JP2021105049A - Oligonucleotide for treatment of muscular dystrophy patients - Google Patents

Abstract

To provide an oligonucleotide and a pharmaceutical composition including the oligonucleotide.SOLUTION: An oligonucleotide is able to bind to a region of a first exon derived from a dystrophin pre-mRNA and to a region of a second exon within the same pre-mRNA, where the region of the second exon has at least 50% identity with the region of the first exon, and the oligonucleotide is suitable for the skipping of the first and second exons of the pre-mRNA, and preferably the entire stretch of exons in between.SELECTED DRAWING: None

Description

The present invention relates to the field of human genetics, more specifically methods for designing single oligonucleotides capable of preferably inducing skipping of two or more exons of pre-mRNA. The present invention further provides the use of said oligonucleotides, pharmaceutical compositions containing said oligonucleotides, and said oligonucleotides identified herein.

Oligonucleotides have emerged in medicine for the treatment of hereditary disorders such as muscular dystrophy. Muscular dystrophy (MD) refers to a hereditary 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 invention. DMD is a severe fatal neuromuscular disorder that results in dependence on wheelchair support before age 12, and DMD patients often die before age 30 due to respiratory or heart failure.

DMD is caused by mutations in the DMD gene, primarily frameshift deletions or replication of one or more exons, small nucleotide insertions or deletions, or nonsense point mutations that typically result in the absence of functional dystrophin. .. Over the last decade, specially induced modifications of splicing to repair the disrupted reading frame of DMD transcripts have emerged as a promising treatment for Duchenne muscular dystrophy (DMD) (van Ommen G. et al. J. et al., Yokota T. et al., Van Deutekom et al., Goemans NM et al.). Using a sequence-specific antisense oligonucleotide (AON) that targets a specific exon that is flanked by or contains the mutation and interferes with its splicing signal, the exon skipping of the processing of the DMD pre-mRNA Can be guided in between. Despite the resulting truncated transcript, the open reading frame is repaired and a protein similar to that found typically in patients with mild Becker muscular dystrophy is introduced. Because AON-induced exon skipping confers mutational properties, it may personalize therapeutic approaches for DMD patients and certain severe BMD patients. Since most mutations are concentrated 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 (about 13%) of patients, including patients with deletions of exons 45-50, 48-50, 50 or 52. For some mutations, more than one exon skipping is needed to repair the open reading frame. For example, in the case of DMD patients with a deletion of exons 46-50 in the DMD gene, skipping both exons 45 and 51 is the only corrective measure. Administration of two oligonucleotides, one targeting exon 45 and the other targeting exon 51, is required to treat these patients. The feasibility of skipping two or more consecutive exons by using a combination of AONs in either cocktails or virally delivered gene constructs has been extensively studied (Artsma-Rus A. et al., et al. 2004; Beroud C. et al .; Van Vliet L. et al.; Yokota T. et al .; Goyenvalle A. et al.). Multi-exon skipping is applicable to a mixed subpopulation of patients, allowing mimicking of deletions known to be associated with a relatively mild phenotype, and of hotspot deletion regions in the DMD gene. It provides a means of dealing with rare mutations on the outside. However, the drawback to developing drugs containing multiple oligonucleotides is that drug regulators may consider different sequences of oligonucleotides to be different drugs, each requiring proof of safe manufacturing, toxicity testing and clinical trials. Is to be. Therefore, there is a need for monomolecular compounds that can induce skipping of at least two exons to facilitate treatment of mixed subpopulations of DMD patients.

The present invention is a method for designing a single oligonucleotide, wherein the oligonucleotide can bind to a region of a first exon and a region of a second exon in the same mRNA precursor, said to the second exon. The region provides a method having at least 50% identity with said region of the first exon. The oligonucleotides that can be obtained by the method can preferably induce skipping of the first exon and the second exon of the pre-mRNA. Skipping of additional exons is also preferably induced, and the additional exons are preferably located between the first exon and the second exon. The resulting transcript of the pre-mRNA, from which the exons are skipped, is in-frame.

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

This oligonucleotide can preferably induce skipping of the first and second exons of the pre-mRNA, more preferably further exon skipping, and said further exons are preferably said first. Located between the exon and the second exon, the resulting transcript is in-frame.

Exon skipping interferes with the natural splicing process that occurs within eukaryotic cells. In higher eukaryotes, genetic information about proteins in cellular DNA is encoded in exons that are separated from each other by intron sequences. These introns are, in some cases, very long. Eukaryotic transcriptional mechanisms produce pre-mRNA containing both exons and introns, whereas splicing mechanisms often already occur during the ongoing production of pre-mRNA with splicing. During the so-called process, the introns present in the pre-mRNA are removed and exons are bound to produce the actual coding mRNA for the protein.

The oligonucleotides of the present invention capable of binding to the first exon region derived from the mRNA precursor and the second exon region in the same pre-mRNA are suitable for binding to the first exon region derived from the mRNA precursor. It is interpreted as an oligonucleotide suitable for binding to the second exon region in the same pre-mRNA. Such oligonucleotides of the present invention are preferably characterized by the binding properties (ie, capable of binding) of the oligonucleotide when used intracellularly with or in combination with pre-mRNA. Attached. Within this context, "capable of" may be replaced with "able to." Therefore, those skilled in the art can bind to the region of the first exon and to the region of the second exon within the same pre-mRNA, and the oligonucleotide defined by the nucleotide sequence structurally defines the oligonucleotide. That is, the oligonucleotide appears to be inversely complementary to the sequence of the region of the first exon and also to the sequence of the region of the second exon of the same pre-mRNA. You will understand that it has a similar sequence. The degree of inverse complementarity of the first exon with the region and / or the second exon with the region required for the oligonucleotide of the present invention may be less than 100%. As further addressed herein, some amount of mismatch or one or two gaps may be tolerated. The nucleotide sequence of the region of the first exon that has at least 50% identity with the region of the second exon to which the oligonucleotide of the invention can bind (or at least 50% of the region of the first exon). The nucleotide sequence of the second exon region with identity) can be designed using the methods of the invention described below. A preferred pre-mRNA is a dystrophin pre-mRNA.

The preferred combinations of the first and second exons of the dystrophin pre-mRNA and the preferred regions of the first and second dystrophin exons are defined in Table 2. The oligonucleotide of the present invention can preferably induce skipping of the first exon and the second exon of the dystrophin pre-mRNA, more preferably further skipping of the exon, and the further exon is the first exon. Is preferably located between the exon and the second exon, and the resulting dystrophin transcript is in-frame, preferably as described in Table 1.

If the transcript has an open reading frame that allows the production of the protein, then the transcript is in-frame. The in-frame status of mRNA can be assessed by sequences known to those of skill in the art and / or RT-PCR analysis. The resulting protein, derived from the translation of the in-frame transcript, can be analyzed by immunofluorescence and / or Western blot analysis using antibodies that cross-react with said protein known to those of skill in the art. Throughout the invention, if the resulting in-frame transcript is identified by RT-PCR and / or sequence analysis, or if the protein obtained from said in-frame transcript is associated in vitro, depending on the identity of the transcript. Or in an in vivo system, the oligonucleotides described herein may be said to be functional when identified by immunofluorescence and / or Western blot analysis. If the transcript is a dystrophin transcript, the relevant system can be muscle cells or myotubes or myofibrils or myofibrils of a healthy donor or DMD patient, as described below.

In one embodiment, the second exon region (located in the same mRNA precursor as the first exon region) is at least 50%, 55%, 60%, 65%, with the first exon region. It has 70%, 75%, 80%, 85%, 90%, 95% or 100% identity (preferred regions of the first dystrophin exon and the second dystrophin exon are listed in Table 2). Identity is exemplified over the entire length of the first exon and / or the 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, Regions of nucleotides 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more Can be evaluated over. It is self-evident to those skilled in the art that the first and second exons described herein are two different exons in one single pre-mRNA or two different exons in the same pre-mRNA. 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 may be said to be the first exon. It may be upstream of the exon.

The first exon is preferably located upstream from the second exon. The oligonucleotides of the present invention can be designed to be capable of primarily binding to a region of a first exon, and the oligonucleotides, given the identity of the regions of the first exon and the second exon. It is self-evident to those skilled in the art that can be secondarily designed to be coupled to said region of said second exon. The reverse design is also possible. That is, the oligonucleotide of the present invention can be designed so as to be capable of primarily binding to the region of the second exon, and in consideration of the identity of the region of the first exon and the region of the second exon, the above-mentioned The oligonucleotide can be designed to secondarily bind to the region of the first exon.

The identity between the region of the first exon and the region of the second exon can be assessed over the region of the first exon, which region is that region to which the oligonucleotide of the invention can bind. It may be shorter, longer, or equal in length than the portion. 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, which defines identity with the region of the second exon, is preferably as long as or longer than the portion of that region to which the oligonucleotides of the invention can bind. It is understood that the oligonucleotides of the present invention can bind to smaller or partially overlapping portions of said regions used to assess sequence identity of the first and / or second exons. It must be. Therefore, it is understood that an oligonucleotide capable of binding to the first exon and the region of the second exon can bind to a part of the region of the first exon and the second exon. The portion may have 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 first exon and / or region of the second exon, said first region. And / or may be on the 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. As long as the sex is at least 50%, it may be a continuous stretch and may be interrupted with a gap of 1, 2, 3, 4 or more.

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

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

It can bind to a region of a first exon and another exon (ie, a second exon) within the same mRNA precursor that has been identified herein (ie, said region of said second exon is said said to be said. The oligonucleotide (having at least 50% identity with said region of one exon) is also preferably at least 80% inversely complementary to said region of said first exon and said of said second exon. It is at least 45% inversely complementary to the region. The oligonucleotide is at least 85%, 90%, 95% or 100% inversely complementary to the region of the first exon and at least 50%, 55%, 60% of the region of the second exon. , 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% inverse complementary. Inverse complementarity is preferably, but not necessarily, evaluated over the length of the oligonucleotide.

The oligonucleotides included in 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 included in 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 may be included. The length of the oligonucleotide of the present invention is defined by the total number of nucleotides contained in the oligonucleotide, regardless of any modifications present in the oligonucleotide. As further described below, nucleotides may contain certain chemical modifications, but such modified nucleotides are still considered nucleotides in the context of the present invention. The optimum length of the oligonucleotide may vary, depending on the chemistry of the oligonucleotide. For example, the length of the 2'-O-methylphosphorothioate oligonucleotide may be up to 15-30. If this oligonucleotide is further modified as illustrated herein, the optimum length may be shortened to 14, 13 or even shorter.

In a preferred embodiment, the oligonucleotides of the invention are intended to limit the potential for reduced synthetic efficiency, yield, purity or extensibility, reduced bioavailability and / or cell uptake and transport, reduced safety. , As well as not longer than 30 nucleotides to limit the cost of the article. In a more preferred embodiment, the oligonucleotide is 15-25 nucleotides. The oligonucleotides included in the present invention most preferably consist 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 the relevant cell culture in vitro, the functionality or activity of the oligonucleotide is the first exon and the first As defined by inducing at least 5% of skipping of two exons (and any exons in between) or by promoting the formation of at least 5% of in-frame transcripts. Is preferable. The assessment of the presence of the transcript has already been described herein. A related cell culture is a cell culture in which the pre-mRNA containing the first exon and the second exon is transcribed into an mRNA transcript and spliced. If the pre-mRNA is a dystrophin pre-mRNA, the associated cell culture comprises (differentiated) muscle cells. In this case, when at least 250 nM of said oligonucleotide is used, at least 20% of the in-frame transcript is formed.

The first 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, 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. It may be defined as%. The region of the first exon can be called the region of identity.

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. It may be defined as. The second exon region can be called the identity region.

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

The oligonucleotide is the entire stretch of the two exons (ie, the first exon and the second exon) and the exon between the first exon and the second exon in the mRNA precursor. More preferably for skipping, it is to eliminate any mutations in the stretch and to obtain a transcript with a repaired open reading frame that allows shorter but protein production.

Although not desired to be bound by any theory, due to at least 50% sequence identity or similarity between the two exons, the single oligonucleotides of the invention are both exons, and preferably. Is believed to be able to bind to the entire stretch of exons in between to obtain shorter transcripts that are in-frame and induce their skipping. Thus, the two exons may be flanked by pre-mRNA, 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 may be separated by 50 exons. The region containing one or more exons that exists between the first exon and the second exon may also be referred to as a (s) exon stretch or a multi-exon stretch. Preferably, the first exon of this multi-exon stretch is the first exon identified above herein, and the last exon of this multi-exon stretch is the first exon identified above herein. It is an exon of 2. The oligonucleotides of the present invention may also be identified as oligonucleotides capable of inducing the skipping of the two exons or the skipping of the (s) exon stretches or the skipping of the multi-exon stretches. In a preferred embodiment, skipping both the first exon and the second exon is induced by using one single oligonucleotide of the invention. In a preferred embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, etc., using one single oligonucleotide. 13 super, 14 super, 15 super, 16 super, 17 super exon, 18 super, 19 super, 20 super, 21 super, 22 super, 23 super, 24 super, 25 super, 26 super, 27 super, 28 super, Over 29, over 30, over 31, over 32, over 33, over 34, over 35, over 36, over 37, over 38, over 39, over 40, over 41, over 42, over 43, over 44, over 45 , 46, 47, 48, 49, and 50 exons skipped, therefore 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 more than 50 exons, this skipping without the use of a mixture of two or more different oligonucleotides or cocktails, or It is performed without the use of two or more different oligonucleotides that can be linked by one or more linkers, or without the use of gene constructs that transcribe two or more different oligonucleotides. Thus, in one embodiment, the single oligonucleotide comprises the first exon and the second exon, provided that the invention comprises one single oligonucleotide and no two or more different oligonucleotides. It can bind to exons and induce skipping of at least the first exon and the second exon in the single mRNA precursor exemplified herein. In this context, one of ordinary skill in the art will understand 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 includes one single oligonucleotide sequence and its use thereof, and does not include two or more different oligonucleotide sequences, wherein the single oligonucleotide sequence is said to be said. It can bind to the first exon and the second exon and induce skipping of at least the first exon and the second exon in the single mRNA precursor exemplified herein. This uses only one single oligonucleotide to treat a disease caused by a (rare) mutation in the gene, provided that different exons in the gene contain regions with at least 50% sequence identity. It was the first invention to allow skipping more than one exon.

The oligonucleotides of the invention are preferably used as part of a therapy based on RNA regulatory activity as defined herein below. Depending on the identity of the transcript in which the first exon and the second exon are present, oligonucleotides for preventing, treating or delaying a given disease can be designed.

Targeting two exons in a single mRNA precursor with a single oligonucleotide capable of binding to both exons results in mRNA lacking the targeted exons and, in addition, 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 may have its own PK profile and thereby they. It should be taken into account that conditions must be found for each of the oligonucleotides of the same to be present in the same cell as well. Therefore, another advantage of using only one single oligonucleotide that can bind to the two different exons identified above is the direct production, toxicity, dose discovery and clinical trials of one single compound. The production and testing can be reduced, which makes them extremely easy.

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

In a preferred embodiment of the invention, the oligonucleotide can bind, target, hybridize, reverse complement, and / or at least the first exon and / or the first exon. It can inhibit the function of an exon and / or at least one splicing control sequence within the second exon and / or affect the structure of at least the first exon and / or the second exon.
The oligonucleotide can bind, target, hybridize, and / or reverse complement to the binding site for the serine-arginine (SR) protein in the first and / or second exon. And / or the oligonucleotide contains 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 bind, target, hybridize, and / or are inversely complementary to.

The oligonucleotide that can bind, target, hybridize, and / or is inversely complementary to the region of the first pre-mRNA exon and / or the region of the second pre-mRNA exon is at least one. More preferably, it can specifically inhibit one splicing control sequence and / or affect the structure of at least the first and / or second exon within the pre-mRNA. Interfering with such splicing control sequences and / or structures has the advantage 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 mechanism 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 enable a more specific and therefore reliable method. The inverse complementarity of the oligonucleotide to the region of the first exon and / or the second exon of the pre-mRNA 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 pharmaceutical use to prevent, delay, ameliorate and / or treat a disease in a subject, said oligonucleotide binding to a first exon region and a second exon region. Yes, 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 Skipping a multi-exon stretch that includes one or more exons, and skipping the entire exon stretch between the first exon and the second exon at most.
In-frame transcripts that allow the production of functional or semi-functional proteins are obtained,
Regarding antisense oligonucleotides.

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

As described herein, the binding of the oligonucleotide is in the mRNA precursor, at least one splicing control sequence in the region of the first exon and the second exon, and / or said. It is more preferred to be able to interfere with the secondary structure of the first exon and / or the second exon and / or at least the secondary structure comprising the first exon and / or the second exon. .. Preferred splicing control sequences are presented herein below.

Therefore, 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 pre-mRNA, the same pre-mRNA. The region of the second exon of is at least 50% identical to the region of the first exon (preferably for DMD as in Table 2) and the oligonucleotide is the mRNA. With respect to oligonucleotides, which can induce skipping of the first and second exons of the precursor, resulting in a transcript that is 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 that is capable of binding, targeting, hybridizing, and / or inversely complementary to the region of the first pre-mRNA exon and / or the second pre-mRNA exon, the first pre-mRNA. A sequence (closed structure) that hybridizes to another part of the pre-mRNA and / or the second pre-mRNA exon, and / or the first pre-mRNA exon and / or the second pre-mRNA exon. Sequences that can bind, target, hybridize, and / or are inversely complementary to the region and do not hybridize in the pre-mRNA (open structure).

For this embodiment, a reference is made to the patent application of WO 2004/083446. RNA molecules exhibit strong secondary structure, largely due to base pairing of inverse-complementary or partially inverse-complementary stretches within the same RNA. It has long been thought that structure in RNA plays a role in RNA function. Although not bound by theory, the secondary structure of exon RNA is thought to play a role in structuring the splicing process. Due to its structure, exons are recognized as part of their need to be included in mRNA. In one embodiment, the oligonucleotide can interfere with the structure of at least the first exon and possibly the second exon and, optionally, the exon stretch in between, thus masking the exon from the splicing mechanism. By doing so, it is possible to prevent splicing of the exon stretches of the first exon and possibly also the second exon and optionally also in between, thereby resulting in skipping of the exon. Although not bound by theory, overlap with open structures improves the efficiency of oligonucleotide entry (ie, increases the efficiency with which oligonucleotides can enter the structure), while overlap with closed structures is exon. It is believed that it will later increase the efficiency of interfering with the secondary structure of RNA. It is found that the length of partial inverse complementarity for both closed and open structures is not extremely limited. We have observed high efficiency with oligonucleotides with variable length inverse complementarity in any structure. The term inverse 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 the bases in the region of inverse complementarity can be paired with the bases in the opposite strand. For example, when designing an oligonucleotide, for example, one or more residues that do not base pair with the base in the inverse complementary strand may be incorporated. Mismatches may be tolerated to some extent if the nucleotide stretch can still hybridize to the inverse complementary moiety in the intracellular environment. In the context of the invention, in one embodiment, the oligonucleotides of the invention are at least 45%, 50%, 55%, 60%, 65% with the region of the first exon and / or the region of the second exon. , 70%, 75%, 80%, 85%, 90%, 95%, 100% are inversely complementary, so the presence of a mismatch in the oligonucleotide is preferred. The presence of a mismatch in the oligonucleotide is a preferred property of the invention, as the oligonucleotide can bind to the region of the first exon and the region of the second exon identified above herein. ..

Other advantages of allowing the presence of mismatches in the antisense oligonucleotides of the invention are defined herein and fluctuate with inosine (hypoxanthine) and / or universal bases and / or modified bases and / or nucleotides. Similar to those 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, quadruple structure. It makes it possible to design oligonucleotides with improved RNA binding kinetics and / or thermodynamic properties.

The inverse complementarity of the oligonucleotide to the region of identity between the first exon and / or the second exon is preferably 45% to 65%, 50% to 75%, but 65% to 100%. Alternatively, it is more preferably 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. Therefore, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21 or 22 in 40 nucleotide oligonucleotides. May have a mismatch of. It is preferred that less than 14 mismatches are present in the 40 nucleotide oligonucleotide. The number of mismatches is that the oligonucleotides of the invention can still bind, hybridize and target the first exon region and the second exon region, thereby at least the first exon and the first exon. It is a number that can induce skipping of 2 exons and induce the production of in-frame transcripts exemplified herein. Production of in-frame transcripts is obtained with an efficiency of at least 5% using at least 100 nM of the oligonucleotide to transfect the relevant cell cultures described above herein in vitro. Is preferable.

The present invention has no mismatch with the region of the first exon and 1, 2, 3, 4, 5, 6, with the corresponding region of the second exon as defined herein. It should be noted that it includes oligonucleotides capable of having 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21 or 22 mismatches. However, the present invention also has no mismatch with the region of the second exon and 1, 2, 3, 4, 5 with the corresponding region of the first exon as defined herein. Also includes oligonucleotides that can have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21 or 22 mismatches. Finally, the present invention relates to the first exon region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19 , 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 includes oligonucleotides that can have one mismatch. Here again, the number of mismatches in the oligonucleotides of the invention hybridizes, allowing the oligonucleotide to still bind to and hybridize to the first exon region and the second exon region as described herein. It is understood that the number can be and can be targeted.

The oligonucleotides of the invention preferably do not intersect the gaps in the alignment of the first exon region and the second exon region identified herein. Alignment is preferably performed using the online tool EMBOSS Matcher described above herein. However, in certain cases, the oligonucleotides of the invention may need to cross the gap, as described herein above. The gap is preferably only one gap. The gap preferably spans less than 3 nucleotides, most preferably only 1 nucleotide. The number and length of gaps allows the oligonucleotides of the invention to still bind, hybridize and target the first exon region and the second exon region, thereby at least the first exon region. The number and length are such that skipping of exons and the second exon can be induced and the production of in-frame transcripts described herein can be induced.

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

One of ordinary skill in the art can predict a predominant exon structure, a given nucleotide sequence, with suitable reproducibility. Optimal predictions can be obtained when implementing such a modeling program using both the exons and adjacent intron sequences. Typically, it is not always necessary to model the structure of the entire pre-mRNA.

The annealing of the oligonucleotides of the present invention can affect the local folding or 3D structure or conformation of the target RNA (ie, the region containing at least the first exon and / or the second exon). Different conformations can result in disruption of the structure recognized by the splicing mechanism. However, when potential (concealed) splice acceptors and / or donor sequences are present within the first and / or second target exons, they occasionally define different (neo) exons, i.e. different 5'. New structures are created with ends, different 3'ends, or both. This type of activity is within the scope of the invention if the target exon is eliminated from the mRNA. The presence of a new exon containing a portion of the first and / or second target exon in the mRNA does not change the fact that the target exon itself is excluded. Neoexson inclusion can be seen as a very 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 mutation. On the one hand, Neo-Exxon works in repairing the reading frame, but on the other hand, the reading frame is not repaired. When selecting an oligonucleotide to repair an open reading frame by multiple exon skipping, under these conditions, exon skipping to repair the open reading frame of a given transcript, with or without neoexson. It is clear that only the oligonucleotides that actually result are selected.

In yet another preferred embodiment, the oligonucleotides of the invention that are capable of binding to a region of a first exon derived from a pre-mRNA and a region of a second exon within the same pre-mRNA are provided. The 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 pre-mRNA. The skipping of the first exon and the second exon of the above can be induced, resulting in a transcript that is in-frame (preferably for DMD as in Table 1 or Table 6). The oligonucleotide provides the individual with a functional or semi-functional protein, which is for one or more binding sites for the serine-arginine (SR) protein in the pre-mRNA exon RNA. It further comprises sequences that can bind, target, hybridize, are inversely complementary, and / or inhibit their function.

In the patent application of WO 2006/11705, we present the effectiveness of the antisense oligonucleotide (AON) inside the exon in inducing exon skipping and the estimation at the target pre-mRNA site of the AON. The existence of a correlation with the presence of SR binding sites was disclosed. Thus, in one embodiment, producing the oligonucleotide as defined herein is one or more (for SR proteins in the RNA of the first exon and / or the second exon). Determining the (estimated) binding site and corresponding at least partially overlapping the (estimated) binding site that can bind, target, hybridize, and / or are inversely complementary to the RNA. Includes producing oligonucleotides. The term "at least partially overlapping" includes the duplication of only a single nucleotide of the SR binding site and the plurality of nucleotides of one or more of the binding sites and the complete duplication of one or more of the binding sites. As defined herein. In this embodiment, 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 above. The step of determining non-hybridizing regions (open structures) in the structure, followed by at least partial overlap with one or more of the (estimated) binding sites and overlap with at least a portion of the closed structure. , Overlapping with at least a portion of the open structure, binding to both the first and second exons, targeting, hybridizing, and / or producing oligonucleotides that are inversely complementary. The steps are preferably further included. Thus, we provide oligonucleotides from pre-mRNA to mRNA that can prevent inclusion of the first and second exons, and, where applicable, the entire stretch of exons in between. Increase the chances of getting. Although not desired to be bound by any theory, the use of oligonucleotides for the SR protein binding site currently causes (at least partial) damage, disruption, or disruption of SR protein binding to said binding site. It is believed that damaged splicing occurs.

The first exon region and / or the second exon region within the same mRNA precursor to which the oligonucleotides of the invention can bind preferably contain an open / closed structure and / or SR protein binding site. It is more preferable that the open structure / closed structure and the SR protein binding site partially overlap, and the open structure / closed structure completely overlaps the SR protein binding site or SR protein binding. It is further preferred that the site completely overlaps the open / closed structure. This allows for a further improved destruction of exon inclusions.

In addition to the consensus splice site sequence, many (but not all) exons contain splicing control sequences such as the exon splicing enhancer (ESE) sequence to facilitate recognition of the true splice site by spliceosomes. 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 recruit other splicing factors such as U1 and U2AF to the (weakly defined) splice site. 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 SF2 / ASF, SC35, SRp40 and SRp55 binding sites in some of the first exons bound, hybridized and / or targeted by the oligonucleotide. There is a correlation between them. In a preferred embodiment, the invention therefore provides an oligonucleotide that binds, hybridizes, targets, and / or is inversely complementary 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 a binding site for the SF2 / ASF, SC35, SRp40 or SRp55 protein in the first exon and a binding site for a different SF2 / ASF, SC35, SRp40 or SRp55 protein in the second exon. On the other hand, it binds, hybridizes, targets, and / or is inversely complementary. In a more preferred embodiment, the oligonucleotide corresponds to the binding site for the SF2 / ASF, SC35, SRp40 or SRp55 protein in the first exon and the similar SF2 / ASF, SC35, SRp40 or SRp55 protein in the second exon. It binds, hybridizes, targets, and / or is inversely complementary to the binding site.

In one embodiment, the patient uses an oligonucleotide capable of binding and targeting a regulatory RNA sequence present in a first exon and / or a second exon required for accurate splicing of said exon in a transcript. By doing so, a functional or semi-functional protein is provided. Some cis-acting RNA sequences require accurate exon splicing in transcripts. In particular, supplementary elements such as exon splicing enhancers (ESEs) have been identified to control the specific and effective splicing of constructive and alternative exons. By using a compound containing an oligonucleotide that binds to or can bind to one of the supplementary elements in the first exon and / or the second exon, their control functions are disrupted so that the exons are skipped. Therefore, in one preferred embodiment, the oligonucleotides of the present invention can bind 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 to said in the second exon. The region has at least 50% identity with the region of the first exon, and the oligonucleotide can induce skipping of the first and second exons of the mRNA precursor, said first. The region of the 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 said oligonucleotide. It can bind, target, inhibit, and / or inversely complement the exon splicing enhancer (ESE), exon recognition sequence (ERS) and / or splicing enhancer sequence (SES).

The following preferred chemical properties of the oligonucleotides of the invention are disclosed.

Oligonucleotides are commonly known as oligomers that have basic hybridization properties similar to those of native nucleic acids. Hybridization is defined in the part assigned to the definition at the end of the description of the invention. As used herein, the terms oligonucleotide and oligomer are used interchangeably. Different types of nucleotides may be used to produce said oligonucleotides of the invention. The oligonucleotide may contain at least one modified internucleoside bond and / or at least one sugar modification and / or at least one base modification as compared to a native ribonucleotide or deoxyribonucleotide-based oligonucleotide.

"Modified nucleoside binding" indicates the presence of a modified form of phosphodiester as naturally occurring in RNA and DNA. Examples of nucleoside binding modifications compatible with the present invention are phosphorothioate (PS), chirally pure phosphorothioate, phosphorodithioate (PS2), phosphonoacetic acid (PACE), phosphonoacetamide (PACA), thiophosphonoacetic acid, thiophospho. Noacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methylphosphonothioate, methyl phosphate, methylphosphorothioate, ethyl phosphate, ethylphosphorothioate, boranophosphate, boranophosphorothioate, methylboranophosphate, methylboranophosphorothioate, Methylboranophosphonate, methylboranophosphonothioate and derivatives thereof. Other modifications include phosphoramidite, phosphoramidate, N3'→ P5' phosphoramidate, phosphorodiamidate, phosphorothioamidate, phosphorothiodiamidate, sulfamic acid, dimethylene sulfoxide, sulfonic acid, methylene. Includes imino (MMI), oxalyl and thioacetamide nucleic acids (TANA) and derivatives thereof. Depending on their 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, It may include 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 skeletal modifications. It is also included in the present invention to introduce more than one different skeletal modification into the oligonucleotide.

Also included in the present invention are oligonucleotides containing internucleoside bonds that may differ with respect to the atoms of the nucleosides that bind to each other as compared to the naturally occurring internucleoside bonds. In this regard, the oligonucleotides of the invention are between at least one nucleoside interpreted as a 3'-3', 5'-5', 2'-3', 2'-5', and 2'-2' binding monomers. It may include a bond. Position numbering may differ from other chemistries, but the idea is within the scope of the present invention.

In one embodiment, the oligonucleotides of the invention comprise 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 sugars, tetrahydropyran, morpholine, 2'-modified sugars, 3'-modified sugars, 4'-modified sugars, 5'-modified sugars and 4'-substituted sugars. Shows the presence of modified forms of naturally occurring ribosyl moieties (ie, furanosyl moieties) in. 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, which is incorporated herein by reference in its entirety. International Publication No. 2013/061295 of the patent application, Universality of the Wittersland), 2'-O- (2- (dimethylamino) ethyl), 2'-O- (haloalkyl) methyl. (Arai K. et al.), For example, 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'-Azide, 2'-Amino and 2'-Substituted Amino, 2'-Halo, eg 2'-F, FANA (2'-F arabinosyl nucleic acid), carbasaccharide and azasaccharide modifications, 3'-O-alkyl, eg 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl, 2', 3 '-Gideoxy and derivatives thereof are included.

Other sugar modifications include, for example, locked nucleic acids (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2'-O, 4'-C constrained ethyl) LNA, cMOEt. (2'-O, 4'-C constrained 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 (described in Kazuyuki Miyashita et al.), Found in CRN described in International Publication No. 2013/036868 of the patent application (which is incorporated herein by reference in its entirety; Marina Biotech). Such as "crosslinked" or "bicyclic" nucleic acid (BNA) modified sugar moieties; as described in US Patent Application Publication No. 2013/0130378 (Alnylam Pharmaceuticals), which is incorporated herein by reference in its entirety. Unlocked Nucleic Acids (UNA) or Other Acyclic Nucleic Acids (UNA); 5'-Methyl Substituted BNA (described in US Patent Application 13/530, 218, which is incorporated by reference in its entirety); Hexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA ( p-DNA); other modified sugar moieties such as morpholino (PMO), cationic morpholino (PMOPlus), PMO-X; tricycloDNA; tricyclo-PS-DNA; and derivatives thereof. BNA derivatives are described, for example, in WO 2011/097641, which is incorporated by reference in its entirety. Examples of PMO-X are described in WO 2011150408, which is incorporated herein by reference in its entirety. Depending on their 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, Even if it contains 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. Incorporating more than one different sugar modification into the oligonucleotide is also included in the present invention.

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, cross-linked nucleotides or BNA. Containing sugar modifications, or oligonucleotides include both bridged nucleotides and 2'-deoxynucleotides (BNA / DNA mixmer). Oligonucleotides containing 2'-fluoro (2-'F) nucleotides have been shown to be able to recruit interleukin enhancer-binding factors 2 and 3 (ILF2 / 3), thereby inducing exon skipping within the targeted mRNA precursor. (Rigo F et al., International Publication No. 2011/097614).

In another embodiment, the oligonucleotides defined herein include or consist of LNAs or derivatives thereof. The overall length of the oligonucleotide according to the present invention is modified by a sugar selected from 2'-O-methyl, 2'-O- (2-methoxy) ethyl, morpholino, bridged nucleic acid (BNA), or BNA / DNA mixmer. It is more preferable to be modified over. In a more preferred embodiment, the oligonucleotides of the invention are completely 2'-O-methyl modified.

In a preferred embodiment, the oligonucleotides of the invention contain at least one sugar modification and at least one modified nucleoside bond. Such modifications include peptide-base nucleic acids (PNAs), boron-cluster modified PNAs, pyrrolidine-based oxy-peptide nucleic acids (POPNAs), glycol-based or glycerol-based nucleic acids (GNA), treose-based nucleic acids (TNAs), and acyclics. Formula Treoninol-based nucleic acid (aTNA), morpholino-based oligonucleotide (PMO, PPMO, PMO-X), cationic morpholino-based oligomer (PMOPlus, PMO-X), oligonucleotide with integrated base and skeleton (ONIB), pyrrolidine- Amidoligonucleotides (POMs) and derivatives thereof are included. In a preferred embodiment, the oligonucleotide of the present invention comprises a peptide nucleic acid backbone and / or a morpholinophosphorodiamidate backbone or a derivative thereof. In a more preferred embodiment, the oligonucleotide according to the invention is a modified 2'-O-methylphosphorothioate, i.e. comprises at least one 2'-O-methylphosphorothioate modification, the oligonucleotide according to the invention is fully. It is preferably a modified 2'-O-methylphosphorothioate. The 2'-O-methylphosphorothioate-modified oligonucleotide or the fully 2'-O-methylphosphorothioate-modified oligonucleotide is preferably an RNA oligonucleotide.

As used herein, the term "base modification" or "modified base" refers to a modification or de novo synthetic base of a naturally occurring base (ie, a pyrimidine or purine base) in RNA and / or DNA. 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 additional modifications such as different types of nucleic acid nucleotide residues or nucleotides described below. Different types of nucleic acid nucleotide residues may be used to produce the oligonucleotides of the invention. The oligonucleotide may have at least one scaffold and / or sugar modification and / or at least one base modification as compared to an RNA or DNA based oligonucleotide.

Oligonucleotides may include the natural bases purine (adenine, guanine) or pyrimidines (cytosine, thymine, uracil) and / or the modified bases defined below. In the present invention, uracil may be replaced with thymine.

Base modifications include modified forms of natural purine and pyrimidine bases (eg, adenin, uracil, guanine, cytosine and thymine) such as hypoxanthin, olotinic acid, agmatidine, lysidine, pseudouracil, pseudothymine, N1-methyl pseudouracil, 2-thiopyrimidine (eg 2-thiouracil, 2-thymine), 2,6-diaminopurine, G-clamp and its derivatives, 5-substituted pyrimidine (eg 5-halouracil, 5-) Methyl uracil, 5-methyl thymine, 5-propynyl uracil, 5-propynyl uchitocin, 5-aminomethyl uracil, 5-hydroxymethyl uracil, 5-aminomethyl thymine, 5-hydroxymethyl thytocin, super T (Super T), 5 -Octylpyrimidine, 5-thiophenyminedin, 5-octin-1-ylpyrimidine, 5-ethynylpyrimidine, 5- (pyridylamide), 5-isobutyl, all of which are incorporated herein by reference. 5-phenyl; 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7, as described in Publication No. 2013/0131141 (RXi). -Dazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A and N4-ethylthytocin or derivatives thereof; N2-cyclopentylguanine (cPent-G) , N2-cyclopentyl-2-aminopurine (cPent-AP) and N2-propyl-2-aminopurine (Pr-AP) or derivatives thereof; and modified or universal bases or debases such as 2,6-difluorotoluene. Absent bases such as sites (eg 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which thymine is replaced by thymine (azaribose)) Examples of derivatives of Super A, Super G and Super T can be found in US Pat. No. 6,683,173 (Epoch Biosciences), which is incorporated herein by reference in its entirety. -G, cPent-AP and Pr-AP reduce immunostimulatory effects when integrated into siRNA It has been shown to reduce (Peacock H et al.) And similar features have been shown for pseudo-uracil and N1-methyl pseudo-uracil (US Patent Application Publication No. 2013 /, which is incorporated herein by reference in its entirety). 0123481, modeRNA Therapeutics). "Thymine" and "5-methyluracil" can be exchanged throughout the specification. Similarly, "2,6-diaminopurine" is identical to "2-aminoadenine" and these terms can be exchanged throughout the 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, thereby said oligonucleotide. At least one of the cytosine nucleobases of is modified by the substitution of a proton at the 5-position of the pyrimidine ring at the methyl group (ie 5-methylcytosine) and / or at least one of the uracil nucleobases of the oligonucleotide. , And / or at least one of the adenine nucleobases of the oligonucleotide is modified by the substitution of a proton at the 5-position of the pyrimidine ring with a methyl group (ie 5-methyluracil). It is understood that each is modified by the substitution of a proton at the 2-position with −diaminopurine). In the present specification, the expression "substitution of a proton at the methyl group at the 5-position of the pyrimidine ring" may be replaced with the expression "substitution of pyrimidine with 5-methylpyrimidine", and pyrimidine is only uracil, cytosine. Refers to only or both. Similarly, in the present specification, the expression "substitution of proton at the amino group at the 2-position of adenine" may be replaced with the expression "substitution of adenine at 2,6-diaminopurine". If the oligonucleotide contains 1,2,3,4,5,6,7,8,9 or more cytosine, uracil and / or adenine, then at least 1,2,3,4,5,6, 7, 8, 9 or more cytosines, uracils and / or adenines may each 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. Be done.

The presence of 5-methylcytosine, 5-methyluracil and / or 2,6-diaminopurine in the oligonucleotides of the present invention has a positive effect on at least one of the parameters of said oligonucleotide or an improvement in at least one parameter. Was discovered. In this context, the parameters include binding affinity and / or kinetics of the oligonucleotide, silencing activity, in vivo stability, (intratissue) distribution, cell uptake and / or cells, as described below. Can include transport and / or immunogenicity.

These modifications are described in the present invention, as some modifications as described above are known to increase Tm values, thereby enhancing the binding of a particular nucleotide to its relatives in its target mRNA. It can be investigated in the context to facilitate the binding of the oligonucleotides of the invention to both the first and second exon regions. The sequences of the oligonucleotides of the invention do not have to be 100% inverse complementary to a given region of the first exon and / or the second exon, so they are cross-linked nucleotides or BNAs (such as LNA) or 5-. Modifications that increase Tm, such as base modifications selected from methylpyrimidines and / or 2,6-diaminopurines, are preferably inversely complementary to the first and / or second regions of the exon. It can be performed at the nucleotide position of interest.

Binding affinity and / or binding or hybridization kinetics depends on the thermodynamic properties of AON. They are the oligonucleotide characterization calculator (http://www.unc.edu/ ~ call / biotool /) for single-stranded RNA using the melting temperature of the oligonucleotide (Tm; eg, basic Tm and adjacent models). (Calculated using oligo / index.html or http: //eu.iddtdna.com/analyzer/Applications/oligoAnalyzer/) and / or (RNA structure version 4.5 or RNA mfold version 3.5) was used. ) Oligonucleotides Determined at least in part by the free energy of the target exon complex. Exon skipping activity is typically increased when Tm is increased, but it is expected that AON will be less sequence-specific when Tm is very high. The Tm and free energy allowed depend on the sequence of oligonucleotides. Therefore, it is difficult to give a preferred range for each of these parameters.

The activity of the oligonucleotides of the invention is preferably defined as:
One or more of the mutations present in the first exon and / or the second exon and / or the mutation-related disorders present in the stretch starting in the first exon and ending in the second exon. To alleviate the symptoms of, preferably to alleviate one or more of the symptoms of DMD or BMD, and / or to alleviate the characteristics of one or more of the patient-derived cells, preferably the patient-derived muscle cells. And / or providing the individual with a functional or semi-functional protein, preferably a functional or semi-functional dystrophin protein, and / or at least partially reducing the production of the abnormal protein in the individual. To reduce the production of abnormal dystrophin protein in the individual at least partially. Each of these features and assays for evaluating them is defined herein below.

Oligonucleotides containing the preferred oligonucleotides of the invention, 5-methylcytosine and / or 5-methyluracil and / or 2,6-diaminopurine base, do not have any 5-methylcytosine, 5-methyl. It is expected to show increased activity compared to the corresponding activity of oligonucleotides that have 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. It can be%, 75%, 80%, 85%, 90%, 95% or 100%. In vivo distribution and in vivo stability are preferably at least partially determined by a valid hybridization ligation assay from Source Yu et al., 2002. In one embodiment, a plasma or homogenized tissue sample is incubated with a specific capture oligonucleotide probe. After separation, the DIG-labeled oligonucleotide is ligated to 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, Harsight, Mountain View, CA). The level of AON (ug) per ml of plasma or 1 mg of tissue is assessed by area under the curve (AUC), peak concentration (Cmax), time vs. peak concentration (Tmax), terminal phase half-life and absorption delay time (tag). Monitored over time to do so. Such preferred assays are disclosed in the experimental section. Oligonucleotides can stimulate the innate immune response 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 on oligodeoxynucleotides (ODNs) by mimicking bacterial DNA that activates the innate immune system by releasing TLR9-mediated cytokines. Occurs. However, 2'-O-methyl modification 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 synchronous immune responses involving innate immunity (macrophages, dendritic cells (DCs) and NK cells) (Krieg AM et al., 1995; Krieg AM. Et al. 2000). Several chemokines and cytokines (Wagner H. et al., 1999; Popovic PJ et al., 2006), such as IP-10, TNFα, IL-6, MCP-1 and IFNα, are involved in this process. Inflammatory cytokines attract additional blood-derived defense cells such as T and B cells. Levels of these cytokines can be determined by in vitro studies. Briefly, whole human blood is incubated with high concentrations of oligonucleotides, after which cytokine levels are determined by standard commercial ELISA kits. The reduced immunogenicity is preferably at least one 5-methylcytosine and compared to cells treated with a corresponding oligonucleotide that does not have 5-methylcytosine, 5-methyluracil or 2,6-diaminopurine. In a detectable reduction in the concentration of at least one of the above-mentioned cytokines compared to the concentration of the corresponding cytokine in the assay of cells treated with oligonucleotides containing / or 5-methyluracil and / or 2,6-diaminopurine. handle.

Therefore, the preferred oligonucleotides of the present invention are acceptable as compared to the corresponding oligonucleotides, which do not have 5-methylcytosine, 5-methyluracil, and 2,6-diaminopurine. It has possible or reduced immunogenicity and / or good biodistribution and / or improved parameters such as acceptable or improved RNA binding kinetics and / or thermodynamic properties. Each of these parameters can be evaluated using assays known to those of skill in the art.

Preferred oligonucleotides of the present invention contain or consist of RNA or modified RNA molecules. In a preferred embodiment, the oligonucleotide is single chain. However, those skilled in the art will appreciate that single-stranded oligonucleotides may form an internal double-stranded structure. However, this oligonucleotide is still named single-stranded oligonucleotide in the context of the present invention. Single-stranded oligonucleotides have several advantages over double-stranded siRNA oligonucleotides: (i) their synthesis is expected to be easier than double-stranded siRNA strands; (ii) intracellular There are a wide range of chemical modifications that can enhance the uptake of, good (physiological) stability, and reduce possible general adverse effects; (iii) siRNAs have non-specific effects (non-target genes). Has a higher likelihood of (including) and excess pharmacology (eg, less controllability of efficacy and selectivity by treatment schedule or dose), and (iv) siRNA is less likely to act in the nucleus. , Cannot be directed at Intron.

In another embodiment, the oligonucleotide of the present invention comprises a debase site or a debase monomer. As used herein, such monomers may be referred to as debase sites or debase monomers. A debased monomer is a nucleotide residue or component that lacks a nucleobase as compared to a corresponding nucleotide residue that contains a nucleobase. Within the present invention, the debase monomer is therefore a component of the oligonucleotide, although it lacks a nucleobase. Such debasement monomers can be present or linked or linked or conjugated to the free end of the oligonucleotide.

In a more preferred embodiment, the oligonucleotides of the invention contain 1 to 10 or more debase monomers. Therefore, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more debase monomers may be present in the oligonucleotides of the present invention.

The debase monomer may be of any type known and conceivable to those skilled in the art. A non-limiting example is shown below.

In the present specification, R ₁ and R ₂ are independently H, oligonucleotide or other debasement site, except that _{both R 1} and R ₂ are not H, and R ₁ and R _{2 are not present.} Not both are oligonucleotides. The debase monomer may be attached to either or both of the terminals of the oligonucleotide previously identified. It should be noted that an oligonucleotide bound to one or two debase sites or debase monomers may 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 debase sites or debase monomers at one or both ends. Other examples of debasement sites included in the present invention are described in US Patent Application Publication No. 2013/0133378 (Alnylam Pharmaceuticals), which is incorporated herein by reference in its entirety.

Depending on their 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, Even if it contains 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. Incorporating more than one different base modification into the oligonucleotide is also included in the present invention.

Therefore, in one embodiment, the oligonucleotide according to the present invention includes:
(A) At least one base modification selected from 2-thiouracil, 2-thiotimine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, and / or (b) at least one sugar modification. And selected from 2'-O-methyl, 2'-O- (2-methoxy) ethyl, 2'-O-deoxy (DNA), 2'-F, morpholino, cross-linked nucleotides or BNA, or said above. Sugar modification and / or (c) at least one skeletal modification selected from phosphorothioate or phosphorodiamidate, wherein the oligonucleotide contains both cross-linked nucleotides and 2'-deoxy-modified nucleotides (BNA / DNA mixmers).

In another embodiment, the oligonucleotides according to the invention include:
(A) At least one base modification selected from 5-methylpyrimidine and 2,6-diaminopurine, and / or (b) at least one sugar modification, which is 2'-O-methyl, and / or (c). ) At least one skeletal modification that is a phosphorothioate.

In one embodiment, the oligonucleotides of the invention have at least one modification, more preferably, compared to a native ribonucleotide or deoxyribonucleotide-based oligonucleotide.
(A) At least one base modification, preferably selected from 2-thiouracil, 2-thiotimine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, 5-methylpyrimidine and 2 , 6-Diaminopurine, a base modification more preferably selected, and / or (b) at least one sugar modification, 2'-O-methyl, 2'-O- (2-methoxy) ethyl, 2 It is preferably selected from'-O-deoxy (DNA), 2'-F, morpholino, crosslinked nucleotides or BNA, or said oligonucleotides contain both crosslinked nucleotides and 2'-deoxy modified nucleotides (BNA / DNA mixmers). Containing, more preferably sugar modification is 2'-O-methyl, and / or (c) at least one skeletal modification, preferably selected from phosphorothioates or phosphorodiamidates, more preferably. Includes skeletal modifications that are phosphorothioates.

Therefore, the oligonucleotide according to this embodiment of the present invention contains the base modification (a), does not contain the sugar modification (b), and does not contain the skeletal modification (c). Another preferred oligonucleotide according to this aspect of the invention comprises sugar modification (b), no base modification (a), and no skeletal modification (c). Another preferred oligonucleotide according to this aspect of the invention comprises skeletal modification (c), no base modification (a), and no sugar modification (b). Also included in the invention are oligonucleotides that do not have any of the modifications described above, as well as two, i.e. (a) and (b), (a) and (c), and / / defined above. Alternatively, an oligonucleotide containing all three of (b) and (c), or modifications (a), (b) and (c) is also included in the present invention.

In a preferred embodiment, the oligonucleotide according to the invention is the same modification selected from (a) one base modification and / or (b) one sugar modification and / or (c) one skeletal modification. One or more of these are modified over their entire length.

With the advent of nucleic acid mimicry technology, it has become possible to produce molecules of the same type, similar, preferably having the same hybridization properties, which are not necessarily the same amount as the nucleic acid itself. Such functional equivalents are, of course, also suitable for use in the present invention.

In another preferred embodiment, oligonucleotides include inosine, hypoxanthine, universal bases, modified bases and / or nucleosides or nucleotides containing bases capable of forming wobble base pairs or functional equivalents thereof. 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 present invention is very attractive as described below. Is. For example, inosine is a known modified base capable of pairing with three bases: uracil, adenine and cytosine. Inosine is a nucleoside formed when hypoxanthine binds to the ribose ring (also known as ribofuranose) via a β [beta] -N9-glycosidic bond. Inosine (I) is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. Wobble base pairs can exist between G and U, or between I on one side and U, A or C on the other. 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 modified base pairs at the first base of the anticodon.

The first advantage of using nucleotides containing inosin (hypoxanthin) and / or universal bases and / or modified bases and / or bases capable of forming a fluctuating base pair in the oligonucleotides of the present invention is that they are derived from pre-mRNA. It is possible to design an oligonucleotide that can bind to the region of one exon and can bind to the region of the second exon in the same pre-mRNA, and the region derived from the second exon is the same as the region of the first exon. Has at least 50% identity. That is, the presence of a nucleotide containing inosine (hypoxanthine) and / or a universal base and / or a modified base and / or a base capable of forming a wobble base pair in the oligonucleotide of the present invention causes the first exon of the pre-mRNA. It is possible to bind the oligonucleotide to the region of No. 1 and the region of the second exon.

The second advantage of using nucleotides containing inosine (hypoxanthin) and / or universal bases and / or modified bases and / or bases capable of forming fluctuating base pairs in the oligonucleotides of the present invention is that the polymorphism is an oligonucleotide. It is possible to design oligonucleotides that supplement single nucleotide polymorphisms (SNPs) without worrying about destroying the annealing effect. Therefore, in the present invention, the use of such bases can be used to design oligonucleotides that can be used in individuals with SNPs within the pre-mRNA stretch targeted by the oligonucleotides of the present invention.

A third advantage of using nucleotides containing inosin (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 described herein. When designing an oligonucleotide to be complementary to a portion of the first pre-mRNA exson, the oligonucleotide usually contains CpG. The presence of CpG in an oligonucleotide is usually associated with increased immunogenicity of said oligonucleotide (Dorn A. and Kippenberger S.). This increased immunogenicity is undesirable as it can induce the breakdown of muscle fibers. Replacing guanine with inosine at one, two or more CpGs in said oligonucleotides is expected to yield oligonucleotides at reduced and / or acceptable levels of immunogenicity. Immunogenicity can be assessed in the animal model by assessing the presence and / or inflammatory mononuclear cell infiltration ^{of CD4 +} and / or CD8 ⁺ cells in animal muscle biopsy. Immunogenicity is an antibody that recognizes the presence and / or said oligonucleotide in the blood of animals or humans treated with the oligonucleotides of the invention using standard immunoassays known to those of skill in the art. Can be evaluated by detecting the presence of. Enhancement of immunogenicity preferably corresponds to a pretreated animal or a nucleotide containing at least one inosine (hypoxanthine) and / or a universal base and / or a modified base and / or a base capable of forming a wobble base pair. Corresponds to a detectable enhancement of at least one of these cell types by comparison with the amount of each cell type during the corresponding muscle biopsy of the animal treated with the nucleotide. Alternatively, immunogenicity enhancement is assessed by using standard immunoassays to detect the presence or increased amount of neutralizing antibodies or antibodies that recognize said oligonucleotides. Can be done. Decreased immunogenicity preferably corresponds to the absence of nucleotides containing pretreatment animals or nucleotides containing inosine (hypoxanthine) and / or universal bases and / or modified bases and / or bases capable of forming wobble base pairs. Corresponds to a detectable reduction in at least one of these cell types by comparison with the amount of the corresponding cell type during the corresponding muscle biopsy of the animal treated with the oligonucleotide. Alternatively, reduced immunogenicity can be assessed using standard immunoassays by the absence of said compounds and / or neutralizing antibodies, or by reduced amounts thereof.

The 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 oligonucleotides. To avoid or reduce multimerization or aggregation. For example, oligonucleotides containing the G-quartet motif 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 is of course undesirable: as a result, the efficiency of oligonucleotides is expected to decrease. Multimerization or aggregation is preferably assessed by standard polyacrylamide non-denaturing gel electrophoresis known to those of skill in the art. In a preferred embodiment, less than 20% or 15%, 10%, 7%, 5% or less of the total number of oligonucleotides of the invention is capable of forming multimers, as assessed using the assays described above. Has the ability to aggregate.

Therefore, the fifth 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 the antithrombotic activity ( Macaya RF et al.) And avoiding quadruple structures associated with binding to macrophages scavenger receptors and inhibition of macrophages scavenger receptors (Suzuki K. et al.).

The sixth 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 RNA binding kinetics and / or It is to enable the design of oligonucleotides with improved thermodynamic properties. RNA binding kinetics and / or thermodynamic properties are determined by the oligonucleotide characterization calculator (http://http: //) for single-stranded RNA using the melting temperature of the oligonucleotide (Tm; basic Tm and closest model). Calculated using www.unc.edu/~cail/biotool/oligo/index.html) and / or at least by the free energy of the AON-target exon complex (using RNA structure version 4.5). Partially determined. If Tm is too high, the oligonucleotide is expected to be less specific. The Tm and free energy allowed depend on the sequence of oligonucleotides. Therefore, it is difficult to give a preferred range for each of these parameters. The permissible Tm can be in the range of 35-85 ° C. and the permissible free energy can be in the range of 15-45 kcal / mol.

Depending on their length, the oligonucleotides of the invention can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 inosine (hypoxanthine) and / or universal base and / or modified. It may include nucleotides containing bases and / or bases capable of forming wobble base pairs or functional equivalents thereof.

Since the RNA / RNA double strand is very stable, it is preferable that the oligonucleotide of the present invention contains 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 contains modifications that impart transport, tissue specificity, etc. to RNA. Preferred modifications are as described above.

Therefore, one embodiment provides an oligonucleotide containing 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-methylphosphorothioate oligonucleotide modification and a bridged nucleic acid modification (BNA exemplified above). In another embodiment of the invention, the oligonucleotide comprises or consists of a hybrid oligonucleotide containing a 2'-O-methoxyethyl phosphorothioate modification and a bridged 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 an equivalent consisting only of bridged nucleic acids and is improved compared to an oligonucleotide consisting of a 2'-O-methylphosphorothioate oligo (deoxy) ribonucleotide modification. Includes effects.

The compounds described in the present invention may preferably have an ionic group. The ionic group may be a base or an acid, may be charged or may be neutral. The ionic group may exist as an ion pair with a suitable counterion having the opposite charge. Examples of cationic counterions are sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium and tetraalkylammonium. Examples of anionic counterions are chlorides, bromides, iodides, lactates, mesylates, acetates, trifluoroacetates, dichloroacetates and citrates. Examples of counterions are described (eg, Kumar L, which is incorporated herein by reference in its entirety). An example of the application of divalent or trivalent counterions, especially Ca2 ⁺ , is to add positive properties to the selected oligonucleotide in US Patent Application Publication No. 2012046348 (Replicaor), which is incorporated by reference in its entirety. It is described as. Preferred divalent or trivalent pair ions are selected from the following description or group: calcium, magnesium, cobalt, iron, manganese, barium, nickel, copper and zinc. The preferred divalent counterion is calcium. Therefore, it is included in the present invention to prepare, obtain and use a composition comprising the oligonucleotide of the present invention and any of the other counterions described above, preferably calcium. Such a method for preparing the composition comprising said oligonucleotide and said counterion, preferably calcium: the oligonucleotide of the present invention may be a pharmaceutically acceptable aqueous excipient. The solution containing the counterion is gradually added to the dissolved oligonucleotide, whereby the oligonucleotide chelate complex remains dissolved.

Thus, in a preferred embodiment, the oligonucleotides of the invention ^{are contacted with such counterions, preferably Ca 2+,} and linked by such counterions as described herein. An oligonucleotide chelate complex containing two or more identical oligonucleotides is formed. Thus, compositions comprising an oligonucleotide chelate complex comprising two or more identical oligonucleotides linked by a counterion, preferably calcium, are included in the present invention.

Methods for Designing Oligonucleotides:
Therefore, in a further aspect of the present invention, there is provided a method for designing an oligonucleotide, which results in the above-mentioned oligonucleotide.

This method involves the following steps:
(A) A step of identifying an in-frame combination of a first exon and a second exon within the same mRNA precursor, wherein the second exon region is at least 50% of the first exon region. Steps, with identity,
(B) The step of designing an oligonucleotide capable of binding to the region of the first exon and the region of the second exon, and (c) the result of the binding, the first exon and the second exon. Skipping, preferably a multi-exon stretch skipping that starts with the first exon and includes one or more exons existing between the first exon and the second exon, and at most. Also a step that results in skipping the entire exon stretch between the first exon and the second exon.

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

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

The oligonucleotides available according to the invention can induce skipping of the first and second exons of the pre-mRNA. Skipping of additional exons is preferably induced, the additional exons are preferably located between the first exon and the second exon, and the resulting mRNA transcript is in-frame. .. Oligonucleotides can preferably induce skipping of the entire exon stretch between the first exon and the second exon. In one embodiment, the region of the first exon and the second exon comprises a splicing control element, whereby the binding of the oligonucleotide is at least in the region of the first exon and the second exon. It can interfere with one splicing control sequence. It also includes the binding of the oligonucleotide interfering with at least the first exon and / or the secondary structure containing the second exon in the mRNA precursor. Preferred splicing control sequences include binding sites for serine-arginine (SR) proteins, exon splicing enhancers (ESE), exon recognition sequences (ERS), and / or exon splicing silencers (ESS).

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

Preferred oligonucleotide:
The oligonucleotide of the present invention is preferably for use as a pharmaceutical, and the compound is more preferably for use in RNA regulatory therapy. It is more preferred that the RNA regulation leads to the correction of the disrupted transcriptional reading frame and / or the repair of the desired or predicted protein expression. Therefore, the methods of the invention and the oligonucleotides of the invention are, in principle, applicable to any disease associated with the presence of frame-destroying mutations that leads to the absence of abnormal transcripts and / or the presence of the abnormal protein. Can be done. In a preferred embodiment, the methods of the invention and the oligonucleotides of the invention are repetitive sequences and thereby relatively high sequence identity (ie, regions of second exons and first exons as defined herein). It is applied to a disease-related gene having a region having at least 50%, 60%, 70%, 80% sequence identity) with the region of. Non-limiting examples are spectroline-like repeats (as the DMD gene involved in Duchenne muscular dystrophy described herein), Kelch-like repeats (KLHL3 gene involved in familial hyperpotassic hypertension (Louis-Dit)). -Picard H et al.), KLHL6 gene involved in chronic lymphocytic leukemia (Puente XS et al.), KLHL7 gene involved in retinal pigment degeneration (Friedman JS et al.), KLHL7 / 12 gene involved in Schegren 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 some cancers (Dhanoa BS et al.), KLHL20 gene involved in promyelocytic leukemia (Dhanoa BS et al.) Or KLHL37 or ENC1 gene involved in brain tumors (Dhanoa BS et al.), FGF-like repeat, EGF-like repeat (NOTCH3 gene involved in CADASIL (Chabrit H et al.)) ), SCUBE gene involved in cancer or metabolic bone disease, neuroxin-1 (NRXN1) gene involved in neuropsychiatric disorders and idiopathic general epilepsy (Moller RS et al.), Del-1 gene, atherosclerosis and Tenesin-C (TNC) gene involved in coronary artery disease (Mollie A et al.), THBS3 gene or fibrillin (FBN1) gene involved in Malfan syndrome (Rantamaki T et al.), Ankyrine-like repeat (involved in dilated myocardial disease) ANKRD1 gene (Dubosq-Bidot L et al.), ANKRD2 gene, ANKRD11 gene involved in KBG syndrome (Sirmaci A et al.), Thrombocytopenia (Noris P et al.) Or ANKRD26 gene involved in diabetes (Raciti GA et al.), Multiple ANKRD55 gene involved in sclerosis (Alloza I et al.) Or TRPV4 gene involved in distal spinal muscle atrophy (Fiorillo C et al.), HEAT-like repeat (httpt gene involved in Huntington's disease, etc.), annexin-like repeat , Leucine-rich repeat (NLRP2 and NLRP7 genes involved in idiopathic recurrent abortion (Hang JY et al.), LRRK2 gene involved in Parkinson's disease (Ab) eliovich A et al.), NLRP3 gene involved in Alzheimer's disease or meningitis (Heneka MT et al.), NALP3 gene involved in renal failure (Knauf F et al.) Or LRIG2 gene (Stuart HM) involved in urological syndrome. Et al.) Or serine protease inhibitor domain (Andrade M. et al.). A. It is a gene having a SPINK5 gene (Hovnanian A et al.) And the like, which are involved in Netherton syndrome (Netherton syndrome), which are described in the above.

In the present invention, the preferred pre-mRNA or transcript is a dystrophin pre-mRNA or transcript. This preferred pre-mRNA or transcript is preferably a human pre-mRNA or transcript. The disease associated with the presence of mutations 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 pre-mRNA 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 within cells, organelles, tissues and / or patients, preferably within BMD or DMD patients or within cells, organs, tissues derived from said patients. Used for. Exons-skipping results in no skipped exons, so that if the exons encode amino acids, they are altered and internally cleaved, but of some to most functional dystrophin products. Mature mRNA is produced that can lead to expression. Techniques for exon skipping are currently directed to the use of AON or AON that transcribes gene constructs. Recently, exon skipping techniques have been investigated to combat genetic muscular dystrophy. Promising results have recently been reported by us and others on AON-induced skipping therapy aimed at repairing the leading frame of intracellular dystrophin mRNA precursors from mdx mice and DMD patients. (Heemskerk H et al .; Cirak S. et al .; Goemans et al.). Targeted skipping of specific exons converts the severe DMD phenotype (lacking functional dystrophin) to the mild BMD phenotype (expressing functional or semi-functional dystrophin). Exon skipping is preferably induced by the binding of AONs that target the exon internal sequences.

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

Patient is preferably intended to mean a patient with DMD or BMD as described below or a patient who is prone to develop DMD or BMD due to his (or her) genetic background. For DMD patients, the compound used preferably corrects mutations such as those present in the patient's DMD gene, and thus preferably creates a dystrophin protein that looks like a dystrophin protein from a BMD patient. The protein is preferably a functional or semi-functional dystrophin described below. For BMD patients, the antisense oligonucleotides of the invention preferably correct mutations such as those present in the BMD gene of the protein, preferably dystrophins that are more functional than the dystrophins initially present in the BMD patients. To create.

As used herein, the functional dystrophin is preferably a wild-type dystrophin corresponding to the protein having the amino acid sequence set forth in SEQ ID NO: 1. Functional dystrophins preferably have binding domains that act on their N-terminal portion (the first 240 amino acids at the N-terminus), the cysteine-rich domain (amino acids 3361 to 3685) and the C-terminal domain (the last 325 amino acids at the C-terminus). And each of these domains is present in wild-type dystrophins known to those skilled in the art. The amino acids shown herein correspond to the amino acids of wild-type dystrophin represented by SEQ ID NO: 1. That is, a functional or semi-functional dystrophin is a dystrophin that exhibits wild-type dystrophin activity, at least to some extent. "At least to some extent" is preferably 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 the dystrophin-related glycoprotein complex (DGC) or (DAGC) (Ehmsen J et al.). Binding of dystrophin to the actin and DGC complex is either immunoco-precipitation using a total protein extract or immunofluorescence analysis of a cross section from a suspected muscular dystrophy known to those skilled in the art. Can be visualized by.

Individuals suffering from DMD typically have mutations in the gene encoding dystrophin that prevents complete protein synthesis (eg, premature arrest prevents C-terminal synthesis). In BMD, the dystrophin gene also contains a mutation, which does not disrupt the open reading frame and the C-terminus is synthesized. The result is a (semi) functional or functional dystrophin protein that is similar in activity to the wild-type protein but not necessarily in similar amounts of activity. The genome of a BMD individual typically contains a dystrophin containing an N-terminal portion (the first 240 amino acids at the N-terminus), a cysteine-rich domain (amino acids 3361 to 3685) and a C-terminal domain (the last 325 amino acids at the C-terminus). It encodes a protein, but in most cases its central terminus can be shorter than that of wild-type dystrophin (Monaco AP et al.). Exon skipping to treat DMD is typically aimed at bypassing premature arrest within the mRNA precursor by skipping exons adjacent to or containing the mutation. This enables the correction of open reading frames and the synthesis of internally cleaved dystrophin proteins, including the C-terminus. In a preferred embodiment, an individual having DMD and treated with the oligonucleotide of the invention synthesizes dystrophin that exhibits similar activity of wild-type dystrophin to at least to some extent. If the individual is or is suspected of being a DMD patient, the (semi) functional dystrophin is more preferably the dystrophin of an individual with BMD: typically the dystrophin is of actin and DGC or DAGC. Can interact with both (Ehmsen J. et al .; Monaco AP et al.).

The central rod-shaped domain of wild-type dystrophin contains 24 spectrin-like repeats (Ehmsen J. et al.). In many cases, the central rod-shaped domain in BMD-like proteins is shorter than that of wild-type dystrophin (Monaco AP et al.). For example, the central rod 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.

Relief of one or more symptoms of DMD or BMD in an individual using the compounds of the invention can be assessed by any of the following assays: prolongation of gait loss time, improvement of muscle strength, ability to lift weights. Improvement, improvement of time to get up from the floor, improvement of walking time of 9 meters, improvement of time to climb 4th 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 of skill in the art. As an example, the publication of Manzur et al. (Manzur AY et al.) Gives a broad description of each of these assays. For each of these assays, as soon as there is a detectable improvement or prolongation of the parameters measured in the assay, preferably one or more symptoms of DMD or BMD are individuals using the compounds of the invention. It means that it is reduced in. The detectable improvement or prolongation is preferably a statistically significant improvement or prolongation as described by Hodgetts et al. (Hodgetts S. et al.). Alternatively, relief of one or more symptoms of DMD or BMD can be assessed by measuring improvements in muscle fiber function, integrity and / or survival. In a preferred method, one or more symptoms of a DMD or BMD patient are alleviated and / or one or more properties of one or more muscle cells from a DMD or BMD patient are improved. Such symptoms or characteristics may be assessed at the cellular, tissue level or on the patient's own.

Mitigation of one or more properties of muscle cells from a DMD or BMD patient can be assessed by one of the following assays for myoblasts or muscle cells from that patient: reduced calcium uptake, reduction by muscle cells. Cellular 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 the cross section of the muscle biopsy.

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

Creatine kinase can be detected in blood as described by Hodgetts et al. (Hodgetts S. et al.). The detectable reduction 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 patients before treatment. , 80%, 90% or more reduction.

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

The detectable increase in muscle fiber diameter uniformity is preferably assessed in the muscle biopsy cross section and more preferably as described by Hodgetts et al. (Hodgetts S. et al.). The increase is measured by comparison with muscle fiber diameter uniformity in the same DMD or BMD patients prior to treatment.

The oligonucleotides of the invention provide the individual with (higher levels) functional and / or (semi) functional dystrophin proteins (both DMD and BMD) and at least produce abnormal dystrophin proteins in the individual. It is preferable that it can be partially reduced. In this context, "one" functional and / or "one" semi-functional dystrophin can mean that several forms of functional and / or semi-functional dystrophin can be produced. This is one in which the region of at least 50% identity between the first exon and the second exon is at least 50% identity between the other first exon and the second exon. Or when overlapping with multiple other regions, and the oligonucleotide can bind to the overlapping portion, thereby skipping some different stretches of exons and producing several different in-frame transcripts. Can be expected if This situation is exemplified in Example 4, and the production of several in-frame transcripts in which one single oligonucleotide (PS816, SEQ ID NO: 1679) according to the present invention shares all exons 10 as a first exon. 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 compared to the 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 dystrophin protein levels. Levels of the functional and / or (semi) functional dystrophin protein are preferably assessed using immunofluorescence or Western blot analysis (protein).

Reducing the 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 that it is still detectable by RT PCR (mRNA) or immunofluorescence or Western blot analysis (protein). Aberrant dystrophin mRNAs or proteins are also herein less functional (compared to the wild-type functional dystrophin proteins defined above) or non-functional dystrophin mRNAs or proteins. Point to. The non-functional dystrophin protein is preferably a dystrophin protein that cannot bind to actin and / or members of the DGC protein complex. Non-functional dystrophin proteins or dystrophin mRNAs typically do not have or encode a dystrophin protein with an intact C-terminus of the protein. In a preferred embodiment, exon skipping (also called RNA regulation or splice switching) techniques are applied.

Increasing the 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 the 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 the aberrant dystrophin protein or mRNA described above herein.

Targeted co-skipping of the two exons (the first exon and the second exon) can induce further exon skipping, which is preferably the first exon. The dystrophin transcript obtained, located between the exon and the second exon, is in-frame (preferably as described in Table 1 or 6), and the DMD or severe BMD phenotype is mild BMD. Or it is further converted to an asymptomatic phenotype. The co-skipping of the two exons is preferably by binding the oligonucleotide to the region of the first exon from the dystrophin mRNA precursor and by binding the oligonucleotide to the region of the second exon within the dystrophin mRNA precursor. Induced by, the region of the second exon has at least 50% identity with the region of the first exon. The first dystrophin exon, the second dystrophin exon, and the region of identity within them are preferably those listed in Table 2 or 6. The oligonucleotide preferably does not show duplication with non-exon sequences. The oligonucleotides preferably do not overlap with the splice site, 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 inversely complementary to the adjacent intron. The exon skipping technique is such that the absence of the two exons in the mRNA produced from the DMD pre-mRNA is preferably located between the additional exons, more preferably between the first and second exons. It is preferred that the absence of exons is applied to produce a more (semi) functional, shorter, but codon region for (higher) expression of the dystrophin protein. In this context (typically a BMD patient), inhibiting inclusion of the two exons, preferably additional exons located between the first exon and the second exon, is preferably. It means the following.
The level of the original abnormal (almost non-functional) dystrophin mRNA is reduced by at least 5% as assessed by RT-PCR, or the corresponding abnormal dystrophin protein level is immunofluorescent or western with anti-dystrophin antibody. A reduction of at least 2% as assessed by blot analysis and / or 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, almost non-functional or non-functional dystrophin protein reductions are 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 inclusion of the two exons, preferably additional exons located between the first and second exons, is preferably (more). It means that (higher levels) more functional or (semi) functional dystrophin protein or mRNA is provided to the individual.

Once a DMD patient is given a (higher level) (higher level) functional or semi-functional dystrophin protein, the cause of DMD is at least partially mitigated. 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 that is directed to both the outer exons of the stretch (or can bind, hybridize, reverse complement, or target). It further provides the insight that skipping from the exon-containing pre-mRNA of the entire exon stretch is induced or enhanced. Throughout the specification in a preferred embodiment, therefore, the oligonucleotides of the invention are at least 80% inversely complementary to the region of the first exon and are defined herein in the second exon. The first exon and the second exon correspond to the outer exon of the skipped exon stretch, which is at least 45% inversely complementary to said region of.

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

The oligonucleotides according to the invention, which are preferably used, are preferably inversely complementary, binding, hybridizing or targeting to the region of the first dystrophin exon, wherein 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, 140, 150, It has 160, 170, 180, 190, 200, or more nucleotides, preferably can be inversely complementary, bindable, hybridizable, or targeted to the region of the second dystrophin exon. The regions are 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, 160, 170, 180, 190, 200, or more nucleotides, said region of said second exon within the same mRNA precursor is at least 50% of said region of said first exon. Is the identity of.

In the present invention, the oligonucleotide may or may contain a functional equivalent of the oligonucleotide. A functional equivalent of an oligonucleotide 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 to some extent. do. The activity of the compound, including the functional equivalent of the oligonucleotide, preferably provides a functional or semi-functional dystrophin protein. Therefore, the activity of said compounds, including functional equivalents of oligonucleotides, is preferably assessed by quantifying the amount of functional or semi-functional dystrophin protein. Functional or semi-functional dystrophins are preferably defined herein as dystrophins that are capable of binding to members of the actin and DGC protein complex and are capable of supporting muscle fibrous membrane structure and flexibility. Assessment of the activity of compounds containing functional oligonucleotides or equivalents 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). The 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 the compound, the activity of which comprises an oligonucleotide from which the functional equivalent is derived. When representing or more, it is preferably retained to at least to some extent. When the term oligonucleotide is used throughout the specification, the term may be replaced by its functional equivalent as defined herein.

Therefore, the use of oligonucleotides or their functional equivalents
Can bind to, target, hybridize, and / or are inversely complementary to the region of the first dystrophin exon.
Can bind to, target, hybridize, and / or are inversely complementary to the region of the second dystrophin exon.
Containing 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.
Relieving one or more symptoms of DMD or BMD and / or relieving one or more properties of patient-derived muscle cells and / or providing the individual with a functional or semi-functional dystrophin protein. The results of DMD treatment by providing and / or reducing the production of abnormal dystrophin protein in the individual at least partially are shown.

Each of these features is already defined herein.

Oligonucleotides contain or consist of sequences that can bind, target, hybridize, and / or are inversely complementary to the region of the first DMD or dystrophin exon, and the second DMD within the same mRNA precursor. Alternatively, the region of dystrophin is preferably at least 50% identical to said region of the first exon. The first exon and the second exon are preferably selected from the group of exons containing exons 8-60, and the exon stretch begins with the first exon and the second exon as the last exon. If included and skipped, it yields an in-frame transcript. Preferred in-frame exon combinations in dystrophin mRNA are given in Tables 1, 2 or 6.

Although not 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 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. It can be 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, those introns can already be an example of a BMD patient expressing a cleaved dystrophin protein, the first, the second and the second exon from the first exon to the second exon. Exon stretch is deleted. Another criterion (criteron) may be the relatively large applicability of skipped exons to the combined subpopulation of DMD (and / or BMD) patients with a particular associated mutation.

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

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

The oligonucleotide of the present invention has an inverse complementary moiety of at least 30%, more preferably at least 40%, still more preferably at least 50%, still more preferably at least 60%, still more preferably at least 30% of the length of the oligonucleotide of the present invention. The first of the dystrophin mRNA precursors is at least 70%, more preferably at least 80%, still more preferably at least 90% or even more preferably at least 95% or even more preferably 98% and most preferably up to 100%. It preferably contains or consists of sequences that can bind, target, hybridize, and / or are inversely complementary to the exon region. In this context, the first exon is preferably the exons 8, 9, 10, 11, 13, 23, 34, 40, 44, 45 or 56 of the dystrophin pre-mRNA as defined herein. .. The oligonucleotide may contain additional flanking sequences. In a more preferred embodiment, the length of the inverse 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 adjacent sequences may be used. Adjacent sequences are preferably used to alter the binding of the protein 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 inversely complementary to the sequence of the dystrophin pre-mRNA that is not present in the exon.

In a preferred embodiment, the oligonucleotide is capable of binding, targeting, hybridizing, and inverse complementary to at least a region of the first dystrophin exon and a region of the second dystrophin exson present in the dystrophin mRNA precursor. The first and second exons are exxons 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 (SEQ ID NO: 1763), SEQ ID NO: 1764), 42 (SEQ ID NO: 11), 44 (SEQ ID NO: 1765), 45 (SEQ ID NO: 12), 47 (SEQ ID NO: 10), 51 (SEQ ID NO: 1760), 53 (SEQ ID NO: 13), 55 (SEQ ID NO: 13) Selected from the group No. 14), 56 (SEQ ID NO: 15), 57 (SEQ ID NO: 1744), or 60 (SEQ ID NO: 16), the region has at least 10 nucleotides. However, the regions are 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 of ordinary skill in the art can also use techniques known in the art to identify regions of the first exon and regions of the second exon. It is more preferred that the online tool EMBOSS Matcher be used as described above. The preferred regions of identity of the first and second dystrophin exons, to which the oligonucleotides of the invention are preferably bound and / or at least partially inversely complementary, are provided in Table 2. Is even more preferable. Here, the inverse 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, hybridizing, targeting, and targeting regions of identity between the first and second exons from Table 2. / Or inversely 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. Has a length of. Here, the inverse 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 listed in Tables 1, 2 or 6, and the oligonucleotides are listed in Table 2 or 6 and sequenced. It is such that it can be coupled to the corresponding first and second exon regions as defined in numbers 17 to 1670, 1742, 1743 or 1766 to 1777.

Preferred oligonucleotides are those disclosed in Table 3 and comprises or consist of SEQ ID NOs: 1671-1741. Other preferred oligonucleotides include or consist of SEQ ID NOs: 1778-1891 disclosed in Table 6. Preferred oligonucleotides include SEQ ID NOs: 1671 to 1741 or 1778 to 1891, with 1, 2, 3, 4 or 5 more nucleotides or 1, 2, 3 than the sequences of those SEQ ID NOs given in Table 3 or 6. It has 4 or 5 fewer nucleotides. These additional nucleotides may be on the 5'or 3'side of the sequence of a given SEQ ID NO:. These missing nucleotides can be nucleotides located on the 5'or 3'side of the sequence of a given SEQ ID NO:. Each of these oligonucleotides may have any of the chemicals described above or a combination thereof. In each of the oligonucleotides identified by SEQ ID NO: herein, 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 comprises SEQ ID NO: 17, and the preferred region of exon 19 comprises SEQ ID NO: 18. Or consists of. Preferred oligonucleotides consist of or include SEQ ID NO: 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 comprises SEQ ID NO: 101 and the preferred region of exon 13 comprises SEQ ID NO: 102. Or consists of.

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

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

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

If 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 sequenced. Includes or consists of 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. Contains a nucleotide sequence having a nucleotide length.

If 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 sequenced. Includes or consists of number 1769. Preferred oligonucleotides are defined in any one of SEQ ID NOs: 1673 or 1674 and include a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

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

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

If the first dystrophin exon is exon 10 and the second dystrophin exon is exon 30, then the preferred region of exon 10 comprises or comprises SEQ ID NO: 127 and the preferred region of exon 30 comprises SEQ ID NO: 128. Or consists of. Preferred oligonucleotides are
A base sequence defined in any one of SEQ ID NOs: 1679 to 1681, 1812, 1813, 1884 to 1886, 1890 or 1891 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or SEQ ID NO: 1814. Defined in, a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or defined in SEQ ID NO: 1675 or 1676, 21, 22, 23, 24, 25, 26, 27, 28, 29 or A base sequence having a length of 30 nucleotides, or a base 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. Defined in one of the base sequences 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or SEQ ID NO: 1786, 1838, 25, A base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or one of SEQ ID NO: 1780, which is defined as 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. It contains a base sequence having a length, or a base sequence defined in any one of SEQ ID NOs: 1785 and 1837 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

If 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 sequenced. Includes or consists of number 1773. Preferred oligonucleotides are
A base 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 NO: 1845, 1846. , 1847, 1848, and a base sequence having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or any one of SEQ ID NOs: 1849, 1850. Defined in any one of the nucleotide sequences 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or SEQ ID NO: 1787, 1851, 26, 27, 28, 29 or 30. Contains a nucleotide sequence having a nucleotide length.

When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 31, the preferred region of exon 10 comprises or comprises SEQ ID NO: 129 and the preferred region of exon 31 comprises SEQ ID NO: 130. Or consists 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 comprises SEQ ID NO: 131 and the preferred region of exon 32 comprises SEQ ID NO: 132. Or consists 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 comprises SEQ ID NO: 137 and the preferred region of exon 35 comprises SEQ ID NO: 138. Or consists of.

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

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

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

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

When the first dystrophin exon is exon 10 and the second dystrophin exon is exon 55, the preferred region of exon 10 comprises or comprises SEQ ID NO: 167 and the preferred region of exon 55 comprises SEQ ID NO: 168. Or consists 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 comprises SEQ ID NO: 169 and the preferred region of exon 57 comprises SEQ ID NO: 170. Or consists 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 comprises SEQ ID NO: 173 and the preferred region of exon 60 comprises SEQ ID NO: 174. Or consists of.

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

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

Preferred oligonucleotides consist of or include SEQ ID NO: 1679 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides. Preferred oligonucleotides include the nucleotide sequence of SEQ ID NO: 1679 and have a length of 25, 26, 27, 28, 29 or 30 nucleotides. Optionally, U 1, 2, 3, 4, 5, 6, 7 of SEQ ID NO: 1679 are replaced / replaced with T. In a preferred embodiment, all U of SEQ ID NO: 1679 have been replaced with T. Preferred oligonucleotides comprising SEQ ID NO: 1679 include any of the chemistries described above: base modification and / or sugar modification and / or skeletal modification.

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

Preferred oligonucleotides consist of or include 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 include 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 include 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 include SEQ ID NO: 1688 and have a length of 26, 27, 28, 29 or 30 nucleotides. Preferred oligonucleotides include the nucleotide sequence of SEQ ID NO: 1688 and have a length of 26, 27, 28, 29 or 30 nucleotides. Optionally, U 1, 2, 3, 4, 5, 6 of SEQ ID NO: 1688 are replaced / replaced with T. In a preferred embodiment, all U of SEQ ID NO: 1688 have been replaced with T. Preferred oligonucleotides comprising SEQ ID NO: 1688 include any of the chemistries defined above herein: base modification and / or sugar modification and / or skeletal modification.

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 exons are exons 13, 14, 15, 18, 20, 27, 30, 31, 32, 35, 42, 44, 47, 48, 55, 57 or 60. This allows the use of any of these oligonucleotides to result in the formation of several in-frame transcripts, each leading to the production of a cleaved but (semi) functional dystrophin protein. 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 comprises SEQ ID NO: 191 and the preferred region of exon 23 comprises SEQ ID NO: 192. Or consists of. Preferred oligonucleotides are
A base 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 NO: 1798, 1865, 1799, 1866. It is defined as any one and contains a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

If the first dystrophin exon is exon 13 and the second dystrophin exon is exon 30, then the preferred region of exon 13 comprises or comprises SEQ ID NO: 285 and the preferred region of exon 30 comprises SEQ ID NO: 286. Or consists of. Preferred oligonucleotides are
Defined in 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. Base sequence,
A base 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 NO: 1856, 1871. , 1860, 1875, and a base sequence having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or any one of SEQ ID NOs: 1857, 1872. And contains 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 comprises SEQ ID NO: 776 and the preferred region of exon 42 comprises SEQ ID NO: 777. Or consists of. Preferred oligonucleotides include or consist of SEQ ID NOs: 1698-1703.

If the first dystrophin exon is exon 34 and the second dystrophin exon is exon 53, then the preferred region of exon 34 comprises or comprises SEQ ID NO: 1294 and the preferred region of exon 53 comprises SEQ ID NO: 1295. Or consists of. Preferred oligonucleotides are
A base 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. Alternatively, it comprises a base 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, the preferred region of exon 40 comprises or comprises SEQ ID NO: 1477 and the preferred region of exon 53 comprises SEQ ID NO: 1478. Or consists of. Preferred oligonucleotides are
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. It is defined as one and contains a base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

If the first dystrophin exon is exon 44 and the second dystrophin exon is exon 56, then the preferred region of exon 44 comprises or comprises SEQ ID NO: 1577 and the preferred region of exon 56 comprises SEQ ID NO: 1558. Or consists 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, the preferred region of exon 45 comprises or comprises SEQ ID NO: 1567 and the preferred region of exon 51 comprises SEQ ID NO: 1568. Or consists of. Preferred oligonucleotides include or consist of SEQ ID NOs: 1730-1731.

If the first dystrophin exon is exon 45 and the second dystrophin exon is exon 53, then the preferred region of exon 45 comprises or comprises SEQ ID NO: 1569 and the preferred region of exon 53 comprises SEQ ID NO: 1570. Or consists of. Preferred oligonucleotides include or consist of SEQ ID NOs: 1732 to 1737.

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

Preferred oligonucleotides consist of or include 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 include 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 include 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 include the nucleotide 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.

If 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 sequenced. Includes or consists of number 1775. Preferred oligonucleotides are defined in any one of SEQ ID NOs: 1790, 1854, 1792, 1855 and include a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides.

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

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

More preferred oligonucleotides include:
(A) A base sequence defined in SEQ ID NO: 1673 or 1674 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 in length as defined in SEQ ID NO: 1677 or 1678. Nucleotide sequence having a nucleotide sequence, or (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 nucleotide sequence. 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 (f) SEQ ID NO: 1688 or 1689. It is defined as either a base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or (g) any one of SEQ ID NOs: 1704 to 1706, 25, 26, 27, 28, 29 or 30. Nucleotide sequence with nucleotide length,
(H) A base 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 base sequence defined in any one of 1717 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

More preferred oligonucleotides include:
(A) Defined in any one of SEQ ID NOs: 1679 to 1681, 1778, 1812, 1813, 1884 to 1886, 1890 or 1891, having a length of 25, 26, 27, 28, 29 or 30 nucleotides. Alternatively, 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 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 defined in SEQ ID NO: 1673 or 1674 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (d) a base sequence. A base sequence defined in 1675 or 1676 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (e) defined in SEQ ID NO: 1677 or 1678, 25, A base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or (f) defined in any one of SEQ ID NOs: 1684-1686, 21, 22, 23, 24, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (g) a base defined in any one of SEQ ID NOs: 1704 to 1706 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence defined in any one of (h) 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 NO: Nucleotide sequence defined in any one of 1710, 1713 to 1717 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (j) SEQ ID NO: 1815 to 1819. Defined in one, a base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (k) any one of SEQ ID NOs: 1820, 1824. It is defined as either a base sequence having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (l) SEQ ID NO: 1826, 1782, 1832. Defined as either a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (m) SEQ ID NO: 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 NOs: 1822, 24, 25, 26, A base sequence having a length of 27, 28, 29 or 30 nucleotides, or (o) defined in any one of SEQ ID NOs: 1823, 1781, 1829, 1830, 1831, 25, 26, 27, 28, 29 or A base sequence having a length of 30 nucleotides, or (p) defined in any one of SEQ ID NOs: 1783, 1833, 1834, 1835, 17, 18, 19, 20, 21, 22, 23, 24, 25, Defined in any one of the base sequences having a length of 26, 27, 28, 29 or 30 nucleotides, or (q) SEQ ID NO: 1887 and having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides. In (r) a base sequence defined in any one of SEQ ID NO: 1888 or 1889 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (s) SEQ ID NO: 1827. Defined in a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (t) SEQ ID NO: 1828, 25, 26, 27, 28, A base sequence having a length of 29 or 30 nucleotides, or (u) defined in any one of SEQ ID NOs: 1784, 1836, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides defined in any one of SEQ ID NOs: 1786 and 1838, or (w). One of the nucleotide sequences defined in any one of SEQ ID NOs: 1780 and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (x) SEQ ID NO: 1785, 1837. Defined in one, a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (y) defined in any one of SEQ ID NOs: 1845, 1846, 1847, 1848, 24, Defined in any one of a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (z) SEQ ID NO: 1849, 1850, 22, 23, 24, 25, 26, 27, Nucleotide sequence having a length of 28, 29 or 30 nucleotides , Or (a1) a base sequence defined in any one of SEQ ID NOs: 1787, 1851 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (b1) SEQ ID NO: 1788, 1852, 1789, 1853. A base sequence defined in any one of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (c1) any one of SEQ ID NOs: 1790, 1854, 1792, 1855. It is defined as either a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (d1) SEQ ID NO: 1794, 1861, 1795, 1862, and is defined as 21, 22, 23. , 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (e1) defined in any one of SEQ ID NOs: 1796, 1863, 1797, 1864, 23, 24, 25. , 26, 27, 28, 29 or 30 nucleotides in length, or (f1) defined in any one of SEQ ID NOs: 1798, 1865, 1799, 1866, 25, 26, 27, 28, 29. Or a base sequence having a length of 30 nucleotides, or (g1) defined in any one of SEQ ID NOs: 1808, 1867, 1809, 1868, 1810, 1869, 1858, 1873, 21, 22, 23, 24, 25. , 26, 27, 28, 29 or 30 nucleotides in length, or (h1) defined in any one of SEQ ID NOs: 1811, 1870, 1859, 1874, 22, 23, 24, 25, 26. , 27, 28, 29 or 30 nucleotides in length, or (i1) defined in any one of SEQ ID NOs: 1856, 1871, 1860, 1875, 23, 24, 25, 26, 27, 28. , A base sequence having a length of 29 or 30 nucleotides, or (j1) a base sequence defined in any one of SEQ ID NOs: 1857, 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 and having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. It is defined in any one of the base sequence or (l1) SEQ ID NO: 1802, 1878, 1803, 1879, and is 23, 24, 25, 26, 27, 28, 29 or 30 nukure. It is defined as either a base sequence having an thiode length or any one of (m1) SEQ ID NOs: 1804 and 1880, and has a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or a base sequence defined in any one of (n1) SEQ ID NOs: 1805 and 1881. (O1) A base sequence defined in any one of SEQ ID NOs: 1806, 1882, 1807, 1883 and having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. ..

An oligonucleotide according to the present invention comprising or consisting of a sequence defined by a SEQ ID NO: means including an oligonucleotide containing the nucleotide sequence defined by the SEQ ID NO:. Also included in the present invention are oligonucleotides having a modified backbone (ie, modified sugar moiety and / or modified nucleoside linkage) with respect to the base sequence defined by SEQ ID NO:. Each base U in the SEQ ID NOs of the oligonucleotides described herein may be modified with T or replaced.

Composition:
In a further embodiment, a composition comprising the oligonucleotides described in the section entitled "oligonucleotides" is provided. The composition preferably comprises or comprises the above oligonucleotides. A preferred composition comprises the one single oligonucleotide described above. Therefore, it is clear that at least the skipping of the first exon and the second exon can be obtained using one single oligonucleotide and without the use of cocktails of different oligonucleotides.

In a preferred embodiment, the composition is for pharmaceutical use. Therefore, the composition is a pharmaceutical composition. Pharmaceutical compositions usually include pharmaceutically acceptable carriers, diluents and / or excipients. In a preferred embodiment, the compositions of the invention comprise the compounds defined herein, which are pharmaceutically acceptable formulations, fillers, preservatives, solubilizers, carriers, diluents, excipients, Additional salts, adjuvants and / or solvents are optionally further included. 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 an acid, may be charged or may be neutral. The ionic group may exist as an ion pair with a suitable counterion having the opposite charge. Examples of cationic counterions are sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium and tetraalkylammonium. Examples of anionic counterions are chlorides, bromides, iodides, lactates, mesylates, acetates, trifluoroacetates, dichloroacetates and citrates. Examples of counterions are described (Kumar L. (the whole of which is incorporated herein by reference)). Thus, in a preferred embodiment, the oligonucleotides of the invention ^{are in contact with a composition containing such an ionic group, preferably Ca 2+} , such a polyvalent cation already defined herein. To form an oligonucleotide chelate complex containing two or more identical oligonucleotides linked by.

The pharmaceutical composition further assists in enhancing the stability, solubility, absorbability, bioavailability, pharmacokinetics and cell uptake of the compound, in particular, in the complex, nanoparticles, microparticles, nanotubes, nanogels, etc. It may be further formulated into a formulation containing an excipient or conjugate 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 include at least one excipient that can further aid 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 include at least one excipient classified as a second type of excipient. The second type of excipient is a target of the compositions and / or oligonucleotides of the invention into tissues and / or cells, such as muscle tissue or muscle cells, and / or into tissues and / or cells. In order to enhance the conversion and / or delivery, the conjugated groups described herein may be included or contained. Both types of excipients may be combined together within one single composition described herein. Preferred conjugate groups are disclosed in the definition part.

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

Such pharmaceutical compositions of the present invention can be administered to an animal, preferably a mammal, at an effective concentration at a set time. More preferred mammals are humans. The compounds or compositions defined herein for use in accordance with the present invention may be suitable for direct in vivo administration to the cells, tissues and / or organs of an individual, said individual to BMD or DMD. It is preferably affected or at risk of developing them and can be administered directly in vivo, ex vivo or in vitro. Administration is via the systemic and / or parenteral route, eg, intravenous, subcutaneous, intraventricular, intrathecal, intramuscular, intranasal, intestinal, intravitreal, intrabrainal, epidural or oral. 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 is preferred that it can be encapsulated in the form of a powder.

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

use:
In a further embodiment, the use of the compositions or compounds described herein for use as pharmaceuticals or as part of therapy, or applications in which the compound imparts its activity intracellularly is provided.

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

Methods for Preventing, Delaying, Ameliorating and / or Treating Diseases:
In a further aspect, methods are provided for preventing, delaying, ameliorating and / or treating the diseases defined herein, preferably DMD or BMD. The disease can be prevented, treated, delayed or ameliorated in an individual, in the cells, tissues or organs of the individual. The method comprises the step of administering the oligonucleotide or composition of the invention to said individual or subject in need thereof.

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

The range of concentrations or doses given above for oligonucleotides or compositions is the preferred concentration or dose for in vitro or exobibo use. A person skilled in the art will use the target cell to be treated, the gene target and its expression level, the medium used and the transfection and incubation conditions, and the concentration or dose of the oligonucleotide used, depending on the identity of the oligonucleotide used. You will understand that may need to be further modified and further optimized.

Definition:
Known in the art, "sequence identity" is the relationship between two or more nucleic acid (polynucleotides or nucleotides) sequences, as determined by comparing the sequences. In the art, "identity" or "similarity" indicates the degree of sequence relevance between nucleic acid sequences, as determined by the coincidence between the strands of such sequences. "Identity" may be replaced herein with "similarity." Identity is preferably determined by comparing all SEQ ID NOs as described herein. However, parts of the sequence can also be used. Here, 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” are, but are not limited to, Computational Molecular Biology, Lesk, A. et al. M. Eds., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. et al. W. Hen, Academic Press, New York, 1993; Computer Analysis of Sexuence Data, Part I, Griffin, A. et al. M. , And Griffin, H. et al. G. Hen, Humana Press, New Jersey, 1994; Sequencing Analysis in Molecular Biology, von Heine, G. et al. , Academic Press, 1987; and Sequence Analysis Primer Gribskov, M. et al. And Devereux, J. et al. Hen, M Stockton Press, New York, 1991; and Carillo, H. et al. , And Lipman, D.I. , SIAM J. Applied Math. , 48: 1073 (1988), which can be readily calculated by known methods.

The preferred method for determining identity is designed to give the maximum match between the sequences tested. Methods for determining identity and similarity are codified in published computer programs. Preferred computer programming methods for determining the identity and similarity between two sequences include, for example, the GCG program package (Deverex, 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 lookups includes, for example, a nucleotide database. BLASTN for nucleotide query sequences to sequences. BLAST 2.0 family programs include NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. et al. . Mol. Biol. 215: 403-410 (1990)). Well-known Smith Waterman algorithms may also be used to determine identity.

Preferred parameters for nucleic acid comparison include: Algorithms: Needleman and Wunsch, J. Mol. 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 manufactured by Genetics Computer Group in Madison, Wisconsin is available. 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. , JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, Time warps, string edits and matrix 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 Matthematic Biology 1992, Vol. 54). pp.521-536; Vingron and Waterman, J. Mol. Biol. 1994; 235 (1), p1-12.]). You can use the following websites: http: // www. ebi. ac. uk / Tools / emboss / align / index. html or html: // emboss. bioinformatics. nl / cgi-bin / emboss / matcher. Definitions of the parameters used in this algorithm can be found at the following website: http: // emboss. Sourceforge. net / docks / themes / AlignFormats. html # id. It is preferred that the default settings (matrix: EDNAFULL, gap_penalty: 16, extension_penalty: 4) are used. Emboss Matcher provides optimal local alignment between two sequences, but alternative alignments are provided as well. Table 2 discloses the optimal local alignment between two different exons, preferably dystrophin exons, used for oligonucleotide design. However, an alternative alignment and thereby an alternative region of identity between the two exons can also be identified and used for oligonucleotide design, which is also part of the invention.

Throughout the specification, the terms "binding," "targeting," and "hybridizing" are used in the context of oligonucleotides that are inversely complementary to the regions of pre-mRNA described herein. If used, it may be used interchangeably. Similarly, the expressions "capable of binding," "targetable," and "hybridizable" indicate that the oligonucleotide has a particular sequence that can bind, target, or hybridize to the target sequence. Can be used interchangeably for. Whenever a target sequence is defined herein or is known in the art, one of ordinary skill in the art can use the concept of inverse complementation to construct all possible structures of said oligonucleotides. In this regard, a limited number of sequence mismatches or gaps between the oligonucleotides of the invention and the target sequences in the first exon and / or the second exon are described above unless binding is affected. It will be understood that it is acceptable, as it is. Therefore, an oligonucleotide that can bind to a particular target sequence can be considered as an oligonucleotide that is inversely complementary to the target sequence. In the context of the present invention, "hybridize" or "hybridize" is used under physiological conditions within cells, preferably human cells unless otherwise noted.

As used herein, "hybridization" refers to the pairing of complementary oligonucleotide compounds (eg, antisense compounds and their target nucleic acids). The most common mechanism of pairing, but not limited by a particular mechanism, is the Watson-click, Hoogsteen or inverted Hoogsteen hydrogen bond between complementary nucleosides or nucleotide bases (nucleobases). Possible, including hydrogen bonds. For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymine, 5-methyluracil and uracil, which are paired by the formation of hydrogen bonds. The natural base guanine is a nucleobase complementary to the natural base cytosine and 5-methylcytosine. Hybridization can occur under variable conditions.

Similarly, "inverse complementary" are two nucleotide sequences that can hybridize to each other, but one of the sequences is oriented from 3'to 5'and the other is oriented in the opposite direction, i.e. 5'to 3'. Used to identify. Therefore, the nucleoside A in the first nucleotide sequence ^{can be paired with the nucleoside A *} in the second nucleotide sequence via their respective nucleic acid bases, and is located at the 5'position from the above-mentioned nucleoside A in the first nucleotide sequence. The nucleoside B located at can be paired ^{with the nucleoside B *} located at the 3'position from the above-mentioned nucleoside A ^{* in the second nucleotide sequence.} In the context of the invention, the first nucleotide sequence is typically the oligonucleotide of the invention, and the second nucleotide sequence portion is an exon derived from a pre-mRNA, preferably a dystrophin pre-mRNA. The oligonucleotide is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% inverse complementary to the region of the first exon and / or the second exon. If so, the oligonucleotide is preferably said to be inversely complementary to the region of an exon (first exon and / or second exon). The inverse complement is preferably at least 80%, 85%, 90%, 95% or 100%.

In the present invention, the excipients include polymers (eg, polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivatives, butylcyanoacrylate (PBCA), hexylcyanoacrylate (PHCA), poly (lactic acid-co-glycol). Acids) (PLGA), polyamines (eg spermin, spermidin, putresin, cadaverine), chitosan, poly (amide amine) (PAMAM), poly (esteramine), polyvinyl ether, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), Cyclodextrin, hyaluronic acid, collomic acid and derivatives thereof), dendrimers (eg, poly (amide amine)), lipids {eg, 1,2-diore oil-3-dimethylammonium propane (DODAP), diole oil dimethylammonium chloride (eg, diole oil dimethylammonium 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 (eg, 1-stearoyl-2-lyso-sn-glycero-3-phosphocholine) S-lyso PC)], sphingomyelin, 2- {3- [bis- (3-amino-propyl) -amino] -propylamino} -N-ditetracedircarbamoylmethylacetamide (RPR209120), phosphoglycerol derivative [eg , 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (DPPG-Na), phosphatid acid derivative [1,2-distearoyl-sn-glycero-3-phosphatidic acid, sodium Salt (DSPA), phosphatidylethanolamine derivatives [eg, dioreoil-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 2-difitanoyl-sn-glycero- 3-phosphoethanolamine (DPhyPE)], N- [1- (2,3-dioreoiloxy) propyl] -N, N, N-trimethylammonium (DOTAP), N- [1- (2,3-2,3-) Dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA), 1,3-di-oleoyloxy-2- (6-carboxy-spermil) -propylamide (DOSPER), (1,2- Jimiristhiol Dimyristyolxypropyl-3-dimethylhydroxyethylammonium (DMRIE), (N1-cholesteryloxycarbonyl-3,7-diazanonan-1,9-diamine (CCAN), dimethyldioctadecylammonium bromide (DDAB), 1-palmitoyl- 2-Oleoil-sn-glycerol-3-phosphocholine (POPC), (b-L-arginine-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (AtuFECT01), N , N-Dimethyl-3-aminopropane derivative [eg, 1,2-distearoyl-N, N-dimethyl-3-aminopropane (DSDMA), 1,2-diorailoxy-N, N-dimethyl-3- Aminopropane (DoDMA), 1,2-dilinoleyloxy-N, N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl [1,3] -dioxolane (DLin-K) -DMA), phosphatidylserine derivatives [1,2-diorail-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) are included.

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 pharmacodynamic 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 radioactive substance labeled moieties. The conjugate group can be optionally protected and may be attached to the oligonucleotide of the invention either directly or via a divalent or multivalent linker.

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, Antimiclob. Agents Chemother. 2009, 53, 525), polyornithins, TAT, TP10, pAntp, polylysine, etc. NLS, Penetratin, MSP, ASSLINIA, MPG, CADY, Pep-1, Pip, SAP, SAP (E), Transportan, Buforin II, Polymixin B, Histatin, CPP5, NickFect, PepFect), Vivo-porter ), Proteins (eg, antibodies, avidin, Ig, transferase, albumin), carbohydrates (eg, glucose, galactose, mannose, maltose, maltriose, ribose, trehalose, glucosamine, N-acetylglucosamine, lactose, sucrose, fucose, arabinose, Thalose, sialic acid, hyaluronic acid, neuromidic acid, ramnorth, quinobos, galactosamine, N-acetylgalactosamine, xylose, lyxose, fructose, mannose-6-phosphate, 2-deoxyribose, glucar, cellulobiose, Chitobiose, chitotriose), polymers (eg, polyethylene glycol, polyethyleneimine, polylactic acid, poly (amide amine)), 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, Bacil, Glycerophospholipid, Glycerolipid, Sphingolipid, Ceramide, Celebroside, Sphingosin, Sterol, Plenol, Elsyl, Alacidonyl, Linoleil, Linolenyl, Alacidyl, Butyryl, Sapeniyl, Elydyl, Lauryl, Behenyl Nonyl, decyl, undecyl, octyl, heptyl, hexyl, pentyl, DOPE, DOTAP, terpenyl, diterpenoid, triterpernoid, polyfluoroalkyl NSAIDs such as moieties (eg, perfluoro [1H, 1H, 2H, 2H] -alkyl), approved drugs with a molecular weight <1500 Da that have affinity for specific proteins (eg, NSAIDs such as ibuprofen (many of which are referred to herein). It is described in US Pat. No. 6,656,730 incorporated in)), antidepressants, antacids, antibiotics, alkylating agents, anti-amoeba agents, antidotes, androgen, ACE inhibitors, Appetite suppressants, antacids, pesticides, anti-angiogenic agents, anti-adrenaline agonists, anti-angina agents, anti-cholinergic agents, anti-coagulants, anti-convulsants, anti-diabetic agents, antidiarrheals, anti-diuretics, Antidotes, antifungal agents, antacids, antidiarrheals, anti-gouts, anti-adotropics, anti-histamines, anti-hyperlipidemic agents, antihypertensive agents, anti-malaria agents (antimalarials), anti-mitral pain agents , Anti-tumor agents, anti-psychiatric agents, anti-rheumatic agents, anti-thyroid agents, anti-toxins, anti-diarrheals, anti-anxiety agents, contraceptives, CNS stimulants, chelating agents, cardiovascular agents, decongestants, skin diseases, diuretics, Antidote, diagnostic agent, gastrointestinal drug, anesthetic, glucocorticoid, anti-arrhythmic agent (antialhytics), immunostimulatory agent, immunosuppressant, antacid, antihansen's disease agent (leprostatics), metabolite, respiratory agent, mucolytic agent, Muscle relaxants, nutritional supplements, vasodilators, thrombolytic agents, uterine contractile agents, pressor agents), natural compounds with a molecular weight <2000 Da (eg, antibiotics, ecosanoids, alkaloids, flavonoids, terpenoids, enzyme cofactors, polyketides) , Steroids (eg prednisone, prednisolone, dexamethasone, lanosterol, antacid, estran, androstan, pregnane, colan, cholesterol, ergosterol, cholesterol, cortisol, cortisol, defrazacoat), pentacyclic triterpenoids (eg 18β-glycyrrhetinic acid, Ursoleic acid, amylin, carbenoloxone, enoxolone, acetoxolone, bethulic acid, Asian acid, erythrodiol, oleanolic acid), polyamine (eg spermin, spermidin, prednisone, cadaberin), fluorescent moiety (eg FAM, carboxyfluoresene) , FITC, TAMRA, JOE, HEX, TET, Rhodamine, Cy3, Cy3.5, Cy5, Cy5.5, CW800, BODIPY, AlexaFluors, Dabcil, DN P), a reporter molecule (e.g., acridine, biotin, digoxigenin, (radioactive) isotopically-labeled portion ^{(e.g., 2 H, 13 C, 14} C, 15 N, 18 O, 18 F, 32 P, 35 S , ⁵⁷ Co, ^{99 m} Tc, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁵³ Gd)) and combinations thereof. Such a conjugate group may be connected to the compound of the present invention either directly or via a linker. The linker may be divalent (which yields a 1: 1 conjugate) or polyvalent which yields an oligomer having more than one conjugate group. Procedures for coupling such conjugate groups to the oligomers of the invention, either directly or via a linker, are known in the art. Also, for example, J.M. Am. Chem. Soc. As described in 2008, 130, 12192 (the whole of which is incorporated herein by reference; Hurst et al.), To the extent such constructs are referred to as spherical nucleic acids (SNAs), the oligos of the invention. The use of nanoparticles covalently bound to nucleotides is also within the context of the present invention.

In the present specification, the verb "to compare" and its use are used in their non-limiting sense to mean that the matters following the term are included, but are described in detail. Matters that are not included are not excluded. Further, the verb "to consist" may be replaced with "to consentially of", and the oligonucleotides or compositions defined herein are specific. It may contain additional components other than those described above, which means that the additional components do not alter the inherent properties of the invention. In addition, there are more than one reference to an element by the indefinite article "one (a)" or "one (an)" unless the context explicitly requires that it be one and only one element. Does not rule out the possibility that the element exists. Therefore, the indefinite article "one (a)" or "one (an)" usually means "at least one."

When used with respect to a number (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 embodiment described herein may be combined together unless otherwise noted. All patents and references cited herein are incorporated herein 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 to 3]
Table 1:
If any exon from exon U + 1 (first exon) to D-1 (second exon) and in between is removed from the transcript, exon U (upstream) is continuous with exon D (downstream). A list of possible exon combinations in DMD gene transcripts with open reading frames.

Table 2:
List of exon areas. Optimal 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 that has (partially) high sequence identity or similarity (at least 50%) between two different dystrophin exons, identified as a pairwise alignment. Skipping these exons and preferably any exons in between yields an in-frame DMD transcript (eg, in Table 1).

Table 3:
A list of preferred nucleotide sequences of oligonucleotides according to the present invention. Oligonucleotides containing these sequences can bind to regions of high similarity (> 50% identity sequence stretch) in two different DMD exons (eg, listed in Table 2) and these two exons. And any exon skipping in between can be induced to produce an in-frame DMD transcript.

＊ Ｄ＝２，６−ジアミノプリン；Ｃ＝５−メチルシトシン；Ｘ＝Ｃ又は５−メチルシトシン；Ｙ＝Ｕ又は５−メチルウラシル；Ｚ＝Ａ又は２，６−ジアミノプリン

* 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:
A list showing the most preferred and / or additional exon combinations, the most preferred and / or additional exon identity regions, and the most preferred and / or additional oligonucleotides, along with their nucleotide sequences, chemical modifications and SEQ ID NOs. .. 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'-fluoronucleotides (eg, SEQ ID NO: 1844).

[Examples 1 to 5]
Materials and methods:
The design of oligonucleotides was based primarily on inverse 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 those of partially open / closed secondary RNA structures in said sequence stretch (inferred by RNA structure version 4.5 or RNA mfold version 3.5 (Zuker, M.)). The presence and / or the presence of a putative SR-protein binding site in said sequence stretch (as inferred by ESE-finder software (Cartegrani L et al., 2002 and Cartegni L et al., 2003)). All AONs are described in Prosensa Therapeutics B. et al. V. Synthesized by (Leiden, The 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 are 2'-O-methylphosphorothioate RNA and were synthesized on a 10 μmol scale using an OP-10 synthesizer (GE / AKTA Oligopilot) using 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 was added to the exchange ions. After evaporation, the compound was redissolved in water, desalted by FPLC and lyophilized. Mass spectrometry confirmed the identity of all compounds and found that the purity (measured by UPLC) was 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 muscle cells (myotubes) were transfected in a 6-well plate at a standard AON concentration of 400 nM according to non-GLP standard working procedures. Polyethylenimine (ExGen500; Fermentas Netherlands) was used for transfection (in 2 μl / μg AON, 0.15 M NaCl). The transfection procedure described above was adapted from previously reported materials and methods (Artsma-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 with 3 μl of cDNA for each sample and included a 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 (Artsma-Rus A. et al., 2002 and Artsma-Rus A. et al., 2003). RT-PCR products were analyzed by gel electrophoresis (2% agarose gel). The obtained RT-PCR fragment was quantified by DNA Lab-on-a-Chip analysis (Agilent Technologies USA; DNA 7500 Lab Chips). Data was processed by the "Agilent 2100 Bioanalyzer" software and Excel 2007. The ratio of the total amount of smaller transcripts (containing the expected multiple exon skippings) to transcripts (percentages representing skipping efficiency) was evaluated and compared directly with that in untransfected cells. PCR fragments were also isolated from agarose gels for sequence validation (Sanger sequencing, BaseClear, Netherlands) (QIA Quick Gel Extraction Kit, Qiagen Netherlands).

Table 4:
PCR primer set used to detect target exon skipping

Table 5:
Primer sequence

result:
Example 1:
Targeting of sequence stretches with high similarity in exons 10 and 18 using AON at different sites.
Based on the high similarity (63%) sequence stretch in Exxon 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110), a series of AONs was designed and Exxon 10 (PS814, SEQ ID NO: 1675; PS815, SEQ ID NO: 1677). Dispersed over said sequence stretch, which is 100% inverse complementary to either PS816, SEQ ID NO: 1679) or Exxon 18 (PS811, SEQ ID NO: 1673). After transfection in healthy human control myotube cultures, RT-PCR analysis demonstrated that all four AONs could induce skipping of exons 10-18 (confirmed by sequence analysis) (FIG. 1). ). PS811 and PS816 had the highest percentage of inverse complementarity in both exons (Fig. 1B) and were most effective with 70% and 66% skipping efficiencies of exons 10-18, respectively (Fig. 1B). 1C). PS814 is the least effective, which can be unique to its position and / or shorter length (21 for 25 nucleotides), and thereby lower binding affinity or stability. No skipping of exons 10-18 was observed in untreated cells (NT). These results are highly reproducible (skipping exons 10-18 by PS816 at 20/20 different transfections), and skipping multi-exon stretches from exons 10-18 has high sequence similarity (63%). ) Can be combined with both exons 10 and 18 in the region with), demonstrating that this is feasible by using a single AON capable of inducing skipping of these exons and all exons in between. Although additional multiple exon skipping fragments were obtained in this transcript region, the resulting transcripts (in-frame transcripts) in which exons 9 were directly spliced to 19 were the most abundant in all experiments.

Example 2:
Targeting of 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 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 muscle cells (ie differentiated myotubes) , RT-PCR analysis demonstrated that all three AONs can induce skipping (confirmed by sequence analysis) of exons 10-18 (FIGS. 2A, B). PS816 and PS1059 were the most effective (68% and 79%, respectively). The shorter PS1050 was less effective (25%). Exon 10-18 skipping was not observed in untreated cells (NT). These results indicate that skipping of multi-exon stretches of exons 10-18 can be performed by using AON of 15, 21 and 25 nucleotides. Longer AONs are expected to work as well. 21-mer PS1059 is the most effective and shortest candidate. 15-mer PS1050 is the least effective, which may be inherent in its low reverse complementarity to exon 18 (47%, FIG. 2B) and / or its low binding affinity or stability to target RNA. In order to improve the biological activity of 15-mer, base modification may be required to improve Tm of 15-mer and thereby binding affinity or double-stranded stability. The most abundant transcript obtained in this experiment was again the exon 9 directly spliced to 19 (in-frame transcript).

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

Example 4:
PS816 induces skipping of other in-frame multi-exon stretches.
The highly similar sequence stretches in Exxon 10 (SEQ ID NO: 109) and 18 (SEQ ID NO: 110) are also exxons 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) are (partially) present (FIGS. 4A, B, Table 2). We have now focused on detecting 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. In fact, using RT-PCR analysis, we have skipped in-frame exons 10-30, exons 10-42, exons 10-47, exons 10-57 and / or exons 10-60 in multiple experiments. Observed (confirmed by sequence analysis), these were not observed in untreated cells. As an example, FIG. 4C shows the skipping of exons 10-18, exons 10-30 and exons 10-47 induced by PS816. Despite additional multi-exon skipping events (either in-frame or out-of-frame), the resulting transcript (in-frame transcript) with exons 9 directly spliced to 19 is the most reproducible and all It seemed to be the most abundant in the experiment. These results allow multiple exon skippings to bind to sequence stretches that are highly similar between two different exons, and skipping these exons and all exons in between to produce an in-frame DMD transcript. It is confirmed that it can be induced across the DMD gene using a single AON capable of inducing.

Example 5:
Targeting of sequence stretches with high similarity in exons 45 and 55 using AON with modified basic chemistries.
Based on the high similarity (80)% sequence stretch in Exxon 45 (SEQ ID NO: 1571) and 55 (SEQ ID NO: 1572), a series of AONs was designed and Exxon 45 (PS1185, SEQ ID NO: 1706; PS1186, SEQ ID NO: 1707). ) Is 100% inverse complementary or 96% inverse complementary to Exxon 55 (with one mismatch) (PS1188, SEQ ID NO: 1713) over the sequence stretch (FIG. 5A). In all three AONs, As was replaced by 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). .. No skipping of exons 45-55 was observed in untreated cells (NT). These results show that skipping 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 in between. It has been demonstrated that this can be done by using a single AON that can induce exon skipping. The resulting transcript, in which exons 44 were directly spliced to 56, was in-frame and was observed in multiple experiments. With respect to exon 10-18 skipping experiments (Example 3), we show here again that AON with modified bases can be effectively applied. Moreover, the results obtained with PS1188 do not require the AON to be 100% inverse complementary to one of the target exons, but there is 100% inverse complementary to any of the target exons. It is shown that it can 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 the sequence stretch, which are highly similar (63%) in Exxon 10 and Exxon 18. Localization of (SEQ ID NO: 1679). B) AON characteristics and efficiency. The inverse complementarity (rev. Comp.) (%) Of each AON to either exon 10 or exon 18 is shown. C) RT-PCR analysis. In healthy human muscle cells (ie differentiated myotubes), all four AONs were effective in inducing the skipping of multi-exon stretches of exons 10 to exon 18. PS811 and PS816 were the 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 muscle cells (ie differentiated myotubes), AONs with different lengths but with the same core target sequence within sequence stretches with high similarity (63%) in exons 10 and 18 were tested. .. PS816 (SEQ ID NO: 1679) (25-mer) and PS1059 (SEQ ID NO: 1684) (21-mer) were the 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 inverse 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 basic chemical properties was tested in healthy human muscle cells (ie differentiated myotubes). PS816 (normal 2'-O-methylphosphorothioate RNA AON; SEQ ID NO: 1679), PS1168 (PS816, but all A have been replaced with 2,6-diaminopurine; SEQ ID NO: 1681), PS1059 (normal 2) '-O-methylphosphorothioate RNA AON; SEQ ID NO: 1684), PS1138 (PS1059, but all Cs have been replaced with 5-methylcytosine; SEQ ID NO: 1685) or PS1170 (PS1059, but all A's 2,6 -Replaced with diaminopurine; SEQ ID NO: 1686). All AONs tested for exon 10-18 skipping were observed. PS816 was the most effective (88%). In these particular sequences, base modification 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 stretches at exons 10, 18, 30 and 47 as multiple targets for PS816. The sequence number numbers refer to the EMBOSS full length exon alignment (as shown in Table 2). In the gray font, the sequence portion was not included in the EMBOSS alignment, but is adjacent to the portion having high inverse complementarity to PS816. B) Overview of exon alignment and PS816 inverse complementarity (rev. Comp.) Percentage. C) RT-PCR analysis. Multiple exon stretch skippings can be induced by PS816 and are 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). The boxes on the left and right sides of the figure represent the content of the PCR-amplified transcript (M: DNA size marker). FIG. 5 (Fig. 5): A) Stations of PS1185 (SEQ ID NO: 1706), PS1186 (SEQ ID NO: 1707) and PS1188 (SEQ ID NO: 1713) in sequence stretch, which are highly similar (80%) in exon 45 and exon 55. Presence. The table summarizes the AON characteristics. The inverse complementarity (rev. Comp.) (%) Of each AON to either exon 45 or exon 55 is shown. B) RT-PCR analysis. In healthy human muscle cells (ie differentiated myotubes), all three AONs were effective in inducing skipping of multi-exon stretches of exons 45-55. 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 pharmaceutical use to prevent, delay, ameliorate and / or treat a disease in a subject.
The oligonucleotide can bind 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 that starts at the first exon and exists between the first exon and the second exon. Or skipping a multi-exon stretch that includes multiple exons, and skipping the entire exon stretch between the first exon and the second exon at most.
In-frame transcripts that allow the production of functional or semi-functional proteins are obtained,
Antisense oligonucleotide.
[2]
The oligonucleotide according to [1], which can induce skipping of the entire stretch of an exon between the first exon and the second exon.
[3]
The binding comprises at least one splicing control sequence in the region of the first exon and the second exon and / or at least the first exon and / or the second exon in the mRNA precursor. The antisense oligonucleotide according to [1] or [2], which is capable of interfering with the secondary structure.
[4]
[3] 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). Antisense oligonucleotide.
[5]
The oligonucleotide according to any one of [1] to [4], which comprises 10 to 40 nucleotides.
[6]
The oligonucleotide according to any one of [1] to [5], which comprises at least one modification as compared with a natural ribonucleotide-based or deoxyribonucleotide-based oligonucleotide, and more preferably.
(A) At least one base modification, preferably selected from 2-thiouracil, 2-thiotimine, 5-methylcytosine, 5-methyluracil, thymine, 2,6-diaminopurine, 5-methylpyrimidin and 2 , 6-Diaminopurine, more preferably a base modification 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 nucleotides or BNA, or said oligonucleotides contain both cross-linked nucleotides and 2'-deoxy modified nucleotides (BNA / DNA mixmers) Containing, more preferably the sugar modification is 2'-O-methyl, the sugar modification and / or (c) at least one skeletal modification, preferably selected from phosphorothioates or phosphorodiamidates, more preferably. Oligonucleotides containing skeletal modifications that are phosphorothioates.
[7]
The oligonucleotide according to any one of [1] to [6].
The oligonucleotide contains one or more conjugate groups and contains one or more conjugate groups.
The conjugate group is optionally protected and contains peptides, proteins, carbohydrates, drugs, target moieties, uptake-enhancing moieties, solubility-enhancing moieties, pharmacodynamic-enhancing moieties, pharmacokinetic-enhancing moieties, polymers, ethylene glycol derivatives, vitamins, lipids. , Polyfluoroalkyl moieties, steroids, cholesterol, fluorescent moieties, reporter molecules, radioactive substance labeled moieties and combinations thereof, and attached to terminal or internal residues directly or via a divalent or polyvalent linker. do,
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], which is selected from 55, 56, 57 or 60 and the disease is DMD or BMD.
[9]
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 , Exon 13-30, Exon 23-42, Exon 34-53, Exon 40-53, Exon 44-56, Exon 45-51, Exon 45-53, Exon 45-55, Exon 45-60 and / or Exon 56. The oligonucleotide according to [8], which can induce skipping of -60.
[10]
A base sequence defined in any one of SEQ ID NOs: 1679, 1688, 1671 to 1678, 1680 to 1687, 1689 to 1741 or 1788 to 1891, the length defined by the number of nucleotides present in the base sequence. Or, it comprises a base sequence having a length of 1, 2, 3, 4 or 5 nucleotides longer or 1, 2, 3, 4 or 5 nucleotides shorter than defined in the base sequence [8] The oligonucleotide according to [9].
[11]
[10] The oligonucleotide according to [10].
(A) Defined in any one of SEQ ID NOs: 1679 to 1681, 1778, 1812, 1813, 1884 to 1886, 1890 or 1891, having a length of 25, 26, 27, 28, 29 or 30 nucleotides. Alternatively, 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 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 defined in SEQ ID NO: 1673 or 1674 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (d) a base sequence. A base sequence defined in 1675 or 1676 and having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (e) defined in SEQ ID NO: 1677 or 1678, 25, A base sequence having a length of 26, 27, 28, 29 or 30 nucleotides, or (f) defined in any one of SEQ ID NOs: 1684-1686, 21, 22, 23, 24, 25, 26, 27, A base sequence having a length of 28, 29 or 30 nucleotides, or (g) a base defined in any one of SEQ ID NOs: 1704 to 1706 and having a length of 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence defined in any one of (h) 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 NO: Nucleotide sequence defined in any one of 1710, 1713 to 1717 and having a length of 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (j) SEQ ID NO: 1815 to 1819. Defined in one, a base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (k) any one of SEQ ID NOs: 1820, 1824. Defined as one of a base sequence having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (l) SEQ ID NO: 1826, 1780, 1782, 1832. , 21, 22, 23, 24, 25, 26, 27, 28, 29 or defined in any one of the base sequences having a length of 29 or 30 nucleotides, or (m) SEQ ID NOs: 1821, 1825. , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (n) defined in any one of SEQ ID NOs: 1822, 24, 25, 26, A base sequence having a length of 27, 28, 29 or 30 nucleotides, or (o) defined in any one of SEQ ID NOs: 1823, 1781, 1829, 1830, 1831, 25, 26, 27, 28, 29 or A base sequence having a length of 30 nucleotides, or (p) defined in any one of SEQ ID NOs: 1783, 1833, 1834, 1835, 17, 18, 19, 20, 21, 22, 23, 24, 25, Defined in any one of the base sequences having a length of 26, 27, 28, 29 or 30 nucleotides, or (q) SEQ ID NO: 1887 and having a length of 24, 25, 26, 27, 28, 29 or 30 nucleotides. In (r) a base sequence defined in any one of SEQ ID NO: 1888 or 1889 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (s) SEQ ID NO: 1827. Defined in a base sequence having a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (t) SEQ ID NO: 1828, 25, 26, 27, 28, A base sequence having a length of 29 or 30 nucleotides, or (u) defined in any one of SEQ ID NOs: 1784, 1836, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides defined in any one of SEQ ID NOs: 1786 and 1838, or (w). One of the nucleotide sequences defined in any one of SEQ ID NOs: 1780 and having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (x) SEQ ID NO: 1785, 1837. Defined in one, a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (y) defined in any one of SEQ ID NOs: 1845, 1846, 1847, 1848, 24, Defined in any one of a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (z) SEQ ID NO: 1849, 1850, 22, 23, 24, 25, 26, 27, Nucleotide sequence having a length of 28, 29 or 30 nucleotides , Or (a1) a base sequence defined in any one of SEQ ID NOs: 1787, 1851 and having a length of 26, 27, 28, 29 or 30 nucleotides, or (b1) SEQ ID NO: 1788, 1852, 1789, 1853. A base sequence defined in any one of 24, 25, 26, 27, 28, 29 or 30 nucleotides, or (c1) any one of SEQ ID NOs: 1790, 1854, 1792, 1855. It is defined as either a base sequence having a length of 25, 26, 27, 28, 29 or 30 nucleotides, or (d1) SEQ ID NO: 1794, 1861, 1795, 1862, and is defined as 21, 22, 23. , 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or (e1) defined in any one of SEQ ID NOs: 1796, 1863, 1797, 1864, 23, 24, 25. , 26, 27, 28, 29 or 30 nucleotides in length, or (f1) defined in any one of SEQ ID NOs: 1798, 1865, 1799, 1866, 25, 26, 27, 28, 29. Or a base sequence having a length of 30 nucleotides, or (g1) defined in any one of SEQ ID NOs: 1808, 1867, 1809, 1868, 1810, 1869, 1858, 1873, 21, 22, 23, 24, 25. , 26, 27, 28, 29 or 30 nucleotides in length, or (h1) defined in any one of SEQ ID NOs: 1811, 1870, 1859, 1874, 22, 23, 24, 25, 26. , 27, 28, 29 or 30 nucleotides in length, or (i1) defined in any one of SEQ ID NOs: 1856, 1871, 1860, 1875, 23, 24, 25, 26, 27, 28. , A base sequence having a length of 29 or 30 nucleotides, or (j1) a base sequence defined in any one of SEQ ID NOs: 1857, 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 and having a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. It is defined in any one of the base sequence or (l1) SEQ ID NO: 1802, 1878, 1803, 1879, and is 23, 24, 25, 26, 27, 28, 29 or 30 nukure. It is defined as either a base sequence having an thiode length or any one of (m1) SEQ ID NOs: 1804 and 1880, and has a length of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. A base sequence having a length of 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or a base sequence defined in any one of (n1) SEQ ID NOs: 1805 and 1881. (O1) A base sequence defined in any one of SEQ ID NOs: 1806, 1882, 1807, 1883 and having a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Oligonucleotides containing.
[12]
A composition containing 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], which comprises two or more identical oligonucleotides according to any one of [1] to [11] linked by a counterion, preferably calcium.
[14]
A method for preventing, delaying, ameliorating and / or treating a disease, preferably BMD or DMD, in an individual, wherein the oligonucleotide according to any one of [1] to [11] or [14 or 13]. A method comprising the step of administering the described composition 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) A step of identifying an in-frame combination of a first exon and a second exon within the same mRNA precursor, wherein the second exon region is at least 50% of the first exon region. Steps, with identity,
(B) The step of designing an oligonucleotide capable of binding to the region of the first exon and the region of the second exon, and (c) the result of the binding, the first exon and the second exon. Skipping, preferably a multi-exon stretch skipping that starts with the first exon and includes one or more exons existing between the first exon and the second exon, and at most. Also a method comprising skipping the entire exon stretch between the first exon and the second exon.

Claims (1)

The invention described in the specification.

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