JP2019030330A - Oligonucleotide for the treatment of muscular dystrophy patients - Google Patents

Abstract

To provide pharmaceutical compositions comprising oligonucleotides which can preferably induce skipping of two or more exons of an mRNA precursor.SOLUTION: An oligonucleotide can bind to a first exon region derived from a dystrophin mRNA precursor and a second exon region in the same mRNA precursor, the region of the second exon has at least 50% identity to the region of the first exon, and the oligonucleotide is suitable for skipping the first exon and the second exon of the mRNA precursor, as well as preferably the entire stretch of exons in between the first and the second exons.SELECTED DRAWING: None

Description

Field of Invention

  The present invention relates to the field of human genetics, more specifically to a method for designing a single oligonucleotide capable of preferably inducing skipping of two or more exons of an mRNA precursor. The present invention further provides the oligonucleotide, a pharmaceutical composition comprising the oligonucleotide, and the use of the oligonucleotide identified herein.

Background of the Invention

  Oligonucleotides are emerging in medicine for treating genetic disorders such as muscular dystrophy. Muscular dystrophy (MD) refers to a genetic disorder characterized by progressive weakness and degeneration of skeletal muscle. Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common pediatric forms of muscular dystrophy and are used herein to illustrate the present invention. DMD is a severely fatal neuromuscular disorder that will depend on wheelchair support before the age of 12, and DMD patients often die before the age of 30 due to breathing or heart failure.

  DMD is caused by mutations in the DMD gene, primarily by frameshift deletions or duplication of one or more exons, small nucleotide insertions or deletions, or typically by nonsense point mutations that result in the absence of functional dystrophin. . During the last decade, a specially induced modification of splicing to repair the disrupted reading frame of the DMD transcript has emerged as a promising treatment for Duchenne muscular dystrophy (DMD). J. et al., Yokota T. et al., Van Deutscheum et al., Goemans NM et al.). Using a sequence-specific antisense oligonucleotide (AON) that targets a specific exon that is adjacent to or contains the mutation and interferes with its splicing signal, skipping that exon is responsible for the processing of the DMD mRNA precursor. Can be induced in between. Despite the resulting truncated transcript, the open reading frame is repaired and proteins similar to those typically found in patients with mild Becker muscular dystrophy are introduced. Since AON-induced exon skipping confers mutational characteristics, it may personalize the therapeutic approach for DMD patients and certain severe BMD patients. Since most mutations are clustered around exons 45-55 in the DMD gene, skipping one particular exon in that region can be a treatment for a subpopulation of patients with various mutations. Exon 51 skipping affects the largest subpopulation of patients (approximately 13%), including patients with deletions of exons 45-50, 48-50, 50 or 52. For some mutations, more than one exon skipping is required to repair the open reading frame. For example, in the case of a DMD patient having a deletion of exon 46 to exon 50 in the DMD gene, only skipping of both exons 45 and 51 is the corrective means. To treat these patients, administration of two oligonucleotides (one targeting exon 45 and the other targeting exon 51) is required. The feasibility of skipping two or more consecutive exons by using a combination of AONs in either cocktail or virally delivered genetic constructs has been extensively studied (Aartsma-Rus A. et al., 2004; Beraud C. et al .; Van Vliet L. et al; Yokota T. et al; Multi-exon skipping can be applied to a mixed subpopulation of patients, allowing for mimicking deletions known to be associated with a relatively mild phenotype, and of hot spot deletion regions in the DMD gene Provides a means to handle rare mutations outside. However, a drawback to developing drugs containing multiple oligonucleotides is that drug regulatory agencies may consider different sequences of oligonucleotides to be different drugs, each requiring safe manufacturing, toxicology and clinical trials. It is to do. Thus, a need exists for a single molecule compound that can induce skipping of at least two exons to facilitate treatment of a mixed subpopulation of DMD patients.

Description of the invention

  The present invention is a method for designing a single oligonucleotide, which can bind to a region of a first exon and a region of a second exon in the same mRNA precursor, A region provides a method wherein the region has at least 50% identity with the region of the first exon. The oligonucleotide obtainable by the method can preferably induce skipping of the first exon and the second exon of the mRNA precursor. Additional exon skipping is also preferably induced, and the additional exon is preferably located between the first exon and the second exon. The resulting transcript of the mRNA precursor in which the exon is skipped is in-frame.

Oligonucleotide:
In the first aspect, the present invention provides an oligonucleotide capable of binding to a region of a first exon and a second exon within the same mRNA precursor, wherein the region of the second exon is the first exon. Relates to oligonucleotides having at least 50% identity with said region of exons.

  The oligonucleotide is preferably capable of inducing skipping of the first exon and second exon of the mRNA precursor, more preferably further exon skipping is induced, wherein the further exon is preferably the first exon. Located between an exon and the second exon, the resulting transcript is in-frame.

  Exon skipping interferes with the natural splicing process that occurs in eukaryotic cells. In higher eukaryotes, genetic information about proteins in cellular DNA is encoded in exons separated from each other by intron sequences. These introns are very long in some cases. Eukaryotic transcription mechanisms generate mRNA precursors that contain both exons and introns, whereas splicing mechanisms are often already involved in splicing and ongoing production of mRNA precursors. During the process called, the actual coding mRNA for the protein is generated by removing introns present in the mRNA precursor and binding exons.

  The oligonucleotide of the present invention capable of binding to a region of a first exon derived from an mRNA precursor and a region of a second exon within the same mRNA precursor is suitable for binding to a region of a first exon derived from an mRNA precursor, Interpreted as an oligonucleotide suitable for binding to a region of a second exon within the same mRNA precursor. Such oligonucleotides of the invention are preferably characterized by the binding properties (ie, capable of binding) of the oligonucleotide when used with or in combination with an mRNA precursor in a cell. Attached. Within this context, “capable of” may be replaced by “able to”. Thus, one skilled in the art can bind to a region of a first exon and can bind to a region of a second exon within the same mRNA precursor, and the oligonucleotide defined by the nucleotide sequence structurally defines the oligonucleotide. That is, the oligonucleotide is reverse complementary to the sequence of the region of the first exon and reverse complementary to the sequence of the region of the second exon of the same mRNA precursor. It will be understood that it has a unique sequence. The degree of reverse complementarity with the region of the first exon and / or the region of the second exon required for the oligonucleotide of the present invention may be less than 100%. As further dealt with herein, some amount of mismatch or one or two gaps may be tolerated. A nucleotide sequence of a region of a first exon having at least 50% identity with said region of said second exon to which an oligonucleotide of the invention can bind (or at least 50% of said region of said first exon) The nucleotide sequence of the region of the second exon having identity) can be designed using the method of the present invention described below. A preferred mRNA precursor is a dystrophin mRNA precursor.

  Preferred combinations of the first and second exons of the dystrophin mRNA precursor and preferred regions of the first and second dystrophin exons are defined in Table 2. The oligonucleotides of the present invention are preferably capable of inducing skipping of the first and second exons of the dystrophin mRNA precursor, more preferably exon skipping is induced, wherein the further exons are Preferably, the dystrophin transcript obtained is located in-frame between the first exon and the second exon, preferably as described in Table 1.

  A transcript is in frame if it has an open reading frame that allows production of the protein. The in-frame state of mRNA can be assessed by sequence and / or RT-PCR analysis known to those skilled in the art. The resulting protein derived from translation of the in-frame transcript can be analyzed by immunofluorescence and / or Western blot analysis using antibodies that cross-react with the protein known to those skilled in the art. Throughout the present invention, when the in-frame transcript obtained is identified by RT-PCR and / or sequence analysis, or the protein obtained from said in-frame transcript is related in vitro depending on the identity of the transcript. Or, in an in vivo system, the oligonucleotides described herein can be said to be functional when identified by immunofluorescence and / or Western blot analysis. When the transcript is a dystrophin transcript, the relevant system can be a muscle cell or myotube or myofiber or myofibril of a healthy donor or DMD patient, as described below.

  In one embodiment, the second exon region (present in the same mRNA precursor as the first exon region) is at least 50%, 55%, 60%, 65% of the first exon region, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity (preferred regions for the first and second dystrophin exons are listed in Table 2). Identity is exemplified over the entire length of said first exon and / or second exon or for dystrophin exons herein, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nucleos It may be evaluated over the area of the de. It will be apparent to those skilled in the art that the first and second exons described herein are two different exons of a single mRNA precursor or two different exons within the same mRNA precursor. The first exon described herein may be located upstream (ie, 5 ′) of the second exon within the same mRNA precursor described herein, or the second exon is the first exon. May be upstream of the exon.

  The first exon is preferably located upstream from the second exon. The oligonucleotide of the present invention can be designed to be able to bind to the first exon region primarily, and considering the identity of the first exon region and the second exon region, the oligonucleotide It will be apparent to those skilled in the art that can be designed to bind secondarily to the region of the second exon. The reverse design is also possible. That is, the oligonucleotide of the present invention can be designed so that it can primarily bind to the region of the second exon, and considering the identity of the region of the first exon and the second exon, Oligonucleotides can be designed to bind secondarily to the region of the first exon.

  Identity between the region of the first exon and the region of the second exon can be assessed over the entire region of the first exon, which region is that of the region to which the oligonucleotide of the invention can bind. It may be shorter, longer or equal in length. In the present invention, the region of the first exon and the region of the second exon can also be specified as an identity region. The region of the first exon that defines identity with the region of the second exon is preferably the same length or longer than the portion of that region to which the oligonucleotide of the invention can bind. It is understood that the oligonucleotides of the invention can bind to smaller or partially overlapping portions of the region used to assess the sequence identity of the first exon and / or the second exon. It must be. Thus, it is understood that an oligonucleotide that can bind to regions of the first exon and the second exon can bind to a portion of the region of the first exon and the second exon. The portion may be the same length as the region of the first exon and / or the second exon. The portion may be shorter or longer than the region of the first exon and / or the second exon. The portion may be included within the region of the first exon and / or the second exon. The portion may overlap the region of the first exon and / or the second exon. This overlap may be 1, 2, 3, 4, 5 or more nucleotides on the 5 'and / or 3' side of the first exon and / or second exon region. The oligonucleotide may be at least 1, 2, 3, 4, 5 or more nucleotides longer or shorter than the region of the first exon and / or the second exon, and the first region And / or 5 ′ or 3 ′ side of the second region.

  13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, used to calculate the identity between the first exon and the second exon 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, Regions that are 76, 77, 78, 79, 80, or up to 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nucleotides are identical throughout the region Sex is at least 50% As long may be a continuous stretch (stretch), it may be interrupted by 1, 2, 3, 4 or more gaps.

  Identity between the region of the first exon and the region of the second exon can be assessed using any program known to those skilled in the art. The identity is preferably evaluated as follows: first exon and second using the online tool EMBOSS Matcher using default settings (matrix: EDNAFULL; gap_penalty: 16; extension_penalty: 4) Optimal pairwise alignment between 2 exons.

  As used herein, an oligonucleotide is preferably capable of binding to, targeting, hybridizing, and / or reverse complementing to a first exon and a second exon region or part of a region within the same mRNA precursor. Refers to the oligomer that is the target.

  Identified herein (ie, capable of binding to a region of a first exon and a region of another exon (ie, a second exon) within the same mRNA precursor, said region of said second exon being said first exon). Having at least 50% identity with said region of one exon), the oligonucleotide is also preferably at least 80% reverse complementary to said region of said first exon, and said region of said second exon Is at least 45% reverse complementary to the region. The oligonucleotide is at least 85%, 90%, 95% or 100% reverse complementary to the region of the first exon and at least 50%, 55%, 60% to the region of the second exon. 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% are more preferably reverse complementary. Reverse complementarity is preferably, but not necessarily, assessed over the length of the oligonucleotide.

  The oligonucleotides encompassed by the present invention are at least 10, 11, 12, 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 or 40 nucleotides. The oligonucleotides encompassed by the present invention are at most 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22 , 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides. The length of the oligonucleotide of the present invention is defined by the total number of nucleotides included in the oligonucleotide, regardless of any modifications present in the oligonucleotide. As described further below, nucleotides may contain certain chemical modifications, but such modified nucleotides are still considered nucleotides in the context of the present invention. Depending on the chemistry of the oligonucleotide, the optimal length of the oligonucleotide may vary. For example, the length of a 2'-O-methyl phosphorothioate oligonucleotide can be up to 15-30. If this oligonucleotide is further modified as exemplified herein, the optimal length may be shortened by 14, 13 or even shorter.

  In preferred embodiments, the oligonucleotides of the invention are used to limit the potential for reduced synthesis efficiency, yield, purity or scalability, reduced bioavailability and / or cellular uptake and transport, reduced safety. As well as to limit the cost of the article, not longer than 30 nucleotides. In a more preferred embodiment, the oligonucleotide is 15-25 nucleotides. Most preferably, the oligonucleotide encompassed by the present invention consists of 20, 21, 22, 23, 24 or 25 nucleotides. The length of the oligonucleotide of the present invention is such that when at least 100 nM of said oligonucleotide is used to transfect cell cultures associated in vitro, the functionality or activity of the oligonucleotide is such that the first exon and the first Appear to be defined by inducing at least 5% skipping of two exons (and any exons in between) or by promoting at least 5% of in-frame transcripts to be formed Is preferred. The assessment of the presence of the transcript has already been described herein. A related cell culture is a cell culture in which an mRNA precursor comprising said first exon and said second exon is transcribed and spliced into an mRNA transcript. If the mRNA precursor is a dystrophin mRNA precursor, the associated cell culture contains (differentiated) muscle cells. In this case, when at least 250 nM of the oligonucleotide is used, at least 20% of the in-frame transcript is formed.

  The region of the first exon is 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, 51, 52, 53, 54, 55, 56, 57, 58 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or up to 90, It may be 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides. The region of the first exon is also at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the length of the exon. % May be defined. The region of the first exon can be referred to as the identity region.

  The second exon region is 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, 51, 52, 53, 54, 55, 56, 57, 58 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or up to 90, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more nucleotides. The region of the second exon is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the length of the exon May be defined to be The region of the second exon can be referred to as the identity region.

  In one embodiment, an oligonucleotide of the invention can bind to a region of exon U + 1 (first exon) from an mRNA precursor and another exon D-1 (second exon) region within the same mRNA precursor is Having at least 50% identity with the region of the (U + 1) exon, and the oligonucleotide is in-frame where exons U and D are spliced together (eg, preferably for DMD as described in Table 1) This is due to the skipping of the U + 1 and D-1 exons of the mRNA precursor (and further exons, preferably located between the first and second exons) to obtain a transcript. The oligonucleotides of the invention are also described herein as compounds. The oligonucleotide of the present invention is preferably an antisense oligonucleotide (ie, AON). The oligonucleotide is preferably for skipping of the two exons of the mRNA precursor (ie the first (U + 1) exon and the second (D-1) exon) and the resulting transcript ( U is spliced directly to D is in-frame (eg, preferably for the DMD described in Table 1). It can be said that the oligonucleotide contains skipping of the two exons in one single mRNA precursor. Optionally, further exon skipping is induced, said additional exon being preferably located between said first exon and said second exon, and the resulting transcript is in frame.

  Oligonucleotides are comprised of the two exons within the mRNA precursor (ie, the first exon and the second exon) and the entire stretch of exons between the first and second exons. More preferably for skipping, to remove any mutations in the stretch and to obtain transcripts with a repaired open reading frame that is shorter but allows protein production.

  While not wishing to be bound by any theory, due to at least 50% sequence identity or similarity between the two exons, a single oligonucleotide of the present invention will have both exons, and preferably Are thought to be able to bind to the entire stretch of exons in order to obtain shorter transcripts that are in-frame and induce their skipping. Thus, the two exons may be adjacent to the mRNA precursor or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 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, or 50 exons. The region encompassing one or more exons present between the first exon and the second exon may also be referred to as exon stretch (s) or multi-exon stretch. Preferably, the first exon of this multi-exon stretch is the first exon identified herein above, and the last exon of this multi-exon stretch is the first exon identified herein above. 2 exons. Oligonucleotides of the invention may also be identified as oligonucleotides capable of inducing skipping of the two exons or skipping of exon stretch (es) or skipping of the multi-exon stretch. In a preferred embodiment, skipping of both the first exon and the second exon is induced by using one single oligonucleotide of the invention. In a preferred embodiment, more than 1, 2, 3, 4, 5, 5, 6, 7, 9, 9, 10, 11, 12 using one single oligonucleotide, Exon of 13 or more, 14 or 15 or 16 or 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, exon skipping is performed, and thus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or more than 50 exons, this skipping is more than 2 Gene construct that transcribes two or more different oligonucleotides without using a mixture or cocktail of different oligonucleotides, or without two or more different oligonucleotides that can be linked by one or more linkers It is carried out without using. Thus, in one embodiment, the single oligonucleotide comprises the first exon and the second, provided that the invention includes one single oligonucleotide and does not include two or more different oligonucleotides. It can bind to an exon and can induce skipping of at least the first exon and the second exon within a single mRNA precursor exemplified herein. In this context, those skilled in the art will appreciate that the term “single” does not refer to the number of molecules required to induce exon skipping. Single refers to a sequence of oligonucleotides: the present invention encompasses one single oligonucleotide sequence and uses thereof, and does not include two or more different oligonucleotide sequences, said single oligonucleotide sequence being It can bind to the first exon and the second exon and can induce skipping of at least the first exon and the second exon within the single mRNA precursor exemplified herein. This uses only one single oligonucleotide to treat diseases caused by (rare) mutations in the gene, provided that different exons in the gene contain regions with at least 50% sequence identity. It is the first invention that allows skipping of more than one exon.

  The oligonucleotides of the invention are preferably used as part of a therapy based on RNA modulating activity as defined herein below. Depending on the identity of the transcript in which the first and second exons are present, oligonucleotides can be designed to prevent, treat or delay a given disease.

  Targeting two exons within a single mRNA precursor using a single oligonucleotide that can bind to both exons results in mRNA lacking the targeted exon, and even the entire stretch of exons in between. It is shown. The advantage of such a single oligonucleotide as defined herein is that defects caused by different mutations within this multi-exon stretch can be treated. If you choose to use two or more different oligonucleotides to induce skipping of two or more exons, for example, each oligonucleotide can have its own PK profile and thereby It should be taken into account that each of the oligonucleotides must be found to be present in the same cell as well. Thus, another advantage of using only one single oligonucleotide capable of binding to the two different exons identified above is that production, toxicity, dose discovery and clinical trials can be performed directly on one single compound. They are much easier because they can be reduced to new production and testing.

  The following additional features of the oligonucleotides of the invention are defined.

In a preferred embodiment of the invention, the oligonucleotide is capable of binding, targeting, hybridizing, reverse complementary and / or at least the first exon and / or the second exon. Can inhibit the function of an exon and / or at least one splicing control sequence within the second exon, and / or affect at least the structure of the first exon and / or the second exon;
The oligonucleotide can bind, target, hybridize and / or reverse complement to a binding site for a serine-arginine (SR) protein in the first exon and / or second exon. And / or the oligonucleotide comprises an exon splicing enhancer (ESE), an exon recognition sequence (ERS), and / or an exon splicing silencer (ESS) in the first exon and / or the second exon. Can be bound, targeted, hybridized, and / or reverse complementary.

  Said oligonucleotide that is capable of binding, targeting, hybridizing and / or reverse complementary to a region of a first mRNA precursor exon and / or a region of a second mRNA precursor exon is at least 1 More preferably, one splicing control sequence can be specifically inhibited and / or can affect the structure of at least the first exon and / or the second exon within the mRNA precursor. Interfering with such splicing control sequences and / or structures has an advantage in that such elements are located within exons. By providing such oligonucleotides as defined herein, the splicing mechanism effectively masks at least the first and second exons, and preferably the entire stretch of exons in between. It becomes possible to do. Therefore, dysfunction of the splicing machinery that recognizes these exons leads to skipping or elimination of these exons from the final mRNA. This embodiment focuses only on the coding sequence. This is believed to allow a more specific and hence reliable method. The reverse complementarity of the oligonucleotide to the region of the first exon and / or second exon of the mRNA precursor is preferably at least 45%, 50%, 55%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95% or 100%.

Therefore, the present invention
An antisense oligonucleotide for use as a medicament for preventing, delaying, ameliorating and / or treating a disease in a subject, said oligonucleotide binding to a region of a first exon and a region of a second exon And the region of the second exon has at least 50% identity with the region of the first exon;
The first exon and the second exon are in the same mRNA precursor in the subject;
As a result of the binding, skipping the first exon and the second exon, and preferably starting with the first exon and existing between the first exon and the second exon 1 Resulting in skipping of a multi-exon stretch encompassing one or more exons, and at most skipping the entire stretch of exons between the first exon and the second exon;
In-frame transcripts are obtained which allow the production of functional or semi-functional proteins,
It relates to an antisense oligonucleotide.

  As described herein, it is preferred that the oligonucleotide is capable of inducing skipping of the entire stretch of exons between the first exon and the second exon.

  As described herein, said binding of said oligonucleotide is within said mRNA precursor, at least one splicing control sequence in said region of said first exon and second exon, and / or said More preferably, the first exon and / or the secondary structure of the second exon, and / or the secondary structure including at least the first exon and / or the second exon can be interfered with. . Preferred splicing control sequences are presented herein below.

Accordingly, one preferred embodiment is an oligonucleotide of the invention capable of binding to a region of a first exon derived from an mRNA precursor and / or a region of a second exon within the same mRNA precursor, wherein the same mRNA precursor Said region of said second exon of said at least 50% identity with said region of said first exon (eg preferably for DMD as in Table 2), said oligonucleotide being said mRNA Relates to oligonucleotides that can induce skipping of said first and second exons of a precursor, resulting in transcripts that are in-frame (eg preferably for DMD as in Table 1 or 6) . The oligonucleotide provides a functional or semi-functional protein to the individual, and the oligonucleotide further comprises:
A sequence capable of binding, targeting, hybridizing and / or reverse complementary to a region of a first mRNA precursor exon and / or a second mRNA precursor exon, wherein the first mRNA A sequence (closed structure) that hybridizes to another part of the precursor exon and / or the second mRNA precursor exon, and / or of the first mRNA precursor exon and / or the second mRNA precursor exon. A sequence (open structure) that can bind, target, hybridize, and / or reverse-complement and does not hybridize in the mRNA precursor.

  With regard to this embodiment, reference is made to the patent application WO 2004/083446. RNA molecules exhibit strong secondary structure, largely due to base pairing of reverse complementary or partially reverse complementary stretches within the same RNA. The structure in RNA has long been thought to play a role in RNA function. Without being bound by theory, it is believed that the exon RNA secondary structure plays a role in structuring the splicing process. Due to its structure, exons are recognized as a part that needs to be included in mRNA. In one embodiment, the oligonucleotide is capable of interfering with at least the structure of the first exon and possibly also the second exon and optionally also the exon stretch, thus masking the exon from the splicing mechanism. By doing so, splicing of the first exon and possibly also the second exon and optionally also the exon stretch can be prevented, thereby causing skipping of the exon. Without being bound by theory, overlap with an open structure improves oligonucleotide entry efficiency (ie, increases the efficiency with which the oligonucleotide can enter the structure), but overlap with a closed structure It is believed that the efficiency of interfering with RNA secondary structure is later increased. It is found that the length of partial reverse complementarity for both closed and open structures is not extremely limited. We observed high efficiency with oligonucleotides with variable length reverse complementarity in any structure. The term reverse complementarity is used herein to refer to a stretch of nucleic acid that can hybridize to another stretch of nucleic acid under physiological conditions. Hybridization conditions are defined herein below. Therefore, it is not always necessary that all of the bases in the region of reverse complementarity can be paired with bases in the opposite strand. For example, when designing an oligonucleotide, one or more residues that do not base pair with a base in the reverse complement may be incorporated, for example. If the stretch of nucleotides can still hybridize to the reverse complement in the intracellular environment, some mismatch may be tolerated. In the context of the present invention, in one embodiment, the oligonucleotide of the invention comprises at least 45%, 50%, 55%, 60%, 65% of the first exon region and / or the second exon region. , 70%, 75%, 80%, 85%, 90%, 95%, 100% are reverse complementary, so the presence of mismatches in the oligonucleotide is preferred. Since the oligonucleotide can bind to the region of the first exon and the region of the second exon identified herein above, the presence of a mismatch in the oligonucleotide is a preferred property of the invention. .

  Other advantages that allow for the presence of mismatches in the antisense oligonucleotides of the present invention are defined herein and include fluctuations in inosine (hypoxanthine) and / or universal bases and / or modified bases and / or nucleotides. Similar to that provided by the presence of nucleotides containing bases capable of forming base pairs, avoiding the presence of CpG, avoiding or reducing potential multimerization or aggregation; Which makes it possible to design oligonucleotides with improved RNA binding kinetics and / or thermodynamic properties.

  The reverse complementarity of the oligonucleotide to the region of identity between the first exon and / or the second exon is preferably 45% -65%, 50% -75%, preferably 65% -100% Or 70% to 90%, or 75% to 85%, or 80% to 95%. In general, this allows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mismatches in a 20 nucleotide oligonucleotide. Thus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, or 22 in a 40 nucleotide oligonucleotide May have a mismatch. Preferably less than 14 mismatches are present in a 40 nucleotide oligonucleotide. The number of mismatches is such that the oligonucleotides of the invention can still bind to, hybridize and target to the first exon region and the second exon region, thereby at least the first exon and the second exon region. A number that can induce skipping of two exons and induce the production of in-frame transcripts exemplified herein. Production of in-frame transcripts can be obtained with an efficiency of at least 5% using at least 100 nM of said oligonucleotide to transfect the relevant cell cultures described hereinabove in vitro. Is preferred.

  The present invention does not have any mismatch with the region of the first exon and is 1, 2, 3, 4, 5, 6, with the corresponding region of the second exon as defined herein. It should be noted that it encompasses oligonucleotides that can have 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21 or 22 mismatches. However, the present invention also does not have any mismatch with the region of the second exon and is 1, 2, 3, 4, 5 with the corresponding region of the first exon as defined herein. , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, or 22 mismatches are also encompassed. Finally, the present invention relates to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 with the region of the first exon. , 20, 21 or 22 mismatches and 22, 21, 20, 19, 18, 17, 16, 15, 14 with the corresponding region of the second exon as defined herein. , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 including oligonucleotides that can have a mismatch. Here again, the number of mismatches in the oligonucleotide of the invention is such that the oligonucleotide can still bind to the region of the first exon and the region of the second exon described herein and hybridizes. Is understood to be a number that can and can be targeted.

  The oligonucleotides of the invention preferably do not cross gaps in the alignment of the first and second exon regions identified herein. The alignment is preferably performed using the online tool EMBOSS Matcher described hereinabove. However, in certain cases, the oligonucleotides of the invention may need to cross a gap, as described herein above. The gap is preferably a single gap. The gap preferably spans less than 3 nucleotides, most preferably only 1 nucleotide. The number and length of the gaps means that the oligonucleotides of the invention can still bind to, hybridize, and target to the first exon region and the second exon region, thereby at least the first exon. A number and length that can induce skipping of the exon and the second exon, and can induce the production of the in-frame transcript described herein.

  The structure (ie open and closed structure) is optimally analyzed in the context of an mRNA precursor in which exons are present. Such structures can be analyzed in actual RNA. However, it is now possible to predict the secondary structure of RNA molecules (with the lowest energy cost) quite well using structural modeling programs. A non-limiting example of a suitable program is the Mfold web server (Zuker, M).

  One skilled in the art can predict the structure of a possible exon, a given nucleotide sequence, with suitable reproducibility. When such a modeling program is implemented using both the exon and adjacent intron sequences, an optimal prediction is obtained. Typically, it is not necessary to model the structure of the entire mRNA precursor.

  Annealing of the oligonucleotides of the invention can affect the local folding or 3D structure or conformation of the target RNA (ie, the region encompassing at least the first and / or second exons). Different conformations can result in the destruction of structures recognized by splicing mechanisms. However, if a potential (hidden) splice acceptor and / or donor sequence is present in the first target exon and / or the second target exon, sometimes a different (neo) exon is defined, ie a different 5 ′ New structures with termini, different 3 ′ termini or both are generated. This type of activity is within the scope of the present invention when the target exon is excluded from the mRNA. The presence of a new exon containing part of the first target exon and / or the second target exon in the mRNA does not change the fact that the target exon itself is eliminated. Inclusion of neoexon can be seen as a rare side effect. There are two possibilities when exon skipping is used to repair the open reading frame of a transcript that is destroyed as a result of a mutation. On the one hand, the neoexon functions in reading frame repair, while in the other, the reading frame is not repaired. When choosing an oligonucleotide to repair an open reading frame by multiple exon skipping, under these conditions, an exon skipping that repairs the open reading frame of a given transcript, regardless of the presence or absence of a neoexon. It is clear that only the oligonucleotides that actually result are selected.

  In yet another preferred embodiment, there is provided an oligonucleotide of the invention capable of binding to a region of a first exon derived from an mRNA precursor and a region of a second exon within the same mRNA precursor, wherein said oligonucleotide of said second exon A region has at least 50% identity with the region of the first exon (preferred regions for dystrophin exons are identified in Table 2 or Table 6), and the oligonucleotide is the mRNA precursor The first exon and the second exon can be skipped, resulting in a transcript that is preferably in-frame (preferably for DMD as in Table 1 or Table 6). The oligonucleotide provides a functional or semi-functional protein to the individual, wherein the oligonucleotide is against one or more binding sites for serine-arginine (SR) protein in the RNA of exon of the mRNA precursor. It further includes sequences that can bind, target, hybridize, reverse complement, and / or inhibit their function.

  In the patent application of WO 2006/112705, the inventors have determined the effectiveness of an antisense oligonucleotide (AON) within an exon in inducing exon skipping and the presumption at the target mRNA precursor site of the AON. The existence of a correlation between the presence of SR binding sites has been disclosed. Thus, in one embodiment, generating the oligonucleotide as defined herein comprises one or more (for SR proteins in the RNA of the first exon and / or the second exon ( (Presumptive) determining a binding site, and corresponding to the RNA, capable of binding, targeting, hybridizing, and / or reverse complementary and at least partially overlapping with the (presumptive) binding site Producing an oligonucleotide. The term “at least partially overlapping” includes a single nucleotide of an SR binding site and a duplication of only one or more nucleotides of the binding site and a complete duplication of one or more of the binding sites. As defined herein. This embodiment comprises a region (closed structure) that hybridizes from the secondary structure of the first exon and / or the second exon to another part of the first exon and / or the second exon, and the Determining a non-hybridized region (open structure) in the structure, and subsequently overlapping at least partly with one or more of the (presumed) binding sites and overlapping at least part of the closed structure. Generating oligonucleotides that overlap with at least a portion of the open structure and bind to, target, hybridize, and / or reverse complement to both the first and second exons. A step is preferably further included. Thus, we have oligonucleotides that can prevent inclusion of the first and second exons and, if applicable, the entire stretch of exons in the mRNA from the mRNA precursor. Increase opportunities to get. While not wishing to be bound by any theory, currently the use of oligonucleotides directed to SR protein binding sites results in (at least partial) damage of SR protein binding to said binding site, resulting in destruction or It is believed that damaged splicing occurs.

  Preferably, the first exon region and / or the second exon region within the same mRNA precursor to which the oligonucleotides of the invention can bind comprises an open / closed structure and / or an SR protein binding site. More preferably, the open / closed structure as well as the SR protein binding site partially overlap, and the open / closed structure completely overlaps with the SR protein binding site or SR protein binding. More preferably, the site completely overlaps with the open / closed structure. This allows for further improved destruction of exon inclusions.

  In addition to consensus splice site sequences, many (but not all) exons contain splicing control sequences such as exon splicing enhancer (ESE) sequences to facilitate recognition of the true splice site by the spliceosome ( Cartegni L et al., 2002 and Cartegni L et al., 2003). A subgroup of splicing factors called SR proteins can bind to these ESEs, and can recruit other splicing factors such as U1 and U2AF to the (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 (Cartegni L et al., 2002 and Cartegni L et al., 2003). The effectiveness of the oligonucleotide and the presence / absence of the SF2 / ASF, SC35, SRp40 and SRp55 binding sites in the part of the first exon bound, hybridized and / or targeted by said oligonucleotide. There is a correlation between them. In preferred embodiments, the present invention therefore provides oligonucleotides that bind, hybridize, target, and / or reverse complement to the binding site for the SR protein. The SR protein is preferably SF2 / ASF or SC35, SRp40 or SRp55. In one embodiment, the oligonucleotide is at the binding site for the SF2 / ASF, SC35, SRp40 or SRp55 protein in the first exon and the binding site for a different SF2 / ASF, SC35, SRp40 or SRp55 protein in the second exon. Bind to, hybridize, target, and / or reverse complement. In a more preferred embodiment, the oligonucleotide is a binding site for the SF2 / ASF, SC35, SRp40 or SRp55 protein in the first exon and the corresponding for the similar SF2 / ASF, SC35, SRp40 or SRp55 protein in the second exon. Bind to, hybridize, target, and / or reverse complement to the binding site.

  In one embodiment, the patient uses an oligonucleotide that can bind and target to a regulatory RNA sequence present in the first and / or second exon required for precise splicing of the exon in the transcript. By doing so, a functional or semi-functional protein is provided. Some cis-acting RNA sequences require precise splicing of exons in the transcript. In particular, supplemental elements such as exon splicing enhancers (ESE) have been identified to control the specific and effective splicing of constitutive and alternative exons. By using a compound comprising an oligonucleotide that binds or can bind to one of the complementary elements in the first exon and / or the second exon, their control functions are broken so that the exons are skipped. Thus, in one preferred embodiment, the oligonucleotide of the invention is capable of binding to a region of a first exon derived from an mRNA precursor and a region of a second exon within the same mRNA precursor, said said exon of said second exon. A region has at least 50% identity with the region of the first exon, and the oligonucleotide is capable of inducing skipping of the first and second exons of the mRNA precursor; The region of exon and / or the region of the second exon comprises an exon splicing enhancer (ESE), an exon recognition sequence (ERS) and / or a splicing enhancer sequence (SES), and / or the oligonucleotide Exon splicing enhancer (ESE), exon recognition sequence ( RS) and / or can bind to a splicing enhancer sequence (SES), can be targeted, inhibition can, and / or a reverse complement.

  The following preferred chemistries of the oligonucleotides of the invention are disclosed.

  Oligonucleotides are generally known as oligomers having the same basic hybridization properties as natural nucleic acids. Hybridization is defined in the portion assigned to the definition at the end of the description of the invention. In this specification, the terms oligonucleotide and oligomer are used interchangeably. Different types of nucleotides may be used to generate the oligonucleotides of the present invention. The oligonucleotide may comprise at least one modified internucleoside linkage and / or at least one sugar modification and / or at least one base modification compared to a natural ribonucleotide-based or deoxyribonucleotide-based oligonucleotide.

  “Modified internucleoside linkage” indicates the presence of a modified form of a phosphodiester as is naturally occurring in RNA and DNA. Examples of internucleoside linkage modifications compatible with the present invention are phosphorothioate (PS), chirally pure phosphorothioate, phosphorodithioate (PS2), phosphonoacetic acid (PACE), phosphonoacetamide (PACA), thiophosphonoacetic acid, thiophosphonoacetic acid Noacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylphosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, Methyl boranophosphonate, methylboranophosphonothioate and their derivatives. Other modifications include phosphoramidite, phosphoramidate, N3 ′ → P5 ′ phosphoramidate, phosphorodiamidate, phosphorothioamidate, phosphorothiodiamidate, sulfamic acid, dimethylene sulfoxide, sulfonic acid, methylene Imino (MMI), oxalyl and thioacetamide nucleic acids (TANA) and their derivatives are included. Depending on its length, the oligonucleotides of the present invention are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 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 or 39 skeletal modifications may be included. It is also encompassed by the present invention to introduce more than one different backbone modification into the oligonucleotide.

  Also encompassed by the present invention are oligonucleotides that contain internucleoside linkages that can differ with respect to the atoms of the nucleosides that bind to each other as compared to naturally occurring internucleoside linkages. In this regard, the oligonucleotides of the invention have at least one internucleoside that is interpreted as a 3′-3 ′, 5′-5 ′, 2′-3 ′, 2′-5 ′, 2′-2 ′ linked monomer. Bonds may be included. Position numbering may differ from other chemistries, but the idea is within the scope of the invention.

  In one embodiment, the oligonucleotide of the invention comprises at least one phosphorothioate modification. In a more preferred embodiment, the oligonucleotide of the invention is a fully modified phosphorothioate.

  “Sugar modification” refers to RNA and DNA such as bicyclic sugar, tetrahydropyran, morpholine, 2′-modified sugar, 3′-modified sugar, 4′-modified sugar, 5′-modified sugar and 4′-substituted sugar Indicates the presence of a modified form of the ribosyl moiety (ie, the furanosyl moiety) as it occurs in nature. Examples of suitable sugar modifications include, but are not limited to, 2′-O-modified RNA nucleotide residues such as 2′-O-alkyl or 2′-O- (substituted) alkyl, such as 2′-O-methyl, 2'-O- (2-cyanoethyl), 2'-O- (2-methoxy) ethyl (2'-MOE), 2'-O- (2-thiomethyl) ethyl, 2'-O-butyryl, 2 ' -O-propargyl, 2'-O-allyl, 2'-O- (2-amino) propyl, 2'-O- (2- (dimethylamino) propyl), 2'-O- (3-amino) propyl 2′-O- (3- (dimethylamino) propyl), 2′-O- (2-amino) ethyl, 2′-O- (3-guanidino) propyl (incorporated herein by reference in its entirety) International Patent Application Publication No. 2013/061295, Univ. rsity of the Witterwaters), 2'-O- (2- (dimethylamino) ethyl), 2'-O- (haloalkyl) methyl (Arai K. et al.), eg 2'-O- (2 -Chloroethoxy) methyl (MCEM), 2'-O- (2,2-dichloroethoxy) methyl (DCEM), 2'-O-alkoxycarbonyl, for example 2'-O- [2- (methoxycarbonyl) ethyl] (MOCE), 2′-O- [2- (N-methylcarbamoyl) ethyl] (MCE), 2′-O- [2- (N, N-dimethylcarbamoyl) ethyl] (DMCE), 2′-O -[Methylaminocarbonyl] methyl, 2'-azido, 2'-amino and 2'-substituted amino, 2'-halo, eg 2'-F, FANA (2'-F arabinosyl nucleus Acid), carbasugar and azasugar modifications, 3'-O-alkyl, such as 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl, 2 ', 3'-dideoxy and their derivatives Is included.

Other sugar modifications include, for example, locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2′-O, 4′-C constrained ethyl) LNA, cMOEt (2′-O, 4′-C-restricted methoxyethyl) LNA, ethylene bridged nucleic acid (ENA), BNA ^NC [N-Me] (Chem. Commun. 2007, which is incorporated herein by reference in its entirety. 3765-3767 Kazuyuki Miyashita et al.), Patent application WO2013 / 036868 (incorporated herein by reference in its entirety; found in CRN described in Marina Biotech). “Bridged” or “bicyclic” nucleic acid (BNA) modified sugar moieties, such as those; Unlocked nucleic acids (UNA) or other acyclic nucleotides such as those described in US Patent Application Publication No. 2013/0130378 (Anylam Pharmaceuticals) incorporated herein; 5′-methyl substituted BNA (its U.S. patent application 13 / 530,218, which is incorporated by reference in its entirety); cyclohexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA ( Other modified sugar moieties such as F-HNA), pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (PMO), cationic morpholino (PMOPlus), PMO-X Tricyclo DNA; tricyclo-PS-DNA; Beauty derivatives thereof. BNA derivatives are described, for example, in WO 2011/097641, which is incorporated by reference in its entirety. An example of PMO-X is described in WO 2011150408, which is incorporated herein by reference in its entirety. Depending on its length, the oligonucleotides of the present invention are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 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 or 40 sugar modifications Good. It is also encompassed by the present invention to incorporate more than one different sugar modification into the oligonucleotide.

  In one embodiment, the oligonucleotide according to the invention is at least one selected from 2′-O-methyl, 2′-O- (2-methoxy) ethyl, 2′-F, morpholino, bridged nucleotide or BNA. Sugar modifications or oligonucleotides contain both bridging nucleotides and 2'-deoxynucleotides (BNA / DNA mixmers). Oligonucleotides containing 2'-fluoro (2-'F) nucleotides can mobilize interleukin enhancer-binding factors 2 and 3 (ILF2 / 3), thereby inducing exon skipping within targeted mRNA precursors (Rigo F et al., International Publication No. 2011/097614).

  In another embodiment, the oligonucleotide as defined herein comprises or consists of LNA or a derivative thereof. The oligonucleotide according to the present invention has its full length due to a sugar modification selected from 2′-O-methyl, 2′-O- (2-methoxy) ethyl, morpholino, cross-linked nucleic acid (BNA), or BNA / DNA mixmer. More preferably, it is modified over the entire range. In a more preferred embodiment, the oligonucleotide of the invention is fully 2'-O-methyl modified.

  In preferred embodiments, the oligonucleotides of the invention comprise at least one sugar modification and at least one modified internucleoside linkage. Such modifications include peptide-base nucleic acid (PNA), boron-cluster modified PNA, pyrrolidine oxy-peptide nucleic acid (POPNA), glycol or glycerol nucleic acid (GNA), threose nucleic acid (TNA), acyclic Threoninol nucleic acid (aTNA), morpholino oligonucleotide (PMO, PPMO, PMO-X), cationic morpholino oligomer (PMOPlus, PMO-X), oligonucleotide with incorporated base and backbone (ONIB), pyrrolidine- Amide oligonucleotides (POM) as well as their derivatives are included. In a preferred embodiment, the oligonucleotide of the invention comprises a peptide nucleic acid backbone and / or a morpholino phosphorodiamidate backbone or derivatives thereof. In a more preferred embodiment, the oligonucleotide according to the invention is a modified 2'-O-methyl phosphorothioate, i.e. comprises at least one 2'-O-methyl phosphorothioate modification, and the oligonucleotide according to the invention is completely It is preferably a modified 2′-O-methyl phosphorothioate. The 2'-O-methyl phosphorothioate modified oligonucleotide or the fully 2'-O-methyl phosphorothioate modified oligonucleotide is preferably an RNA oligonucleotide.

  As used herein, the term “base modification” or “modified base” refers to a modified or de novo synthesized base of a base that is naturally present in RNA and / or DNA (ie, a pyrimidine or purine base). Such de novo synthetic bases can be identified as “modified” by comparison with existing bases.

  In addition to the above modifications, the oligonucleotides of the invention may include further modifications such as the different types of nucleic acid nucleotide residues or nucleotides described below. Different types of nucleic acid nucleotide residues may be used to generate the oligonucleotides of the invention. The oligonucleotide may have at least one backbone and / or sugar modification and / or at least one base modification compared to an RNA or DNA-based oligonucleotide.

  The oligonucleotide may comprise the natural bases purines (adenine, guanine) or pyrimidines (cytosine, thymine, uracil) and / or modified bases as defined below. In the present invention, uracil may be replaced with thymine.

  Base modifications include modified forms of natural purine and pyrimidine bases (eg, adenine, uracil, guanine, cytosine and thymine) such as hypoxanthine, orotic acid, agmatidine, ricidin, pseudouracil, pseudothymine, N1-methylpseudouracil, 2-thiopyrimidine (eg 2-thiouracil, 2-thiothymine), 2,6-diaminopurine, G-clamp and its derivatives, 5-substituted pyrimidines (eg 5-halouracil, 5- Methyluracil, 5-methylcytosine, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, super T (S per T), 5-octylpyrimidine, 5-thiophenypyrimidine, 5-oct-1-ylpyrimidine, 5-ethynylpyrimidine, 5- (pyridylamide), 5-isobutyl, which is incorporated herein by reference in its entirety. 5-phenyl; 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, described in U.S. Patent Application Publication No. 2013/0131141 (RXi), 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A and N4-ethylcytosine or their derivatives; N2-cyclopentylguanine (CPent-G), N2-cyclopentyl-2-aminopurine (cP nt-AP) and N2-propyl-2-aminopurine (Pr-AP) or derivatives thereof; and denatured or universal bases such as 2,6-difluorotoluene or absent bases such as abasic sites (eg 1- Deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced by nitrogen (azaribose) Super A, Super G and Examples of derivatives of Super T can be found in US Patent No. 6,683,173 (Epoch Biosciences), which is incorporated herein by reference in its entirety, cPent-G, cPent-AP and Pr-AP May reduce immune activation when incorporated into siRNA. (Peacock H et al.) And similar features have been shown for pseudouracil and N1-methyl pseudouracil (US 2013/0123481, which is incorporated herein by reference in its entirety). modeRNA Therapeutics). “Thymine” and “5-methyluracil” may be interchanged throughout this specification. Similarly, “2,6-diaminopurine” is identical to “2-aminoadenine” and these terms can be interchanged throughout this specification.

  In a preferred embodiment, the oligonucleotide of the invention comprises at least one 5-methylcytosine and / or at least one 5-methyluracil and / or at least one 2,6-diaminopurine base, whereby said oligonucleotide At least one of the cytosine nucleobases is modified by substitution of a proton at the 5-position of the pyrimidine ring with a methyl group (ie 5-methylcytosine) and / or at least one of the uracil nucleobases of the oligonucleotide Has been modified by substitution of a proton at the 5-position of the pyrimidine ring with a methyl group (ie 5-methyluracil) and / or at least one of the adenine nucleobases of said oligonucleotide is an amino group (ie 2,6 -Diaminopurine) It is understood respectively that has been modified by substitution of protons in the 2-position. In the present specification, the expression “substitution of a proton with a methyl group at the 5-position of a pyrimidine ring” may be replaced with the expression “substitution of a pyrimidine with 5-methylpyrimidine”. Refers to only or both. Similarly, in the present specification, the expression “substitution of proton with amino group at the 2-position of adenine” may be replaced with the expression “substitution of adenine with 2,6-diaminopurine”. When the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more cytosines, uracils and / or adenines, at least 1, 2, 3, 4, 5, 6, Each of 7, 8, 9 or more cytosines, uracils and / or adenines may be modified in this way. In a preferred embodiment, all cytosines, all uracils and / or all adenines are thus modified or replaced with 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine, respectively. It is done.

  The presence of 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine in the oligonucleotide of the invention has a positive effect on at least one parameter of said oligonucleotide or an improvement of at least one parameter. It was discovered. In this context, the parameters include, as described below, the binding affinity and / or kinetics of the oligonucleotide, silencing activity, in vivo stability, (intra-tissue) distribution, cell uptake and / or cell Transportation and / or immunogenicity may be included.

  Some modifications as described above are known to increase Tm values, thereby enhancing the binding of specific nucleotides to their counterparts in their target mRNA, so that these modifications are of the present invention. In context, it can be investigated to promote binding of the oligonucleotides of the invention to both the first and second exon regions. Since the sequence of the oligonucleotide of the invention may not be 100% reverse complementary to a given region of the first exon and / or the second exon, a bridging nucleotide or BNA (such as LNA) or 5- Modifications that increase Tm, such as base modifications selected from methylpyrimidine and / or 2,6-diaminopurine, are preferably reverse complementary to the first region and / or the second region of the exon Can be performed at nucleotide positions that are specific.

  The binding affinity and / or binding or hybridization kinetics depends on the thermodynamic properties of the AON. They can be used to determine the oligonucleotide characterization calculator (http://www.unc.edu/˜cail/biotool/) for single stranded RNA using the melting temperature of the oligonucleotide (Tm; eg, basic Tm and adjacent model). oligo / index.html or http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/)) and / or (RNA structure version 4.5 or RNA mfold version 3.5 was used) ) Determined at least in part by the free energy of the oligonucleotide target exon complex. As Tm increases, exon skipping activity typically increases, but when Tm is very high, AON is expected to be less sequence specific. The allowed Tm and free energy depend on the sequence of the oligonucleotide. Therefore, it is difficult to give a preferred range for each of these parameters.

The activity of the oligonucleotide of the invention is preferably defined as follows:
One or more of the diseases associated with mutations present in the first exon and / or the second exon and / or mutations present in the stretch starting in the first exon and ending in the second exon Alleviating one or more symptoms of DMD or BMD, and / or reducing one or more characteristics of patient-derived cells, preferably patient-derived muscle cells And / or providing said individual with a functional or semi-functional protein, preferably a functional or semi-functional dystrophin protein, and / or at least partially reducing abnormal protein production in said individual, preferably At least partially reduces abnormal dystrophin protein production in the individual Be. Each of these features and the assays for evaluating them are defined herein below.

  Oligonucleotides comprising 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine base, which are preferred oligonucleotides of the present invention, have no 5-methylcytosine, It is expected to show an increased activity compared to the corresponding activity of an oligonucleotide that has no uracil and no 2,6-diaminopurine base. This difference in activity is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95% or 100%. Biodistribution and in vivo stability are preferably determined, at least in part, by the effective hybridization ligation assay of source Yu et al., 2002. In one embodiment, plasma or a homogenized tissue sample is incubated with a specific capture oligonucleotide probe. After separation, the DIG-labeled oligonucleotide is ligated with the complex, followed by detection using anti-DIG antibody-linked peroxidase. Non-compartmental pharmacokinetic analysis is performed using the WINNONLIN software package (Model 200, version 5.2, Pharsight, Mountainview, CA). AON level (ug) per ml plasma or 1 mg tissue evaluates the area under the curve (AUC), peak concentration (Cmax), time versus peak concentration (Tmax), terminal phase half-life and absorption delay time (tag) To be monitored over time. Such preferred assays are disclosed in the experimental part. Oligonucleotides can stimulate innate immune responses by activating Toll-like receptors (TLRs), including TLR9 and TLR7 (Krieg AM et al., 1995). Activation of TLR9 is typically due to the presence of unmethylated CG sequences present in oligodeoxynucleotides (ODN) by mimicking bacterial DNA that activates the innate immune system by TLR9-mediated cytokine release It occurs as a result. However, 2'-O-methyl modifications can significantly reduce such possible effects. TLR7 has been described to recognize uracil repeats in RNA (Diebold SS et al., 2006).

  Activation of TLR9 and TLR7 results in a series of synchronized immune responses including innate immunity (macrophages, dendritic cells (DCs) and NK cells) (Krieg AM et al., 1995; Krieg AM. Et al., 2000). Several chemokines and cytokines such as IP-10, TNFα, IL-6, MCP-1 and IFNα (Wagner H. et al., 1999; Popovic PJ et al., 2006) are involved in this process. Inflammatory cytokines attract additional protective cells from blood such as T and B cells. The levels of these cytokines can be examined by in vitro tests. Briefly, human whole blood is incubated with a high concentration of oligonucleotides, after which cytokine levels are determined by standard commercial ELISA kits. The reduced immunogenicity is preferably at least one 5-methylcytosine compared to cells treated with 5-methylcytosine, 5-methyluracil or the corresponding oligonucleotide without 2,6-diaminopurine and A detectable decrease in the concentration of at least one of the above cytokines compared to the concentration of the corresponding cytokine in an assay of cells treated with oligonucleotides comprising / or 5-methyluracil and / or 2,6-diaminopurine Correspond.

  Accordingly, preferred oligonucleotides of the invention are permissive compared to the corresponding oligonucleotides without 5-methylcytosine, without 5-methyluracil, and without 2,6-diaminopurine. Having improved parameters such as possible or reduced immunogenicity and / or good biodistribution and / or acceptable or improved RNA binding kinetics and / or thermodynamic properties. Each of these parameters can be evaluated using assays known to those skilled in the art.

  Preferred oligonucleotides of the invention comprise or consist of RNA molecules or modified RNA molecules. In a preferred embodiment, the oligonucleotide is single stranded. However, one skilled in the art will understand that single stranded oligonucleotides may be capable of forming internal double stranded structures. However, this oligonucleotide is still named a single stranded oligonucleotide in the context of the present invention. Single stranded oligonucleotides have several advantages compared to double stranded siRNA oligonucleotides: (i) Its synthesis is expected to be easier than two complementary siRNA strands; (ii) intracellular There is a wide range of chemical modifications that can enhance uptake, good (physiological) stability, and reduce possible common adverse effects; (iii) siRNAs are non-specific And (iv) siRNAs are less likely to act in the nucleus; and (iv) siRNAs are less likely to act in the nucleus Can't be directed to the intron.

  In another embodiment, the oligonucleotide of the invention comprises an abasic site or abasic monomer. Herein, such monomers can be referred to as abasic sites or abasic monomers. An abasic monomer is a nucleotide residue or component that lacks a nucleobase as compared to the corresponding nucleotide residue that contains the nucleobase. Within the present invention, an abasic monomer therefore lacks a nucleobase but is a component part of an oligonucleotide. Such abasic monomers can be present or linked or conjugated or conjugated at the free end of the oligonucleotide.

  In a more preferred embodiment, the oligonucleotide of the invention comprises 1-10 or more abasic monomers. Thus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more abasic monomers may be present in the oligonucleotides of the invention.

  The abasic monomer may be of any type known and conceivable to those skilled in the art. Non-limiting examples are shown below.

As used herein, R ₁ and R ₂ are independently H, an oligonucleotide or other abasic site, provided that both R ₁ and R ₂ are not H, but R ₁ and R ₂ Both are not oligonucleotides. The abasic monomer may be attached to either or both of the ends of the previously specified oligonucleotide. It should be noted that oligonucleotides attached to one or two abasic sites or abasic monomers can contain less than 10 nucleotides. In this regard, the oligonucleotide according to the invention may comprise at least 10 nucleotides optionally comprising one or more abasic sites or abasic monomers at one or both ends. Other examples of abasic sites encompassed by the present invention are described in US Patent Application Publication No. 2013/013378 (Anylam Pharmaceuticals), which is incorporated herein by reference in its entirety.

  Depending on its length, the oligonucleotides of the present invention are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Including 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 base modifications Good. It is also encompassed by the invention to incorporate more than one different base modification into the oligonucleotide.

Accordingly, in one embodiment, the oligonucleotide according to the present invention comprises:
(A) at least one base modification selected from 2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, and / or (b) at least one sugar modification Selected from 2'-O-methyl, 2'-O- (2-methoxy) ethyl, 2'-O-deoxy (DNA), 2'-F, morpholino, bridged nucleotide or BNA, or Sugar modifications wherein the oligonucleotide comprises both bridging nucleotides and 2′-deoxy modified nucleotides (BNA / DNA mixmers), and / or (c) at least one backbone modification selected from phosphorothioates or phosphorodiamidates.

In another embodiment, the oligonucleotide according to the invention comprises:
(A) at least one base modification selected from 5-methylpyrimidine and 2,6-diaminopurine, and / or (b) at least one sugar modification being 2′-O-methyl, and / or (c) ) At least one backbone modification that is a phosphorothioate.

In one embodiment, the oligonucleotide of the invention has at least one modification, more preferably, compared to a natural or deoxyribonucleotide-based oligonucleotide,
(A) at least one base modification, preferably selected from 2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, and 5-methylpyrimidine and 2 Base modification more preferably selected from 1,6-diaminopurine, and / or (b) at least one sugar modification, 2′-O-methyl, 2′-O- (2-methoxy) ethyl, 2 Preferably selected from '-O-deoxy (DNA), 2'-F, morpholino, cross-linked nucleotide or BNA, or the oligonucleotide contains both cross-linked nucleotide and 2'-deoxy modified nucleotide (BNA / DNA mixmer) More preferably, the sugar modification is 2′-O-methyl, and / or (c) at least one skeleton A decoration, preferably from phosphorothioate or phosphorodiamidate is selected, including backbone modification more preferably a phosphorothioate.

  Thus, the oligonucleotide according to this embodiment of the invention contains a base modification (a), no sugar modification (b), and no backbone modification (c). Another preferred oligonucleotide according to this aspect of the invention comprises a sugar modification (b), no base modification (a) and no backbone modification (c). Another preferred oligonucleotide according to this aspect of the invention includes a backbone modification (c), no base modification (a), and no sugar modification (b). Also, oligonucleotides that do not have any of the above modifications are included in the present invention and are similarly two, ie, (a) and (b), (a) and (c), and / or as defined above. Alternatively, oligonucleotides comprising all three of (b) and (c) or modifications (a), (b) and (c) are also included in the present invention.

  In a preferred embodiment, the oligonucleotide according to the invention has the same modification selected from (a) one of the base modifications and / or (b) one of the sugar modifications and / or (c) one of the backbone modifications. One or more of which are modified throughout their length.

  With the advent of nucleic acid mimicry technology, it is possible to generate molecules of the same type but not necessarily the same amount as the nucleic acid itself, but with similar and preferably the same hybridization properties. Such functional equivalents are of course also suitable for use in the present invention.

  In another preferred embodiment, the oligonucleotide comprises nucleosides or nucleotides or functional equivalents containing inosine, hypoxanthine, universal bases, modified bases and / or bases capable of forming wobble base pairs. The use of nucleotides containing inosine (hypoxanthine) and / or universal bases and / or modified bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the invention is very attractive as explained below. It is. For example, inosine is a known modified base that can pair with three bases: uracil, adenine and cytosine. Inosine is a nucleoside that is formed when hypoxanthine binds to the ribose ring (also known as ribofuranose) via a β [beta] -N9-glycoside bond. Inosine (I) is generally found in tRNA and is essential for proper translation of the genetic code in wobble base pairs. Fluctuating base pairs can exist between G and U, or between I on one side and U, A or C on the other side. These are the basis for the formation of RNA secondary structure. Its thermodynamic stability is comparable to that of Watson-Crick base pairs. The genetic code compensates for the difference in the number of amino acids (20) for the triplet codon (64) by using a modified base pair at the first base of the anticodon.

  The first advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or denatured bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the present invention is that An oligonucleotide capable of binding to a region of one exon and capable of binding to a region of a second exon in the same mRNA precursor, wherein the region derived from the second exon is combined with the region of the first exon; Have at least 50% identity. That is, in the oligonucleotide of the present invention, inosine (hypoxanthine) and / or a universal base and / or a denatured base and / or a nucleotide containing a base capable of forming a wobble base pair, the first exon of the mRNA precursor is present. The oligonucleotide can be bound to the region and the region of the second exon.

  A second advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or modified bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the invention is that the polymorphism is An oligonucleotide that compensates for a single nucleotide polymorphism (SNP) can be designed without worrying about destroying the annealing effect. Thus, in the present invention, the use of such bases allows the design of oligonucleotides that can be used for individuals having SNPs in the mRNA precursor stretch targeted by the oligonucleotides of the present invention.

A third advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or denatured bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the invention is described herein. When designing an oligonucleotide to be complementary to a portion of the first mRNA precursor exon, the oligonucleotide usually contains CpG. The presence of CpG in the oligonucleotide is usually associated with the increased immunogenicity of the oligonucleotide (Dorn A. and Kippenberger S.). This increased immunogenicity is undesirable because it can induce muscle fiber breakdown. It is expected that replacing guanine with inosine in one, two or more CpGs in the oligonucleotide will result in oligonucleotides with reduced and / or acceptable levels of immunogenicity. Immunogenicity can be assessed in the animal model by assessing the presence of CD4 ⁺ and / or CD8 ⁺ cells and / or inflammatory mononucleate infiltration in an animal muscle biopsy. Immunogenicity is also confirmed in the presence of neutralizing antibodies and / or antibodies that recognize the oligonucleotides in animal or human blood treated with the oligonucleotides of the invention using standard immunoassays known to those skilled in the art. Can be evaluated by detecting the presence of. Enhanced immunogenicity preferably corresponds to an animal prior to treatment or at least one inosine (hypoxanthine) and / or a nucleotide containing a universal base and / or a denatured base and / or a base capable of forming a wobble base pair. Corresponding to at least one detectable enhancement of these cell types by comparison with the amount of each cell type in the corresponding muscle biopsy of the animals treated with the nucleotides. Alternatively, enhanced immunogenicity is assessed by using standard immunoassays to detect the presence of neutralizing antibodies or antibodies that recognize the oligonucleotide, or an increase in their amounts. Can be done. The reduced immunogenicity preferably corresponds to an animal prior to treatment or no inosine (hypoxanthine) and / or nucleotides containing universal bases and / or denatured bases and / or bases capable of forming wobble base pairs. It corresponds to a detectable decrease in at least one of these cell types by comparison with the amount of the corresponding cell type in the corresponding muscle biopsy of the animal treated with the oligonucleotide. Alternatively, a decrease in immunogenicity can be assessed by the absence of the compounds and / or neutralizing antibodies, or by their reduced amounts, using standard immunoassays.

  A fourth advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or modified bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the present invention is the potential of the oligonucleotide. To avoid or reduce multimerization or aggregation. For example, oligonucleotides containing G-quartet motifs are known to have a tendency to form quadruplexes, multimers or aggregates formed by Hoogsteen base pairing of four single-stranded oligonucleotides. (Cheng AJ and Van Dyke MW), which of course is undesirable: as a result, the efficiency of the oligonucleotide is expected to decrease. Multimerization or aggregation is preferably assessed by standard polyacrylamide non-denaturing gel electrophoresis methods known to those skilled in the art. In preferred embodiments, the ability to form multimers, wherein less than 20% or 15%, 10%, 7%, 5% or less of the total number of oligonucleotides of the invention is assessed using the above assay or Has the ability to aggregate.

  Therefore, a fifth advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or modified bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the present invention is the antithrombotic activity ( Macaya R.F. et al.) And avoiding the quadruple structure associated with binding to macrophage scavenger receptor and inhibition of macrophage scavenger receptor (Suzuki K. et al.).

  The sixth advantage of using nucleotides containing inosine (hypoxanthine) and / or universal bases and / or denatured bases and / or bases capable of forming wobble base pairs in the oligonucleotides of the invention is that RNA binding kinetics and / or It allows the design of oligonucleotides with improved thermodynamic properties. RNA binding kinetics and / or thermodynamic properties can be determined by using oligonucleotide melting temperature (Tm; basic Tm and nearest neighbor models) for single-stranded RNA using an oligonucleotide property calculator (http: /// at least by the free energy of the AON-targeted exon complex (using RNA structure version 4.5)) and / or (calculated using www.unc.edu/~cail/biotool/oligo/index.html)) Partially determined. If the Tm is too high, the oligonucleotide is expected to be less specific. The allowed Tm and free energy depend on the sequence of the oligonucleotide. It is therefore difficult to give a preferred range for each of these parameters. Acceptable Tm can be in the range of 35-85 ° C. and acceptable free energy can be in the range of 15-45 kcal / mol.

  Depending on its length, the oligonucleotides of the present invention have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 inosine (hypoxanthine) and / or universal bases and / or denaturation. Nucleotides containing bases and / or bases capable of forming wobble base pairs or functional equivalents thereof may be included.

  Since RNA / RNA duplexes are very stable, it is preferred that the oligonucleotide of the present invention comprises RNA. RNA oligonucleotides have additional properties such as resistance to endonucleases, exonucleases and RNase H, additional hybridization strength, increased stability (eg in body fluids), increased or decreased flexibility, decreased toxicity, increased intracellular It preferably includes modifications that impart transport, tissue specificity, etc. to the RNA. Preferred modifications are as described above.

  Thus, one embodiment provides an oligonucleotide comprising at least one modification. A preferred modified oligonucleotide is fully modified 2'-O-methyl. In one embodiment of the invention, the oligonucleotide comprises or consists of a hybrid oligonucleotide comprising a 2'-O-methyl phosphorothioate oligonucleotide modification and a bridging nucleic acid modification (BNA exemplified above). In another embodiment of the invention, the oligonucleotide comprises or consists of a hybrid oligonucleotide comprising a 2'-O-methoxyethyl phosphorothioate modification and a crosslinked nucleic acid (BNA exemplified above). In another embodiment of the invention, the oligonucleotide comprises or consists of a hybrid oligonucleotide comprising a bridged nucleic acid (BNA exemplified above) modification and an oligodeoxyribonucleotide modification. This particular combination contains better sequence specificity compared to equivalents consisting only of bridged nucleic acids and is improved compared to oligonucleotides consisting of 2'-O-methyl phosphorothioate oligo (deoxy) ribonucleotide modifications. Including effects.

The compounds described in the present invention may preferably have an ionic group. The ionic group may be a base or acid, and may be charged or neutral. The ionic group may be present as an ion pair with a suitable counter ion having the opposite charge. Examples of cationic counter ions are sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium and tetraalkylammonium. Examples of anionic counterions are chloride, bromide, iodide, lactate, mesylate, acetate, trifluoroacetate, dichloroacetate and citrate. Examples of counter ions have been described (eg Kumar L, which is hereby incorporated by reference in its entirety). An example of the application of a divalent or trivalent counterion, in particular Ca ²⁺ , adds positive properties to selected oligonucleotides in US Patent Application Publication No. 20120346348 (Replicator), which is incorporated by reference in its entirety. It is described as. Preferred divalent or trivalent counterions are selected from the following description or group: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper and zinc. A preferred divalent counterion is calcium. Thus, it is encompassed by the present invention to prepare, obtain and use a composition comprising an oligonucleotide of the invention and any other counterion described above, preferably calcium. Such a method for preparing the composition comprising the oligonucleotide and the counterion, preferably calcium, may be as follows: The oligonucleotide of the invention is a pharmaceutically acceptable aqueous excipient The solution containing the counter ion is gradually added to the dissolved oligonucleotide, so that the oligonucleotide chelate complex remains dissolved.

Thus, in a preferred embodiment, the oligonucleotides of the invention are linked by such counter ions, preferably in contact with a composition comprising Ca ²⁺ , by such counter ions as described herein. An oligonucleotide chelate complex is formed comprising two or more identical oligonucleotides. Thus, a composition comprising an oligonucleotide chelate complex comprising two or more identical oligonucleotides linked by a counter ion, preferably calcium, is encompassed by the present invention.

Methods for designing oligonucleotides:
Accordingly, in a further aspect of the invention, there is provided a method for designing an oligonucleotide, resulting in the oligonucleotide described above.

The method includes the following steps.
(A) identifying an in-frame combination of a first exon and a second exon within the same mRNA precursor, wherein the region of the second exon is at least 50% of the region of the first exon. Having identity, steps,
(B) designing an oligonucleotide capable of binding to the region of the first exon and the region of the second exon; and (c) as a result of the binding, the first exon and the second exon. Preferably skipping a multi-exon stretch starting with the first exon and including one or more exons present between the first exon and the second exon, and at most Also causing skipping of the entire stretch of exons between the first exon and the second exon.

  In step b) of such a method, said oligonucleotide preferably binds its binding to at least one splicing control sequence in said region of said first exon and / or second exon in said mRNA. Designed to do.

  Alternatively, or in combination with splicing control sequence interference, the binding of the oligonucleotide preferably interferes with a secondary structure comprising at least the first exon and / or the second exon within the mRNA precursor. To do.

  Oligonucleotides obtainable according to the present invention can induce skipping of the first and second exons of the mRNA precursor. It is preferred that additional exon skipping is induced, said additional exon being preferably located between said first exon and said second exon, and the resulting mRNA transcript is in-frame . The oligonucleotide is preferably capable of inducing skipping of the entire stretch of exon between the first exon and the second exon. In one embodiment, the region of the first exon and the second exon includes a splicing control element, whereby binding of the oligonucleotide is at least in the region of the first exon and the second exon. Can interfere with one splicing control sequence. It is also encompassed that the binding of the oligonucleotide interferes with a secondary structure including at least the first exon and / or the second exon in the mRNA precursor. Preferred splicing control sequences include a binding site for a serine-arginine (SR) protein, an exon splicing enhancer (ESE), an exon recognition sequence (ERS), and / or an exon splicing silencer (ESS).

  Each feature of this method has already been described herein or is known to those skilled in the art.

Preferred oligonucleotides:
The oligonucleotide of the invention is preferably for use as a medicament, and more preferably the compound is for use in RNA modulation therapy. More preferably, said RNA modulation leads to the correction of disrupted transcription reading frames and / or the repair of desired or predicted protein expression. Thus, the methods of the invention and the oligonucleotides of the invention apply in principle to any disease associated with the presence of a frame disruption mutation that leads to the absence of an aberrant transcript and / or the coding protein and / or the presence of the aberrant protein. Can be done. In a preferred embodiment, the method of the invention and the oligonucleotide of the invention comprise a repetitive sequence and thereby a relatively high sequence identity (i.e. the region of the second exon and the first exon as defined herein). Applied to disease-related genes having regions having at least 50%, 60%, 70%, 80% sequence identity). Non-limiting examples include spectrin-like repeats (as the DMD gene involved in Duchenne muscular dystrophy described herein), Kelch-like repeats (KLHL3 gene (Louis-Dit involved in familial hyperkalemia) -Picard H et al., KLHL6 gene involved in chronic lymphocytic leukemia (Puente XS et al.), KLHL7 gene involved in retinitis pigmentosa (Friedman JS et al.), KLHL7 / 12 gene involved in Sjogren's syndrome (Uchida K et al.) KLHL9 gene involved in peripheral myopathy (Cirak S et al.), KLHL16 or GAN gene involved in giant axonal neuropathy (Bomont P et al.), KLHL19 or KEAP1 gene involved in several cancers (Dhanoa B S et al., KLHL20 gene (Dhanoa BS et al.) Involved in promyelocytic leukemia, or KLHL37 or ENC1 gene (Dhanoa BS et al.) Involved in brain tumor), FGF-like repeat, EGF-like repeat (NOTCH3 gene involved in CADASIL) (Chabriat H et al.), SCUBE gene involved in cancer or metabolic bone disease, Neulexin-1 (NRXN1) gene (Moller RS et al.) Involved in neuropsychiatric disorders and idiopathic generalized epilepsy, Del-1 gene, atheromatous Tenascin-C (TNC) gene (Mollie A et al.) Involved in arteriosclerosis and coronary artery disease, THBS3 gene or fibrillin (FBN1) gene (Rantamaki T et al.) Involved in Marfan syndrome), ankyrin-like repeat (ANKRD1 gene (Dubosq-Bidot L et al.) Involved in dilated cardiomyopathy, ANKRD2 gene, ANKRD11 gene (Sirmaci A et al.) Involved in KBG syndrome, thrombocytopenia (Noris P et al.) Or diabetes (Raciti GA et al.) ANKRD26 gene involved, ANKRD55 gene involved in multiple sclerosis (Alloza I et al.) Or TRPV4 gene involved in distal spinal muscular atrophy (Fiillolo C et al.), HEAT-like repeats (htt involved in Huntington's disease) Genes), annexin-like repeats, leucine-rich repeats (NLRP2 and NLRP7 genes (Huang JY et al.) Involved in idiopathic recurrent miscarriage, LRRK2 gene (Abeliovic A et al.) Involved in Parkinson's disease, a NLRP3 gene (Heneka MT et al.) Involved in Luzheimer's disease or meningitis, NALP3 gene (Knauf F et al.) Involved in renal failure or LRIG2 gene (Stuart HM et al.) Involved in urofial syndrome (such as) Serine protease inhibitor domain (Andrade M. et al. A. And a gene having a SPINK5 gene (Hovnanian A et al.) Involved in the Netherton syndrome.

  In the present invention, a preferred mRNA precursor or transcript is a dystrophin mRNA precursor or transcript. This preferred mRNA precursor or transcript is preferably a human mRNA precursor or transcript. The disease associated with the presence of the mutation present in the dystrophin mRNA precursor is DMD or BMD, depending on the mutation. Preferred combinations and regions of the first and second exons derived from the dystrophin mRNA precursor used in the context of the present invention are listed in Table 2.

  In a preferred embodiment, the compounds of the invention induce exon-skipping in cells, organs, tissues and / or patients, preferably in BMD or DMD patients or in cells, organs, tissues from said patients. Used for. As a result of exon-skipping, it does not contain a skipped exon, so that if the exon encodes an amino acid, it is altered and internally cleaved, but of partial to most functional dystrophin products. A mature mRNA is generated that can guide expression. Techniques for exon skipping are currently directed to the use of AON or AON to transcribe gene constructs. Recently, exon skipping techniques have been investigated to address genetic muscular dystrophy. Promising results have recently been reported by the inventors and others for AON-induced skipping therapy aimed at repairing the reading frame of dystrophin mRNA precursors in cells from mdx mice and DMD patients. (Heemskerk H et al .; Kirak S. et al .; Goemans et al.). By targeted skipping of specific exons, a severe DMD phenotype (devoid of functional dystrophin) is converted to a mild BMD phenotype (expressing functional or semi-functional dystrophin). Exon skipping is preferably induced by the binding of AONs targeting exon internal sequences.

  In the following, the present invention is exemplified by a mutant dystrophin mRNA precursor in which a first exon and a second exon are present. In the present specification, the dystrophin mRNA precursor preferably means the mRNA precursor of the DMD gene encoding dystrophin protein. Mutant dystrophin mRNA precursors are BMDs with mutations compared to unaffected human wild-type DMD mRNA precursors that result in (reduced levels) abnormal protein (BMD) or absence of functional dystrophin (DMD) Or it corresponds to the mRNA precursor of DMD patients. Dystrophin mRNA precursor is also termed DMD mRNA precursor. The dystrophin gene can also be named DMD gene. Dystrophin and DMD can be used interchangeably throughout this specification.

  A patient is preferably intended to mean a patient having DMD or BMD, described below, or a patient susceptible to developing DMD or BMD due to his (or her) genetic background. In the case of a DMD patient, the compound used preferably corrects the mutation as present in the DMD gene of the patient and thus preferably creates a dystrophin protein that looks like a dystrophin protein from a BMD patient. The protein is preferably a functional or semifunctional dystrophin as described below. In the case of BMD patients, the antisense oligonucleotides of the present invention preferably correct mutations such as those present in the BMD gene of the protein, and are preferably dystrophin that is more functional than dystrophin originally present in the BMD patient. Create.

  In the present specification, the functional dystrophin is preferably a wild-type dystrophin corresponding to a protein having the amino acid sequence set forth in SEQ ID NO: 1. The functional dystrophin preferably acts on its N-terminal portion (first 240 amino acids at the N-terminus), cysteine-rich domain (amino acids 3361 to 3675) and C-terminal domain (last 325 amino acids at the C-terminus) binding domain And each of these domains is present in wild-type dystrophin known to those skilled in the art. The amino acids set forth herein correspond to the wild-type dystrophin amino acid represented by SEQ ID NO: 1. That is, a functional or semi-functional dystrophin is a dystrophin that exhibits the activity of wild-type dystrophin to at least some extent. “At least to some extent” preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the activity of the corresponding wild-type functional dystrophin. Or it means 100%. In this context, the activity of functional dystrophin preferably binds to actin and interacts with dystrophin-related glycoprotein complex (DGC) or (DAGC) (Ehmsen J et al.). The binding of dystrophin to actin and DGC complexes is either co-immunoprecipitation using whole protein extracts or cross-section immunofluorescence analysis from muscle biopsy suspected of being dystrophic, known to those skilled in the art Can be visualized.

  Individuals suffering from DMD typically have a mutation in the gene encoding dystrophin that prevents complete protein synthesis (eg, premature termination prevents C-terminal synthesis). In BMD, the dystrophin gene also contains a mutation that does not destroy the open reading frame and the C-terminus is synthesized. As a result, a (semi) functional or functional dystrophin protein is produced that is not necessarily a similar amount of activity but is homologous to the wild-type protein and has similar activity. The genome of a BMD individual typically contains a dystrophin comprising an N-terminal portion (first 240 amino acids at the N-terminus), a cysteine-rich domain (amino acids 3361 to 3585) and a C-terminal domain (last 325 amino acids at the C-terminus). It encodes a protein, but in most cases its central rod domain can be shorter than that of wild type dystrophin (Monaco AP et al.). Exon skipping to treat DMD is typically directed to bypassing premature arrest in the mRNA precursor by skipping exons that are adjacent to or contain the mutation. This makes it possible to correct the open reading frame and to synthesize a dystrophin protein that contains the C-terminus but is cleaved internally. In a preferred embodiment, an individual having DMD and being treated with an oligonucleotide of the invention synthesizes dystrophin that exhibits at least some similar activity of wild-type dystrophin. Where the individual is a DMD patient or suspected of being a DMD patient, it is more preferred that the (semi) functional dystrophin is a dystrophin of an individual with BMD: typically the dystrophin is actin and DGC or DAGC Can interact with both (Ehmsen J. et al .; Monaco AP et al.).

  The central rod-like domain of wild-type dystrophin contains 24 spectrin-like repeats (Ehmsen J. et al.). In many cases, the central rod-like domain in BMD-like proteins is shorter than that of wild-type dystrophin (Monaco AP et al.). For example, the central rod-like domain of dystrophin provided herein may contain 5-23, 10-22, or 12-18 spectrin-like repeats as long as it can bind to actin and DGC. Good.

  Reduction of one or more symptoms of DMD or BMD in an individual using a compound of the present invention can be assessed by any of the following assays: prolonged gait loss, improved muscle strength, ability to lift weight. Improvement, improvement of time required to get up from the floor, improvement of 9-meter walking time, improvement of time required to climb the fourth floor, improvement of malignancy of foot function, improvement of lung function, improvement of heart function, life Quality improvement. Each of these assays is known to those skilled in the art. As an example, the publication of Manzur et al. (Manzur AY et al.) Gives a comprehensive description of each of these assays. For each of these assays, as soon as a detectable improvement or prolongation of the parameter measured in the assay is seen, preferably one or more symptoms of DMD or BMD are individuals using the compounds of the invention. It means that it is mitigated. The detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described in Hodgetts et al. (Hodgetts S. et al.). Alternatively, the alleviation of one or more symptoms of DMD or BMD can be assessed by measuring an improvement in muscle fiber function, integrity and / or survival. In preferred methods, one or more symptoms of a DMD or BMD patient are alleviated and / or one or more properties of one or more myocytes from the DMD or BMD patient are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient himself.

  Reduction in one or more properties of a myocyte from a DMD or BMD patient can be assessed by any of the following assays on myogenic or myocyte from that patient: reduced calcium uptake by the myocyte, reduction Collagen synthesis, altered morphology, altered lipid biosynthesis, reduced oxidative stress and / or improved muscle fiber function, integrity and / or survival. These parameters are usually assessed using immunofluorescence and / or histochemical analysis of cross sections of muscle biopsy.

  Improvements in muscle fiber function, integrity and / or survival can be assessed using at least one of the following assays: detectable reduction of creatine kinase in blood, biopsy of muscle suspected of being dystrophic A detectable decrease in muscle fiber necrosis in the cross section and / or a detectable increase in muscle fiber diameter uniformity in a biopsy section of muscle suspected of being dystrophic. Each of these assays is known to those skilled in the art.

  Creatine kinase can be detected in blood as described by Hodgetts et al. (Hodgetts S. et al.). The detectable decrease in creatine kinase is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% compared to the concentration of creatine kinase in the same DMD or BMD patient before treatment , 80%, 90% or more reduction.

  The detectable reduction in muscle fiber necrosis is preferably assessed in muscle biopsy, more preferably as described in Hodgetts et al. (Hodgetts S. et al.) Using biopsy sections. . A detectable decrease in necrosis is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the area where necrosis is identified using a biopsy section. % Or more reduction. Reduction is measured by comparison with necrosis assessed in the same DMD or BMD patient before treatment.

  Detectable increase in muscle fiber diameter uniformity is preferably assessed in muscle biopsy sections, and more preferably as described in Hodgetts et al. (Hodgetts S. et al.). The increase is measured by comparison with muscle fiber diameter uniformity in the same DMD or BMD patient before treatment.

  The oligonucleotides of the present invention provide the individual with (higher levels) of functional and / or (semi) functional dystrophin proteins (both DMD and BMD) and at least produce abnormal dystrophin protein in the individual. Preferably it can be partially reduced. In this context, “single” functional and / or “single” semi-functional dystrophin may mean that several forms of functional and / or semi-functional dystrophin can be generated. This is because a region of at least 50% identity between the first exon and the second exon is one of at least 50% identity between the other first exon and the second exon. Or if it overlaps with other regions, and the oligonucleotide can bind to the overlapping part, thereby skipping several different stretches of exons and producing several different in-frame transcripts Can be expected. This situation is illustrated in Example 4, where one single oligonucleotide according to the invention (PS816, SEQ ID NO: 1679) produces several in-frame transcripts that all share exon 10 as the first exon. And the second exon may be 13, 14, 15, 18, 20, 27, 30, 31, 32, 35, 42, 44, 47, 48 or 55.

  Higher levels are functional and / or (semi) functional as compared to corresponding levels of functional and / or (semi) functional dystrophin protein in patients prior to initiation of treatment with the oligonucleotides of the invention. Refers to an increase in target dystrophin protein levels. The level of said functional and / or (semi) functional dystrophin protein is preferably assessed using immunofluorescence or Western blot analysis (protein).

  Reducing production of abnormal dystrophin mRNA or abnormal dystrophin protein preferably means 90%, 80%, 70%, 60%, 50%, 40% of the initial amount of abnormal dystrophin mRNA or abnormal dystrophin protein. %, 30%, 20%, 10%, 5% or less means still detectable by RT PCR (mRNA) or immunofluorescence or Western blot analysis (protein). An aberrant dystrophin mRNA or protein is also used herein as being hardly functional (as compared to the wild-type functional dystrophin protein as defined herein above) or non-functional dystrophin mRNA or protein. Point to. The non-functional dystrophin protein is preferably a dystrophin protein that cannot bind to members of the actin and / or DGC protein complex. A non-functional dystrophin protein or dystrophin mRNA typically does not have or encode a dystrophin protein with the intact C-terminus of the protein. In a preferred embodiment, exon skipping (also called RNA regulation or splice switching) technology is applied.

  Increasing production of functional and / or semi-functional dystrophin mRNA and / or protein preferably means that such functional and / or semi-functional dystrophin mRNA and / or protein is detectable, Or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% compared to a detectable amount of said mRNA and / or protein detectable at the start of treatment , 90% or more. The detection can be performed using RT-PCR (mRNA) or immunofluorescence or Western blot analysis (protein).

  In another embodiment, the compounds of the invention provide the individual with a functional or semi-functional dystrophin protein. This functional or semi-functional dystrophin protein or mRNA can be detected as an abnormal dystrophin protein or mRNA as described herein above.

Targeted co-skiping of the two exons (the first exon and the second exon) can induce further exon skipping, and the further exon is preferably the first exon And the resulting dystrophin transcript is in-frame (preferably as described in Table 1 or 6) and DMD or severe BMD phenotype is mild BMD Or further converted to an asymptomatic phenotype. The co-skiping of the two exons is preferably by binding of the oligonucleotide to the region of the first exon from the dystrophin mRNA precursor and to the binding of the oligonucleotide to the region of the second exon in the dystrophin mRNA precursor. And the region of the second exon has at least 50% identity with the region of the first exon. The first dystrophin exon and the second dystrophin exon and the identity region therein are preferably those described in Table 2 or 6. Said oligonucleotide preferably does not show any overlap with non-exon sequences. The oligonucleotides preferably do not overlap with the splice sites at least as long as they are present in the intron. The oligonucleotide is preferably directed to an exon internal sequence that does not contain a sequence that is reverse complementary to an adjacent intron. The exon skipping technique is such that in the mRNA produced from the DMD mRNA precursor, the absence of the two exons is preferably located between further exons, more preferably between the first exon and the second exon. It is preferred that the absence of exons be applied to generate a codon region for (higher) expression of the dystrophin protein, although shorter (semi) functional. In this context (typically BMD patients), inhibiting the inclusion of the two exons, preferably further exons located between the first exon and the second exon, preferably Means the following:
The level of the original abnormal (little functional) dystrophin mRNA is reduced by at least 5% as assessed by RT-PCR, or the corresponding abnormal dystrophin protein level is immunofluorescence or Western using an anti-dystrophin antibody A decrease of at least 2% as assessed by blot analysis and / or whether an in-frame transcript encoding a semi-functional or functional dystrophin protein is detectable or its level is RT-PCR (mRNA level) Increase by at least 5% as assessed by or by at least 2% as assessed by immunofluorescence or Western blot using anti-dystrophin antibody (protein level).

  Abnormal, hardly functional or non-functional dystrophin protein reduction is preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% Or 100%, preferably consistent with or similar to the detection or increased production of more functional or semi-functional dystrophin transcripts or proteins. In this context (typically DMD patients), inhibiting the inclusion of the two exons, preferably further exons located between the first exon and the second exon, is preferably (more It means that a higher level (more) or (semi) function dystrophin protein or mRNA is provided to the individual.

  Once a DMD patient is given (higher) (more) functional or semi-functional dystrophin protein, the cause of DMD is at least partially alleviated. Therefore, it is then expected that the symptoms of DMD will be sufficiently reduced. The present invention uses at least two dystrophins when using a single oligonucleotide directed (or capable of binding or hybridizing or reverse complementary or targeting) to both outer exons of the stretch. It further provides the insight that skipping of the entire exon stretch from the mRNA precursor containing the exon is induced or enhanced. Throughout this specification in a preferred embodiment, therefore, the oligonucleotide of the invention is at least 80% reverse complementary to the region of the first exon and the second exon as defined herein. The first exon and the second exon correspond to the outer exons of the exon stretch to be skipped.

  The oligonucleotide is at least 85%, 90%, 95% or 100% reverse complementary to the first region and at least 50%, 55%, 60%, 65% to the region of the second exon. More preferably it is%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reverse complementary. Enhanced skipping frequency also increases the level of more (semi) functional dystrophin protein produced in the muscle cells of DMD or BMD individuals.

  The oligonucleotide according to the present invention, which is preferably used, is preferably reverse-complementary or capable of binding or hybridizing or targeting to a region of the first dystrophin exon, said region comprising 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 110, 120, 130, 140, 150, 1 Has 0, 170, 180, 190, 200, or more nucleotides, and is preferably reverse-complementary or capable of binding or hybridizing or targeting to a region of the second dystrophin exon; The region is 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 110, 120, 130 , 14 150, 160, 170, 180, 190, 200, or more nucleotides, and the region of the second exon within the same mRNA precursor is at least 50% identical to the region of the first exon It is sex.

  In the present invention, the oligonucleotide may comprise or consist of a functional equivalent of the oligonucleotide. Oligonucleotide functional equivalent preferably means an oligonucleotide as defined herein, in which one or more nucleotides are substituted and the activity of the functional equivalent is retained to at least some extent. To do. Preferably, the activity of the compound comprising a functional equivalent of an oligonucleotide provides a functional or semi-functional dystrophin protein. Accordingly, the activity of the compound comprising a functional equivalent of an oligonucleotide is preferably assessed by quantifying the amount of functional or semi-functional dystrophin protein. A functional or semi-functional dystrophin is preferably defined herein as a dystrophin capable of binding to members of the actin and DGC protein complex and supporting muscle fiber membrane structure and flexibility. The assessment of the activity of a compound comprising a functional oligonucleotide or equivalent is preferably RT-PCR (to detect exon skipping at the RNA level) and / or immunofluorescence or Western blot analysis (protein expression and localization). To detect). Said activity is at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% of the corresponding activity of said compound comprising the oligonucleotide from which the functional equivalent is derived. When it represents or more, it is preferably retained at least to some extent. Throughout this specification, where the term oligonucleotide is used, the term may be replaced by its functional equivalent as defined herein.

Thus, the use of oligonucleotides or functional equivalents thereof is
Capable of binding to, targeting, hybridizing and / or reverse complementary to a region of the first dystrophin exon;
Capable of binding to, targeting, hybridizing and / or reverse complementary to a region of the second dystrophin exon;
Comprising or consisting of an array,
The region of the second exon within the dystrophin mRNA precursor has at least 50% identity with the region of the first exon;
Reducing one or more symptoms of DMD or BMD, and / or reducing one or more characteristics of a patient-derived muscle cell, and / or providing a functional or semi-functional dystrophin protein to said individual FIG. 6 shows DMD treatment results by providing and / or at least partially reducing abnormal dystrophin protein production in the individual.

  Each of these features has already been defined herein.

  The oligonucleotide comprises a sequence comprising or consisting of a sequence that can bind, target, hybridize, and / or reverse complement to a region of the first DMD or dystrophin exon, and a second DMD within the same mRNA precursor. Alternatively, the region of dystrophin is preferably at least 50% identical to the region of the first exon. The first and second exons are preferably selected from the group of exons comprising exons 8-60, and the exon stretch starts with the first exon and the second exon as the last exon. If included and skipped, an in-frame transcript is produced. Preferred in-frame exon combinations in dystrophin mRNA are given in Tables 1, 2 or 6.

  Without wishing to be bound by any theory, the identity of the first dystrophin exon and the second dystrophin exon can be determined by one or more of the following aspects. In one embodiment, the one or more introns present between the first exon and the second exon are not very large. In this context, very large introns in the dystrophin gene are 70, 80, 90, 100, 200 kb or more; for example, intron 1 (about 83 kb), intron 2 (about 170 kb), intron 7 (about 110 kb), intron 43 (about 70 kb), intron 44 (about 248 kb), intron 55 (about 119 kb) or intron 60 (about 96 kb). In addition to further embodiments, the introns may already be an example of a BMD patient that expresses a truncated dystrophin protein, the first, second, and second exons from the first exon to the second exon. Exon stretch is deleted. Another criterion may be the relatively large applicability of skipped exons for a combined subpopulation of DMD (and / or BMD) patients with certain associated mutations.

In a preferred embodiment, the in-frame stretch of the DMD exon is skipped (more preferably skipped completely within one transcript) and the outer exon is defined by the first and second exons as follows: The
The first exon is exon 8 and the second exon is exon 19 (applicable to about 7% DMD patients);
The first exon is exon 9 and the second exon is exon 22 (applicable to about 11% DMD patients);
The first exon is exon 9 and the second exon is exon 30 (applicable to about 14% DMD patients);
The first exon is exon 10 and the second exon is exon 18 (applicable to about 5% DMD patients);
The first exon is exon 10 and the second exon is exon 30 (applicable to about 13% DMD patients);
The first exon is exon 10 and the second exon is exon 42 (applicable to about 16% DMD patients);
The first exon is exon 10 and the second exon is exon 47 (applicable to about 29% DMD patients);
The first exon is exon 10 and the second exon is exon 57 (applicable to about 72% DMD patients);
The first exon is exon 10 and the second exon is exon 60 (applicable to about 72% DMD patients);
The first exon is exon 11 and the second exon is exon 23 (applicable to about 8% DMD patients);
The first exon is exon 13 and the second exon is exon 30 (applicable to about 10% DMD patients);
The first exon is exon 23 and the second exon is exon 42 (applicable to about 7% DMD patients);
The first exon is exon 34 and the second exon is exon 53 (applicable to about 42% DMD patients);
The first exon is exon 40 and the second exon is exon 53 (applicable to about 38% DMD patients);
The first exon is exon 44 and the second exon is exon 56 (applicable to about 40% DMD patients);
The first exon is exon 45 and the second exon is exon 51 (applicable to about 17% DMD patients);
The first exon is exon 45 and the second exon is exon 53 (applicable to about 28% DMD patients);
The first exon is exon 45 and the second exon is exon 55 (applicable to about 33% DMD patients);
The first exon is exon 45 and the second exon is exon 60 (applicable to about 37% of DMD patients); or the first exon is exon 56 and the second exon is exon 60 Yes (applicable to about 2% DMD patients).

  Thus, in one embodiment, an oligonucleotide of the invention comprises the following dystrophin exon skipping: exons 8-19, exons 9-22, exons 9-30, exons 10-18, exons 10-30, exons 10 42, exon 10 to 47, exon 10 to 57, exon 10 to 60, exon 11 to 23, exon 13 to 30, exon 23 to 42, exon 34 to 53, exon 40 to 53, exon 44 to 56, exon 45 51, exons 45 to 53, exons 45 to 55, exons 45 to 60, or exons 56 to 60.

  In the oligonucleotide of the present invention, the reverse complementary portion is at least 30% of the length of the oligonucleotide of the present invention, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably The first of the dystrophin mRNA precursors is such that it is at least 70%, more preferably at least 80%, more preferably at least 90% or more preferably at least 95% or more preferably 98% and most preferably at most 100%. It preferably comprises or consists of a sequence that can bind to, target, hybridize and / or reverse complement to a region of an exon. In this context, the first exon is preferably the exon 8, 9, 10, 11, 13, 23, 34, 40, 44, 45 or 56 of the dystrophin mRNA precursor as defined herein. . Said oligonucleotide may comprise further flanking sequences. In a more preferred embodiment, the length of the reverse complementary portion of the oligonucleotide is at least 10, 11, 12, 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 or 40 nucleotides. Several types of flanking sequences may be used. It is preferred that flanking sequences are used to alter protein binding to the oligonucleotide or to alter the thermodynamic properties of the oligonucleotide, more preferably to alter the target RNA binding affinity. In another preferred embodiment, the additional flanking sequence is reverse complementary to the sequence of a dystrophin mRNA precursor that is not present in the exon.

  In a preferred embodiment, the oligonucleotide is capable of binding to, targeting, hybridizing and reverse complementary to at least a region of the first dystrophin exon and a region of the second dystrophin exon present in the dystrophin mRNA precursor. The first exon and the second exon comprise or consist of exons 8 (SEQ ID NO: 2), 9 (SEQ ID NO: 3), 10 (SEQ ID NO: 4), 11 (SEQ ID NO: 1761), 13 (SEQ ID NO: 1762), 18 (SEQ ID NO: 5), 19 (SEQ ID NO: 6), 22 (SEQ ID NO: 7), 23 (SEQ ID NO: 8), 30 (SEQ ID NO: 9), 34 (SEQ ID NO: 1763), 40 ( Sequence number 1764), 42 (sequence number 11), 44 (sequence number 1765), 45 (sequence number 12), 47 (sequence number 10), 51 (sequence number) 1760), 53 (SEQ ID NO: 13), 55 (SEQ ID NO: 14), 56 (SEQ ID NO: 15), 57 (SEQ ID NO: 1744), or 60 (SEQ ID NO: 16), wherein the region is at least 10 Has nucleotides. However, the region is also at least 11, 12, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, It may have 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides. For the preferred exons described above, one skilled in the art can also identify the region of the first exon and the region of the second exon using techniques known in the art. More preferably, the online tool EMBOSS Matcher is used as described above. Preferred regions of identity of the first dystrophin exon and the second dystrophin exon to which the oligonucleotides of the present invention preferably bind and / or are at least partially reverse complementary are provided in Table 2. Is more preferable. Here, reverse complementary is preferably at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. . Preferred oligonucleotide sequences used in the present invention are preferably capable of binding to, hybridizing, targeting to the region of identity between the first exon and the second exon from Table 2, and And / or reverse complementary, at least 10, 11, 12, 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 or 40 nucleotides, more preferably less than 40 nucleotides or more preferably less than 30 nucleotides, even more preferably less than 25 nucleotides and most preferably 20 to 25 nucleotides Have a length of Here, reverse complementary is preferably at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. .

  In one embodiment, preferred oligonucleotides are those in which the first dystrophin exon and the second dystrophin exon are described in Table 1, 2 or 6, wherein the oligonucleotide is described in Table 2 or 6 and the sequence Such that it can bind to the region of the corresponding first exon and second exon defined in the numbers 17 to 1670, 1742, 1743 or 1766 to 1777.

  Preferred oligonucleotides are those disclosed in Table 3 and comprise or consist of SEQ ID NOs: 1671-1741. Other preferred oligonucleotides comprise or consist of SEQ ID NOs: 1778-1891 disclosed in Table 6. Preferred oligonucleotides comprise SEQ ID NOs: 1671-1741 or 1778-1891, 1, 2, 3, 4 or 5 more nucleotides than those given in Tables 3 or 6, or 1, 2, 3 Has 4 or 5 fewer nucleotides. These additional nucleotides can be 5 'or 3' to the sequence of a given SEQ ID NO. These missing nucleotides can be nucleotides present 5 'or 3' of the sequence of a given SEQ ID NO. Each of these oligonucleotides may have any of the chemicals described above or combinations thereof. In each of the oligonucleotides identified herein by SEQ ID NO, U may be replaced with T.

  More preferred oligonucleotides are shown below.

  When the first dystrophin exon is exon 8 and the second dystrophin exon is exon 19, the preferred region of exon 8 comprises or consists of SEQ ID NO: 17, and the preferred region of exon 19 comprises SEQ ID NO: 18. Or consist of. Preferred oligonucleotides consist of or comprise SEQ ID NOs: 1722 or 1723 and have a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 13, the preferred region of exon 10 comprises or consists of SEQ ID NO: 101, and the preferred region of exon 13 comprises SEQ ID NO: 102 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 14, the preferred region of exon 10 comprises or consists of SEQ ID NO: 103, and the preferred region of exon 14 comprises SEQ ID NO: 104 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 15, the preferred region of exon 10 comprises or consists of SEQ ID NO: 105, and the preferred region of exon 15 comprises SEQ ID NO: 106 Or consist of.

When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 18, the preferred region of exon 10 comprises or consists of SEQ ID NO: 109, and the preferred region of exon 18 comprises SEQ ID NO: 110 Or consist of. Preferred oligonucleotides include:
A nucleotide sequence defined in any one of SEQ ID NOs: 1679-1681, 1778, 1812, 1813, 1884-1886, 1890 or 1891 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or a sequence Defined as No. 1814 and having a length of 26, 27, 28, 29 or 30 nucleotides, or as defined in any one of SEQ ID Nos. 1815 to 1819, and 22, 23, 24, 25, 26, 27 , 28, 29 or 30 nucleotides in length, or as defined in any one of SEQ ID NOs: 1820, 1824, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or A nucleotide sequence having a length of 30 nucleotides, or any of SEQ ID NOs: 1826, 1782, 1832 Any one of base sequences having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or SEQ ID NO: 1821, 1825, 1780. Defined as any one of the base sequences having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or SEQ ID NO: 1822, and 24, 25, 26, 27, A nucleotide sequence having a length of 28, 29 or 30 nucleotides, or a nucleotide sequence as defined in any one of SEQ ID NOs: 1823, 1781, 1829, 1830, 1831 A nucleotide sequence having
Defined as any one of SEQ ID NO: 1887 and defined as any one of base sequences having a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any one of SEQ ID NO: 1888 or 1889; , 27, 28, 29 or 30 nucleotides in length, or as defined in SEQ ID NO: 1827 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides The base sequence or the base sequence defined in SEQ ID NO: 1828 and having a length of 25, 26, 27, 28, 29, or 30 nucleotides.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 18, another preferred region of exon 10 comprises or consists of SEQ ID NO: 1766, and another preferred region of exon 18 is the sequence Contains or consists of the number 1767. Preferred oligonucleotides are defined in any one of SEQ ID NOs: 1783, 1833, 1834, 1835 and are 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 It includes a base sequence having a nucleotide length.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 18, another preferred region of exon 10 comprises or consists of SEQ ID NO: 1768, and another preferred region of exon 18 is the sequence Contains or consists of the number 1769. Preferred oligonucleotides comprise a base sequence as defined in any one of SEQ ID NOs: 1673 or 1674 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 20, the preferred region of exon 10 comprises or consists of SEQ ID NO: 111, and the preferred region of exon 20 comprises SEQ ID NO: 112 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 27, the preferred region of exon 10 comprises or consists of SEQ ID NO: 121, and the preferred region of exon 27 comprises SEQ ID NO: 122 Or consist of.

When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 30, the preferred region of exon 10 comprises or consists of SEQ ID NO: 127, and the preferred region of exon 30 comprises SEQ ID NO: 128. Or consist of. Preferred oligonucleotides are
A nucleotide sequence defined in any one of SEQ ID NOS: 1679-1681, 1812, 1813, 1884-1886, 1890 or 1891 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or SEQ ID NO: 1814 Or a nucleotide sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or as defined in SEQ ID NO: 1675 or 1676, 21, 22, 23, 24, 25, 26, 27, 28, 29 or A nucleotide sequence having a length of 30 nucleotides, or a nucleotide sequence defined in SEQ ID NO: 1677 or 1678 and having a length of 25, 26, 27, 28, 29, or 30 nucleotides, or any one of SEQ ID NOs: 1784, 1836 21, 22, 23, 24, 25, 26, 27 A base sequence having a length of 28, 29 or 30 nucleotides, or a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in any one of SEQ ID NOs: 1786, 1838, Or a nucleotide sequence defined in any one of SEQ ID NO: 1780 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any one of SEQ ID NOs: 1785, 1837 And includes a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 30, another preferred region of exon 10 comprises or consists of SEQ ID NO: 1772, and another preferred region of exon 30 is the sequence Contains or consists of the number 1773. Preferred oligonucleotides are
A nucleotide sequence defined in any one of SEQ ID NOs: 1688, 1689, 1839, 1840, 1841, 1842, 1843 or 1844 and having a length of 26, 27, 28, 29 or 30 nucleotides, or SEQ ID NOs: 1845, 1846 , 1847, 1848, a nucleotide sequence having a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any one of SEQ ID NOs: 1849, 1850, A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or defined as any one of SEQ ID NOs: 1787, 1851, and 26, 27, 28, 29, or 30 It includes a base sequence having a nucleotide length.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 31, a preferred region of exon 10 comprises or consists of SEQ ID NO: 129, and a preferred region of exon 31 comprises SEQ ID NO: 130. Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 32, the preferred region of exon 10 comprises or consists of SEQ ID NO: 131, and the preferred region of exon 32 comprises SEQ ID NO: 132 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 35, the preferred region of exon 10 comprises or consists of SEQ ID NO: 137, and the preferred region of exon 35 comprises SEQ ID NO: 138. Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 42, a preferred region of exon 10 comprises or consists of SEQ ID NO: 151, and a preferred region of exon 42 comprises SEQ ID NO: 152 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 44, a preferred region of exon 10 comprises or consists of SEQ ID NO: 153, and a preferred region of exon 44 comprises SEQ ID NO: 154 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 47, a preferred region of exon 10 comprises or consists of SEQ ID NO: 157, and a preferred region of exon 47 comprises SEQ ID NO: 158 Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 48, a preferred region of exon 10 comprises or consists of SEQ ID NO: 159, and a preferred region of exon 48 comprises SEQ ID NO: 160. Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 55, a preferred region of exon 10 comprises or consists of SEQ ID NO: 167, and a preferred region of exon 55 comprises SEQ ID NO: 168. Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 57, the preferred region of exon 10 comprises or consists of SEQ ID NO: 169, and the preferred region of exon 57 comprises SEQ ID NO: 170. Or consist of.

  When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 60, the preferred region of exon 10 comprises or consists of SEQ ID NO: 173, and the preferred region of exon 60 comprises SEQ ID NO: 174. Or consist of.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1673 and have a length of 25, 26, 27, 28, 29, or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1675 and have a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1677 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1679 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides. Preferred oligonucleotides comprise the base sequence of SEQ ID NO: 1679 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides. Optionally, U's 1, 2, 3, 4, 5, 6, 7 of SEQ ID NO: 1679 are replaced / replaced by T. In a preferred embodiment, all U of SEQ ID NO: 1679 are replaced with T. Preferred oligonucleotides comprising SEQ ID NO: 1679 comprise any of the above chemistries: base modifications and / or sugar modifications and / or backbone modifications.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1681 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1684 and have a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1685 and have a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1686 and have a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1688 and have a length of 26, 27, 28, 29 or 30 nucleotides. Preferred oligonucleotides comprise the base sequence of SEQ ID NO: 1688 and have a length of 26, 27, 28, 29 or 30 nucleotides. Optionally, U's 1, 2, 3, 4, 5, 6 of SEQ ID NO: 1688 are replaced / replaced by T. In a preferred embodiment, all U of SEQ ID NO: 1688 are replaced with T. Preferred oligonucleotides comprising SEQ ID NO: 1688 comprise any of the chemistries defined herein above: base modifications and / or sugar modifications and / or backbone modifications.

  For each of the oligonucleotides represented by SEQ ID NOs: 1673, 1675, 1677, 1679, 1681, 1684, 1685, 1686 and 1688, the first dystrophin exon is exon 10. However, the second dystrophin exon is exon 13, 14, 15, 18, 20, 27, 30, 31, 32, 35, 42, 44, 47, 48, 55, 57 or 60. This means that by using any of these oligonucleotides, several formations of in-frame transcripts can occur, each leading to the production of a (semi-) functional dystrophin protein that has been cleaved. Means that.

When the first dystrophin exon is exon 11 and the second dystrophin exon is exon 23, the preferred region of exon 23 comprises or consists of SEQ ID NO: 191 and the preferred region of exon 23 comprises SEQ ID NO: 192 Or consist of. Preferred oligonucleotides are
A nucleotide sequence defined in any one of SEQ ID NOs: 1794, 1861, 1795, 1862 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or SEQ ID NO: 1796 , 1863, 1797, 1864, a base sequence having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or SEQ ID NOs: 1798, 1865, 1799, 1866 It includes a base sequence defined as any one and having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

When the first dystrophin exon is exon 13 and the second dystrophin exon is exon 30, the preferred region of exon 13 comprises or consists of SEQ ID NO: 285, and the preferred region of exon 30 comprises SEQ ID NO: 286. Or consist of. Preferred oligonucleotides are
Defined as any one of SEQ ID NOs: 1808, 1867, 1809, 1868, 1810, 1869, 1858, 1873 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides Having a nucleotide sequence,
A nucleotide sequence defined in any one of SEQ ID NOs: 1811, 1870, 1859, 1874 and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or SEQ ID NOs: 1856, 1871 , 1860, 1875, a base sequence having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or defined as any one of SEQ ID NOs: 1857, 1872 And includes a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides.

  When the first dystrophin exon is exon 23 and the second dystrophin exon is exon 42, the preferred region of exon 23 comprises or consists of SEQ ID NO: 776 and the preferred region of exon 42 comprises SEQ ID NO: 777. Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1698-1703.

When the first dystrophin exon is exon 34 and the second dystrophin exon is exon 53, a preferred region of exon 34 comprises or consists of SEQ ID NO: 1294, and a preferred region of exon 53 comprises SEQ ID NO: 1295 Or consist of. Preferred oligonucleotides are
A nucleotide sequence defined in any one of SEQ ID NOs: 1800, 1876, 1801, 1877 and having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides; Or a nucleotide sequence defined in any one of SEQ ID NOs: 1802, 1878, 1803, 1879 and having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

When the first dystrophin exon is exon 40 and the second dystrophin exon is exon 53, a preferred region of exon 40 comprises or consists of SEQ ID NO: 1477, and a preferred region of exon 53 comprises SEQ ID NO: 1478 Or consist of. Preferred oligonucleotides are
A nucleotide sequence defined in any one of SEQ ID NOs: 1804 and 1880 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any of SEQ ID NOs: 1805 and 1881 Or a base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

  When the first dystrophin exon is exon 44 and the second dystrophin exon is exon 56, a preferred region of exon 44 comprises or consists of SEQ ID NO: 1577, and a preferred region of exon 56 comprises SEQ ID NO: 1558 Or consist of. Preferred oligonucleotides are defined in any one of SEQ ID NOs: 1806, 1882, 1807, 1883 and have a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides Contains the base sequence.

  When the first dystrophin exon is exon 45 and the second dystrophin exon is exon 51, a preferred region of exon 45 comprises or consists of SEQ ID NO: 1567, and a preferred region of exon 51 comprises SEQ ID NO: 1568 Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1730-1731.

  When the first dystrophin exon is exon 45 and the second dystrophin exon is exon 53, a preferred region of exon 45 comprises or consists of SEQ ID NO: 1569, and a preferred region of exon 53 comprises SEQ ID NO: 1570 Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1732-1737.

  When the first dystrophin exon is exon 45 and the second dystrophin exon is exon 55, a preferred region of exon 45 comprises or consists of SEQ ID NO: 1571 and a preferred region of exon 55 comprises SEQ ID NO: 1572 Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1704-1719, 1788, 1852, 1789, 1853.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1706 and have a length of 25, 26, 27, 28, 29, or 30 nucleotides. A preferred oligonucleotide comprising SEQ ID NO: 1706 and having a length of 25 nucleotides consists of SEQ ID NO: 1706.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1707 and have a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. A preferred oligonucleotide comprising SEQ ID NO: 1707 and having a length of 25 nucleotides consists of SEQ ID NO: 1706.

  Preferred oligonucleotides consist of or comprise SEQ ID NO: 1713 and have a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. A preferred oligonucleotide comprising SEQ ID NO: 1713 and having a length of 25 nucleotides consists of SEQ ID NO: 1710.

  Preferred oligonucleotides comprise the base sequence defined in any one of SEQ ID NOs: 1788, 1852, 1789, 1853 and have a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides.

  When the first dystrophin exon is exon 45 and the second dystrophin exon is exon 55, another preferred region of exon 45 comprises or consists of SEQ ID NO: 1774, and another preferred region of exon 55 is the sequence Contains or consists of the number 1775. Preferred oligonucleotides comprise a base sequence defined in any one of SEQ ID NOs: 1790, 1854, 1792, 1855 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

  When the first dystrophin exon is exon 45 and the second dystrophin exon is exon 60, a preferred region of exon 45 comprises or consists of SEQ ID NO: 1577, and a preferred region of exon 60 comprises SEQ ID NO: 1578 Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1738-1741.

  When the first dystrophin exon is exon 56 and the second dystrophin exon is exon 60, a preferred region of exon 56 comprises or consists of SEQ ID NO: 1742, and a preferred region of exon 60 comprises SEQ ID NO: 1743 Or consist of. Preferred oligonucleotides comprise or consist of SEQ ID NOs: 1720-1721.

More preferred oligonucleotides include:
(A) a nucleotide sequence defined in SEQ ID NO: 1673 or 1673 and having a length of 25, 26, 27, 28, 29, or 30 nucleotides; or (b) defined in SEQ ID NO: 1675 or 1676, 21, 22, 23 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (c) 25, 26, 27, 28, 29 or 30 nucleotides long as defined in SEQ ID NO: 1677 or 1678 (D) a nucleotide sequence defined in any one of SEQ ID NOS: 1679 to 1681 and having a length of 25, 26, 27, 28, 29, or 30 nucleotides, or (e) a SEQ ID NO: 1684 to 1686, 21, 22, 23, 24, 25, 26, 27, 28, 29 Or a base sequence having a length of 30 nucleotides, or (f) a base sequence defined by SEQ ID NO: 1688 or 1689 and having a length of 26, 27, 28, 29, or 30 nucleotides, or (g) SEQ ID NO: 1704 A base sequence defined in any one of -1706 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides;
(H) a nucleotide sequence defined in any one of SEQ ID NOs: 1707 to 1709 and having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (i) SEQ ID NOs: 1710, 1713 A nucleotide sequence defined in any one of -1717 and having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

Further preferred oligonucleotides include:
(A) is defined in any one of SEQ ID NOS: 1679-1681, 1778, 1812, 1813, 1884-1886, 1890 or 1891 and has a length of 25, 26, 27, 28, 29 or 30 nucleotides, Or a nucleotide sequence defined in SEQ ID NO: 1814 and having a length of 26, 27, 28, 29, or 30 nucleotides, or (b) defined in SEQ ID NO: 1688, 1689 or 1839-1844, 26, 27, 28, 29 Or a base sequence having a length of 30 nucleotides, or (c) a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in SEQ ID NO: 1673 or 1674, or (d) a SEQ ID NO: 1675 or 1676, 21, 22, 23, 24, 25, 26, 27, 28, 2 Or a nucleotide sequence having a length of 30 nucleotides, or (e) a nucleotide sequence defined in SEQ ID NO: 1677 or 1678 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (f) a SEQ ID NO. A base sequence defined in any one of 1684 to 1686 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (g) of SEQ ID NOs: 1704 to 1706 A base sequence defined in any one and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (h) defined in any one of SEQ ID NOS: 1707 to 1709, 23, 24, A base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (i) any one of SEQ ID NOs: 1710 and 1713 to 1717 A base sequence having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (j) defined in any one of SEQ ID NOs: 1815-1819, 22, 23 , 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (k) defined in any one of SEQ ID NOs: 1820, 1824 and 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 nucleotides in length, or (l) defined in any one of SEQ ID NOs: 1826, 1782, 1832 and 21, 22, 23, 24, 25 , 26, 27, 28, 29 or 30 nucleotides in length, or (m) defined in any one of SEQ ID NOS: 1821, 1825, 1780, 22, 23 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or (n) defined in any one of SEQ ID NO: 1822, 24, 25, 26, 27, 28, 29 Or a base sequence having a length of 30 nucleotides, or (o) a length of 25, 26, 27, 28, 29, or 30 nucleotides as defined in any one of SEQ ID NOs: 1823, 1781, 1829, 1830, 1831 (P) defined in any one of SEQ ID NOs: 1783, 1833, 1834, 1835, and 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29 or 30 nucleotides in length, or (q) any one of SEQ ID NO: 1887, 24, 25, 26, 27, 28, 29 or Is a nucleotide sequence having a length of 30 nucleotides, or (r) a nucleotide sequence defined in any one of SEQ ID NOs: 1888 or 1889 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (s ) As defined in SEQ ID NO: 1827 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (t) as defined in SEQ ID NO: 1828 as 25, 26 27, 28, 29, or 30 nucleotides in length, or (u) defined in any one of SEQ ID NOs: 1784, 1836, 21, 22, 23, 24, 25, 26, 27, 28 , 29 or 30 nucleotides in length, or (v) defined in any one of SEQ ID NOs: 1786, 1838, 25, 26, 27, 28, 2 Or a base sequence having a length of 30 nucleotides, or (w) defined in any one of SEQ ID NO: 1780 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides A base sequence, or (x) a base sequence defined in any one of SEQ ID NOs: 1785, 1837 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (y) a SEQ ID NO: 1845, 1846 , 1847, 1848, a base sequence having a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (z) any one of SEQ ID NOs: 1849, 1850 A nucleotide sequence defined and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (a1) SEQ ID NO: 1787, 851 and a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or (b1) defined as any one of SEQ ID NOs: 1788, 1852, 1789, 1853, A base sequence having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (c1) defined in any one of SEQ ID NOs: 1790, 1854, 1792, 1855, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (d1) defined in any one of SEQ ID NOs: 1794, 1861, 1795, 1862, 21, 22, 23, 24, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (e1) any of SEQ ID NOs: 1796, 1863, 1797, 1864 A base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (f1) defined in any one of SEQ ID NOs: 1798, 1865, 1799, 1866 Or a base sequence having a length of 25, 26, 27, 28, 29, or 30 nucleotides, or (g1) defined in any one of SEQ ID NOs: 1808, 1867, 1809, 1868, 1810, 1869, 1858, 1873 Or a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (h1) defined in any one of SEQ ID NOS: 1811, 1870, 1859, 1874 A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (i1) A nucleotide sequence defined in any one of column numbers 1856, 1871, 1860, 1875 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (j1) SEQ ID NO: 1857, Defined in any one of 1872 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (k1) defined in any one of SEQ ID NOs: 1800, 1876, 1801, 1877, A base sequence having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (l1) any one of SEQ ID NOs: 1802, 1878, 1803, 1879 Or a base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (m1) a sequence number A base sequence defined in any one of 1804 and 1880 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (n1) of SEQ ID NOs: 1805 and 1881 A base sequence defined in any one and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (o1) any one of SEQ ID NOs: 1806, 1882, 1807, 1883 A base sequence defined as one and having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

  An oligonucleotide according to the invention comprising or consisting of a sequence defined by a SEQ ID NO is meant to encompass an oligonucleotide comprising a base sequence defined by a SEQ ID NO. In addition, an oligonucleotide having a modified backbone (that is, a modified sugar moiety and / or a modified internucleoside linkage) with respect to the base sequence defined by the SEQ ID NO is included in the present invention. Each base U in the oligonucleotide SEQ ID NOs described herein may be modified with T or replaced.

Composition:
In a further aspect, a composition comprising the oligonucleotide described in the section entitled “Oligonucleotide” is provided. This composition preferably comprises or consists of an oligonucleotide as described above. A preferred composition comprises one single oligonucleotide as described above. Thus, it is clear that at least the skipping of the first exon and the second exon can be obtained using one single oligonucleotide and no cocktail of different oligonucleotides.

In a preferred embodiment, the composition is for use as a medicament. Thus, the composition is a pharmaceutical composition. The pharmaceutical composition usually comprises a pharmaceutically acceptable carrier, diluent and / or excipient. In a preferred embodiment, the composition of the invention comprises a compound as defined herein and is a pharmaceutically acceptable formulation, filler, preservative, solubilizer, carrier, diluent, excipient, Optionally further comprises a salt, an adjuvant and / or a solvent. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, diluents, salts, adjuvants, solvents and / or excipients are found, for example, in Remington. The compounds described in the present invention have at least one ionic group. The ionic group may be a base or acid, and may be charged or neutral. The ionic group may be present as an ion pair with a suitable counter ion having the opposite charge. Examples of cationic counter ions are sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium and tetraalkylammonium. Examples of anionic counterions are chloride, bromide, iodide, lactate, mesylate, acetate, trifluoroacetate, dichloroacetate and citrate. Examples of counter ions have been described (Kumar L., which is incorporated herein by reference in its entirety). Thus, in a preferred embodiment, the oligonucleotide of the invention is contacted with a composition comprising such an ionic group, preferably Ca ²⁺ , such a polyvalent cation as already defined herein. To form an oligonucleotide chelate complex comprising two or more identical oligonucleotides linked by

  In particular, the pharmaceutical composition may further assist in enhancing the stability, solubility, absorbability, bioavailability, pharmacokinetics and cellular uptake of said compound, in particular complex, nanoparticle, microparticle, nanotube, nanogel, It may be further formulated into a formulation comprising excipients or conjugates capable of forming hydrogels, poloxamers or pluronics, polymersomes, colloids, microbubbles, vesicles, micelles, lipoplexes and / or liposomes. Examples of nanoparticles include polymer nanoparticles, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, protein nanoparticles and peptide nanoparticles.

  Preferred compositions comprise at least one excipient that may further assist in enhancing the targeting and / or delivery of the composition and / or the oligonucleotide into muscle and / or cells. The cell may be a muscle cell.

  Another preferred composition may comprise at least one excipient classified as a second type of excipient. The second type of excipient is a target of the composition and / or oligonucleotide of the present invention to and / or into tissues and / or cells, eg muscle tissue or muscle cells In order to enhance the synthesis and / or delivery, it may include or contain a conjugate group as described herein. Both types of excipients may be combined together in one single composition as described herein. Preferred conjugate groups are disclosed in the definition part.

  One skilled in the art can select, combine and / or select one or more of the above or other alternative excipients and delivery systems to formulate and deliver a compound for use in the present invention. Can be adapted.

  Such a pharmaceutical composition of the present invention can be administered to an animal, preferably a mammal, in an effective concentration at a set time. A more preferred mammal is a human. A compound or composition as defined herein for use in accordance with the present invention may be suitable for in vivo direct administration to an individual's cells, tissues and / or organs, said individual receiving BMD or DMD It is preferred that they are affected or at risk of developing them and can be administered directly in vivo, ex vivo or in vitro. Administration is via systemic and / or parenteral routes, for example, intravenous, subcutaneous, intraventricular, intrathecal, intramuscular, intranasal, intestinal, intravitreal, intracerebral, epidural or oral routes. There may be. Such pharmaceutical compositions of the present invention may be in the form of emulsions, suspensions, pills, tablets, capsules or soft gels for oral delivery, or aerosols or dried for delivery to the respiratory tract and lungs. It can be preferably encapsulated in the form of a powder.

  In one embodiment, the compounds of the invention may be used in conjunction with other compounds that are already known to be used in the treatment of the diseases. Such other compounds may be used to slow disease progression, for example to reduce abnormal behavior or behavior, to reduce muscle tissue inflammation, to improve muscle fiber function, integrity and / or survival, And / or can be used to improve, increase or repair cardiac function. Examples include but are not limited to steroids, preferably (carbohydrate) corticosteroids, ACE inhibitors (preferably perindopril), angiotensin II type 1 receptor blockers (preferably losartan), tumor necrosis factor-alpha (TNFα). Inhibitor, TGFβ inhibitor (preferably decorin), human recombinant biglycan, mIGF-1 source, myostatin inhibitor, mannose-6-phosphate, antioxidant, ion channel inhibitor, protease inhibitor, phosphodiesterase inhibitor ( PDE5 inhibitors such as sildenafil or tadalafil), L-arginine, dopamine blockers, amantadine, tetrabenazine and / or coenzyme Q10. This combined use may be sequential use: each component is administered in a different composition. Alternatively, each compound may be used together in a single composition.

use:
In a further aspect, there is provided the use of a composition or compound described herein for use as a medicament or as part of a therapy, or an application in which the compound confers its activity intracellularly.

  In a preferred embodiment, a compound or composition of the invention is for use as a medicament, which medicament prevents, delays, ameliorates and / or prevents a disease as defined herein, preferably DMD or BMD. To treat.

Methods for preventing, delaying, ameliorating and / or treating a disease:
In a further aspect, methods are provided for preventing, delaying, ameliorating and / or treating a disease as defined herein, preferably DMD or BMD. The disease can be prevented, treated, delayed or ameliorated in an individual in the cell, tissue or organ of the individual. The method includes the step of administering an oligonucleotide or composition of the invention to said individual or subject in need thereof.

  The method according to the invention provides for the in vivo of an oligonucleotide or composition as defined herein in an individual, preferably an individual suffering from or at risk of developing such a disease. Can be suitable for administration to cells, tissues and / or organs and can be administered in vivo, ex vivo or in vitro. The individual or subject in need is preferably a mammal, more preferably a human.

  In one embodiment, in the methods of the invention, the concentration of oligonucleotide or composition ranges from 0.01 nM to 1 μM. The concentration used is more preferably 0.02 to 400 nM, 0.05 to 400 nM or 0.1 to 400 nM, even more preferably 0.1 to 200 nM.

  The dose range of the oligonucleotides or compositions according to the invention is preferably designed based on increasing dose studies in clinical trials (in vivo use) where strict protocol requirements exist. Oligonucleotides as defined herein may be from 0.01 to 200 mg / kg or from 0.05 to 100 mg / kg or from 0.1 to 50 mg / kg or from 0.1 to 20 mg / kg, preferably from 0.5 It can be used at doses ranging from 10 mg / kg.

  The concentration or dose range of oligonucleotides or compositions given above is a preferred concentration or dose for in vitro or ex vivo use. The skilled person will depend on the identity of the oligonucleotide used, the target cell to be treated, the gene target and its expression level, the medium used and the transfection and incubation conditions, the concentration or dose of said oligonucleotide used. It will be appreciated that may vary further and may require further optimization.

Definition:
“Sequence identity” as known in the art is a relationship between two or more nucleic acid (polynucleotide or nucleotide) sequences, as determined by comparing the sequences. In the art, “identity” or “similarity” refers to the degree of sequence relatedness between nucleic acid sequences as determined by match between strands of such sequences. “Identity” may be replaced herein by “similarity”. Preferably, identity is determined by comparing all SEQ ID NOs described herein. However, part of the sequence can also be used. Wherein a portion of the sequence is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of a given sequence or SEQ ID NO Can mean.

  “Identity” and “similarity” include, but are not limited to, Computational Molecular Biology, Lesk, A. et al. M.M. Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. et al. W. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A .; M.M. And Griffin, H .; G. Ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G. , Academic Press, 1987; and Sequence Analysis Primer Griskov, M .; And Devereux, J. et al. Ed., M Stockton Press, New York, 1991; , And Lipman, D .; SIAM J .; Applied Math. 48: 1073 (1988) and can be readily calculated by known methods.

  Preferred methods for determining identity are designed to give the greatest match between the sequences tested. Methods for determining identity and similarity are codified in published computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit and FASTA. (Altschul, SF, et al., J. Mol. Biol. 215: 403-410 (1990). The BLAST 2.0 family of programs that can be used for database similarity searches include, for example, nucleotide databases. BLASTN for nucleotide query sequences against sequences is included The BLAST 2.0 family program is NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI N M NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)) The well-known Smith Waterman algorithm is also used to determine identity. May be.

  Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. et al. Mol. Biol. 48: 443-453 (1970); comparison matrix: match = + 10, mismatch = 0; gap penalty: 50; gap length penalty: 3. As a Gap program, a Gap program made by Genetics Computer Group in Madison, Wisconsin can be used. Given above are the default parameters for nucleic acid comparisons.

  Another preferred method for determining sequence similarity and identity is the algorithm Needleman-Wunsch (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453, Kruskal. , J. B. (1983) An overview of sequence comparision in D. Sankoff and J. B. Kruskal ed., Time warps, string edits and macromolecules: the theority and ce p By using.

  Another preferred method for determining sequence similarity and identity is the EMBOSS Matcher and Waterman-Eggert algorithm (local alignment of two sequences; [Schoniger and Waterman, Bulletin of Mathematical Biology 1992, Vol. 54, 4). pp. 521-536; Vingron and Waterman, J. Mol. Biol. 1994; 235 (1), p1-12.]). The following website can be used: http: // www. ebi. ac. uk / Tools / emboss / align / index. html or http: // emboss. bioinformatics. nl / cgi-bin / emboss / matcher. Definitions of parameters used in this algorithm can be found at the following website: http: // emboss. sourceforge. net / docs / themes / AlignFormats. html # id. The default settings (matrix: EDNAFULL, gap_penalty: 16, extension_penalty: 4) are preferably used. Emboss Matcher provides optimal local alignment between the two sequences, but alternative alignments are provided as well. Table 2 discloses the optimal local alignment between two different exons, preferably dystrophin exons, preferably used for oligonucleotide design. However, alternative alignments and thereby alternative identity regions between two exons can also be identified and used for oligonucleotide design, which is also part of the present invention.

  Throughout this specification, the terms “bind”, “target”, “hybridize” are in the context of oligonucleotides that are reverse complementary to the regions of the mRNA precursor described herein. If used, it can be used interchangeably. Similarly, the expressions "can bind", "can be targeted" and "can hybridize" indicate that the oligonucleotide has a particular sequence that can bind, target or hybridize to the target sequence. Can be used interchangeably. Whenever a target sequence is defined herein or known in the art, one of skill in the art can construct all possible structures of the oligonucleotide using the concept of reverse complement. In this regard, a limited number of sequence mismatches or gaps between the oligonucleotide of the invention and the target sequence in the first exon and / or the second exon are described above unless binding is affected. As will be appreciated, it is acceptable. Thus, an oligonucleotide that can bind to a particular target sequence can be considered an oligonucleotide that is reverse complementary to the target sequence. In the context of the present invention, “hybridize” or “capable of hybridizing” is used under physiological conditions in cells, preferably human cells unless otherwise specified.

  As used herein, “hybridization” refers to the pairing of complementary oligonucleotide compounds (eg, an antisense compound and its target nucleic acid). Although not limited by a particular mechanism, the most common mechanism of pairing is Watson-Crick, Hoogsteen or inverted Hoogsteen hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases). Possible hydrogen bonds. For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymine, 5-methyluracil and uracil that pair by hydrogen bond formation. The natural base guanine is a nucleobase complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur under variable conditions.

Similarly, “reverse complementary” is a two nucleotide sequence that can hybridize to each other, but one of the sequences is oriented 3 ′ to 5 ′ and the other is oriented in the opposite direction, ie 5 ′ to 3 ′. Used to identify Thus, nucleoside A in the first nucleotide sequence can be paired with nucleoside A ^* in the second nucleotide sequence via their respective nucleobases, and 5 ′ position from nucleoside A described above in the first nucleotide sequence. The nucleoside B located at can be paired with the nucleoside B ^* located 3 ′ from the nucleoside A ^* described above in the second nucleotide sequence. In the context of the present invention, the first nucleotide sequence is typically an oligonucleotide of the present invention and the second nucleotide sequence portion is an exon derived from an mRNA precursor, preferably a dystrophin mRNA precursor. The oligonucleotide is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% reverse complementary to the region of the first and / or second exon. In some cases, the oligonucleotide is preferably said to be reverse complementary to a region of an exon (first exon and / or second exon). The reverse complement is preferably at least 80%, 85%, 90%, 95% or 100%.

  In the present invention, the excipient includes a polymer (for example, polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivative, butyl cyanoacrylate (PBCA), hexyl cyanoacrylate (PHCA), poly (lactic acid-co-glycol). Acid) (PLGA), polyamines (eg, spermine, spermidine, putrescine, cadaverine), chitosan, poly (amidoamine) (PAMAM), poly (ester amine), polyvinyl ether, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), Cyclodextrin, hyaluronic acid, colominic acid and their derivatives), dendrimers (eg poly (amidoamine)), lipids {eg 1,2-dioleoyl-3-dimethylammoniumpropane (DO AP), dioleoyldimethylammonium chloride (DODAC), phosphatidylcholine derivatives [eg 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)], lyso-phosphatidylcholine derivatives [eg 1-stearoyl-2- Lyso-sn-glycero-3-phosphocholine (S-lysoPC)], sphingomyelin, 2- {3- [bis- (3-amino-propyl) -amino] -propylamino} -N-ditetracedylcarbamoylmethyl Acetamide (RPR209120), phosphoglycerol derivatives [eg 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na), phosphatidic acid derivatives [1,2-distear Royl-sn-glycero-3-phosphatidic acid, sodium salt (DSPA), phosphatidylethanolamine derivatives [eg dioleoyl-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE)], N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium (DOTAP) ), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA), 1,3-di-oleoyloxy-2- (6-carboxy-spermyl) -Propylamide (DOSPER), (1,2-Dimyristiolki Propyl (dimyristolxypropyl) -3-dimethylhydroxyethylammonium (DMRIE), (N1-cholesteryloxycarbonyl-3,7-diazanonan-1,9-diamine (CDAN), dimethyldioctadecylammonium bromide (DDAB), 1-palmitoyl- 2-oleoyl-sn-glycerol-3-phosphocholine (POPC), (b-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (AtuFECT01), N , N-dimethyl-3-aminopropane derivatives [for example, 1,2-distearoyl-N, N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N, N-dimethyl-3- Aminopropane ( oDMA), 1,2-dilinoleyloxy-N, N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl [1,3] -dioxolane (DLin-K-DMA) Phosphatidylserine derivatives [1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS)], cholesterol}, proteins (eg albumin, gelatin, atelocollagen) and peptides (eg , Protamine, PepFect, NickFect, polyarginine, polylysine, CADY, MPG).

  In the present invention, the conjugate group is selected from the group consisting of a target moiety, a stability enhancing moiety, an uptake enhancing moiety, a solubility enhancing moiety, a pharmacokinetic enhancing moiety, a pharmacodynamic enhancing moiety, an activity enhancing moiety, a reporter molecule and a drug, These moieties may be peptides, proteins, carbohydrates, polymers, ethylene glycol derivatives, vitamins, lipids, polyfluoroalkyl moieties, steroids, cholesterol, fluorescent moieties and radiolabeled moieties. The conjugate group can optionally be protected and may be linked directly or via a divalent or multivalent linker to the oligonucleotide of the invention.

Conjugate groups include moieties that enhance targeting, uptake, solubility, activity, pharmacodynamics, pharmacokinetics, and reduce toxicity. Examples of such groups include peptides (eg, glutamine, polyarginine, RXR peptides (see, eg, Antimicrob. Agents Chemother. 2009, 53, 525), polyornithine, TAT, TP10, pAntp, polylysine, NLS, penetratin, MSP, ASSLNIA, MPG, CADY, Pep-1, Pip, SAP, SAP (E), transportan, buforin II, polymyxin B, histatin, CPP5, NickFect, PepFect), vivo-porter (vivo-porter) ), Proteins (eg, antibodies, avidin, Ig, transferrin, albumin), carbohydrates (eg, glucose, galactose, mannose, maltose, maltriose, ribose, Trehalose, glucosamine, N-acetylglucosamine, lactose, sucrose, fucose, arabinose, talose, sialic acid, hyaluronic acid, neuramidinic acid, rhamnose, quinobose, galactosamine, N-acetylgalactosamine, xylose, lyxose, fructose, mannose -6-phosphate, 2-deoxyribose, glucal, cellulobiose, chitobiose, chitotriose), polymers (eg, polyethylene glycol, polyethyleneimine, polylactic acid, poly (amidoamine)), ethylene glycol derivatives (eg, triethylene glycol) , Tetraethylene glycol), water-soluble vitamins (eg, vitamins B, B1, B2) B3, B5, B6, B7, B9, B12, C), fat-soluble vitamins (eg, vitamins A, D, D2, D3, E, K1, K2, K3), lipids (eg, palmityl, myristyl, oleyl, Stearyl, batyl, glycerophospholipid, glycerolipid, sphingolipid, ceramide, cerebroside, sphingosine, sterol, prenol, erucyl, arachidonyl, linoleyl, linolenyl, arachidyl, butyryl, sapeinyl, elaidyl, lauryl, beryl , Decyl, undecyl, octyl, heptyl, hexyl, pentyl, DOPE, DOTAP, terphenyl, diterpenoid, triterpernoid, polyfluoroalkyl moiety (Eg perfluoro [1H, 1H, 2H, 2H] -alkyl), approved drugs with a molecular weight <1500 Da with affinity for specific proteins (eg NSAIDs such as ibuprofen (many are hereby incorporated by reference) Incorporated in US Pat. No. 6,656,730))), antidepressants, antiviral agents, antibiotics, alkylating agents, anti-amoeba agents, analgesics, androgens, ACE inhibitors, appetite Depressant, Antacid, Antiparasitic, Antiangiogenic, Antiadrenergic, Antianginal, Anticholinergic, Anticoagulant, Anticonvulsant, Antidiabetic, Antidiarrheal, Antidiuretic, Detoxification Agents, antifungal agents, antiemetics, anti-angiogenic agents, anti-gout agents, antigonadotropics, antihistamines, antihyperlipidemic agents, antihypertensive agents, antimalarial agents antimalarial), antimigraine, antitumor, antipsychotic, antirheumatic, antithyroid, antitoxin, antitussive, anxiolytic, contraceptive, CNS stimulant, chelating agent, cardiovascular agent, decongestant, Dermatological agent, diuretic agent, expectorant agent, diagnostic agent, gastrointestinal agent, anesthetic agent, glucocorticoid, antiarrhythmic agent (antiarrhytics), immunostimulant, immunosuppressant, laxative, antiprophylactic agent (leprostatics), metabolic agent, respiratory Organs, mucolytic agents, muscle relaxants, nutritional supplements, vasodilators, thrombolytic agents, uterine contractors, vasopressors), natural compounds with molecular weight <2000 Da (eg antibiotics, eicosanoids, alkaloids, flavonoids, terpenoids) , Enzyme cofactors, polyketides), steroids (eg prednisone, prednisolone, dexamethasone, lanostere) Roll, cholic acid, estrane, androstane, pregnane, colan, cholestane, ergosterol, cholesterol, cortisol, cortisone, deflazacoat), pentacyclic triterpenoids (eg, 18β-glycyrrhetinic acid, ursolic acid, amylin, carbenoloxone, enoxolone , Acetoxolone, betulinic acid, asiatic acid, erythrodiol, oleanolic acid), polyamine (eg, spermine, spermidine, putrescine, cadaverine), fluorescent moiety (eg, FAM, carboxyfluorescein, FITC, TAMRA, JOE, HEX, TET, Rhodamine, Cy3, Cy3.5, Cy5, Cy5.5, CW800, BODIPY, AlexaFluors, Dabcyl, DNP Reporter molecule (e.g., acridine, biotin, digoxigenin, (portions labeled with radioactive) isotopes ^{(e.g., 2 H, 13 C, 14} C, 15 N, 18 O, 18 F, 32 P, 35 S, 57 Co, ^99m Tc, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁵³ Gd)) and combinations thereof. Such conjugate groups may be connected directly or via a linker to the compounds of the invention. This linker may be divalent (resulting in a 1: 1 conjugate) or multivalent resulting in an oligomer having more than one conjugate group. Procedures for coupling such conjugate groups to oligomers according to the present invention, either directly or via a linker, are known in the art. Also, for example, J. Org. Am. Chem. Soc. 2008, 130, 12192 (incorporated herein by reference in their entirety; Hurst et al.), To the extent such constructs are referred to as spherical nucleic acids (SNA), the oligos of the invention The use of nanoparticles to which nucleotides are covalently bound is also within the context of the present invention.

  In this specification, the verb “to comprise” and its conjugation are used in its non-limiting sense to mean that the latter part of the term is included, but is described in detail. Items that are not excluded are not excluded. Further, the verb “to consist” may be replaced with “to consist essentially of” and oligonucleotides or compositions as defined herein are specifically Additional components other than those specifically described may be included, which means that the additional components do not alter the intrinsic properties of the present invention. Further, reference to an element by the indefinite article “a” or “an” is more than one unless the context clearly requires that it be one and only one element. Does not exclude the possibility that an element exists. Thus, the indefinite article “a” or “an” usually means “at least one”.

  When used in reference to a numerical value (about 10), the term “about” or “approximately” preferably means that the value can be a given value that is 1% more or less than 10 of that value.

  Each of the embodiments described herein may be combined together unless otherwise specified. All patents and references cited herein are hereby incorporated by reference in their entirety.

  The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[Tables 1-3]
Table 1:
Exon U (upstream) is continuous with exon D (downstream) if any exon from exon U + 1 (first exon) to D-1 (second exon) and any in between are removed from the transcript. List of possible exon combinations in DMD gene transcripts with open reading frames.

Table 2:
A list of exon areas. Optimized by EMBOSS Matcher using default settings (matrix: EDNAFULL; gap_penalty: 16; extension_penalty: 4) (http://www.ebi.ac.uk/tools/psa/emboss_matcher/nucleotide.html) A sequence stretch with (partially) high sequence identity or similarity (at least 50%) between two different dystrophin exons, identified as a pairwise alignment. Skipping of these exons and preferably any intervening exons results in in-frame DMD transcripts (eg, in Table 1).

Table 3:
The list | wrist of the preferable base sequence of the oligonucleotide which concerns on this invention. Oligonucleotides containing these base sequences can bind to regions of high similarity (sequence stretches with> 50% identity) in two different DMD exons (eg as described in Table 2), and these two exons And any exon skipping in between can be induced to generate in-frame DMD transcripts.

* D = 2,6-diaminopurine; C = 5-methylcytosine; X = C or 5-methylcytosine; Y = U or 5-methyluracil; Z = A or 2,6-diaminopurine

Table 6:
List showing most preferred and / or further exon combinations, most preferred and / or further exon identity regions, and most preferred and / or further oligonucleotides, along with their base sequence, chemical modification and sequence identification number . In the list: ^* = LNA; C = 5-methylcytosine; U = 5-methyluracil; D = 2,6-diaminopurine; X = C or 5-methylcytosine; Y = U or 5-methyluracil; Z = A or 2,6-diaminopurine; <> are all 2′-fluoro nucleotides (eg, SEQ ID NO: 1844).

[Examples 1 to 5]
Materials and methods:
Oligonucleotide design was based primarily on reverse complementarity to specific and highly similar sequence stretches in two different DMD exons identified by EMBOSS Matcher and disclosed in Table 2. Additional sequence parameters to be considered are of the partially open / closed secondary RNA structure in the sequence stretch (inferred by RNA structure version 4.5 or RNA mfold version 3.5 (Zuker, M.)) Presence and / or presence of putative SR-protein binding sites in the sequence stretch (estimated by ESE-finder software (Cartegni L et al., 2002 and Cartegni L et al., 2003)). All AONs are the same as Prosensa Therapeutics B. V. (Leiden, Netherlands) or obtained from a commercial source (ChemGenes, US) and contains a 2′-O-methyl RNA and a full length phosphorothioate (PS) backbone. All oligonucleotides were 2′-O-methyl phosphorothioate RNA and were synthesized on a 10 μmol scale using an OP-10 synthesizer (GE / AKTA Oligopilot) according to standard phosphoramidite protocols. Oligonucleotides were cleaved in a two step sequence (DIEA followed by concentrated NH ₄ OH treatment), deprotected, purified by HPLC, dissolved in water, and excess NaCl added to the exchange ion. After evaporation, the compound was redissolved in water, desalted with FPLC and lyophilized. Mass spectrometry confirmed the identity of all compounds and the purity (measured by UPLC) was found to be acceptable (> 80%) for all compounds. For the in vitro experiments described herein, a 50 μM working solution of AON was prepared in phosphate buffered saline (pH 7.0).

Tissue culture, transfection and RT-PCR analysis:
Differentiated human healthy control myocytes (myotubes) were transfected in 6-well plates at a standard AON concentration of 400 nM according to non-GLP standard operating procedures. Polyethyleneimine (ExGen500; Fermentas Netherlands) was used for transfection (2 μl / μg AON in 0.15 M NaCl). The transfection procedure described above was adapted from previously reported materials and methods (Aartsma-Rus A. et al., 2003). Twenty-four hours after transfection, RNA was isolated and analyzed by RT-PCR. Briefly, a random hexamer mixture (1.6 μg / μl, Roche Netherlands) was used in a reverse transcriptase (RT) reaction with an input of 500-1000 ng of RNA to generate cDNA. Subsequently, PCR analysis was performed on 3 μl of cDNA for each sample and included the first PCR and nested PCR using DMD gene specific primers (see Tables 4 and 5). RNA isolation and RT-PCR analysis were performed according to non-GLP standard operating procedures adapted from previously reported materials and methods (Aartsma-Rus A. et al., 2002 and Aartsma-Rus A. et al., 2003). RT-PCR products were analyzed by gel electrophoresis (2% agarose gel). The resulting RT-PCR fragments were quantified by DNA Lab-on-a-Chip analysis (Agilent Technologies USA; DNA 7500 LabChips). Data were processed by “Agilent 2100 Bioanalyzer” software and Excel 2007. The ratio of the smaller transcript (containing the predicted multiple exon skipping) to the total amount of transcript was evaluated (expressing the skipping efficiency in percent) and directly compared to that in untransfected cells. PCR fragments were also isolated from agarose gels (QIA quick gel extraction kit, Qiagen Netherlands) for sequence verification (Sanger sequencing, BaseClear, Netherlands).

Table 4:
PCR primer set used for detection of target exon skipping

Table 5:
Primer sequence

result:
Example 1:
Targeting sequence stretches with high similarity in exons 10 and 18 with AON at different sites.
A series of AONs were designed based on the high similarity (63%) sequence stretch in exons 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110), and exon 10 (PS814, SEQ ID NO: 1675; PS815, SEQ ID NO: 1677). Dispersed over the sequence stretch that is 100% reverse complementary to either PS816, SEQ ID NO: 1679) or exon 18 (PS811, SEQ ID NO: 1673); After transfection in healthy human control myotube cultures, RT-PCR analysis demonstrated that all four AONs can induce exon 10-18 skipping (confirmed by sequence analysis) (FIG. 1). ). PS811 and PS816 had the highest percentage of reverse complementarity in both exons (FIG. 1B) and were most effective with skipping efficiencies of exons 10-18 of 70% and 66%, respectively (FIG. 1C). PS814 is least effective, which may be inherent in its position and / or shorter length (21 versus 25 nucleotides), and may thus be lower binding affinity or stability. Exon 10-18 skipping was not observed in untreated cells (NT). These results are highly reproducible (skip exons 10-18 by PS816 in 20/20 different transfections), and skipping of exon stretches from exon 10-18 is highly sequence similar (63% ) Has been demonstrated to be feasible by using a single AON that can bind to both exons 10 and 18 in the region having) and can induce skipping of these exons and all intervening exons. Multiple additional exon skipping fragments were obtained in this transcript region, but the resulting transcript in which exon 9 was spliced directly to 19 (in-frame transcript) was most abundant in all experiments.

Example 2:
Targeting sequence stretches with high similarity in exons 10 and 18 using different lengths of AON.
Within the highly similar sequence stretches in exons 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110), we have identified AONs with different lengths to identify the most effective minimum length. The effect of was evaluated. After transfection of PS816 (25-mer, SEQ ID NO: 1679), PS1059 (21-mer, SEQ ID NO: 1684) or PS1050 (15-mer, SEQ ID NO: 1682) in healthy human control myocytes (ie differentiated myotubes) RT-PCR analysis demonstrated that all three AONs can induce exon 10-18 skipping (confirmed by sequence analysis) (FIGS. 2A, B). PS816 and PS1059 were the most effective (68% and 79% respectively). The shorter PS1050 was not very effective (25%). Skipping of exons 10 to 18 was not observed in untreated cells (NT). These results indicate that exon 10-18 multi-exon stretch skipping can be performed by using 15, 21, and 25 nucleotide AONs. Longer AONs are expected to work as well. 21-mer PS1059 is the most effective and the shortest candidate. 15-mer PS1050 is least effective, which may be inherent to its low reverse complementarity to exon 18 (47%, FIG. 2B) and / or low binding affinity or stability to the target RNA. In order to improve the biological activity of the 15-mer, base modifications may be required to improve the 15-mer Tm and thereby the binding affinity or duplex stability. The most abundant transcript obtained in this experiment was again the one in which exon 9 was spliced directly to 19 (in-frame transcript).

Example 3:
Targeting sequence stretches with high similarity in exons 10 and 18 using AON with modified base chemistry.
The specific properties of the selected AON chemistry will at least partially affect the delivery of AON to the target transcript: route of administration, biological stability, biodistribution, tissue distribution, and cellular uptake and transport. Furthermore, further optimization of the oligonucleotide chemistry can improve binding affinity and stability, increase activity, improve safety, and / or reduce length or improve synthesis and It is envisaged to reduce the cost of the article by a purification method. Within highly similar sequence stretches in exons 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110), we have 2 different bases (such as 5-substituted pyrimidines and 2,6-diaminopurines). The effect of '-O-methyl RNA AON was evaluated. PS816 (normal 2′-O-methyl RNA AON; SEQ ID NO: 1679), PS1168 (PS816, but all A replaced with 2,6-diaminopurine in healthy human control myocytes (ie differentiated myotubes) SEQ ID NO: 1681), PS1059 (normal 2′-O-methyl phosphorothioate RNA AON; SEQ ID NO: 1684), PS1138 (PS1059, but all Cs have been replaced with 5-methylcytosine; SEQ ID NO: 1685) Or after transfection of PS1170 (PS1059 but all A replaced by 2,6-diaminopurine; SEQ ID NO: 1686) (FIG. 3B), all five AONs were found to be exon 10 by RT-PCR analysis. ~ 18 skipping (confirmed by sequence analysis) It was demonstrated that it can be induced (FIG. 3). PS816 was the most effective (88%). Although base modifications in these specific sequences did not further improve biological activity, they can have a more positive effect on biodistribution, stability and / or safety, so Less effective compounds are still suitable for clinical development. These results indicate that skipping of exon 10-18 multi-exon stretch can be performed by using AON with modified base. The most abundant transcript obtained in this experiment was again the one in which exon 9 was spliced directly to 19 (in-frame transcript).

Example 4:
PS816 induces skipping of other in-frame multi-exon stretches.
Highly similar sequence stretches in exons 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110) are also shown in exons 30 (SEQ ID NO: 128), 31 (SEQ ID NO: 130), 32 (SEQ ID NO: 132), 42 (SEQ ID NO: 132). 152), 47 (SEQ ID NO: 158), 48 (SEQ ID NO: 160), 57 (SEQ ID NO: 170), and 60 (SEQ ID NO: 174) (partially) (Figure 4A, B, Table 2). We now focused on the detection of different multi-exon stretch skipping after transfection of 400 nM PS816 (SEQ ID NO: 1679). We used different primer sets (Tables 4 and 5) for that purpose. Indeed, using RT-PCR analysis, we skipped in-frame exons 10-30, exons 10-42, exons 10-47, exons 10-57 and / or exons 10-60 in multiple experiments. (Confirmed by sequence analysis) were observed and these were not observed in untreated cells. As an example, FIG. 4C shows exon 10-18, exon 10-30 and exon 10-47 skipping induced by PS816. Despite additional multi-exon skipping events (either in-frame or out-of-frame), the resulting transcript in which exon 9 was spliced directly to 19 (in-frame transcript) was the most reproducible, It seemed most abundant in the experiment. These results allow multiple exon skippings to bind to sequence stretches that are highly similar between two different exons, skipping these exons and all the exons in between to produce an in-frame DMD transcript. It is confirmed that a single AON can be induced across the DMD gene using a single AON.

Example 5:
Targeting sequence stretches with high similarity in exons 45 and 55 using AON with modified base chemistry.
Based on the high similarity (80)% sequence stretch in exons 45 (SEQ ID NO: 1571) and 55 (SEQ ID NO: 1572), a series of AONs were designed and exon 45 (PS1185, SEQ ID NO: 1706; PS1186, SEQ ID NO: 1707). ) Or 100% reverse complementary to exon 55 (with one mismatch) (PS1188, SEQ ID NO: 1713) distributed over the sequence stretch (FIG. 5A). In all three AONs, As was replaced with 2,6-diaminopurine. After transfection in healthy human control myotube cultures, RT-PCR analysis demonstrated that PS1185, PS1186 and PS1188 can induce exon 45-55 skipping (confirmed by sequence analysis) (FIG. 5B). . Exon 45-55 skipping was not observed in untreated cells (NT). These results show that skipping of another multi-exon stretch of exons 45-55 can bind to both exons 45 and 55 in regions with high sequence similarity (80%), and all these exons and all in between It has been demonstrated that it can be done by using a single AON that can induce exon skipping. The resulting transcript in which exon 44 was spliced directly to 56 was in-frame and was observed in several experiments. Regarding the exon 10-18 skipping experiment (Example 3), we again show here that AON with modified bases can be effectively applied. Furthermore, the results obtained with PS1188 do not require that AON be 100% reverse complementary to one of the target exons, but there is 100% reverse complement to either target exon. It shows that it can also be designed as a hybrid structure that does not.

FIG. 1 (FIG. 1): A) PS811 (SEQ ID NO: 1673), PS814 (SEQ ID NO: 1675), PS815 (SEQ ID NO: 1677) and PS816 in a sequence stretch that is highly similar (63%) in exon 10 and exon 18 Localization of (SEQ ID NO: 1679). B) AON characteristics and efficiency. The reverse complementarity (rev. Comp.) (%) Of each AON to either exon 10 or exon 18 is shown. C) RT-PCR analysis. In healthy human myocytes (ie differentiated myotubes), all four AONs were effective in inducing skipping of exon 10 to exon 18 multi-exon stretches. PS811 and PS816 were most effective. The box on the left side of the figure represents the content of the PCR amplified transcript (M: DNA size marker; NT: untreated cells). FIG. 2 (FIG. 2): A) RT-PCR analysis. In healthy human myocytes (ie differentiated myotubes), AONs with the same core target sequence within a sequence stretch with different lengths but high similarity (63%) in exon 10 and exon 18 were tested. . PS816 (SEQ ID NO: 1679) (25-mer) and PS1059 (SEQ ID NO: 1684) (21-mer) were most effective (68% and 79% exon 10-18 skipping, respectively). 15-mer PS1050 (SEQ ID NO: 1682) had little effect. The box on the left side of the figure represents the content of the PCR amplified transcript (M: DNA size marker; NT: untreated cells). B) AON characteristics and efficiency. The reverse complementarity (rev. Comp.) (%) Of each AON to either exon 10 or exon 18 is shown. FIG. 3 (FIG. 3): A) RT-PCR analysis. AON with different base chemistry was tested in healthy human myocytes (ie differentiated myotubes). PS816 (normal 2′-O-methyl phosphorothioate RNA AON; SEQ ID NO: 1679), PS1168 (PS816, but all A are replaced by 2,6-diaminopurine; SEQ ID NO: 1681), PS1059 (normal 2 '-O-methyl phosphorothioate RNA AON; SEQ ID NO: 1684), PS1138 (PS1059, but all C has been replaced with 5-methylcytosine; SEQ ID NO: 1685) or PS1170 (PS1059, all A being 2,6 -Replaced by diaminopurine; SEQ ID NO: 1686). Observations were made for all AONs tested for skipping of exons 10-18. PS816 was most effective (88%). In these specific sequences, base modifications did not further improve biological activity. The box on the left side of the figure shows the PCR amplified transcript (M: DNA size marker; NT: untreated cells). B) AON characteristics and efficiency. FIG. 4 (FIG. 4): A) Highly similar sequence stretch in exons 10, 18, 30 and 47 as multiple targets for PS816. The sequence number numbers refer to the EMBOSS full length exon alignment (as in Table 2). In the gray font, the sequence portion was not included in the EMBOSS alignment, but is adjacent to the portion with high reverse complementarity to PS816. B) Summary of exon alignment and percent PS816 reverse complementation (rev. Comp.). C) RT-PCR analysis. Multiple exon stretch skipping can be induced by PS816, identified here as exon 10-18, exon 10-30 and exon 10-47 skipping. The resulting transcript is in-frame. These were not detected in untreated cells (NT). Boxes on the left and right sides of the figure represent the content of PCR amplified transcripts (M: DNA size marker). Figure 5 (Fig. 5): A) Stations of PS1185 (SEQ ID NO: 1706), PS1186 (SEQ ID NO: 1707) and PS1188 (SEQ ID NO: 1713) in a sequence stretch that is highly similar (80%) in exon 45 and exon 55 Presence. The table summarizes the AON characteristics. The reverse complementarity (rev. Comp.) (%) Of each AON to either exon 45 or exon 55 is shown. B) RT-PCR analysis. In healthy human myocytes (ie differentiated myotubes), all three AONs were effective in inducing skipping of the multi-exon stretch of exon 45 to exon 55. The box on the left side of the figure represents the content of the PCR amplified transcript (M: DNA size marker; NT: untreated cells).

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Preferred embodiments of the present invention are as follows.
[1]
An antisense oligonucleotide for use as a medicament for preventing, delaying, ameliorating and / or treating a disease in a subject comprising:
The oligonucleotide is capable of binding to a region of a first exon and a region of a second exon, the region of the second exon having at least 50% identity with the region of the first exon;
The first exon and the second exon are in the same mRNA precursor in the subject;
As a result of the binding, skipping of the first exon and the second exon, preferably one starting between the first exon and existing between the first exon and the second exon Or skipping a multi-exon stretch that includes multiple exons, and at most skipping the entire stretch of exons between the first exon and the second exon;
In-frame transcripts are obtained which allow the production of functional or semi-functional proteins,
Antisense oligonucleotide.
[2]
The oligonucleotide according to [1], which is capable of inducing skipping of the entire exon stretch between the first exon and the second exon.
[3]
The binding includes, within the mRNA precursor, at least one splicing control sequence in the region of the first exon and second exon, and / or at least the first exon and / or the second exon. The antisense oligonucleotide according to [1] or [2], which can interfere with a secondary structure.
[4]
The splicing control sequence comprises a binding site for a serine-arginine (SR) protein, an exon splicing enhancer (ESE), an exon recognition sequence (ERS), and / or an exon splicing silencer (ESS) according to [3]. Antisense oligonucleotide.
[5]
The oligonucleotide according to any one of [1] to [4], comprising 10 to 40 nucleotides.
[6]
The oligonucleotide according to any one of [1] to [5], comprising at least one modification compared to a natural ribonucleotide-based or deoxyribonucleotide-based oligonucleotide, more preferably
(A) at least one base modification, preferably selected from 2-thiouracil, 2-thiothymine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, and 5-methylpyrimidine and 2 Base modification more preferably selected from 1,6-diaminopurine, and / or (b) at least one sugar modification, 2′-O-methyl, 2′-O- (2-methoxy) ethyl, 2 Preferably selected from '-O-deoxy (DNA), 2'-F, morpholino, cross-linked nucleotide or BNA, or the oligonucleotide contains both cross-linked nucleotide and 2'-deoxy modified nucleotide (BNA / DNA mixmer) More preferably, the sugar modification is 2′-O-methyl, and / or (c) at least one backbone modification There are, preferably from phosphorothioate or phosphorodiamidate is selected oligonucleotides and more preferably from backbone modification is a phosphorothioate.
[7]
The oligonucleotide according to any one of [1] to [6],
The oligonucleotide comprises one or more conjugate groups;
The conjugate group is optionally protected, peptide, protein, carbohydrate, drug, target moiety, uptake enhancing moiety, solubility enhancing moiety, pharmacodynamic enhancing moiety, pharmacokinetic enhancing moiety, polymer, ethylene glycol derivative, vitamin, lipid , Selected from the group consisting of polyfluoroalkyl moieties, steroids, cholesterol, fluorescent moieties, reporter molecules, radioactive labeling moieties and combinations thereof, and attached to a terminal or internal residue directly or via a divalent or multivalent linker To
Oligonucleotide.
[8]
The first exon and the second exon are dystrophin exons 10, 18, 30, 8, 9, 11, 13, 19, 22, 23, 34, 40, 42, 44, 45, 47, 51, 53, The oligonucleotide according to any one of [1] to [6], wherein the oligonucleotide is selected from 55, 56, 57, or 60, and the disease is DMD or BMD.
[9]
Dystrophin exons 10-18, exons 10-30, exons 10-42, exons 10-47, exons 10-57, exons 10-60, exons 8-19, exons 9-22, exons 9-30, exons 11-23 , Exons 13-30, exons 23-42, exons 34-53, exons 40-53, exons 44-56, exons 45-51, exons 45-53, exons 45-55, exons 45-60 and / or exons 56 The oligonucleotide according to [8], which can induce skipping of ˜60.
[10]
A nucleotide sequence defined in any one of SEQ ID NOs: 1679, 1688, 1671 to 1678, 1680 to 1687, 1689 to 1741, or 1788 to 1891, and a length defined by the number of nucleotides present in the nucleotide sequence Or comprises a base sequence having a length of 1, 2, 3, 4 or 5 nucleotides longer than defined in the base sequence, or a length of 1, 2, 3, 4 or 5 nucleotides shorter [8] or [9] The oligonucleotide according to [9].
[11]
[10] the oligonucleotide according to
(A) is defined in any one of SEQ ID NOS: 1679-1681, 1778, 1812, 1813, 1884-1886, 1890 or 1891 and has a length of 25, 26, 27, 28, 29 or 30 nucleotides, Or a nucleotide sequence defined in SEQ ID NO: 1814 and having a length of 26, 27, 28, 29, or 30 nucleotides, or (b) defined in SEQ ID NO: 1688, 1689 or 1839-1844, 26, 27, 28, 29 Or a base sequence having a length of 30 nucleotides, or (c) a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in SEQ ID NO: 1673 or 1674, or (d) a SEQ ID NO: 1675 or 1676, 21, 22, 23, 24, 25, 26, 27, 28, 2 Or a nucleotide sequence having a length of 30 nucleotides, or (e) a nucleotide sequence defined in SEQ ID NO: 1677 or 1678 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (f) a SEQ ID NO. A base sequence defined in any one of 1684 to 1686 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (g) of SEQ ID NOs: 1704 to 1706 A base sequence defined in any one and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (h) defined in any one of SEQ ID NOS: 1707 to 1709, 23, 24, A base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (i) any one of SEQ ID NOs: 1710 and 1713 to 1717 A base sequence having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (j) defined in any one of SEQ ID NOs: 1815-1819, 22, 23 , 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (k) defined in any one of SEQ ID NOs: 1820, 1824 and 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 nucleotides in length, or (l) defined in any one of SEQ ID NOs: 1826, 1780, 1782, 1832, 21, 22, 23, 24 25, 26, 27, 28, 29 or 30 nucleotides in length, or (m) defined in any one of SEQ ID NOS: 1821 and 1825, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or (n) defined in any one of SEQ ID NO: 1822, 24, 25, 26, 27, 28, 29 Or a base sequence having a length of 30 nucleotides, or (o) a length of 25, 26, 27, 28, 29, or 30 nucleotides as defined in any one of SEQ ID NOs: 1823, 1781, 1829, 1830, 1831 (P) defined in any one of SEQ ID NOs: 1783, 1833, 1834, 1835, and 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29 or 30 nucleotides in length, or (q) any one of SEQ ID NO: 1887, 24, 25, 26, 27, 28, 29 or Is a nucleotide sequence having a length of 30 nucleotides, or (r) a nucleotide sequence defined in any one of SEQ ID NOs: 1888 or 1889 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (s ) As defined in SEQ ID NO: 1827 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (t) as defined in SEQ ID NO: 1828 as 25, 26 27, 28, 29, or 30 nucleotides in length, or (u) defined in any one of SEQ ID NOs: 1784, 1836, 21, 22, 23, 24, 25, 26, 27, 28 , 29 or 30 nucleotides in length, or (v) defined in any one of SEQ ID NOs: 1786, 1838, 25, 26, 27, 28, 2 Or a base sequence having a length of 30 nucleotides, or (w) defined in any one of SEQ ID NO: 1780 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides A base sequence, or (x) a base sequence defined in any one of SEQ ID NOs: 1785, 1837 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (y) a SEQ ID NO: 1845, 1846 , 1847, 1848, a base sequence having a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (z) any one of SEQ ID NOs: 1849, 1850 A nucleotide sequence defined and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (a1) SEQ ID NO: 1787, 851 and a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or (b1) defined as any one of SEQ ID NOs: 1788, 1852, 1789, 1853, A base sequence having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (c1) defined in any one of SEQ ID NOs: 1790, 1854, 1792, 1855, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (d1) defined in any one of SEQ ID NOs: 1794, 1861, 1795, 1862, 21, 22, 23, 24, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (e1) any of SEQ ID NOs: 1796, 1863, 1797, 1864 A base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (f1) defined in any one of SEQ ID NOs: 1798, 1865, 1799, 1866 Or a base sequence having a length of 25, 26, 27, 28, 29, or 30 nucleotides, or (g1) defined in any one of SEQ ID NOs: 1808, 1867, 1809, 1868, 1810, 1869, 1858, 1873 Or a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (h1) defined in any one of SEQ ID NOS: 1811, 1870, 1859, 1874 A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (i1) A nucleotide sequence defined in any one of column numbers 1856, 1871, 1860, 1875 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (j1) SEQ ID NO: 1857, Defined in any one of 1872 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (k1) defined in any one of SEQ ID NOs: 1800, 1876, 1801, 1877, A base sequence having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (l1) any one of SEQ ID NOs: 1802, 1878, 1803, 1879 Or a base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (m1) a sequence number A base sequence defined in any one of 1804 and 1880 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (n1) of SEQ ID NOs: 1805 and 1881 A base sequence defined in any one and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (o1) any one of SEQ ID NOs: 1806, 1882, 1807, 1883 An oligonucleotide comprising a base sequence defined in one and having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
[12]
A composition comprising the oligonucleotide according to any one of [1] to [11], wherein a pharmaceutically acceptable carrier, diluent, excipient, salt, adjuvant and / or solvent is optionally selected A composition further comprising:
[13]
The composition according to [12], comprising two or more identical oligonucleotides according to any one of [1] to [11] linked by a counter ion, preferably calcium.
[14]
A method for preventing, delaying, ameliorating and / or treating a disease in an individual, preferably BMD or DMD, wherein the oligonucleotide according to any one of [1] to [11] or [14 or 13] Administering a composition as described to said individual.
[15]
The oligonucleotide according to any one of [1] to [11] or the composition according to [12] or [13] for preventing, delaying, ameliorating and / or treating a disease, preferably BMD or DMD Use of things.
[16]
A method for designing the oligonucleotide according to any one of [1] to [11],
(A) identifying an in-frame combination of a first exon and a second exon within the same mRNA precursor, wherein the region of the second exon is at least 50% of the region of the first exon. Having identity, steps,
(B) designing an oligonucleotide capable of binding to the region of the first exon and the region of the second exon; and (c) the first exon and the second exon as a result of the binding. Preferably skipping a multi-exon stretch starting with the first exon and including one or more exons present between the first exon and the second exon, and at most Also causing skipping of the entire stretch of exons between the first exon and the second exon.

Claims (8)

An antisense oligonucleotide for use as a medicament to prevent, delay, ameliorate and / or treat Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) in a subject comprising:
The oligonucleotide comprises 15-40 nucleotides, wherein the oligonucleotide can bind to a region within a first exon and a region within a second exon, wherein the region of the second exon is a region of the first exon. Having at least 50% identity with said region;
The first exon and the second exon are in the same dystrophin mRNA precursor of the subject, and the binding of the oligonucleotide is to at least one splicing control sequence in the region of the first and second exons. And / or can interfere with a secondary structure comprising at least the first exon and / or the second exon in the mRNA precursor, and the splicing control sequence comprises an exon splicing enhancer (ESE), An exon recognition sequence (ERS) and / or a serine-arginine (SR) protein binding site, and / or the binding of the oligonucleotide results in exon skipping, wherein the exon skipping comprises the first exon and Before skipping the second exon From the first exon and the second exon and skipping of a multi-exon stretch comprising one or more exons existing between the first exon and the second exon or from the first exon Skipping the entire stretch of exon up to the second exon, resulting in an in-frame transcript that allows for the production of at least partially functional protein, wherein the oligonucleotide is linked with one or more linkers Not part of a cocktail or combination of two or more different oligonucleotides that may be linked,
Oligonucleotide.   2. The oligonucleotide of claim 1, wherein the oligonucleotide cannot bind to non-exon sequences. The oligonucleotide according to claim 1 or 2,
A nucleotide sequence defined in any one of SEQ ID NOs: 1679, 1688, 1671 to 1678, 1680 to 1687, 1689 to 1741, or 1778 to 1891, and a length defined by the number of nucleotides present in the nucleotide sequence Or an oligonucleotide comprising a base sequence having a length 1, 2, 3, 4 or 5 nucleotides longer than that. The oligonucleotide according to any one of claims 1 to 3,
Comprising a portion of the base sequence defined in any one of SEQ ID NOs: 1679, 1688, 1671 to 1678, 1680 to 1687 or 1689 to 1741, wherein the portion is 1 from the length defined in the base sequence; 2. An oligonucleotide corresponding to a base sequence defined in any of the above sequences, 2, 3, 4 or 5 nucleotides short. The oligonucleotide according to claim 4, wherein
(A) is defined in any one of SEQ ID NOS: 1679-1681, 1778, 1812, 1813, 1884-1886, 1890 or 1891 and has a length of 25, 26, 27, 28, 29 or 30 nucleotides, Or a base sequence defined in SEQ ID NO: 1814 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (b) defined in any one of SEQ ID NOS: 1688, 1689 or 1839-1844, A base sequence having a length of 27, 28, 29 or 30 nucleotides, or (c) a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in SEQ ID NO: 1673 or 1674, or (D) defined in SEQ ID NO: 1675 or 1676, 21, 22, 23, 24, 25, 26, A base sequence having a length of 7, 28, 29 or 30 nucleotides, or (e) a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in SEQ ID NO: 1677 or 1678, or (F) a nucleotide sequence defined in any one of SEQ ID NOs: 1684 to 1686 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (g) a sequence Defined as any one of Nos. 1704 to 1706 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (h) defined as any one of SEQ ID Nos. 1707 to 1709 , 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (i) SEQ ID NO: 1710 or 1713-17 Or a base sequence having a length of 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (j) defined in any one of SEQ ID NOS: 1815-1819 A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (k) defined in SEQ ID NO: 1820 or 1824, and 20, 21, 22, 23, 24 25, 26, 27, 28, 29 or 30 nucleotides in length, or (l) defined in any one of SEQ ID NOs: 1826, 1780, 1782 or 1832, 21, 22, 23, 24 25, 26, 27, 28, 29 or 30 nucleotides in length, or (m) defined in SEQ ID NO: 1821 or 1825, 22, 23, 24 , 25, 26, 27, 28, 29 or 30 nucleotides in length, or (n) defined in SEQ ID NO: 1822 and having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides Or (o) a nucleotide sequence defined in any one of SEQ ID NOS: 1823, 1781, 1829, 1830 or 1831 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or ( p) as defined in any one of SEQ ID NOs: 1783, 1833, 1834 or 1835, and a length of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides (Q) defined in SEQ ID NO: 1887 and having a length of 24, 25, 26, 27, 28, 29, or 30 nucleotides (R) a nucleotide sequence defined in SEQ ID NO: 1888 or 1889 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (s) a nucleotide sequence defined in SEQ ID NO: 1827, A base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (t) a length of 25, 26, 27, 28, 29 or 30 nucleotides as defined in SEQ ID NO: 1828 (U) a base sequence defined in SEQ ID NO: 1784 or 1836 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (v) A nucleotide sequence defined in SEQ ID NO: 1786 or 1838 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (w) SEQ ID NO: 17 A base sequence defined as 0 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (x) defined in SEQ ID NO: 1785 or 1837, 25, 26, 27, A nucleotide sequence having a length of 28, 29 or 30 nucleotides, or (y) defined in any one of SEQ ID NOs: 1845, 1846, 1847 or 1848, 24, 25, 26, 27, 28, 29 or 30 nucleotides (Z) a nucleotide sequence defined in SEQ ID NO: 1849 or 1850 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (a1 ) A nucleotide sequence defined in SEQ ID NO: 1787 or 1851 and having a length of 26, 27, 28, 29 or 30 nucleotides; or (b1) a sequence A nucleotide sequence defined in any one of Nos. 1788, 1852, 1789 or 1853 and having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (c1) SEQ ID NOs: 1790, 1854, 1792 Or a base sequence having a length of 25, 26, 27, 28, 29, or 30 nucleotides, or (d1) any one of SEQ ID NOs: 1794, 1861, 1795, or 1862 Or a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (e1) any one of SEQ ID NOs: 1796, 1863, 1797 or 1864 A nucleotide sequence defined and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or ( f1) a base sequence defined in any one of SEQ ID NOs: 1798, 1865, 1799 or 1866 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (g1) SEQ ID NOs: 1808, 1867, A nucleotide sequence defined in any one of 1809, 1868, 1810, 1869, 1858 or 1873 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides; h1) a nucleotide sequence defined in any one of SEQ ID NOs: 1811, 1870, 1859 or 1874 and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (i1) Defined in any one of SEQ ID NOs: 1856, 1871, 1860 or 1875, 23, 24, 25, 26, 27 A base sequence having a length of 28, 29 or 30 nucleotides, or (j1) a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides defined in SEQ ID NO: 1857 or 1872, or (k1) a sequence A nucleotide sequence defined in any one of the numbers 1800, 1876, 1801 or 1877 and having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (L1) a base sequence defined in any one of SEQ ID NOs: 1802, 1878, 1803 or 1879 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (m1) sequence Number 1804 or 1880 and is 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides (N1) a nucleotide sequence defined by SEQ ID NO: 1805 or 1881 and having a length of 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or (o1) sequence An oligonucleotide comprising a base sequence defined in any one of numbers 1806, 1882, 1807 or 1883 and having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides .   A composition comprising the oligonucleotide according to any one of claims 1 to 5, optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient, salt, adjuvant and / or solvent. A composition comprising.   7. A composition according to claim 6, comprising two or more identical oligonucleotides as defined in any one of claims 1 to 5 linked by a counter ion.   8. A composition according to claim 6 or 7 for preventing, delaying, ameliorating and / or treating BMD or DMD in an individual.