Antisense oligonucleotide rescues aberrant splicing of abca4

Abstract

To provide a novel antisense oligonucleotide that can be used in treatment, prevention and / or delaying of Stargardt disease.SOLUTION: Provided is an antisense oligonucleotide for controlling splicing which binds to a specific polynucleotide, and / or is complementary with a polynucleotide having a specific sequence, where the antisense oligonucleotide binds to a polynucleotide selected from the group consisting of a specific sequence, or complementary with a polynucleotide selected from a specific sequence.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] [Field of the Invention] The present invention relates to the fields of medicine and immunology. In particular, the present invention relates to novel antisense oligonucleotides that can be used in the treatment, prevention and / or delay of ABCA4-related conditions.

[0002] [Background of the invention] Autosomal recessive mutations in ABCA4 cause Stargardt disease, a progressive disorder characterized by loss of central vision and often leading to total blindness. A typical hallmark of Stargardt disease is the presence of numerous yellow spots (specks) distributed throughout the fundus of the patient's eye. The ABCA4 gene consists of 50 exons and encodes a protein of 2273 amino acids. This protein is expressed in the outer segments of cone and rod photoreceptors and plays an important role in the removal of waste products after phototransduction.

[0003] Besides STGD1 , variants in ABCA4 can also result in other subtypes of retinal disease, ranging from target maculopathy to autosomal recessive cone-rod dystrophy (arCRD; Cremers et al., 1998 ; Maugeri et al., 2000 ) and panretinal dystrophy ( Cremers et al., 1998 ; Martinez-Mir et al., 1998 ), depending on the severity of the allele.

[0004] Biallelic ABCA4 variants can be identified in approximately 80% of cases due to STGD1 (Allikmets et al., 1997; Fujinami et al., 2013; Lewis et al., 1999; Maugeri et al., 1999; Rivera et al., 2000; Schulz et al., 2017; Webster et al., 2001; Zernant et al., 2011; Zernant et al., 2017) and 30% of cases due to arCRD (Maugeri et al., 2000, supra) after sequencing the coding region and flanking splice sites. Generally, individuals with arCRD or panretinal dystrophy carry two severe ABCA4 alleles, while individuals with STGD1 carry two moderately severe variants or a combination of a mild and a severe variant (Maugeri et al., 1999; van Driel et al., 1998). The majority of unknown ABCA4 variants in STGD1 patients are hypothesized to reside in the intronic regions of the gene, and indeed, over the past few years, several groups have demonstrated the existence of such deep intronic variants (Bauwens et al., 2015; Bax et al., 2015; Braun et al., 2013; Lee et al., 2016; Schulz et al., 2017; Albert et al., 2018; Sangermano et al., 2019; and Bauwens et al., 2019). Many of these deep intronic mutations activate cryptic splice acceptor or donor sites or alter exonic splice enhancer or silencer motifs, all of which result in the inclusion of pseudoexons (PEs) in a significant proportion of ABCA4 transcripts. The extent of PE insertions may vary for each ABCA4 variant but also depends on which tissue is studied; i.e., the degree of PE inclusion may be proportionally higher in "retinal-like" tissues. Although the majority of ABCA4 mutations are spread throughout the ABCA4 gene, there are some mutations that tend to cluster together, including a series of variants in intron 36 of the gene.In particular, applicants identified four intron 36 variants (c.5196+10134>G; c.5196+10564>G; c.5196+1137G>A; c.5196+12164>G) that result in the inclusion of a pseudoexon in the ABCA4 pre-mRNA and, consequently, the predicted loss of ABCA4 protein function.

[0005] The fact that a significant amount of mutations in ABCA4 affect ABCA4 pre-mRNA splicing makes this mutation an attractive target for antisense oligonucleotide (AON)-based splice modulation therapy. Therefore, there is a need to develop AONs for splice modulation of the ABCA4 gene to enable the expression of functional ABCA4 protein in subjects with Stargardt disease.

[0006] [Summary of the Invention] In a first aspect, the present invention relates to an antisense oligonucleotide for controlling splicing that binds to and / or is complementary to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 4. Preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 8, 9 and 10. More preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 5, 6 and 7. Even more preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 and 42.

[0007] In a second aspect, the present invention relates to a viral vector that expresses an antisense oligonucleotide for controlling splicing as defined herein when placed under conditions that promote expression of said molecule.

[0008] In a third aspect, the present invention relates to a pharmaceutical composition comprising the antisense oligonucleotide for regulating splicing according to the present invention or the viral vector according to the present invention, and a pharmaceutically acceptable excipient.

[0009] In a fourth aspect, the present invention relates to an antisense oligonucleotide for controlling splicing according to the present invention, a vector according to the present invention, or a pharmaceutical composition according to the present invention for use as a pharmaceutical, preferably for treating an ABCA4-associated disease or condition that requires modulation of ABCA4 splicing. Preferably, the ABCA4-associated disease or condition is Stargardt disease.

[0010] In a fifth aspect, the present invention relates to the antisense oligonucleotide for regulating splicing according to the present invention, the vector according to the present invention, or the pharmaceutical composition according to the present invention, for treating an ABCA4-related disease or condition that requires modulation of ABCA4 splicing.

[0011] In a sixth aspect, the present invention relates to a method for modulating the splicing of ABCA4 in a cell, the method comprising the step of contacting said cell with an antisense oligonucleotide for controlling splicing as defined herein, a vector according to the invention or a pharmaceutical composition according to the invention.

[0012] [Detailed Description of the Invention] By definition, antisense oligonucleotides (AONs) are substantially complementary (antisense) to their targets, allowing them to bind to the corresponding pre-mRNA molecule, thereby preventing the binding of proteins essential for splicing, without wishing to be bound by theory. As the inventors have previously shown for several mutations in ABCA4 (WO 2018 / 109011), the lack of such binding usually results in skipping of the targeted exon.

[0013] In addition, AONs can regulate the splicing machinery toward adjacent splice acceptor or donor sites. This led us to select ABCA4 mutations that may be suitable for AON-based splice modulation therapy. All of these mutations are deep intronic variants that create novel splice acceptor, splice donor, or exon splice enhancer binding sites, resulting in the inclusion of pseudoexons in the mRNA of the corresponding gene. AONs may be used to block the recognition of the pseudoexons (and thereby induce their skipping), thereby fully restoring wild-type transcripts and corresponding protein function.

[0014] The following mutations were selected: c.5196+1013A>G. This mutation results in the inclusion of a 129-nt pseudoexon between exons 36 and 37 of ABCA4.

[0015] c.5196+1056A>G. This mutation results in the inclusion of a 177-nt pseudoexon between exons 36 and 37 of ABCA4.

[0016] c.5196+1137G>A. This mutation results in the inclusion of a 73-nt pseudoexon between exons 36 and 37 of ABCA4.

[0017] c.5196+1216A>G. This mutation results in the inclusion of a 73-nt pseudoexon between exons 36 and 37 of ABCA4.

[0018] These four ABCA4 variants in intron 36 have been identified in several patients; in particular, the c.5196+1137G>A variant was found in heterozygous form in 15 reported and 19 unpublished STGD1 cases. Two of the four variants generate similar pseudoexons, and thus the same AON molecule can correct defects caused by two different mutations. Here, we show that specific AONs can restore aberrant ABCA4 splicing caused by intron 36 variants.

[0019] The inventors have provided AONs for modulating splicing for the classes of mutations delineated herein above; the terms "modulating splicing" and "controlling splicing" are used interchangeably herein and encompass AON-based splice modulation therapy for the mutations delineated herein above. The term "controlling splicing" is defined herein as controlling ABCA4 pre-mRNA splicing to obtain the original transcript.

[0020] Therefore, the present invention provides an antisense oligonucleotide for controlling splicing, which binds to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 and / or is complementary to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4, preferably an antisense oligonucleotide that binds to a polynucleotide selected from the group consisting of SEQ ID NOs: 8, 9 and 10 or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 8, 9 and 10, more preferably an antisense oligonucleotide that binds to a polynucleotide selected from the group consisting of SEQ ID NOs: 5, 6 and 7 or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 5, 6 and 7. and even more preferably, antisense oligonucleotides that bind to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 and 42 or that are complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 and 42.

[0021] The terms "antisense oligonucleotide" or "AON" are used interchangeably herein and are understood to refer to an oligonucleotide molecule comprising a nucleotide sequence that is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heteronuclear RNA), or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that the molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. Binding of an AON to its target can be readily assessed by one skilled in the art using techniques known in the art, such as the gel mobility shift assay described in EP 1619249.

[0022] The term "complementary" as used in the context of the present invention indicates that some mismatches in the antisense sequence are tolerated as long as functionality, i.e., splicing control, is achieved. Preferably, the complementarity is 90% to 100%. Generally, this allows for one or two mismatches in a 20-nucleotide AON, one, two, three, or four mismatches in a 40-nucleotide AON, or one, two, three, four, five, or six mismatches in a 60-nucleotide AON, etc. Optionally, the AON can be further tested by transfection into retinal-like cells of a patient. The complementary region is preferably designed to be specific for the pseudoexon in the pre-mRNA when combined. Such specificity depends on the actual sequence in other (pre-)mRNA molecules in the system, and therefore can be generated by complementary regions of various lengths. The risk that an AON may hybridize to one or more other pre-mRNA molecules decreases with increasing AON size. It is clear that AONs containing mismatches in the regions of complementarity but retaining the ability to hybridize and / or bind to targeted region(s) in pre-mRNA can be used in the present invention. However, since AONs lacking mismatches in the complementary portions typically have higher efficiency and higher specificity than AONs with such mismatches in one or more complementary regions, preferably at least the complementary portions do not contain such mismatches. Higher hybridization strength (i.e., an increased number of interactions with the opposing strand) is believed to be advantageous in increasing the efficiency of the process of interfering with the splicing machinery of this system.

[0023] The AONs of the present invention preferably do not contain a stretch of CpGs, more preferably, do not contain any CpGs. The presence of CpGs or stretches of CpGs in oligonucleotides is usually associated with increased immunogenicity of the oligonucleotides (Dorn and Kippenberger, 2008). This increased immunogenicity is undesirable because it can induce damage to the treated tissue, i.e., the eye. Immunogenicity can be assessed in animal models by assessing the presence of CD4+ and / or CD8+ cells and / or inflammatory mononuclear cell infiltration. Immunogenicity can also be assessed in the blood of animals or humans treated with the AONs of the present invention by detecting the presence of neutralizing antibodies and / or antibodies recognizing the AON using standard immunoassays known to those skilled in the art. Inflammatory responses, type I-like interferon production, IL-12 production, and / or increased immunogenicity can be assessed by detecting the presence or increased amount of neutralizing antibodies or antibodies recognizing the AON using standard immunoassays. Even more preferably, AONs of the present invention have acceptable RNA-binding kinetics and / or thermodynamic properties. RNA-binding kinetics and / or thermodynamic properties are determined, at least in part, by the melting temperature (Tm; calculated by the Oligonucleotide Properties Calculator for single-stranded RNA (www.unc.edu / -cail / biotool / oligo / index) using a basic Tm and nearest neighbor model) of the oligonucleotide and / or the free energy of the AON-target exon complex (using RNA structure version 4.5). If the Tm is too high, the AON is expected to have low specificity. Acceptable Tm and free energy depend on the sequence of the AON. Therefore, it is difficult to give a preferred range for each of these parameters. Acceptable Tm may range between 35 and 70°C, and acceptable free energy may range between 15 and 45 kcal / mol.

[0024] In all embodiments, the nucleotides in the antisense oligonucleotides of the present invention can be RNA residues, DNA residues, or nucleotide analogs or equivalents, or combinations thereof.

[0025] Preferred AONs for controlling splicing according to the present invention have a length of about 8 to about 40 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to about 23 nucleotides, such as 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides.

[0026] In one embodiment, the antisense oligonucleotide for controlling splicing of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35 and 39, or consists of a sequence selected from the group consisting of SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35 and 39.

[0027] In a preferred embodiment, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1013A>G mutation comprises or consists of SEQ ID NO:27 or SEQ ID NO:31.

[0028] In another preferred embodiment, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1056A>G mutation comprises or consists of an AON selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35 and SEQ ID NO: 39. More preferably, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1056A>G mutation is selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 31 and SEQ ID NO: 35.

[0029] In another preferred embodiment, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1137G>A mutation comprises an AON selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 23, or consists of an AON selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 23. More preferably, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1137G>A mutation comprises or consists of SEQ ID NO: 15 or SEQ ID NO: 19.

[0030] In yet another preferred embodiment, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1216C>A mutation comprises or consists of an AON selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 23. More preferably, the AON for regulating aberrant splicing of ABCA4 caused by the c.5196+1216C>A mutation consists of SEQ ID NO: 11 or SEQ ID NO: 15.

[0031] Preferably, the AON for controlling splicing according to the present invention comprises one or more residues modified to increase nuclease resistance and / or to increase the affinity of the antisense oligonucleotide for the target sequence. Thus, in a preferred embodiment, the AON comprises at least one nucleotide analog or equivalent, where a nucleotide analog or equivalent is defined as a residue having a modified base and / or a modified backbone and / or a non-natural internucleoside linkage, or a combination of these modifications.

[0032] In a preferred embodiment, the nucleotide analog or equivalent comprises a modified backbone.Examples of such backbone include morpholino backbone, carbamate backbone, siloxane backbone, sulfide, sulfoxide and sulfone backbone, formacetyl and thioformacetyl backbone, methyleneformacetyl backbone, riboacetyl backbone, alkene-containing backbone, sulfamate, sulfonate and sulfonamide backbone, methyleneimino and methylenehydrazino backbone, and amide backbone.Phosphorodiamidate morpholino oligomer is a modified backbone oligonucleotide that has been previously investigated as an antisense agent.

[0033] Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugars of DNA are replaced by six-membered rings and the phosphodiester linkages are replaced by phosphorodiamidate linkages. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by inhibiting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by physically disrupting the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient delivery method; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. Recent reports have demonstrated triplex formation by morpholino oligonucleotides, and due to the non-ionic backbone, these studies have shown that morpholino oligonucleotides are capable of triplex formation in the absence of magnesium.

[0034] It is further preferred that the linkages between residues in the backbone do not contain phosphorus atoms, such as linkages formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages.

[0035] Preferred nucleotide analogs or equivalents include peptide nucleic acids (PNAs) with modified polyamide backbones (Nielsen et al., 1991). PNA-based molecules are true mimics of DNA molecules in terms of base pair recognition. The PNA backbone consists of N-(2-aminoethyl)-glycine units linked by peptide bonds, and the nucleobases are linked to the backbone by methylene carbonyl bonds. Alternative backbones include one-carbon extended pyrrolidine PNA monomers (Govindaraju and Kumar, 2005). Because the backbone of PNA molecules does not contain charged phosphate groups, PNA-RNA hybrids are typically more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al., 1993). Further preferred backbones include morpholino nucleotide analogs or equivalents, in which the ribose or deoxyribose sugar is replaced by a six-membered morpholino ring. The most preferred nucleotide analogs or equivalents include phosphorodiamidate morpholino oligomers (PMOs), in which the ribose or deoxyribose sugar is replaced by a six-membered morpholino ring and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

[0036] In yet another embodiment, the nucleotide analogs or equivalents of the present invention contain a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base pairing but adds significant resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, H-phosphonates, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, or boranophosphates.

[0037] Further preferred nucleotide analogs or equivalents according to the present invention comprise one or more sugar moieties mono- or di-substituted at the 2', 3', and / or 5' positions, such as -OH; -F; substituted or unsubstituted, straight-chain or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, aryl, or aralkyl, optionally interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-, S-, or N-aryl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or a derivative thereof, or a deoxypyranose or a derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Preferred derivatized sugar moieties include locked nucleic acids (LNAs), in which the 2'-carbon atom is linked to the 3'- or 4'-carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. Preferred LNAs include 2'-O,4'-C-ethylene-bridged nucleic acids (Morita et al., 2001). These substitutions render the nucleotide analogs or equivalents RNase H and nuclease resistant and increase affinity for target RNA.

[0038] In another embodiment, a nucleotide analog or equivalent of the present invention comprises one or more base modifications or substitutions. Modified bases include synthetic and natural bases such as inosine, xanthine, hypoxanthine, and other aza, deaza, hydroxy, halo, thio, thiol, alkyl, alkenyl, alkynyl, thioalkyl derivatives of pyrimidine and purine bases, as are known or will become known in the art.

[0039] Those skilled in the art will understand that not all positions in an AON need be uniformly modified. In addition, more than one of the above analogs or equivalents can be incorporated into a single AON, or even at a single position within an AON. In certain embodiments, the AONs of the present invention have at least two different types of analogs or equivalents.

[0040] Thus, in a preferred embodiment, the antisense oligonucleotide for controlling splicing according to the present invention comprises at least one 2'-O-alkyl phosphorothioate antisense oligonucleotide, such as a 2'-O-methyl modified ribose, a 2'-O-ethyl modified ribose, a 2'-O-propyl modified ribose and / or substituted derivatives thereof, such as halogenated derivatives, having these modifications, and preferably the antisense oligonucleotide comprises at least one 2'-O-propyl modified ribose.

[0041] In another preferred embodiment, the antisense oligonucleotides for controlling splicing according to the present invention comprise 2'-O-alkyl phosphorothioate antisense oligonucleotides, such as 2'-O-methyl modified ribose, 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose and / or substituted derivatives thereof, such as halogenated derivatives, having these modifications, preferably the antisense oligonucleotides comprise 2'-O-propyl modified ribose.

[0042] In a preferred embodiment, the antisense oligonucleotide for controlling splicing according to the present invention comprises a phosphorothioate backbone.

[0043] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 11 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0044] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 15 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0045] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 19 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0046] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 23 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0047] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 27 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0048] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 31 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0049] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 35 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0050] In one embodiment, an AON for regulating splicing according to the present invention comprises or consists of SEQ ID NO: 39 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone.

[0051] Those skilled in the art will also understand that different antisense oligonucleotides can be combined for efficient splicing control. In a preferred embodiment, the present invention comprises a set of AONs for controlling ABCA4 splicing according to the present invention, and preferably, such a set comprises at least two AONs for controlling splicing according to the present invention. Preferably, such a set comprises at least two, at least three, or at least four antisense oligonucleotides selected from the group consisting of SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, and 39.

[0052] The splicing-control AONs of the present invention can be administered indirectly using suitable means known in the art. AONs can be administered to an individual or to cells, tissues, or organs of the individual, for example, as so-called "naked" AONs. AONs can also be administered in the form of expression vectors, which encode RNA transcripts comprising the sequences of the AONs of the present invention. Expression vectors are preferably introduced into cells, tissues, organs, or individuals via gene delivery vehicles. In a preferred embodiment, a viral-based expression vector is provided that includes an expression or transcription cassette that drives the expression or transcription of the splicing-control AONs of the present invention. Thus, the present invention provides viral vectors that express the splicing-control antisense oligonucleotides of the present invention when placed under conditions conducive to expression of the molecule.

[0053] The AON for splicing control of the present invention can be delivered to cells via plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus or adeno-associated virus-based vectors. Expression can be driven by an RNA polymerase II promoter (Pol II), such as the U7 RNA promoter, or an RNA polymerase III (Pol III) promoter, such as the U6 RNA promoter. Preferred delivery vehicles are viral vectors, such as adeno-associated virus vectors (AAV) or retroviral vectors, such as lentiviral vectors. Plasmids, artificial chromosomes, and plasmids that can be used for targeted homologous recombination and integration into the human genome of cells can also be used appropriately for delivering the AON for splicing control of the present invention. Preferred vectors for the present invention are those in which transcription is driven from a Pol III promoter and / or in which the transcript is in the form of a fusion with a U1 or U7 transcript, which produces excellent results for delivering small transcripts. Designing suitable transcripts is within the skill of those skilled in the art. Pol III-driven transcripts, preferably in the form of a fusion transcript with a U1 or U7 transcript, are preferred. Such fusions can be made as previously described (Gorman et al., 1998).

[0054] A preferred expression system for the AON for splicing regulation according to the present invention is an adeno-associated virus (AAV)-based vector. Single- and double-stranded AAV-based vectors have been developed that can be used for the prolonged expression of antisense nucleotide sequences for highly efficient splicing regulation. Preferred AAV-based vectors contain, for example, an expression cassette driven by an RNA polymerase III promoter (Pol III) or an RNA polymerase II promoter (Pol II). Preferred RNA promoters include, for example, the Pol III U6 RNA promoter or the Pol II U7 RNA promoter.

[0055] Thus, the present invention provides a viral-based vector comprising a Pol II or Pol III promoter-driven expression cassette for expression of an AON for controlling splicing according to the present invention.

[0056] The AAV vector of the present invention is a recombinant AAV vector and refers to an AAV vector comprising a portion of an AAV genome containing an encoded AON for controlling splicing of the present invention, encapsidated in a protein shell of capsid proteins derived from an AAV serotype described elsewhere herein. The portion of the AAV genome can contain inverted terminal repeats (ITRs) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, and others. The protein shell composed of capsid proteins can be derived from an AAV serotype, such as AAV1, 2, 3, 4, 5, 8, 9, and others. The protein shell can also be referred to as a capsid protein shell. The AAV vector can be deleted for one or preferably all wild-type AAV genes but still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for replication, rescue, and packaging of AAV virions. The ITR sequences can be wild-type sequences, or can have at least 80%, 85%, 90%, 95%, or 100% sequence identity to the wild-type sequence, or can be altered, for example, by nucleotide insertion, mutation, deletion, or substitution, so long as they maintain functionality. In this context, functionality refers to the ability to direct packaging of the genome into a capsid shell and subsequently enable expression in the host or target cell to be infected. In the context of the present invention, the capsid protein shell can be of a different serotype from the AAV vector genome ITRs. Thus, while the AAV vector of the present invention can be composed of a capsid protein shell, i.e., an icosahedral capsid, containing the capsid proteins (VP1, VP2, and / or VP3) of one AAV serotype, e.g., AAV serotype 2, the ITR sequences contained in this AAV5 vector can be of any of the AAV serotypes listed above, including AAV2 vectors.Thus, an "AAV2 vector" comprises an AAV serotype 2 capsid protein shell, while, for example, an "AAV5 vector" comprises an AAV serotype 5 capsid protein shell, either of which may encapsidate any AAV vector genome ITR of the present invention.

[0057] Preferably, a recombinant AAV vector of the invention comprises an AAV serotype 2, 5, 8, or AAV serotype 9 capsid protein shell, and the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8, or AAV serotype 9; such AAV vectors are referred to as AAV2 / 2, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV5 / 2, AAV5 / 5, AAV5 / 8, AAV5 / 9, AAV8 / 2, AAV8 / 5, AAV8 / 8, AAV8 / 9, AAV9 / 2, AAV9 / 5, AAV9 / 8, or AAV9 / 9 vectors.

[0058] More preferably, a recombinant AAV vector of the present invention comprises a capsid protein shell of AAV serotype 2, and the AAV genome or ITRs present in the vector are derived from AAV serotype 5; such a vector is referred to as an AAV2 / 5 vector.

[0059] More preferably, a recombinant AAV vector of the present invention comprises a capsid protein shell of AAV serotype 2, and the AAV genome or ITRs present in the vector are derived from AAV serotype 8; such a vector is referred to as an AAV2 / 8 vector.

[0060] More preferably, a recombinant AAV vector of the present invention comprises a capsid protein shell of AAV serotype 2, and the AAV genome or ITRs present in the vector are derived from AAV serotype 9; such a vector is referred to as an AAV2 / 9 vector.

[0061] More preferably, a recombinant AAV vector of the present invention comprises an AAV serotype 2 capsid protein shell, and the AAV genome or ITRs present in the vector are derived from AAV serotype 2; such vectors are referred to as AAV2 / 2 vectors.

[0062] The nucleic acid molecule encoding the AON for controlling splicing of the present invention, represented by the selected nucleic acid sequence, is preferably inserted between the AAV genome or ITR sequences identified above, e.g., an expression construct comprising expression control elements operably linked to the coding sequence and 3' termination sequence.

[0063] "AAV helper functions" generally refer to the corresponding AAV functions required for AAV replication and packaging that are supplied to an AAV vector in trans. AAV helper functions complement AAV functions lacking in the AAV vector, but lack the AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV, i.e., the rep and cap coding regions, or functionally substantially identical sequences thereof. The Rep and Cap regions are well known in the art; see, for example, U.S. Pat. No. 5,139,941, incorporated herein by reference. AAV helper functions can be provided in an AAV helper construct, which can be a plasmid. Introduction of the helper construct into a host cell can occur prior to or simultaneously with introduction of the AAV genome present in the AAV vector identified herein, for example, by transformation, transfection, or transduction. Thus, the AAV helper constructs of the present invention can be selected to produce a desired combination of serotypes for the capsid protein shell of the AAV vector, on the one hand, and the AAV genome present during said AAV vector replication and packaging, on the other hand.

[0064] An "AAV helper virus" provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV types 1 and 2), and vaccinia virus. The additional functions provided by the helper virus can also be introduced into the host cell via a vector, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference.

[0065] Preferably, the AAV genome present in a recombinant AAV vector of the present invention does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. The AAV genome may further contain marker or reporter genes, such as, for example, antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products (e.g., lacZ, aph, etc.) known in the art.

[0066] Preferably, the AAV vectors of the present invention are constructed and produced according to the method by Garanto et al., 2016, which is incorporated herein by reference.

[0067] A preferred AAV vector according to the present invention is an AAV vector, preferably an AAV2 / 5, AAV2 / 8, AAV2 / 9 or AAV2 / 2 vector, expressing an AON for controlling splicing according to the present invention, which AON comprises, or preferably consists of, a sequence complementary to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 4, and preferably the antisense oligonucleotide binds to a polynucleotide selected from the group consisting of SEQ ID NOs: 8, 9 and 10, or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 8, 9 and 10. More preferably, the antisense oligonucleotide binds to a polynucleotide selected from the group consisting of SEQ ID NOs: 5, 6 and 7, or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 5, 6 and 7. Even more preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 and 42.

[0068] In view of the progress already achieved to date, improvements are expected in the means for delivering the splicing-controlling AONs of the present invention to an individual or the cells, tissues, or organs of the individual. Naturally, such future improvements can be incorporated to achieve the effects mentioned in the reconstitution of mRNA using the methods of the present invention. The splicing-controlling AONs of the present invention can be delivered to an individual or the cells, tissues, or organs of the individual as "naked" AONs, etc. When administering the splicing-controlling AONs of the present invention, it is preferable that the molecules are dissolved in a solution compatible with the delivery method. Plasmids for expressing antisense oligonucleotides can be delivered to retinal cells by providing the plasmids in aqueous solution.

[0069] Alternatively, a preferred delivery method for AONs for controlling splicing or plasmids for expressing such AONs is a viral vector or nanoparticle. Preferably, the viral vector or nanoparticle is delivered to the retina or other relevant cells. Such delivery to retinal cells or other relevant cells can be in vivo, in vitro, or ex vivo; see, for example, Garanto et al., 2016, which is incorporated herein by reference.

[0070] Alternatively, the plasmids can be provided by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal, and / or intracerebroventricular administration, it is preferred that the solution is physiological saline. Particularly preferred in the present invention is the use of excipients or transfection agents that will aid in the delivery of each of the constructs defined herein to and / or into cells, preferably retinal cells. Preferred are excipients or transfection agents that can form complexes, nanoparticles, micelles, vesicles, and / or liposomes that deliver each of the constructs defined herein complexed or entrapped in vesicles or liposomes through the cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agents include polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers including polypropylenimine or polyethylenimine copolymers (PEC) and derivatives, synthetic amphiphiles (SAINT-18), lipofectin™, DOTAP, and / or viral capsid proteins capable of self-assembling into particles capable of delivering each of the constructs defined herein to cells, preferably retinal cells. Such excipients have been shown to efficiently deliver oligonucleotides, such as AONs, to a wide variety of cultured cells, including retinal cells. Their high transfection potential is combined with expected low-to-moderate toxicity in terms of overall cell survival. The ease of structural modification allows for further modification and characterization of their (in vivo) nucleic acid transfer characteristics and toxicity.

[0071] Lipofectin is an example of a liposomal transfection agent. Lipofectin consists of two lipid components: the cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP, which is the methyl sulfate salt) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates intracellular release. Another group of delivery systems are polymeric nanoparticles.

[0072] Polycations such as diethylaminoethylaminoethyl (DEAE)-dextran, a well-known DNA transfection agent, can be combined with butyl cyanoacrylate (PBCA) and hexyl cyanoacrylate (PHCA) to formulate cationic nanoparticles capable of delivering the constructs defined herein, preferably the AONs of the present invention, across cell membranes and into cells.

[0073] In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach for formulating oligonucleotides in colloids. This colloidal nanoparticle system can be prepared by a simple self-assembly process to form so-called proticles that can package oligonucleotides and mediate their intracellular release. One skilled in the art can select and adapt any of the above-mentioned or other commercially available alternative excipients and delivery systems to package and deliver exon-retaining molecules for use in the present invention for the prevention, treatment, or delay of ABCA4-related diseases or conditions. "Prevention, treatment, or delay of ABCA4-related diseases or conditions" is preferably defined herein as preventing, halting, terminating the progression, or reversing partial or complete vision impairment or blindness caused by a genetic defect in the ABCA4 gene.

[0074] Additionally, the AONs for controlling splicing of the present invention can be covalently or non-covalently linked to targeting ligands specifically designed to facilitate uptake into cells, the cytoplasm, and / or their nuclei. Such ligands can include (i) compounds (including, but not limited to, peptide(-like) structures) that recognize cell-, tissue-, or organ-specific elements to facilitate cellular uptake, and / or (ii) chemicals that can facilitate the intracellular uptake of oligonucleotides in vesicles, e.g., endosomes or lysosomes, and / or intracellular release from such vesicles, e.g., endosomes or lysosomes.

[0075] Therefore, in a preferred embodiment, the AON for controlling splicing according to the present invention is formulated into a composition or medicament or composition comprising at least an excipient and / or a targeting ligand for delivery and / or a delivery device thereof to a cell and / or one that enhances its intracellular delivery.

[0076] It should be understood that when a composition comprises additional components, such as auxiliary compounds as defined later in this specification, each component of the composition does not have to be formulated in a single combination or composition or preparation. Depending on their identity and specific characteristics, a person skilled in the art will know which type of formulation is most appropriate for each component as defined herein. In a preferred embodiment, the present invention provides a composition or preparation in the form of a kit of parts comprising an AON for controlling splicing according to the present invention and further auxiliary compounds as defined later in this specification.

[0077] If required and / or desired, the AON for controlling splicing according to the present invention, or the vector of the present invention, preferably a viral vector, expressing the AON for controlling splicing according to the present invention, can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.

[0078] Thus, the present invention also provides a composition, preferably a pharmaceutical composition, comprising an antisense oligonucleotide for controlling splicing according to the present invention or a viral vector according to the present invention and a pharmaceutically acceptable excipient. Such a composition may contain a single AON or viral vector for controlling splicing according to the present invention, but may also contain multiple separate AONs or viral vectors for controlling splicing according to the present invention. Such a pharmaceutical composition may contain any pharmaceutically acceptable excipient, including carriers, fillers, preservatives, adjuvants, solubilizers, and / or diluents. Such pharmaceutically acceptable carriers, fillers, preservatives, adjuvants, solubilizers, and / or diluents can be found, for example, in Remington, 2000. Each feature of the composition is defined earlier in this specification.

[0079] The preferred route of administration is via intravitreal injection of an aqueous solution or specially adapted formulation for intraocular administration. EP 2425814 discloses an oil-in-water emulsion specifically adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. Because this emulsion has a lower density than the vitreous humor, the emulsion floats on the vitreous, preventing the injected drug from impairing vision. Thus, in one embodiment, a pharmaceutical composition suitable for intravitreal administration is provided, administered in an amount of total antisense oligonucleotide ranging from 0.01 to 20 mg / kg per eye, preferably 0.05 to 20 mg / kg per eye. Suitable intravitreal doses are provided, containing between 0.05 mg and 5 mg per eye, preferably between 0.1 and 1 mg, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg per eye.

[0080] A preferred AON for controlling splicing according to the present invention is for treating an ABCA4-related disease or condition in an individual. In all embodiments of the present invention, the term "treatment" is understood to include prevention and / or delay of an ABCA4-related disease or condition. Individuals who can be treated using an AON for controlling splicing according to the present invention may already be diagnosed with an ABCA4-related disease or condition.

[0081] Alternatively, an individual who can be treated using the AON for regulating splicing of the present invention may not yet have been diagnosed with an ABCA4-related disease or condition, but may be an individual who, taking into account the individual's genetic background, has an increased risk of developing an ABCA4-related disease or condition in the future. Preferred individuals are humans. In all embodiments of the present invention, the ABCA4-related disease or condition is preferably Stargardt disease.

[0082] Thus, the present invention further provides an antisense oligonucleotide for controlling splicing according to the present invention, or a viral vector according to the present invention, or a (pharmaceutical) composition according to the present invention, for use as a medicament, preferably for the treatment of an ABCA4-related disease or condition which requires modulation of ABCA4 splicing, and for use as a medicament for the prevention, treatment or delay of an ABCA4-related disease or condition. Each feature of any medical use embodiment herein is as defined hereinbefore, and preferably is such a feature as defined hereinbefore.

[0083] The present invention further provides a use of an AON for controlling splicing according to the present invention, a vector according to the present invention or a (pharmaceutical) composition according to the present invention for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. Each feature of any medical use embodiment herein is as defined hereinbefore, and preferably is such a feature as defined hereinbefore.

[0084] The present invention further provides a method for treating an ABCA4-related disease or condition requiring modulation of ABCA4 splicing, comprising contacting cells of said individual with an AON for controlling splicing according to the present invention, a vector according to the present invention, or a (pharmaceutical) composition according to the present invention. Each feature of any medical use embodiment herein is as defined hereinbefore, and preferably is such a feature as defined hereinbefore.

[0085] The present invention further provides the use of an AON for controlling splicing according to the present invention, a vector according to the present invention or a (pharmaceutical) composition according to the present invention for the preparation of a medicament for the treatment of an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. Each feature of any medical use embodiment herein is as defined hereinbefore, and preferably is such a feature as defined hereinbefore.

[0086] The present invention further provides the antisense oligonucleotide for regulating splicing according to the present invention, the use according to the present invention or the method according to the present invention, wherein the ABCA4-related disease or condition is Stargardt disease.

[0087] Treatment in the uses or methods of the present invention is preferably at least once and preferably lasts for at least one week, one month, several months, one year, two, three, four, five, six years, or more, or even lifelong. Each AON or its equivalent for controlling splicing of the present invention defined herein for use in the present invention can be suitable for direct administration to cells, tissues, and / or organs in vivo of an individual already suffering from or at risk of developing an ABCA4-related disease or condition, and can be administered directly in vivo, ex vivo, or in vitro. The frequency of administration of the AON, composition, compound, or ancillary compound of the present invention may depend on several parameters, such as the severity of the disease, the patient's age, the patient's mutation, the number of AONs for controlling splicing of the present invention (i.e., dose), the formulation of the AON, composition, compound, or ancillary compound of the present invention, the route of administration, etc. The administration frequency can vary between daily, weekly, at least once every two, three, four, five, or more weeks, or longer.

[0088] The dose range of the AON, composition, compound, or ancillary compound of the present invention is preferably designed based on ascending dose studies in clinical trials (in vivo use) with strict protocol requirements. The AON of the present invention can be used at doses ranging from 0.01 to 20 mg / kg, preferably 0.05 to 20 mg / kg. A suitable intravitreal dose will be about: between 0.05 mg and 5 mg per eye, preferably between 0.1 and 1 mg per eye, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg per eye.

[0089] In a preferred embodiment, a concentration of oligonucleotide as defined herein in the range of 0.1 nM to 1 μM is used. Preferably, this range is for in vitro use in cell models such as retinal cells or tissue. More preferably, the concentration used is in the range of 1 to 400 nM, even more preferably 10 to 200 nM, and even more preferably 50 to 100 nM. When multiple separate AONs are used, this concentration or dose can refer to the total concentration or dose of the AONs or the concentration or dose of each AON added.

[0090] In a preferred embodiment, the viral vectors described hereinabove, preferably AAV vectors, as delivery vehicles for the molecules of the present invention, are administered in a dose of 1 x 10 per injection. 9 ~1×10 17 viral particles, more preferably 1 x 10 per injection 10 ~1×10 12 The vaccine is administered at a dose ranging from 100 to 1000 viral particles.

[0091] The AON concentration or dosage ranges depicted above are preferred concentrations or dosages for in vivo, in vitro, or ex vivo use. Those skilled in the art will understand that depending on the AON used, the target cell to be treated, the gene target and its expression level, the medium used, and the transfection and incubation conditions, the AON concentration or dosage used may further vary and may require further optimization.

[0092] The AON for controlling splicing of the present invention, the viral vector for controlling splicing of the present invention, or the composition for controlling splicing of the present invention for use in the present invention can be administered to cells, tissues, and / or organs in vivo of an individual already suffering from or at risk of developing an ABCA4-related disease or condition, and can be administered in vivo, ex vivo, or in vitro. The AON for controlling splicing of the present invention, the viral vector for controlling splicing of the present invention, or the composition for controlling splicing of the present invention can be administered directly or indirectly to cells, tissues, and / or organs in vivo of an individual already suffering from or at risk of developing an ABCA4-related disease or condition, and can be administered directly or indirectly in vivo, ex vivo, or in vitro. Because Stargardt disease has a pronounced phenotype in retinal cells, it is preferred that the targeted cells are retinal cells, and more preferably that the tissue is the retina, and even more preferably that the organ comprises or consists of the eye.

[0093] The present invention further provides a method for modulating ABCA4 splicing in a cell, the method comprising contacting the cell, preferably a retinal cell, with an antisense oligonucleotide for controlling splicing of the present invention, a vector for controlling splicing of the present invention, or a pharmaceutical composition for controlling splicing of the present invention. Features of this aspect are preferably those defined earlier in this specification. The step of contacting the cell with an AON for controlling splicing of the present invention, a viral vector for controlling splicing of the present invention, or a composition for controlling splicing of the present invention can be carried out by any method known to those skilled in the art. This includes the use of methods for delivery of AONs, viral vectors, and compositions for controlling splicing described earlier in this specification. The contacting step can be direct or indirect, and can be in vivo, ex vivo, or in vitro.

[0094] Unless otherwise stated, each embodiment described herein can be combined with any other embodiment described herein.

[0095] [Definition] In this document and its claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that items preceding this word are included, but items not specifically mentioned are not excluded. In addition, referring to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one of the element is present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0096] The word "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably means that the value may be 5% more or less than the given value (10).

[0097] The sequence information provided herein should not be interpreted so narrowly as to require the inclusion of erroneously identified bases. Those skilled in the art will be able to identify such erroneously identified bases and will know how to correct such errors. In the event of a sequence error, the sequence of the polypeptide obtainable by expression of the gene present in SEQ ID NO: 1 containing the nucleic acid sequence encoding the polypeptide shall prevail.

[0098] All patent and literature references cited herein are hereby incorporated by reference in their entirety. [Brief explanation of the drawings]

[0099] [Figure 1] Figure 1 is a schematic diagram of the intron 36 variants in ABCA4 that underlie STGD1 and the PE insertions they result in. The c.5196+1013A>G and c.5196+1056A>G variants result in duplicated PEs with the same splice acceptor but different splice donor sites. [Figure 2]Figure 2 shows an overview of the PE insertions introduced by different ABCA4 mutations and their relative locations in the eight designed AONs. Note that AON7 was specifically designed for the 177-nt PE inserted by the c.5196+1056A>G mutation and therefore contains a mismatch to the 129-nt PE inserted by the c.5196+1013A>G variant. [Figure 3] Figure 3 shows RT-PCR analysis of HEK293T cells transfected with either wild-type or mutant ABCA4 midigenes, with or without AON or sense oligonucleotides (SON, negative control). RHO was amplified to demonstrate equal transfection efficiency of the midigenes. MQ: MilliQ water. A) c.5196+1216A, B) c.5196+1056G, C) c.5196+1013G. [Figure 4] Figure 4 shows RT-PCR analysis of photoreceptor precursor cells (PPCs) transfected with or without AON or SON. STGD1 patients were compound heterozygous for the c.5196+1137G>A variant, along with a deletion and a missense mutation in the second allele. Actin was amplified to reveal equal cDNA input. CHX: cycloheximide. MQ: Milli-Q water.

[0100] [Array Description] [Table 1-1] [Table 1-2] [Example]

[0101] We evaluated the in vitro efficacy of a number of AONs to control ABCA4 splicing in cells. To do this, we used midigene constructs, i.e., plasmids containing a portion of the ABCA4 gene sequence, typically a region of interest with or without a mutation, flanked on both sides by at least 100 bp of wild-type ABCA4 sequence. In addition to midigene assays, for the c.5196+1137G>A mutation, we also used iPSC technology to evaluate the efficacy of AONs targeting the effect of this mutation.

[0102] Materials and Methods AON design For each mutation, a smaller region of interest was selected to design the AONs. For the mutation c.5196+1013A>G, the target region is represented by SEQ ID NO: 8; for the mutation c.5196+1056A>G, ​​the target region is represented by SEQ ID NO: 9; and for the mutations c.5196+1137G>A and c.5196+1217C>A, the target region is represented by SEQ ID NO: 10. All oligonucleotides were subjected to in silico RNA structure prediction, and from the results, eight AONs were designed and ordered (Table 2).

[0103] [Table 2]

[0104] AON testing in HEK293T After AON design, HEK293T cells were transfected with midigenes containing either a portion of the wild-type ABCA4 gene (SEQ ID NO: 44) or mutant forms (SEQ ID NOs: 45, 46, and 47) with different targeted intron 36 mutations, i.e., c.5196+1013A>G, c.5196+1056A>G, ​​and c.5196+1216C>A, as listed in Table 2. For each mutation, wild-type or mutant midigene-transfected cells were treated either without AON (NT), with AONs designed against specific PEs (AONs 1–4 for c.5196+1216C>A and AONs 5–8 for c.5196+1013A>G and c.5196+1056A>G), or with a sense oligonucleotide (SON) (SEQ ID NO: 43) serving as a negative control. Forty-eight hours after transfection, cells were harvested and RNA was isolated. RT-PCR analysis was then performed to determine which AONs were able to correct the splice defect.

[0105] RT-PCR analysis Total RNA was isolated using the NucleoSpin RNA Clean-up Kit (Cat. No. 740955-50; Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. RNA was quantified, and cDNA was synthesized from 1 μg of RNA using the iScript cDNA Synthesis Kit (Cat. No. 1708891; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Finally, the efficacy of the AON was evaluated by nested PCR using the ABCA4 primers represented by SEQ ID NO:48 and SEQ ID NO:49. To evaluate transfection efficiency, exon 5 of rhodopsin was amplified using the following primer pair: SEQ ID NO:50 and SEQ ID NO:51.

[0106] result As shown in Figure 3, for each PE insertion, AONs could completely or at least partially restore the splicing defect. More specifically, for the c.5196+1216C>A variant, both AON1 and AON2 completely converted the aberrantly spliced ​​midigene into a correctly spliced ​​version, while AON3 and AON4 substantially restored the splicing defect (Figure 3A). For the c.5196+1056A>G change, AON5 appeared to be the most potent AON. For AON7, and to a somewhat lesser extent for AON6, the majority of PE-containing transcripts were also restored. AON8 was able to partially restore the splicing defect (Figure 3B). Finally, for the c.5196_1013A>G change, AON5 and AON6 appeared to be the most potent AONs. AON7 was still able to at least partially control splicing despite a single-nucleotide mismatch between the AON (designed against other variants) and the PE. For the c.5196_1013A>G change, AON8 did not show any efficacy, a result that is not entirely unexpected given that its position lies outside the 129-nt PE inserted by this variant (Figure 3C).

[0107] AON inspection Because the resulting c.5196+1137G>A PE variant was nearly undetectable in midigene assays, we used iPSC technology to evaluate the efficacy of AONs targeting this PE. Blood cells from a patient with STGD1 carrying the c.5196+1137G>A variant in combination with a partial ABCA4 deletion and a missense mutation (c.[2918+775_3328+640del;4462T>C]p.[Ser974Glnfs*64;Cys1488Arg]) in the other allele were reprogrammed into iPSCs (Sangermano et al., 2016) and subsequently differentiated into photoreceptor progenitor cells (PPCs) for 30 days. On day 28, AONs 1–4 (or SON) were added to the cells at a final concentration of 1 μM. On day 29, cycloheximide (CHX) was added to block nonsense-mediated decay of transcripts with premature termination codons. Finally, on day 30, cells were harvested and subjected to RNA analysis (as described above). In this case, actin (ACTB) was amplified using a forward primer in exon 3 represented by SEQ ID NO: 52 and a reverse primer in exon 4 represented by SEQ ID NO: 53 to normalize the samples.

[0108] result As can be seen in Figure 4, PE insertions were more readily detected in PPCs. In addition, two of the four AONs tested (AON2 and 3) successfully converted the PE-containing ABCA4 transcript into a correctly spliced ​​product.

[0109] conclusion For all of the different PEs (77-nt, 129-nt, and 177-nt), AONs were identified that could effectively restore the splice defect. For c.5196+1013A>G, AON5 and AON6 appeared to be the most effective AONs, while for c.5196+1056A>G, ​​the PE insertion appeared to be best prevented by AON5, AON6, or AON7. For c.5196+1137G>A, AON2 and AON3 appeared to be the most potent, at least in the PPC. Finally, for c.5196+1216C>A, AON1 and AON2 completely restored the splice defect, but AON3 and AON4 also showed splicing correction.

[0110] References Albert, S., Garanto, A., Sangermano, R., Khan, M., Bax, NM, Hoyng, CB, Zernant, J., Lee, W., Allikmets, R., Collin, RW and Cremers, FP, 2018. Identification and rescue of splice defects caused by two neighboring deep-intronic ABCA4 mutations underlying Stargardt disease. The American Journal of Human Genetics, 102(4), pp.517-527. Allikmets, R., Singh, N., Sun, H., Shroyer, N. F., Hutchinson, A., Chidambaram, A., Gerrard, B., Baird, L., Stauffer, D., Peiffer, A., Rattner, A., Smallwood, P., Li, Y., Anderson, K. L., Lewis, R. A., Nathans, J., Leppert, M., Dean, M. & Lupski, J. R. A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat. Genet. 15, 236-246 (1997), doi:10.1038 / ng0397-236. Bauwens, M., De Zaeytijd, J., Weisschuh, N., Kohl, S., Meire, F., Dahan, K., Depasse, F., De Jaegere, S., De Ravel, T., De Rademaeker, M., Loeys, B., Coppieters, F., Leroy, B. P. & De Baere, E. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutat. 36, 39-42 (2015), doi:10.1002 / humu.22716. Bauwens, M., Garanto, A., Sangermano, R., Naessens, S., Weisschuh, N., De Zaeytijd, J., Khan, M., Sadler, F., Balikova, I., Van Cauwenbergh, C. and Rosseel, T., 2019. ABCA4-associated missingness model for autoimmune disease assessment disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants. Genetics in Medicine, p.1. Bax, NM, Sangermano, R., Roosing, S., Thiadens, AA, Hoefsloot, LH, van den Born, LI, Phan, M., Klevering, BJ, Westeneng-van Haaften, C., Braun, TA, Zonneveld-Vrieling, MN, de Wijs, I., Mut, M., Den Hollander, EM, Den Stone, EM. Klaver, CC, Hoyng, CB & Cremers, FPM Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum. Mutated. 36, 43-47 (2015), doi:10.1002 / humu.22717. 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ABCA4 gene screening by next-generation sequencing in a British cohort. Invest. Ophthalmol. Vis. Sci. 54, 6662-6674 (2013), doi:10.1167 / iovs.13-12570. Garanto A, Chung DC, Duijkers L, Corral-Serrano JC, Messchaert M, Xiao R, Bennett J, Vandenberghe LH, Collin RWJ (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet, 25: 2552-2563. Lewis, R. A., Shroyer, N. F., Singh, N., Allikmets, R., Hutchinson, A., Li, Y., Lupski, J. R., Leppert, M. & Dean, M. Genotype / Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am. J. Hum. Genet. 64, 422-434 (1999), doi:10.1086 / 302251. Maugeri, A., van Driel, M. A., van de Pol, D. J., Klevering, B. J., van Haren, F. J., Tijmes, N., Bergen, A. A., Rohrschneider, K., Blankenagel, A., Pinckers, A. J., Dahl, N., Brunner, H. G., Deutman, A. F., Hoyng, C. B. & Cremers, F. P. M. 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Claims

1. An antisense oligonucleotide for controlling splicing that binds to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:4 and / or is complementary to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:4, preferably an antisense oligonucleotide that binds to a polynucleotide selected from the group consisting of SEQ ID NOs:8, 9 and 10 or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs:8, 9 and 10, more preferably an antisense oligonucleotide that binds to a polynucleotide selected from the group consisting of SEQ ID NOs:5, 6 and 7 or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs:5, 6 and 7. and even more preferably, an antisense oligonucleotide that binds to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41, and 42, or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 12, 13, 14, 16, 17, 18, 20, 21, 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41, and 42.

2. The antisense oligonucleotide for controlling splicing according to claim 1, wherein the nucleotide in the antisense oligonucleotide can be an RNA residue, a DNA residue, or a nucleotide analog or equivalent.

3. The antisense oligonucleotide for controlling splicing according to claim 1, having a length of about 8 to about 40 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to about 23 nucleotides, such as 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides.

4. An antisense oligonucleotide for controlling splicing according to any one of claims 1 to 3, comprising a sequence selected from the group consisting of SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35 and 39, or consisting of a sequence selected from the group consisting of SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35 and 39.

5. 5. An antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4, comprising at least one 2'-O-alkyl phosphorothioate antisense oligonucleotide, such as a 2'-O-methyl modified ribose, a 2'-O-ethyl modified ribose, a 2'-O-propyl modified ribose and / or substituted derivatives thereof, such as halogenated derivatives, having these modifications, preferably comprising at least one 2'-O-propyl modified ribose.

6. The antisense oligonucleotide for controlling splicing according to any one of claims 1 to 5, which comprises a phosphorothioate backbone.

7. A viral vector that expresses the antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4 when placed under conditions that promote expression of the molecule.

8. A pharmaceutical composition comprising the antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4 or the viral vector according to claim 7, and a pharmaceutically acceptable excipient.

9. 9. The pharmaceutical composition of claim 8, which is for intravitreal administration and is administered in an amount of total antisense oligonucleotide ranging from 0.05 mg to 5 mg.

10. The pharmaceutical composition according to claims 8 and 9, which is for intravitreal administration and is administered in an amount of a total antisense oligonucleotide for controlling splicing in the range of 0.1 to 1 mg per eye, for example, an amount of a total antisense oligonucleotide for controlling splicing of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg per eye.

11. The antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4, the vector according to claim 7 or the pharmaceutical composition according to any one of claims 8 to 10, for use as a medicament, preferably for use as a medicament for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4.

12. Use of the antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4, the vector according to claim 7, or the pharmaceutical composition according to any one of claims 8 to 10, for treating an ABCA4-related disease or condition that requires regulating ABCA4 splicing.

13. A method for regulating ABCA4 splicing in a cell, the method comprising a step of contacting the cell with an antisense oligonucleotide for controlling splicing described in any one of claims 1 to 4, a vector described in claim 7, or a pharmaceutical composition described in any one of claims 8 to 10.

14. 14. The antisense oligonucleotide for controlling splicing for the use according to claim 11, the use according to claim 12 or the method according to claim 13, wherein the ABCA4-related disease or condition is Stargardt disease.

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