUUUGCAAUGCUGCUGUCUU	SEQ ID NO 453	AUGAAUAAUGUCAAUCCGACCUGAG
SEQ ID NO 423	UGCACUUUGCAAUGCUGCUGUCUUC	SEQ ID NO 454	UGAAUAAUGUCAAUCCGACCUGAGC
SEQ ID NO 424	GCACUUUGCAAUGCUGCUGUCUUCU	SEQ ID NO 455	GAAUAAUGUCAAUCCGACCUGAGCU
SEQ ID NO 425	CACUUUGCAAUGCUGCUGUCUUCUU	SEQ ID NO 456	AAUAAUGUCAAUCCGACCUGAGCUU
SEQ ID NO 426	ACUUUGCAAUGCUGCUGUCUUCUUG	SEQ ID NO 457	AUAAUGUCAAUCCGACCUGAGCUUU
SEQ ID NO 427	CUUUGCAAUGCUGCUGUCUUCUUGC	SEQ ID NO 458	UAAUGUCAAUCCGACCUGAGCUUUG
SEQ ID NO 428	UUUGCAAUGCUGCUGUCUUCUUGCU	SEQ ID NO 459	AAUGUCAAUCCGACCUGAGCUUUGU
SEQ ID NO 429	UUGCAAUGCUGCUGUCUUCUUGCUA	SEQ ID NO 460	AUGUCAAUCCGACCUGAGCUUUGUU
SEQ ID NO 430	UGCAAUGCUGCUGUCUUCUUGCUAU	SEQ ID NO 461	UGUCAAUCCGACCUGAGCUUUGUUCG
SEQ ID NO 431	GCAAUGCUGCUGUCUUCUUGCUAUG	SEQ ID NO 462	GUCAAUCCGACCUGAGCUUUGUUGU
SEQ ID NO 432	CAAUGCUGCUGUCUUCUUGCUAUGA	SEQ ID NO 463	UCAAUCCGACCUGAGCUUUGUUGUA
SEQ ID NO 433	AAUGCUGCUGUCUUCUUGCUAUGAA	SEQ ID NO 464	CAAUCCGACCUGAGCUUUGUUGUAG
SEQ ID NO 434	AUGCUGCUGUCUUCUUGCUAUGAAU	SEQ ID NO 465	AAUCCGACCUGAGCUUUGUUGUAGA
SEQ ID NO 435	UGCUGCUGUCUUCUUGCUAUGAAUA	SEQ ID NO 466	AUCCGACCUGAGCUUUGUUGUAGAC
SEQ ID NO 436	GCUGCUGUCUUCUUGCUAUGAAUAA	SEQ ID NO 467	UCCGACCUGAGCUUUGUUGUAGACU
SEQ ID NO 437	CUGCUGUCUUCUUGCUAUGAAUAAU	SEQ ID NO 468	CCGACCUGAGCUUUGUUGUAGACUA
SEQ ID NO 438	UGCUGUCUUCUUGCUAUGAAUAAUG	SEQ ID NO 469	CGACCUGAGCUUUGUUGUAG
SEQ ID NO 439	GCUGUCUUCUUGCUAUGAAUAAUGU	SEQ ID NO 470	CGACCUGAGCUUUGUUGUAGACUAU
SEQ ID NO 440	CUGUCUUCUUGCUAUGAAUAAUGUC	SEQ ID NO 471	GACCUGAGCUUUGUUGUAGACUAUC
SEQ ID NO 441	UGUCUUCUUGCUAUGAAUAAUGUCA	SEQ ID NO 472	ACCUGAGCUUUGUUGUAGACUAUCA
SEQ ID NO 442	GUCUUCUUGCUAUGAAUAAUGUCAA	SEQ ID NO 473	CCUGA GCUUU GUUGU AGACU AUC


SEQ ID NO 474	CAUUUUUGACCUACAUGUGG	SEQ ID NO 479	AUUUUUGACCUACAUGGGAAAG
SEQ ID NO 475	UUUGACCUACAUGUGGAAAG	SEQ ID NO 480	UACGAGUUGAUUGUCGGACCCAG
SEQ ID NO 476		SEQ ID NO 481	GUGGUCUCCUUACCUAUGACUGUGG
SEQ ID NO 477	GGUCUCCUUACCUAUGA	SEQ ID NO 482	UGUCUCAGUAAUCUUCUUACCUAU
SEQ ID NO 478	UCUUACCUAUGACUAUGGAUGAGA


SEQ ID NO 483	UGCAUGUUCCAGUCGUUGUGUGG	SEQ ID NO 485	AUUUACCAACCUUCAGGAUCGAGUA
SEQ ID NO 484	CACUAUUCCAGUCAAAUAGGUCUGG	SEQ ID NO 486	GGCCUAAAACACAUACACAUA


SEQ ID NO 487	GAUAGGUGGUAUCAACAUCUGUAA	SEQ ID NO 490	UGUUGUUGUUUAUGCUCAUU
SEQ ID NO 488	GAUAGGUGGUAUCAACAUCUG	SEQ ID NO 491	GUACAUUAAGAUGGACUUC
SEQ ID NO 489	CUUCCUGGAUGGCUUGAAU


SEQ ID NO 492	CUGUUGCAGUAAUCUAUGAG	SEQ ID NO 495	UGCCAUUGUUUCAUCAGCUCUUU
SEQ ID NO 493	UGCAGUAAUCUAUGAGUUUC	SEQ ID NO 496	UCCUGUAGGACAUUGGCAGU
SEQ ID NO 494	GAGUCUUCUAGGAGCCUU	SEQ ID NO 497	CUUGGAGUCUUCUAGGAGCC


SEQ ID NO 498		SEQ ID NO 500	GAAAAUUGUGCAUUUACCCAUUUU
SEQ ID NO 499	CCCAUUUUGUGAAUGUUUUCUUUU	SEQ ID NO 501	UUGUGCAUUUACCCAUUUUGUG


SEQ ID NO 502	CCCUGAGGCAUUCCCAUCUUGAAU	SEQ ID NO 504	CUUGAAUUUAGGAGAUUCAUCUG
SEQ ID NO 503	AGGACUUACUUGCUUUGUUU	SEQ ID NO 505	CAUCUUCUGAUAAUUUUCCUGUU


SEQ ID NO 506	CCAUUACAGUUGUCUGUGUU	SEQ ID	UAAUCUGCCUCUUCUUUUGG
SEQ ID NO 507	UGACAGCCUGUGAAAUCUGUGAG


SEQ ID NO 509	CAGCAGUAGUUGUCAUCUGC	SEQ ID NO 511
SEQ ID NO 510		SEQ ID NO 512	UCUGCUGGCAUCUUGC


SEQ ID NO 513	GCCGGUUGACUUCAUCCUGUGC	SEQ ID NO 516	CUGCAUCCAGGAACAUGGGUCC
SEQ ID NO 514	GUCUGCAUCCAGGAACAUGGGUC	SEQ ID NO 517	GUUGAAGAUCUGAUAGCCGGUUGA
SEQ ID NO 515	UACUUACUGUCUGUAGCUCUUUCU


SEQ ID NO 518	UAGGUGCCUGCCGGCUU	SEQ ID NO 520	CUGAACUGCUGGAAAGUCGCC
SEQ ID NO 519	UUCAGCUGUAGCCACACC	SEQ ID NO 521


SEQ ID NO 522	CAAUUUUUCCCACUCAGUAUU	SEQ ID NO 524	UCCUCAGGAGGCAGCUCUAAAU
SEQ ID NO 523	UUGAAGUUCCUGGAGUCUU


SEQ ID NO 525	UGGCUCUCUCCCAGGG	NO 527	GGGCACUUUGUUUGGCG
SEQ ID NO 526


SEQ ID NO 528	GGUCCCAGCAAGUUGUUUG	SEQ ID NO 530	GUAGAGCUCUGUCAUUUUGGG
SEQ ID NO 529	UGGGAUGGUCCCAGCAAGUUGUUUG


SEQ ID NO 531	GCUCAAGAGAUCCACUGCAAAAAAC	SEQ ID NO 533	UCUGCAGGAUAUCCAUGGGCUGGUC
SEQ ID NO 532


SEQ ID NO 534


SEQ ID NO 535	UGCUUUAGACUCCUGUACCUGAUA


SEQ ID NO 536	GGCGGCCUUUGUGUUGAC	SEQ ID NO 538	CCUUUAUGUUCGUGCUGCU
SEQ ID NO 537	GGACAGGCCUUUAUGUUCGUGCUGC

Human IGF-1 Isoform 4 amino acid sequence

SEQ ID NO 577:

SEQUENCE LISTING

<110> Prosensa Technologies B.V.
<120> Oligonucleotide comprising an inosine for treating DMD by exon skipping
<130> P6024692PCT
<150> US 61/172,506 <151> 2009-04-24
<150> EP 09158731.1 <151> 2009-04-24
<160> 577
<170> PatentIn version 3.3
<210> 1 <211> 3685 <212> PRT <213> Homo sapiens
<400> 1
<210> 2 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 2 guaccuccaa caucaaggaa gaugg 25
<210> 3 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 3 uaccuccaac aucaaggaag auggc 25
<210> 4 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 4 accuccaaca ucaaggaaga uggca 25
<210> 5 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 5 ccuccaacau caaggaagau ggcau 25
<210> 6 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 6 cuccaacauc aaggaagaug gcauu 25
<210> 7 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 7 uccaacauca aggaagaugg cauuu 25
<210> 8 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 8 ccaacaucaa ggaagauggc auuuc 25
<210> 9 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 9 caacaucaag gaagauggca uuucu 25
<210> 10 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 10 aacaucaagg aagauggcau uucua 25
<210> 11 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 11 acaucaagga agauggcauu ucuag 25
<210> 12 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 12 caucaaggaa gauggcauuu cuagu 25
<210> 13 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 13 aucaaggaag auggcauuuc uaguu 25
<210> 14 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 14 ucaaggaaga uggcauuucu aguuu 25
<210> 15 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 15 caaggaagau ggcauuucua guuug 25
<210> 16 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 16 aaggaagaug gcauuucuag uuugg 25
<210> 17 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 17 aggaagaugg cauuucuagu uugga 25
<210> 18 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 18 ggaagauggc auuucuaguu uggag 25
<210> 19 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 19 gaagauggca uuucuaguuu ggaga 25
<210> 20 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 20 aagauggcau uucuaguuug gagau 25
<210> 21 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 21 agauggcauu ucuaguuugg agaug 25
<210> 22 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 22 gauggcauuu cuaguuugga gaugg 25
<210> 23 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 23 auggcauuuc uaguuuggag auggc 25
<210> 24 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 24 uggcauuucu aguuuggaga uggca 25
<210> 25 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 25 ggcauuucua guuuggagau ggcag 25
<210> 26 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 26 gcauuucuag uuuggagaug gcagu 25
<210> 27 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 27 cauuucuagu uuggagaugg caguu 25
<210> 28 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 28 auuucuaguu uggagauggc aguuu 25
<210> 29 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 29 uuucuaguuu ggagauggca guuuc 25
<210> 30 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 30 uucuaguuug gagauggcag uuucc 25
<210> 31 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 31 ucuaguuugg agauggcagu uuccu 25
<210> 32 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 32 cuaguuugga gauggcaguu uccuu 25
<210> 33 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 33 uaguuuggag auggcaguuu ccuua 25
<210> 34 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 34 aguuuggaga uggcaguuuc cuuag 25
<210> 35 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 35 guuuggagau ggcaguuucc uuagu 25
<210> 36 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 36 uuuggagaug gcaguuuccu uagua 25
<210> 37 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 37 uuggagaugg caguuuccuu aguaa 25
<210> 38 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 38 uggagauggc aguuuccuua guaac 25
<210> 39 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 39 gagauggcag uuuccuuagu aacca 25
<210> 40 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 40 agauggcagu uuccuuagua accac 25
<210> 41 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 41 gauggcaguu uccuuaguaa ccaca 25
<210> 42 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 42 auggcaguuu ccuuaguaac cacag 25
<210> 43 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 43 uggcaguuuc cuuaguaacc acagg 25
<210> 44 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 44 ggcaguuucc uuaguaacca 25
<210> 45 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 45 gcaguuuccu uaguaaccac agguu 25
<210> 46 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 46 caguuuccuu aguaaccaca gguug 25
<210> 47 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 47 aguuuccuua guaaccacag guugu 25
<210> 48 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 48 guuuccuuag uaaccacagg uugug 25
<210> 49 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 49 uuuccuuagu aaccacaggu ugugu 25
<210> 50 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 50 uuccuuagua accacagguu guguc 25
<210> 51 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 51 uccuuaguaa ccacagguug uguca 25
<210> 52 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 52 ccuuaguaac cacagguugu gucac 25
<210> 53 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 53 cuuaguaacc acagguugug ucacc 25
<210> 54 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 54 uuaguaacca cagguugugu cacca 25
<210> 55 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 55 uaguaaccac agguuguguc accag 25
<210> 56 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 56 aguaaccaca gguuguguca ccaga 25
<210> 57 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 57 guaaccacag guugugucac cagag 25
<210> 58 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 58 uaaccacagg uugugucacc agagu 25
<210> 59 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 59 aaccacaggu ugugucacca gagua 25
<210> 60 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 60 accacagguu gugucaccag aguaa 25
<210> 61 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 61 ccacagguug ugucaccaga guaac 25
<210> 62 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 62 cacagguugu gucaccagag uaaca 25
<210> 63 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 63 acagguugug ucaccagagu aacag 25
<210> 64 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 64 cagguugugu caccagagua acagu 25
<210> 65 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 65 agguuguguc accagaguaa caguc 25
<210> 66 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 66 gguuguguca ccagaguaac agucu 25
<210> 67 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 67 guugugucac cagaguaaca gucug 25
<210> 68 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 68 uugugucacc agaguaacag ucuga 25
<210> 69 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 69 ugugucacca gaguaacagu cugag 25
<210> 70 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 70 gugucaccag aguaacaguc ugagu 25
<210> 71 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 71 ugucaccaga guaacagucu gagua 25
<210> 72 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 72 gucaccagag uaacagucug aguag 25
<210> 73 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 73 ucaccagagu aacagucuga guagg 25
<210> 74 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 74 caccagagua acagucugag uagga 25
<210> 75 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 75 accagaguaa cagucugagu aggag 25
<210> 76 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 76 uuugccgcug cccaaugcca uccug 25
<210> 77 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 77 auucaauguu cugacaacag uuugc 25
<210> 78 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 78 ccaguugcau ucaauguucu gacaa 25
<210> 79 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 79 caguugcauu caauguucug ac 22
<210> 80 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 80 aguugcauuc aauguucuga 20
<210> 81 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 81 gauugcugaa uuauuucuuc c 21
<210> 82 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 82 gauugcugaa uuauuucuuc cccag 25
<210> 83 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 83 auugcugaau uauuucuucc ccagu 25
<210> 84 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 84 uugcugaauu auuucuuccc caguu 25
<210> 85 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 85 ugcugaauua uuucuucccc aguug 25
<210> 86 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 86 gcugaauuau uucuucccca guugc 25
<210> 87 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 87 cugaauuauu ucuuccccag uugca 25
<210> 88 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 88 ugaauuauuu cuuccccagu ugcau 25
<210> 89 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 89 gaauuauuuc uuccccaguu gcauu 25
<210> 90 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 90 aauuauuucu uccccaguug cauuc 25
<210> 91 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 91 auuauuucuu ccccaguugc auuca 25
<210> 92 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 92 uuauuucuuc cccaguugca uucaa 25
<210> 93 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 93 uauuucuucc ccaguugcau ucaau 25
<210> 94 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 94 auuucuuccc caguugcauu caaug 25
<210> 95 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 95 uuucuucccc aguugcauuc aaugu 25
<210> 96 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 96 uucuucccca guugcauuca auguu 25
<210> 97 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 97 ucuuccccag uugcauucaa uguuc 25
<210> 98 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 98 cuuccccagu ugcauucaau guucu 25
<210> 99 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 99 uuccccaguu gcauucaaug uucug 25
<210> 100 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 100 uccccaguug cauucaaugu ucuga 25
<210> 101 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 101 ccccaguugc auucaauguu cugac 25
<210> 102 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 102 cccaguugca uucaauguuc ugaca 25
<210> 103 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 103 ccaguugcau ucaauguucu gacaa 25
<210> 104 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 104 caguugcauu caauguucug acaac 25
<210> 105 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 105 aguugcauuc aauguucuga caaca 25
<210> 106 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 106 uccuguagaa uacuggcauc 20
<210> 107 <211> 27 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 107 ugcagaccuc cugccaccgc agauuca 27
<210> 108 <211> 34 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 108 uugcagaccu ccugccaccg cagauucagg cuuc 34
<210> 109 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 109 guugcauuca auguucugac aacag 25
<210> 110 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 110 uugcauucaa uguucugaca acagu 25
<210> 111 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 111 ugcauucaau guucugacaa caguu 25
<210> 112 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 112 gcauucaaug uucugacaac aguuu 25
<210> 113 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 113 cauucaaugu ucugacaaca guuug 25
<210> 114 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 114 auucaauguu cugacaacag uuugc 25
<210> 115 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 115 ucaauguucu gacaacaguu ugccg 25
<210> 116 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 116 caauguucug acaacaguuu gccgc 25
<210> 117 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 117 aauguucuga caacaguuug ccgcu 25
<210> 118 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 118 auguucugac aacaguuugc cgcug 25
<210> 119 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
400> 119 uguucugaca acaguuugcc gcugc 25
<210> 120 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 120 guucugacaa caguuugccg cugcc 25
<210> 121 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 121 uucugacaac aguuugccgc ugccc 25
<210> 122 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 122 ucugacaaca guuugccgcu gccca 25
<210> 123 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 123 cugacaacag uuugccgcug cccaa 25
<210> 124 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 124 ugacaacagu uugccgcugc ccaau 25
<210> 125 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 125 gacaacaguu ugccgcugcc caaug 25
<210> 126 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 126 acaacaguuu gccgcugccc aaugc 25
<210> 127 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 127 caacaguuug ccgcugccca augcc 25
<210> 128 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 128 aacaguuugc cgcugcccaa 25
<210> 129 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 129 acaguuugcc gcugcccaau gccau 25
<210> 130 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 130 caguuugccg cugcccaaug ccauc 25
<210> 131 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 131 aguuugccgc ugcccaaugc caucc 25
<210> 132 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 132 guuugccgcu gcccaaugcc auccu 25
<210> 133 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 133 uuugccgcug cccaaugcca uccug 25
<210> 134 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 134 uugccgcugc ccaaugccau ccugg 25
<210> 135 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 135 ugccgcugcc caaugccauc cugga 25
<210> 136 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 136 gccgcugccc aaugccaucc uggag 25
<210> 137 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 137 ccgcugccca augccauccu ggagu 25
<210> 138 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 138 cgcugcccaa ugccauccug gaguu 25
<210> 139 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 139 uguuuuugag gauugcugaa 20
<210> 140 <211> 40 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 140 uguucugaca acaguuugcc gcugcccaau gccauccugg 40
<210> 141 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 141 cucuggccug uccuaagacc ugcuc 25
<210> 142 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 142 ucuggccugu ccuaagaccu gcuca 25
<210> 143 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 143 cuggccuguc cuaagaccug cucag 25
<210> 144 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 144 uggccugucc uaagaccugc ucagc 25
<210> 145 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 145 ggccuguccu aagaccugcu cagcu 25
<210> 146 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 146 gccuguccua agaccugcuc agcuu 25
<210> 147 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 147 ccuguccuaa gaccugcuca gcuuc 25
<210> 148 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 148 cuguccuaag accugcucag cuucu 25
<210> 149 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 149 uguccuaaga ccugcucagc uucuu 25
<210> 150 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 150 guccuaagac cugcucagcu ucuuc 25
<210> 151 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 151 uccuaagacc ugcucagcuu cuucc 25
<210> 152 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 152 ccuaagaccu gcucagcuuc uuccu 25
<210> 153 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 153 cuaagaccug cucagcuucu uccuu 25
<210> 154 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 154 uaagaccugc ucagcuucuu ccuua 25
<210> 155 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 155 aagaccugcu cagcuucuuc cuuag 25
<210> 156 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 156 agaccugcuc agcuucuucc uuagc 25
<210> 157 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 157 gaccugcuca gcuucuuccu uagcu 25
<210> 158 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 158 accugcucag cuucuuccuu agcuu 25
<210> 159 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 159 ccugcucagc uucuuccuua gcuuc 25
<210> 160 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 160 cugcucagcu ucuuccuuag cuucc 25
<210> 161 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 161 ugcucagcuu cuuccuuagc uucca 25
<210> 162 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 162 gcucagcuuc uuccuuagcu uccag 25
<210> 163 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 163 cucagcuucu uccuuagcuu ccagc 25
<210> 164 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 164 ucagcuucuu ccuuagcuuc cagcc 25
<210> 165 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 165 cagcuucuuc cuuagcuucc agcca 25
<210> 166 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 166 agcuucuucc uuagcuucca gccau 25
<210> 167 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 167 gcuucuuccu uagcuuccag ccauu 25
<210> 168 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 168 cuucuuccuu agcuuccagc cauug 25
<210> 169 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 169 uucuuccuua gcuuccagcc auugu 25
<210> 170 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 170 ucuuccuuag cuuccagcca uugug 25
<210> 171 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 171 cuuccuuagc uuccagccau ugugu 25
<210> 172 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 172 uuccuuagcu uccagccauu guguu 25
<210> 173 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 173 uccuuagcuu ccagccauug uguug 25
<210> 174 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 174 ccuuagcuuc cagccauugu guuga 25
<210> 175 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 175 cuuagcuucc agccauugug uugaa 25
<210> 176 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 176 uuagcuucca gccauugugu ugaau 25
<210> 177 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 177 uagcuuccag ccauuguguu gaauc 25
<210> 178 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 178 agcuuccagc cauuguguug aaucc 25
<210> 179 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 179 gcuuccagcc auuguguuga auccu 25
<210> 180 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 180 cuuccagcca uuguguugaa uccuu 25
<210> 181 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 181 uuccagccau uguguugaau ccuuu 25
<210> 182 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 182 uccagccauu guguugaauc cuuua 25
<210> 183 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 183 ccagccauug uguugaaucc uuuaa 25
<210> 184 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 184 cagccauugu guugaauccu uuaac 25
<210> 185 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 185 agccauugug uugaauccuu uaaca 25
<210> 186 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 186 gccauugugu ugaauccuuu aacau 25
<210> 187 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 187 ccauuguguu gaauccuuua acauu 25
<210> 188 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 188 cauuguguug aauccuuuaa cauuu 25
<210> 189 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 189 ucagcuucug uuagccacug 20
<210> 190 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 190 uucagcuucu guuagccacu 20
<210> 191 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 191 uucagcuucu guuagccacu g 21
<210> 192 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 192 ucagcuucug uuagccacug a 21
<210> 193 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 193 uucagcuucu guuagccacu ga 22
<210> 194 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 194 ucagcuucug uuagccacug a 21
<210> 195 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 195 uucagcuucu guuagccacu ga 22
<210> 196 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 196 ucagcuucug uuagccacug au 22
<210> 197 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 197 uucagcuucu guuagccacu gau 23
<210> 198 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 198 ucagcuucug uuagccacug auu 23
<210> 199 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 199 uucagcuucu guuagccacu gauu 24
<210> 200 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 200 ucagcuucug uuagccacug auua 24
<210> 201 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 201 uucagcuucu guuagccacu gaua 24
<210> 202 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 202 ucagcuucug uuagccacug auuaa 25
<210> 203 <211> 26 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 203 uucagcuucu guuagccacu gauuaa 26
<210> 204 <211> 26 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 204 ucagcuucug uuagccacug auuaaa 26
<210> 205 <211> 27 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 205 uucagcuucu guuagccacu gauuaaa 27
<210> 206 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 206 cagcuucugu uagccacug 19
<210> 207 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 207 cagcuucugu uagccacuga u 21
<210> 208 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 208 agcuucuguu agccacugau u 21
<210> 209 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 209 cagcuucugu uagccacuga uu 22
<210> 210 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 210 agcuucuguu agccacugau ua 22
<210> 211 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 211 cagcuucugu uagccacuga uua 23
<210> 212 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 212 agcuucuguu agccacugau uaa 23
<210> 213 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 213 cagcuucugu uagccacuga uuaa 24
<210> 214 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 214 agcuucuguu agccacugau uaaa 24
<210> 215 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 215 cagcuucugu uagccacuga uuaaa 25
<210> 216 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 216 agcuucuguu agccacugau uaaa 24
<210> 217 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 217 agcuucuguu agccacugau 20
<210> 218 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 218 gcuucuguua gccacugauu 20
<210> 219 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 219 agcuucuguu agccacugau u 21
<210> 220 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 220 gcuucuguua gccacugauu a 21
<210> 221 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 221 agcuucuguu agccacugau ua 22
<210> 222 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 222 gcuucuguua gccacugauu aa 22
<210> 223 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 223 agcuucuguu agccacugau uaa 23
<210> 224 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 224 gcuucuguua gccacugauu aaa 23
<210> 225 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 225 agcuucuguu agccacugau uaaa 24
<210> 226 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 226 gcuucuguua gccacugauu aaa 23
<210> 227 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 227 ccauuuguau uuagcauguu ccc 23
<210> 228 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 228 agauaccauu uguauuuagc 20
<210> 229 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 229 gccauuucuc aacagaucu 19
<210> 230 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 230 gccauuucuc aacagaucug uca 23
<210> 231 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 231 auucucagga auuugugucu uuc 23
<210> 232 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 232 ucucaggaau uugugucuuu c 21
<210> 233 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 233 guucagcuuc uguuagcc 18
<210> 234 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 234 cugauuaaau aucuuuauau c 21
<210> 235 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 235 gccgccauuu cucaacag 18
<210> 236 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 236 guauuuagca uguuccca 18
<210> 237 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 237 caggaauuug ugucuuuc 18
<210> 238 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 238 gcuuuucuuu uaguugcugc ucuuu 25
<210> 239 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 239 cuuuucuuuu aguugcugcu cuuuu 25
<210> 240 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 240 uuuucuuuua guugcugcuc uuuuc 25
<210> 241 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 241 uuucuuuuag uugcugcucu uuucc 25
<210> 242 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 242 uucuuuuagu ugcugcucuu uucca 25
<210> 243 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 243 ucuuuuaguu gcugcucuuu uccag 25
<210> 244 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 244 cuuuuaguug cugcucuuuu ccagg 25
<210> 245 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 245 uuuuaguugc ugcucuuuuc caggu 25
<210> 246 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 246 uuuaguugcu gcucuuuucc agguu 25
<210> 247 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 247 uuaguugcug cucuuuucca gguuc 25
<210> 248 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 248 uaguugcugc ucuuuuccag guuca 25
<210> 249 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 249 aguugcugcu cuuuuccagg uucaa 25
<210> 250 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 250 guugcugcuc uuuuccaggu ucaag 25
<210> 251 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 251 uugcugcucu uuuccagguu caagu 25
<210> 252 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 252 ugcugcucuu uuccagguuc aagug 25
<210> 253 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 253 gcugcucuuu uccagguuca agugg 25
<210> 254 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 254 cugcucuuuu ccagguucaa guggg 25
<210> 255 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 255 ugcucuuuuc cagguucaag uggga 25
<210> 256 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 256 gcucuuuucc agguucaagu gggac 25
<210> 257 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 257 cucuuuucca gguucaagug ggaua 25
<210> 258 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 258 ucuuuuccag guucaagugg gauac 25
<210> 259 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 259 ucuuuuccag guucaagugg 20
<210> 260 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 260 cuuuuccagg uucaaguggg auacu 25
<210> 261 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 261 uuuuccaggu ucaaguggga uacua 25
<210> 262 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 262 uuuccagguu caagugggau acuag 25
<210> 263 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 263 uuccagguuc aagugggaua cuagc 25
<210> 264 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 264 uccagguuca agugggauac uagca 25
<210> 265 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 265 ccagguucaa gugggauacu agcaa 25
<210> 266 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 266 cagguucaag ugggauacua gcaau 25
<210> 267 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 267 agguucaagu gggauacuag caaug 25
<210> 268 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 268 gguucaagug ggauacuagc aaugu 25
<210> 269 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 269 guucaagugg gauacuagca auguu 25
<210> 270 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 270 uucaaguggg auacuagcaa uguua 25
<210> 271 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 271 ucaaguggga uacuagcaau guuau 25
<210> 272 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 272 caagugggau acuagcaaug uuauc 25
<210> 273 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 273 aagugggaua cuagcaaugu uaucu 25
<210> 274 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 274 agugggauac uagcaauguu aucug 25
<210> 275 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 275 gugggauacu agcaauguua ucugc 25
<210> 276 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 276 ugggauacua gcaauguuau cugcu 25
<210> 277 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 277 gggauacuag caauguuauc ugcuu 25
<210> 278 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 278 ggauacuagc aauguuaucu gcuuc 25
<210> 279 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 279 gauacuagca auguuaucug cuucc 25
<210> 280 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 280 auacuagcaa uguuaucugc uuccu 25
<210> 281 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 281 uacuagcaau guuaucugcu uccuc 25
<210> 282 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 282 acuagcaaug uuaucugcuu ccucc 25
<210> 283 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 283 cuagcaaugu uaucugcuuc cucca 25
<210> 284 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 284 uagcaauguu aucugcuucc uccaa 25
<210> 285 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 285 agcaauguua ucugcuuccu ccaac 25
<210> 286 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 286 gcaauguuau cugcuuccuc caacc 25
<210> 287 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 287 caauguuauc ugcuuccucc aacca 25
<210> 288 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 288 aauguuaucu gcuuccucca accau 25
<210> 289 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 289 auguuaucug cuuccuccaa ccaua 25
<210> 290 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 290 uguuaucugc uuccuccaac cauaa 25
<210> 291 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 291 agccucuuga uugcuggucu uguuu 25
<210> 292 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 292 gccucuugau ugcuggucuu guuuu 25
<210> 293 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 293 ccucuugauu gcuggucuug uuuuu 25
<210> 294 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 294 ccucuugauu gcuggucuug 20
<210> 295 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 295 cucuugauug cuggucuugu uuuuc 25
<210> 296 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 296 ucuugauugc uggucuuguu uuuca 25
<210> 297 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 297 cuugauugcu ggucuuguuu uucaa 25
<210> 298 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 298 uugauugcug gucuuguuuu ucaaa 25
<210> 299 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 299 ugauugcugg ucuuguuuuu caaau 25
<210> 300 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 300 gauugcuggu cuuguuuuuc aaauu 25
<210> 301 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 301 gauugcuggu cuuguuuuuc 20
<210> 302 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 302 auugcugguc uuguuuuuca aauuu 25
<210> 303 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 303 uugcuggucu uguuuuucaa auuuu 25
<210> 304 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 304 ugcuggucuu guuuuucaaa uuuug 25
<210> 305 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 305 gcuggucuug uuuuucaaau uuugg 25
<210> 306 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 306 cuggucuugu uuuucaaauu uuggg 25
<210> 307 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 307 uggucuuguu uuucaaauuu ugggc 25
<210> 308 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 308 ggucuuguuu uucaaauuuu gggca 25
<210> 309 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 309 gucuuguuuu ucaaauuuug ggcag 25
<210> 310 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 310 ucuuguuuuu caaauuuugg gcagc 25
<210> 311 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 311 cuuguuuuuc aaauuuuggg cagcg 25
<210> 312 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 312 uuguuuuuca aauuuugggc agcgg 25
<210> 313 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 313 uguuuuucaa auuuugggca gcggu 25
<210> 314 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 314 guuuuucaaa uuuugggcag cggua 25
<210> 315 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 315 uuuuucaaau uuugggcagc gguaa 25
<210> 316 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 316 uuuucaaauu uugggcagcg guaau 25
<210> 317 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 317 uuucaaauuu ugggcagcgg uaaug 25
<210> 318 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 318 uucaaauuuu gggcagcggu aauga 25
<210> 319 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 319 ucaaauuuug ggcagcggua augag 25
<210> 320 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 320 caaauuuugg gcagcgguaa ugagu 25
<210> 321 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 321 aaauuuuggg cagcgguaau gaguu 25
<210> 322 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 322 aauuuugggc agcgguaaug aguuc 25
<210> 323 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 323 auuuugggca gcgguaauga guucu 25
<210> 324 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 324 uuuugggcag cgguaaugag uucuu 25
<210> 325 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 325 uuugggcagc gguaaugagu ucuuc 25
<210> 326 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 326 uugggcagcg guaaugaguu cuucc 25
<210> 327 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 327 ugggcagcgg uaaugaguuc uucca 25
<210> 328 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 328 gggcagcggu aaugaguucu uccaa 25
<210> 329 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 329 ggcagcggua augaguucuu ccaac 25
<210> 330 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 330 gcagcgguaa ugaguucuuc caacu 25
<210> 331 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 331 cagcgguaau gaguucuucc aacug 25
<210> 332 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 332 agcgguaaug aguucuucca acugg 25
<210> 333 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 333 gcgguaauga guucuuccaa cuggg 25
<210> 334 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 334 cgguaaugag uucuuccaac ugggg 25
<210> 335 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 335 gguaaugagu ucuuccaacu gggga 25
<210> 336 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 336 gguaaugagu ucuuccaacu gg 22
<210> 337 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 337 guaaugaguu cuuccaacug gggac 25
<210> 338 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 338 uaaugaguuc uuccaacugg ggacg 25
<210> 339 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 339 aaugaguucu uccaacuggg gacgc 25
<210> 340 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 340 augaguucuu ccaacugggg acgcc 25
<210> 341 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 341 ugaguucuuc caacugggga cgccu 25
<210> 342 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 342 gaguucuucc aacuggggac gccuc 25
<210> 343 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 343 aguucuucca acuggggacg ccucu 25
<210> 344 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 344 guucuuccaa cuggggacgc cucug 25
<210> 345 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 345 uucuuccaac uggggacgcc ucugu 25
<210> 346 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 346 ucuuccaacu ggggacgccu cuguu 25
<210> 347 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 347 cuuccaacug gggacgccuc uguuc 25
<210> 348 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 348 uuccaacugg ggacgccucu guucc 25
<210> 349 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 349 uccaacuggg gacgccucug uucca 25
<210> 350 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 350 ccaacugggg acgccucugu uccaa 25
<210> 351 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 351 caacugggga cgccucuguu ccaaa 25
<210> 352 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 352 aacuggggac gccucuguuc caaau 25
<210> 353 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 353 acuggggacg ccucuguucc aaauc 25
<210> 354 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 354 cuggggacgc cucuguucca aaucc 25
<210> 355 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 355 uggggacgcc ucuguuccaa auccu 25
<210> 356 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 356 ggggacgccu cuguuccaaa uccug 25
<210> 357 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 357 gggacgccuc uguuccaaau ccugc 25
<210> 358 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 358 ggacgccucu guuccaaauc cugca 25
<210> 359 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 359 gacgccucug uuccaaaucc ugcau 25
<210> 360 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 360 ccaauagugg ucaguccagg agcua 25
<210> 361 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 361 caauaguggu caguccagga gcuag 25
<210> 362 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 362 aauagugguc aguccaggag cuagg 25
<210> 363 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 363 auagugguca guccaggagc uaggu 25
<210> 364 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 364 auagugguca guccaggagc u 21
<210> 365 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 365 uaguggucag uccaggagcu agguc 25
<210> 366 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 366 aguggucagu ccaggagcua gguca 25
<210> 367 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 367 guggucaguc caggagcuag gucag 25
<210> 368 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 368 uggucagucc aggagcuagg ucagg 25
<210> 369 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 369 ggucagucca ggagcuaggu caggc 25
<210> 370 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 370 gucaguccag gagcuagguc aggcu 25
<210> 371 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 371 ucaguccagg agcuagguca ggcug 25
<210> 372 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 372 caguccagga gcuaggucag gcugc 25
<210> 373 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 373 aguccaggag cuaggucagg cugcu 25
<210> 374 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 374 guccaggagc uaggucaggc ugcuu 25
<210> 375 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 375 uccaggagcu aggucaggcu gcuuu 25
<210> 376 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 376 ccaggagcua ggucaggcug cuuug 25
<210> 377 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 377 caggagcuag gucaggcugc uuugc 25
<210> 378 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 378 aggagcuagg ucaggcugcu uugcc 25
<210> 379 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 379 ggagcuaggu caggcugcuu ugccc 25
<210> 380 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 380 gagcuagguc aggcugcuuu gcccu 25
<210> 381 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 381 agcuagguca ggcugcuuug cccuc 25
<210> 382 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 382 gcuaggucag gcugcuuugc ccuca 25
<210> 383 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 383 cucagcucuu gaaguaaacg guuua 25
<210> 384 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 384 cagcucuuga aguaaacggu uuacc 25
<210> 385 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 385 gcucuugaag uaaacgguuu accgc 25
<210> 386 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 386 cuaggucagg cugcuuugcc cucag 25
<210> 387 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 387 uaggucaggc ugcuuugccc ucagc 25
<210> 388 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 388 aggucaggcu gcuuugcccu cagcu 25
<210> 389 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 389 ggucaggcug cuuugcccuc agcuc 25
<210> 390 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 390 gucaggcugc uuugcccuca gcucu 25
<210> 391 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 391 ucaggcugcu uugcccucag cucuu 25
<210> 392 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 392 caggcugcuu ugcccucagc ucuug 25
<210> 393 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 393 aggcugcuuu gcccucagcu cuuga 25
<210> 394 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 394 ggcugcuuug cccucagcuc uugaa 25
<210> 395 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 395 gcugcuuugc ccucagcucu ugaag 25
<210> 396 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 396 cugcuuugcc cucagcucuu gaagu 25
<210> 397 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 397 ugcuuugccc ucagcucuug aagua 25
<210> 398 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 398 gcuuugcccu cagcucuuga aguaa 25
<210> 399 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 399 cuuugcccuc agcucuugaa guaaa 25
<210> 400 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 400 uuugcccuca gcucuugaag uaaac 25
<210> 401 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 401 uugcccucag cucuugaagu aaacg 25
<210> 402 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 402 ugcccucagc ucuugaagua aacgg 25
<210> 403 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 403 gcccucagcu cuugaaguaa acggu 25
<210> 404 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 404 cccucagcuc uugaaguaaa cgguu 25
<210> 405 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 405 ccucagcucu ugaaguaaac 20
<210> 406 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 406 ccucagcucu ugaaguaaac g 21
<210> 407 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 407 cucagcucuu gaaguaaacg 20
<210> 408 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 408 ccucagcucu ugaaguaaac gguuu 25
<210> 409 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 409 ucagcucuug aaguaaacgg uuuac 25
<210> 410 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 410 agcucuugaa guaaacgguu uaccg 25
<210> 411 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 411 cucuugaagu aaacgguuua ccgcc 25
<210> 412 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 412 ccacaggcgu ugcacuuugc aaugc 25
<210> 413 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 413 cacaggcguu gcacuuugca augcu 25
<210> 414 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 414 acaggcguug cacuuugcaa ugcug 25
<210> 415 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 415 caggcguugc acuuugcaau gcugc 25
<210> 416 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 416 aggcguugca cuuugcaaug cugcu 25
<210> 417 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 417 ggcguugcac uuugcaaugc ugcug 25
<210> 418 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 418 gcguugcacu uugcaaugcu gcugu 25
<210> 419 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 419 cguugcacuu ugcaaugcug cuguc 25
<210> 420 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 420 cguugcacuu ugcaaugcug cug 23
<210> 421 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 421 guugcacuuu gcaaugcugc ugucu 25
<210> 422 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 422 uugcacuuug caaugcugcu gucuu 25
<210> 423 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 423 ugcacuuugc aaugcugcug ucuuc 25
<210> 424 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 424 gcacuuugca augcugcugu cuucu 25
<210> 425 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 425 cacuuugcaa ugcugcuguc uucuu 25
<210> 426 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 426 acuuugcaau gcugcugucu ucuug 25
<210> 427 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 427 cuuugcaaug cugcugucuu cuugc 25
<210> 428 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 428 uuugcaaugc ugcugucuuc uugcu 25
<210> 429 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 429 uugcaaugcu gcugucuucu ugcua 25
<210> 430 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 430 ugcaaugcug cugucuucuu gcuau 25
<210> 431 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 431 gcaaugcugc ugucuucuug cuaug 25
<210> 432 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 432 caaugcugcu gucuucuugc uauga 25
<210> 433 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 433 aaugcugcug ucuucuugcu augaa 25
<210> 434 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 434 augcugcugu cuucuugcua ugaau 25
<210> 435 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 435 ugcugcuguc uucuugcuau gaaua 25
<210> 436 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 436 gcugcugucu ucuugcuaug aauaa 25
<210> 437 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 437 cugcugucuu cuugcuauga auaau 25
<210> 438 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 438 ugcugucuuc uugcuaugaa uaaug 25
<210> 439 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 439 gcugucuucu ugcuaugaau aaugu 25
<210> 440 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 440 cugucuucuu gcuaugaaua auguc 25
<210> 441 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 441 ugucuucuug cuaugaauaa uguca 25
<210> 442 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 442 gucuucuugc uaugaauaau gucaa 25
<210> 443 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 443 ucuucuugcu augaauaaug ucaau 25
<210> 444 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 444 cuucuugcua ugaauaaugu caauc 25
<210> 445 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 445 uucuugcuau gaauaauguc aaucc 25
<210> 446 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 446 ucuugcuaug aauaauguca auccg 25
<210> 447 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 447 cuugcuauga auaaugucaa uccga 25
<210> 448 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 448 uugcuaugaa uaaugucaau ccgac 25
<210> 449 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 449 ugcuaugaau aaugucaauc cgacc 25
<210> 450 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 450 gcuaugaaua augucaaucc gaccu 25
<210> 451 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 451 cuaugaauaa ugucaauccg accug 25
<210> 452 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 452 uaugaauaau gucaauccga ccuga 25
<210> 453 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 453 augaauaaug ucaauccgac cugag 25
<210> 454 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 454 ugaauaaugu caauccgacc ugagc 25
<210> 455 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 455 gaauaauguc aauccgaccu gagcu 25
<210> 456 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 456 aauaauguca auccgaccug agcuu 25
<210> 457 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 457 auaaugucaa uccgaccuga gcuuu 25
<210> 458 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 458 uaaugucaau ccgaccugag cuuug 25
<210> 459 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 459 aaugucaauc cgaccugagc uuugu 25
<210> 460 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 460 augucaaucc gaccugagcu uuguu 25
<210> 461 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 461 ugucaauccg accugagcuu uguug 25
<210> 462 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 462 gucaauccga ccugagcuuu guugu 25
<210> 463 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 463 ucaauccgac cugagcuuug uugua 25
<210> 464 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 464 caauccgacc ugagcuuugu uguag 25
<210> 465 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 465 aauccgaccu gagcuuuguu guaga 25
<210> 466 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 466 auccgaccug agcuuuguug uagac 25
<210> 467 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 467 uccgaccuga gcuuuguugu agacu 25
<210> 468 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 468 ccgaccugag cuuuguugua gacua 25
<210> 469 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 469 cgaccugagc uuuguuguag 20
<210> 470 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 470 cgaccugagc uuuguuguag acuau 25
<210> 471 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 471 gaccugagcu uuguuguaga cuauc 25
<210> 472 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 472 accugagcuu uguuguagac uauca 25
<210> 473 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 473 ccugagcuuu guuguagacu auc 23
<210> 474 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 474 cauuuuugac cuacaugugg 20
<210> 475 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 475 uuugaccuac auguggaaag 20
<210> 476 <211> 26 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 476 uacauuuuug accuacaugu ggaaag 26
<210> 477 <211> 17 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 477 ggucuccuua ccuauga 17
<210> 478 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 478 ucuuaccuau gacuauggau gaga 24
<210> 479 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 479 auuuuugacc uacaugggaa ag 22
<210> 480 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 480 uacgaguuga uugucggacc cag 23
<210> 481 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 481 guggucuccu uaccuaugac ugugg 25
<210> 482 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 482 ugucucagua aucuucuuac cuau 24
<210> 483 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 483 ugcauguucc agucguugug ugg 23
<210> 484 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 484 cacuauucca gucaaauagg ucugg 25
<210> 485 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 485 auuuaccaac cuucaggauc gagua 25
<210> 486 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 486 ggccuaaaac acauacacau a 21
<210> 487 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 487 gauagguggu aucaacaucu guaa 24
<210> 488 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 488 gauagguggu aucaacaucu g 21
<210> 489 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 489 cuuccuggau ggcuugaau 19
<210> 490 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 490 uguuguuguu uaugcucauu 20
<210> 491 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 491 guacauuaag auggacuuc 19
<210> 492 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 492 cuguugcagu aaucuaugag 20
<210> 493 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 493 ugcaguaauc uaugaguuuc 20
<210> 494 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 494 gagucuucua ggagccuu 18
<210> 495 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 495 ugccauuguu ucaucagcuc uuu 23
<210> 496 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 496 uccuguagga cauuggcagu 20
<210> 497 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 497 cuuggagucu ucuaggagcc 20
<210> 498 <211> 30 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 498 ccauuuugug aauguuuucu uuugaacauc 30
<210> 499 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 499 cccauuuugu gaauguuuuc uuuu 24
<210> 500 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 500 gaaaauugug cauuuaccca uuuu 24
<210> 501 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 501 uugugcauuu acccauuuug ug 22
<210> 502 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 502 cccugaggca uucccaucuu gaau 24
<210> 503 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 503 aggacuuacu ugcuuuguuu 20
<210> 504 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 504 cuugaauuua ggagauucau cug 23
<210> 505 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 505 caucuucuga uaauuuuccu guu 23
<210> 506 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 506 ccauuacagu ugucuguguu 20
<210> 507 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 507 ugacagccug ugaaaucugu gag 23
<210> 508 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 508 uaaucugccu cuucuuuugg 20
<210> 509 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 509 cagcaguagu ugucaucugc 20
<210> 510 <211> 27 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 510 gccugagcug aucugcuggc aucuugc 27
<210> 511 <211> 31 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 511 gccugagcug aucugcuggc aucuugcagu u 31
<210> 512 <211> 16 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 512 ucugcuggca ucuugc 16
<210> 513 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 513 gccgguugac uucauccugu gc 22
<210> 514 <211> 23 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 514 gucugcaucc aggaacaugg guc 23
<210> 515 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 515 uacuuacugu cuguagcucu uucu 24
<210> 516 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 516 cugcauccag gaacaugggu cc 22
<210> 517 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 517 guugaagauc ugauagccgg uuga 24
<210> 518 <211> 17 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 518 uaggugccug ccggcuu 17
<210> 519 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 519 uucagcugua gccacacc 18
<210> 520 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 520 cugaacugcu ggaaagucgc c 21
<210> 521 <211> 30 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 521 cuggcuucca aaugggaccu gaaaaagaac 30
<210> 522 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 522 caauuuuucc cacucaguau u 21
<210> 523 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 523 uugaaguucc uggagucuu 19
<210> 524 <211> 22 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 524 uccucaggag gcagcucuaa au 22
<210> 525 <211> 16 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 525 uggcucucuc ccaggg 16
<210> 526 <211> 27 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 526 gagauggcuc ucucccaggg acccugg 27
<210> 527 <211> 17 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 527 gggcacuuug uuuggcg 17
<210> 528 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 528 ggucccagca aguuguuug 19
<210> 529 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 529 ugggaugguc ccagcaaguu guuug 25
<210> 530 <211> 21 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 530 guagagcucu gucauuuugg g 21
<210> 531 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 531 gcucaagaga uccacugcaa aaaac 25
<210> 532 <211> 26 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 532 gccauacgua cguaucauaa acauuc 26
<210> 533 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 533 ucugcaggau auccaugggc ugguc 25
<210> 534 <211> 27 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 534 gauccucccu guucgucccc uauuaug 27
<210> 535 <211> 24 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 535 ugcuuuagac uccuguaccu gaua 24
<210> 536 <211> 18 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 536 ggcggccuuu guguugac 18
<210> 537 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 537 ggacaggccu uuauguucgu gcugc 25
<210> 538 <211> 19 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 538 ccuuuauguu cgugcugcu 19
<210> 539 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<400> 539 ucaaggaaga uggcauuucu 20
<210> 540 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (5)..(5) <223> I
<220> <221> misc_feature <222> (5)..(5) <223> n is a, c, g, or u
<400> 540 ucaangaaga uggcauuucu 20
<210> 541 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (6)..(6) <223> I
<220> <221> misc_feature <222> (6)..(6) <223> n is a, c, g, or u
<400> 541 ucaagnaaga uggcauuucu 20
<210> 542 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (9) .. (9) <223> I
<220> <221> misc_feature <222> (9) .. (9) <223> n is a, c, g, or u
<400> 542 ucaaggaana uggcauuucu 20
<210> 543 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (12)..(12) <223> I
<220> <221> misc_feature <222> (12)..(12) <223> n is a, c, g, or u
<400> 543 ucaaggaaga ungcauuucu 20
<210> 544 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (13)..(13) <223> I
<220> <221> misc_feature <222> (13)..(13) <223> n is a, c, g, or u
<400> 544 ucaaggaaga ugncauuucu 20
<210> 545 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (1)..(1) <223> I
<220> <221> misc_feature <222> (1)..(1) <223> n is a, c, g, or u
<400> 545 ncaaggaaga uggcauuucu 20
<210> 546 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (11)..(11) <223> I
<220> <221> misc_feature <222> (11)..(11) <223> n is a, c, g, or u
<400> 546 ucaaggaaga nggcauuucu 20
<210> 547 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (16)..(16) <223> I
<220> <221> misc_feature <222> (16)..(16) <223> n is a, c, g, or u
<400> 547 ucaaggaaga uggcanuucu 20
<210> 548 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (17)..(17) <223> I
<220> <221> misc_feature <222> (17)..(17) <223> n is a, c, g, or u
<400> 548 ucaaggaaga uggcaunucu 20
<210> 549 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (18)..(18) <223> I
<220> <221> misc_feature <222> (18)..(18) <223> n is a, c, g, or u
<400> 549 ucaaggaaga uggcauuncu 20
<210> 550 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (20)..(20) <223> I
<220> <221> misc_feature <222> (20)..(20) <223> n is a, c, g, or u
<400> 550 ucaaggaaga uggcauuucn 20
<210> 551 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (3)..(3) <223> I
<220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g, or u
<400> 551 ucnaggaaga uggcauuucu 20
<210> 552 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (4)..(4) <223> I
<220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, or u
<400> 552 ucanggaaga uggcauuucu 20
<210> 553 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (7)..(7) <223> I
<220> <221> misc_feature <222> (7)..(7) <223> n is a, c, g, or u
<400> 553 ucaaggnaga uggcauuucu 20
<210> 554 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (8).. (8) <223> I
<220> <221> misc_feature <222> (8)..(8) <223> n is a, c, g, or u
<400> 554 ucaagganga uggcauuucu 20
<210> 555 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (10)..(10) <223> I
<220> <221> misc_feature <222> (10)..(10) <223> n is a, c, g, or u
<400> 555 ucaaggaagn uggcauuucu 20
<210> 556 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (15)..(15) <223> I
<220> <221> misc_feature <222> (15)..(15) <223> n is a, c, g, or u
<400> 556 ucaaggaaga uggcnuuucu 20
<210> 557 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (7).. (7) <223> I
<220> <221> misc_feature <222> (7)..(7) <223> n is a, c, g, or u
<400> 557 uuugccncug cccaaugcca uccug 25
<210> 558 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (10)..(10) <223> I
<220> <221> misc_feature <222> (10)..(10) <223> n is a, c, g, or u
<400> 558 uuugccgcun cccaaugcca uccug 25
<210> 559 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (17)..(17) <223> I
<220> <221> misc_feature <222> (17)..(17) <223> n is a, c, g, or u
<400> 559 uuugccgcug cccaauncca uccug 25
<210> 560 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (4)..(4) <223> I
<220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, or u
<400> 560 uuunccgcug cccaaugcca uccug 25
<210> 561 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (25) .. (25) <223> I
<220> <221> misc_feature <222> (25) .. (25) <223> n is a, c, g, or u
<400> 561 uuugccgcug cccaaugcca uccun 25
<210> 562 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (1)..(1) <223> I
<220> <221> misc_feature <222> (1)..(1) <223> n is a, c, g, or u
<400> 562 nuugccgcug cccaaugcca uccug 25
<210> 563 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (2)..(2) <223> I
<220> <221> misc_feature <222> (2) .. (2) <223> n is a, c, g, or u
<400> 563 unugccgcug cccaaugcca uccug 25
<210> 564 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (3)..(3) <223> I
<220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g, or u
<400> 564 uungccgcug cccaaugcca uccug 25
<210> 565 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (9) .. (9) <223> I
<220> <221> misc_feature <222> (9) .. (9) <223> n is a, c, g, or u
<400> 565 uuugccgcng cccaaugcca uccug 25
<210> 566 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (15)..(15) <223> I
<220> <221> misc_feature <222> (15)..(15) <223> n is a, c, g, or u
<400> 566 uuugccgcug cccanugcca uccug 25
<210> 567 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (20)..(20) <223> I
<220> <221> misc_feature <222> (20)..(20) <223> n is a, c, g, or u
<400> 567 uuugccgcug cccaaugccn uccug 25
<210> 568 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (4)..(4) <223> I
<220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, or u
<220> <221> modified_base <222> (7).. (7) <223> I
<220> <221> misc_feature <222> (7)..(7) <223> n is a, c, g, or u
<400> 568 uuunccncug cccaaugcca uccug 25
<210> 569 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (16)..(16) <223> I
<220> <221> misc_feature <222> (16)..(16) <223> n is a, c, g, or u
<400> 569 uuugccgcug cccaangcca uccug 25
<210> 570 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (21) .. (21) <223> I
<220> <221> misc_feature <222> (21) .. (21) <223> n is a, c, g, or u
<400> 570 uuugccgcug cccaaugcca nccug 25
<210> 571 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (24) .. (24) <223> I
<220> <221> misc_feature <222> (24) .. (24) <223> n is a, c, g, or u
<400> 571 uuugccgcug cccaaugcca uccng 25
<210> 572 <211> 25 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (14)..(14) <223> I
<220> <221> misc_feature <222> (14)..(14) <223> n is a, c, g, or u
<400> 572 uuugccgcug cccnaugcca uccug 25
<210> 573 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (10)..(10) <223> I
<220> <221> misc_feature <222> (10)..(10) <223> n is a, c, g, or u
<400> 573 ucagcuucun uuagccacug 20
<210> 574 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (14)..(14) <223> I
<220> <221> misc_feature <222> (14)..(14) <223> n is a, c, g, or u
<400> 574 ucagcuucug uuanccacug 20
<210> 575 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (4)..(4) <223> I
<220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, or u
<400> 575 ucancuucug uuagccacug 20
<210> 576 <211> 20 <212> RNA <213> Artificial
<220> <223> oligonucleotide
<220> <221> modified_base <222> (20)..(20) <223> I
<220> <221> misc_feature <222> (20)..(20) <223> n is a, c, g, or u
<400> 576 ucagcuucug uuagccacun 20
<210> 577 <211> 153 <212> PRT <213> Homo sapiens
<400> 577

Claims

An antisense oligonucleotide comprising at least one inosine, wherein the oligonucleotide, comprises a sequence which targets at least part of a dystrophin pre-mRNA exon part being a contiguous stretch comprising at least 13 nucleotides and wherein the antisense oligonucleotide is capable of exon-skipping.
An oligonucleotide according to claim 1, wherein the contiguous stretch comprises between 13 and 50 nucleotides, preferably between 14 and 25 nucleotides, of RNA of an exon of a dystrophin pre-mRNA.
An oligonucleotide according to claim 1 or 2, wherein said exon comprises exon 51, 45, 53, 44, 46, 52, 50 and/or 55.
An oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide comprises RNA and wherein preferably said RNA contains a modification.
An oligonucleotide according to claim 4, wherein the modification is selected from a 2'-O-methyl modified ribose and a 2'-deoxy modified ribose, preferably the modification is a 2'-O-methyl modified ribose.
An oligonucleotide according to claim 4, wherein said modification comprises a 2'-O-methyl-phosphorothioate modification.
An oligonucleotide according to any one of claims 1 to 6, wherein said oligonucleotide comprises a peptide nucleic acid, a locked nucleic acid, a morpholino phosphorodiamidate, or any combination thereof, most preferably morpholino phosphorodiamidate.
An oligonucleotide according to any one of claims 1 to 7 for use as a medicament, preferably for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual.
An oligonucleotide according to claim 8, wherein the medicament provides the individual with a functional dystrophin protein and/or at least in part decreases the production of an aberrant dystrophin protein and/or increases the production of a functional or a more functional dystrophin protein.
An oligonucleotide according claim 8 or 9, wherein the medicament alleviates one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in said individual.
An oligonucleotide according to any one of claims 1 to 10, wherein the oligonucleotide is represented by a sequence that comprises or consists of any one of the base sequence SEQ ID NO:2 to 486 or 539 wherein at least one inosine is present in said sequence.
An oligonucleotide according to any one of claims 1 to 10, wherein said oligonucleotide comprises or consists of the base or nucleotide sequence 5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO:557).
An antisense oligonucleotide consisting of the base sequence 5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO:557), wherein the oligonucleotide contains 2'-O-methyl ribose moieties and has a full-length phosphorothioate backbone.
A composition comprising at least two distinct oligonucleotides as defined in any one of claims 1 to 7, or 11 to 13, wherein the composition is preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.
Use of the oligonucleotide as defined in any one of claims 1 to 7 or 11 or of the composition as defined in claim 14 for the manufacturing of a medicament for preventing or treating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in an individual.
An in vitro method for alleviating one or more symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in a cell, the method comprising administering to said cell an oligonucleotide as defined in any one of claims 1 to 13 or a composition as defined in claim 14.