Antisense oligonucleotides rescue aberrant splicing of ABCA4

Abstract

To provide novel antisense oligonucleotides that may be used in the treatment, prevention and / or delay of an ABCA4-associated condition.SOLUTION: The present invention provides an antisense oligonucleotide for controlling the splicing of ABCA4, a viral vector expressing the antisense oligonucleotide, and a pharmaceutical composition comprising the antisense oligonucleotide or the viral vector and a pharmaceutically acceptable excipient.SELECTED DRAWING: None

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 resulting in total blindness. A typical hallmark of Stargardt disease is the presence of many yellow spots (specks) distributed throughout the fundus of the patient's eye. The ABCA4 gene is composed 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 targeted 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 with 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 with arCRD (Maugeri et al., 2000) after sequencing the coding regions and flanking splice sites. In general, individuals with arCRD or panretinal dystrophy carry two severe ABCA4 alleles, while individuals with STGD1 carry two moderately severe variants or a combination of mild and severe variants (Maugeri et al., 1999; van Driel et al., 1998). It has been hypothesized that the majority of unknown ABCA4 variants in STGD1 patients reside in intronic regions of the gene, and indeed, over the last few years, several groups have demonstrated the presence 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). A mutation frequently present in STGDI patients (approximately 2-3% of all STGDI cases in Western countries harbor this mutation) is c.768G>T, a variant affecting the last nucleotide of exon 6. This mutation weakens the splice donor site of exon 6, leading to the use of an alternative splice donor site downstream in intron 6 and a subsequent extension of the transcript by 35 nucleotides. This extension is therefore predicted to result in a premature termination of ABCA4 protein synthesis within a frameshift.

[0005] The fact that a significant amount of STGD1 cases carry the c.768G>T variant makes this variant 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 suffering from Stargardt disease, especially with respect to the c.768G>T mutation.

[0006] [Summary of the Invention] In a first aspect, the present invention provides an antisense oligonucleotide for redirecting splicing, which binds to and / or is complementary to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:4, preferably an antisense oligonucleotide which binds to or is complementary to a polynucleotide having SEQ ID NO:5, more preferably an antisense oligonucleotide which binds to or is complementary to a polynucleotide having SEQ ID NO:80, even more preferably an antisense oligonucleotide which binds to a polynucleotide selected from the group consisting of SEQ ID NOs:7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21, or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs:7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21.

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

[0008] In a third aspect, the present invention provides a pharmaceutical composition comprising an antisense oligonucleotide for controlling splicing as defined herein or a viral vector as defined herein and a pharma- ceutically acceptable excipient.

[0009] In a fourth aspect, the present invention provides an antisense oligonucleotide for controlling splicing as defined herein 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.

[0010] [Detailed Description of the Invention] By definition, antisense oligonucleotides (AONs) are substantially complementary (antisense) to their target, 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 some mutations in ABCA4 (WO 2018 / 109011), the lack of such binding usually results in the skipping of the targeted exon.

[0011] Some mutations create novel splice acceptor, splice donor or exon splice enhancer binding sites, which lead to the inclusion of pseudoexons in the mRNA of the corresponding gene. The c.768G>T mutation weakens the splice donor site in exon 6 and leads to the use of an alternative splice donor site downstream in intron 6, followed by a 35-nucleotide extension of the transcript. To restore splicing in individuals carrying the c.768G>T mutation, we designed an AON that specifically blocks the newly used alternative splice donor site in intron 6, thereby controlling the splicing machinery back to the original site at c.768.

[0012] Thus, in a first aspect, the present invention provides an antisense oligonucleotide for controlling splicing, which binds to and / or is complementary to a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4. Preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide having SEQ ID NO: 5. More preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide having SEQ ID NO: 80. Even more preferably, the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs: 7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21.

[0013] The term "antisense oligonucleotide" or "AON" is used interchangeably herein and is understood to refer to an oligonucleotide molecule that comprises a nucleotide sequence that is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogeneous nuclear 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. The binding of an AON to its target can be easily assessed by a person skilled in the art using techniques known in the art, such as the gel mobility shift assay described in EP1619249.

[0014] 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., controlling splicing, is achieved. Preferably, the complementarity is between 90% and 100%. In general, this allows for 1 or 2 mismatches in a 20 nucleotide AON, or 1, 2, 3 or 4 mismatches in a 40 nucleotide AON, or 1, 2, 3, 4, 5 or 6 mismatches in a 60 nucleotide AON, etc. Optionally, the AON can be further tested by transfection into retinal cells of the patient. The complementary regions can be preferably designed such that, when combined, they are specific for an exon in the pre-mRNA. Such specificity depends on the actual sequence in the other (pre)mRNA molecules in the system, and therefore can be created by complementary regions of various lengths. The risk that the AON may hybridize to one or more other pre-mRNA molecules decreases with increasing size of the AON. It is clear that AONs that contain mismatches in the region of complementarity but retain the ability to hybridize and / or bind to the targeted region(s) in the pre-mRNA can be used in the present invention. However, since AONs that lack mismatches in the complementary portion typically have higher efficiency and higher specificity than AONs that have such mismatches in one or more complementary regions, preferably at least the complementary portion does not contain such mismatches. It is believed that a higher hybridization strength (i.e., an increased number of interactions with the opposing strand) is advantageous in increasing the efficiency of the process of interfering with the splicing mechanism of this system.

[0015] The terms "modulate splicing" and "control splicing" are used interchangeably herein and encompass AON-based splice modulation therapy for the c.768G>T mutation. The term "control splicing" is defined herein as controlling ABCA4 pre-mRNA splicing to obtain the original transcript.

[0016] The AONs of the present invention preferably do not contain a stretch of CpG, more preferably they do not contain any CpG. The presence of CpG or stretches of CpG in an oligonucleotide is usually associated with increased immunogenicity of said oligonucleotide (Dorn and Kippenberger, 2008). This increased immunogenicity is undesirable as it may induce damage to the tissue to be treated, i.e. the eye. Immunogenicity can be evaluated in animal models by assessing the presence of CD4+ and / or CD8+ cells, and / or inflammatory mononuclear cell infiltration. Immunogenicity can also be evaluated 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 said AON using standard immunoassays known to those skilled in the art. Inflammatory response, type I-like interferon production, IL-12 production and / or increased immunogenicity can be evaluated by detecting the presence or increased amount of neutralizing antibodies or antibodies recognizing said AON using standard immunoassays. AONs according to the invention even more preferably have acceptable RNA binding kinetics and / or thermodynamic properties. The 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 the 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. It is therefore difficult to give a preferred range for each of these parameters. Acceptable Tm may range between 35-70°C and acceptable free energy may range between 15-45 kcal / mol.

[0017] In all embodiments, the nucleotides in the antisense oligonucleotides of the present invention may be RNA residues, DNA residues, or nucleotide analogs or equivalents.

[0018] 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 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, AONs according to the present invention have a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.

[0019] In a preferred embodiment, the AON for controlling splicing according to the present invention comprises or consists of SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77. It was found that these AONs were highly efficient in controlling the aberrant splicing of ABCA4 caused by the c.768G>T mutation. These preferred AONs preferably comprise from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably comprise or consist of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0020] It is preferred that the AONs for controlling splicing according to the present invention comprise 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 AONs comprise at least one nucleotide analogue or equivalent, where a nucleotide analogue 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.

[0021] In a preferred embodiment, nucleotide analog or equivalent comprises modified backbone.Examples of such backbone are provided by 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 antisense drug.

[0022] 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 methods that physically disrupt 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 because of the non-ionic backbone, these studies have shown that morpholino oligonucleotides are capable of triplex formation in the absence of magnesium.

[0023] It is further preferred that the linkages between the 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.

[0024] 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 backbone of PNA 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 usually 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 linkages between adjacent morpholino rings are replaced by nonionic phosphorodiamidate linkages.

[0025] In yet another embodiment, the nucleotide analog or equivalent of the present invention comprises 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.

[0026] Further preferred nucleotide analogs or equivalents according to the 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, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, aryl or aralkyl, which may be 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.

[0027] In another embodiment, the nucleotide analogs or equivalents of the present invention contain 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.

[0028] 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 in 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.

[0029] Thus, in a 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 (RNA), 2'O-ethyl modified ribose, 2'O-methoxyethyl modified ribose, 2'-O-propyl modified ribose and / or substituted derivatives, such as halogenated derivatives, having these modifications.

[0030] In a preferred embodiment, the AON for controlling splicing according to the present invention comprises or consists of SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77 and comprises a 2'-O-methyl modified ribose (RNA) and a phosphorothioate backbone. In another preferred embodiment, the AON for controlling splicing according to the present invention comprises or consists of SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77 and comprises a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0031] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:6 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0032] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 10 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0033] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 14 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0034] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 18 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0035] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 22 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0036] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 26 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0037] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 30 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0038] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 47 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0039] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 48 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0040] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 49 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0041] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 50 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0042] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:51 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0043] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:52 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0044] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 53 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0045] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:54 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0046] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 55 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0047] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 56 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0048] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:57 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0049] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:58 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0050] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:59 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0051] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 60 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0052] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:61 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0053] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:62 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0054] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 63 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0055] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:64 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0056] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 65 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0057] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 66 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0058] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:67 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0059] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:68 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0060] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO:69 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0061] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 70 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0062] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 71 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0063] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 72 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0064] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 73 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0065] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 74 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0066] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 75 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0067] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 76 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0068] In one embodiment, the AON for controlling splicing of the present invention comprises or consists of SEQ ID NO: 77 and comprises a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose (RNA) and a phosphorothioate backbone.

[0069] The AON for controlling splicing according to the present invention can be administered indirectly using suitable means known in the art. The AON can be given to an individual or a cell, tissue or organ of said individual, for example as a so-called "naked" AON. The AON can also be administered in the form of an expression vector, which encodes an RNA transcript comprising the sequence of said AON according to the present invention. The expression vector is preferably introduced into a cell, tissue, organ or individual by a gene delivery vehicle. In a preferred embodiment, a viral-based expression vector is provided that comprises an expression cassette or transcription cassette that drives the expression or transcription of the AON for controlling splicing according to the present invention. Thus, the present invention provides a viral vector that expresses an antisense oligonucleotide for controlling splicing according to the present invention when placed under conditions that promote expression of said molecule.

[0070] AONs for controlling splicing according to the invention can be provided to cells by plasmid-derived antisense oligonucleotide expression or viral expression provided by vectors based on adenovirus or adeno-associated virus. 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. A preferred delivery vehicle is a viral vector, such as an adeno-associated virus vector (AAV), or a retroviral vector, such as a lentiviral vector, among others. Also, plasmids, artificial chromosomes, plasmids available for targeted homologous recombination and integration in the human genome of cells can be appropriately applied for delivery of AONs for controlling splicing according to the invention. Vectors in which transcription is driven from a PolIII promoter and / or in which the transcript is in the form of a fusion with U1 or U7 transcripts, which produce excellent results for delivering small transcripts, are preferred for the present invention. Designing suitable transcripts is within the skill of the person skilled in the art. PolIII-driven transcripts, preferably in the form of a fusion transcript with U1 or U7 transcripts, are preferred. Such fusions can be made as previously described (Gorman et al., 1998).

[0071] A preferred expression system for the AONs for controlling splicing according to the present invention is an adeno-associated virus (AAV)-based vector. Single-stranded and double-stranded AAV-based vectors have been developed that can be used for the prolonged expression of antisense nucleotide sequences for controlling splicing with high efficiency. Preferred AAV-based vectors include, for example, expression cassettes driven by an RNA polymerase III promoter (Pol III) or an RNA polymerase II promoter (Pol II). Preferred RNA promoters are, for example, the Pol III U6 RNA promoter or the Pol II U7 RNA promoter.

[0072] 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.

[0073] The AAV vector of the present invention is a recombinant AAV vector, and refers to an AAV vector that includes a portion of the AAV genome that includes the encoded AON for controlling splicing of the present invention, encapsidated in a protein shell of capsid proteins derived from an AAV serotype depicted 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 named capsid protein shell. The AAV vector can be deleted of 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 sequence can be a wild-type sequence, or can have at least 80%, 85%, 90%, 95 or 100% sequence identity with the wild-type sequence, or can be modified, for example, by insertion, mutation, deletion or substitution of nucleotides, so long as it maintains functionality. In this context, functionality refers to the ability to direct packaging of the genome into a capsid shell and then allow 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 than the AAV vector genome ITR. Thus, while the AAV vector of the present invention can be composed of a capsid protein shell, i.e., an icosahedral capsid, comprising the capsid proteins (VP1, VP2 and / or VP3) of one AAV serotype, for example, AAV serotype 2, the ITR sequence contained in this AAV5 vector can be any of the AAV serotypes described above, including the AAV2 vector.Thus, an "AAV2 vector" comprises a capsid protein shell of AAV serotype 2, whereas, for example, an "AAV5 vector" comprises a capsid protein shell of AAV serotype 5, either of which may encapsidate any AAV vector genomic ITR of the invention.

[0074] 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.

[0075] More preferably, a recombinant AAV vector of the 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.

[0076] 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.

[0077] More preferably, a recombinant AAV vector of the 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.

[0078] 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 a vector is referred to as an AAV2 / 2 vector.

[0079] 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 the 3' termination sequence.

[0080] "AAV helper functions" generally refer to the corresponding AAV functions required for AAV replication and packaging that are provided in trans to the AAV vector. AAV helper functions complement AAV functions that are missing 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, namely 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, (Chiorini et al., 1999) or U.S. Pat. No. 5,139,941, which are 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 the 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 for the AAV genome present during said AAV vector replication and packaging, on the other hand.

[0081] 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.

[0082] Preferably, the AAV genome present in a recombinant AAV vector of the invention does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. The AAV genome can further include 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.

[0083] Preferably, the AAV vector of the present invention is constructed and produced according to the method by Garanto et al., 2016, which is incorporated herein by reference.

[0084] A preferred AAV vector according to the 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 invention, which is an AON comprising or preferably consisting of the following sequence: A sequence complementary or substantially complementary to a nucleotide sequence consisting of SEQ ID NO: 4, preferably the AON is complementary to a polynucleotide having SEQ ID NO: 5, more preferably the AON is complementary to a polynucleotide having SEQ ID NO: 80, even more preferably a sequence complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21. Even more preferably, the AON comprises or consists of a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, and SEQ ID NO:30, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, or most preferably, selected from the group consisting of SEQ ID NOs:34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75, and 77.

[0085] Considering the progress already achieved to date, improvements in the means for providing an individual or a cell, tissue or organ of said individual with the AON for controlling splicing according to the present invention are expected. Of course, such future improvements can be incorporated to achieve the effects mentioned in the reconstruction of mRNA using the method according to the present invention. The AON for controlling splicing according to the present invention can be delivered to an individual, a cell, tissue or organ of said individual as a "naked" AON or the like. When administering the AON for controlling splicing according to the present invention, it is preferred that the molecule is dissolved in a solution that is compatible with the delivery method. The retinal cells can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution.

[0086] Alternatively, the preferred delivery method for AONs or plasmids for expressing such AONs to control splicing is a viral vector or nanoparticle.Preferably, the viral vector or nanoparticle is delivered to the retina or other relevant cells.Such delivery to retina 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.

[0087] Alternatively, the plasmids can be given by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal and / or intracerebroventricular administration, it is preferred that the solution is a physiological saline solution. 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 the respective 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 an expected low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to further modify and analyze their further (in vivo) nucleic acid transfer characteristics and toxicity.

[0088] Lipofectin represents 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.

[0089] 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.

[0090] In addition to these common nanoparticle materials, the cationic peptide protamine provides an alternative approach to formulate oligonucleotides with colloids. This colloidal nanoparticle system can be prepared by a simple self-assembly process to form a so-called proticle that can package oligonucleotides and mediate their intracellular release. A person skilled in the art can select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver exon-retaining molecules for use in the present invention to prevent, treat or delay 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.

[0091] In addition, the AONs for controlling splicing according to the present invention can be covalently or non-covalently linked to targeting ligands specifically designed to facilitate uptake into the cell, cytoplasm and / or its nucleus. 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 the oligonucleotide in vesicles, e.g., endosomes or lysosomes, and / or the intracellular release from said vesicles, e.g., endosomes or lysosomes.

[0092] Thus, 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.

[0093] It should be understood that when the composition comprises additional components, such as auxiliary compounds as defined later in this specification, each component of the composition may not be appropriately formulated in one single combination or composition or preparation. Depending on their identity and specific features, the skilled artisan 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.

[0094] 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 pharma- ceutical active mixture by adding a pharma- ceutical acceptable carrier.

[0095] 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 pharma- ceutically acceptable excipient. Such a composition may comprise a single AON or viral vector for controlling splicing according to the present invention, but may also comprise a plurality of separate AONs or viral vectors for controlling splicing according to the present invention. Such a pharmaceutical composition may comprise any pharma- ceutically acceptable excipient, including carriers, fillers, preservatives, adjuvants, solubilizers and / or diluents. Such pharma- ceutical 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 the present specification.

[0096] A preferred route of administration is by intravitreal injection of an aqueous solution or a formulation specially adapted for intraocular administration. EP2425814 discloses an oil-in-water emulsion specially adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. As the emulsion is less dense than the vitreous humor, the emulsion floats on the vitreous, avoiding the injected drug from impairing vision. Thus, in one embodiment, a pharmaceutical composition is provided which is suitable for intravitreal administration and is dosed 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. A suitable intravitreal dose is given and comprises between 0.05 mg and 5 mg per eye, preferably between 0.1 and 1 mg of total antisense oligonucleotide, 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.

[0097] A preferred AON for controlling splicing according to the present invention is for the treatment of an ABCA4-related disease or condition in an individual. In any embodiment of the present invention, the term "treatment" is understood to include prevention and / or delay of an ABCA4-related disease or condition. An individual 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.

[0098] Alternatively, an individual who can be treated using the AON for controlling splicing according to the present invention may not yet be 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. A preferred individual is a human. In any embodiment of the present invention, the ABCA4-related disease or condition is preferably Stargardt's disease.

[0099] 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 modulating the splicing of ABCA4, 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, preferably as such feature as defined hereinbefore.

[0100] 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 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 such a feature is as defined hereinbefore.

[0101] The present invention further provides a method for the treatment of an ABCA4-related disease or condition which requires modulating the splicing of ABCA4, comprising contacting a cell 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 such a feature is as defined hereinbefore.

[0102] 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 such a feature is as defined hereinbefore.

[0103] 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.

[0104] The treatment in the use or method according to the invention is preferably at least once and preferably lasts for at least one week, one month, several months, one year, 2, 3, 4, 5, 6 years or more, lifelong. Each AON or its equivalent for controlling splicing according to the invention defined herein for use according to the invention may 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 may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of the AON, composition, compound or auxiliary compound according to the invention may depend on several parameters such as the severity of the disease, the age of the patient, the mutation of the patient, the number of AONs for controlling splicing according to the invention (i.e., dose), the formulation of the AON, composition, compound or auxiliary compound according to the invention, the route of administration, etc. The frequency of administration may vary between daily, weekly, at least once every 2 weeks or 3 weeks or 4 weeks or 5 weeks or longer.

[0105] 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) where strict protocol requirements exist. The AON of the present invention can be used at a dose ranging from 0.01 to 20 mg / kg, preferably 0.05 to 20 mg / kg. A suitable intravitreal dose would be about: between 0.05 mg and 5 mg per eye, preferably between 0.1 to 1 mg, 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.

[0106] 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 tissues. More preferably, the concentration used is in the range of 1 to 400 nM, even more preferably, 10 to 200 nM, 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.

[0107] In a preferred embodiment, the viral vector described hereinbefore, preferably the AAV vector, as a delivery vehicle for the molecules according to the invention, is administered at a dose of 1×10 per injection. 9 ~1×10 17 10 viral particles, more preferably 1 x 10 per injection 10 ~1×10 12 The vaccine is administered at a dose ranging from 10 to 15 viral particles.

[0108] The ranges of concentrations or dosages of AONs 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 AONs used, the target cells to be treated, the gene targets and their expression levels, the medium used, and the transfection and incubation conditions, the concentrations or dosages of AONs used may further vary and may require further optimization to be performed.

[0109] The AON for controlling splicing according to the invention or the viral vector according to the invention or the composition according to the invention for use according to the 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 according to the invention or the viral vector according to the invention or the composition according to the 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. Since Stargardt's disease has a prominent phenotype in retinal cells, it is preferred that the targeted cells are retinal cells, more preferably that the tissue is the retina, and even more preferably that the organ comprises or consists of the eye.

[0110] The present invention further provides a method for modulating the splicing of ABCA4 in a cell, comprising contacting the cell, preferably a retinal cell, with an antisense oligonucleotide for controlling splicing according to the present invention, a vector for controlling splicing according to the present invention or a pharmaceutical composition for controlling splicing according to the present invention. The features of this aspect are preferably those defined hereinbefore. The step of contacting the cell with an AON for controlling splicing according to the present invention or a viral vector for controlling splicing according to the present invention or a composition for controlling splicing according to the present invention can be carried out by any method known to the skilled person. The use of the methods for delivery of AON, viral vector and composition for controlling splicing described hereinbefore is included. The step of contacting can be direct or indirect, and can be in vivo, ex vivo or in vitro.

[0111] Unless otherwise stated, each embodiment described herein can be combined with any other embodiment described herein.

[0112] [Definition] In this document and the claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the items preceding this word are included, but items not specifically mentioned are not excluded. In addition, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that only one of the elements is present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0113] 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).

[0114] 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 are able to identify such erroneously identified bases and know how to correct such errors. In case of sequence errors, 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 should be given priority.

[0115] All patent and literature references cited herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0116] [Figure 1A] A) Bioinformatics prediction of splice donor site strength for the native exon 6 splice donor site and for the alternative splice donor site in intron 6, both for the wild-type (top sequence) and c.768G>T mutant (bottom sequence) contexts. Usage of the alternative splice site in the mutant context is predicted to result in a 35-nt expansion of the ABCA4 transcript (shown in grey, red in the color version). [Figure 1B] B) RT-PCR analysis of ABCA4 in RNA from fibroblasts of a control (ctrl) individual, an STGD1 patient with compound heterozygosity (comp.het.) for the c.768G>T change, and an STGD1 patient with homozygosity (hom.) for the c.768G>T mutation. - or + indicates the absence or presence of cycloheximide. M: 100-bp ladder. MQ: milliQ water. [Diagram 2] Schematic representation of the arrangement of the c.768G>T variant, the native and alternative splice donor sites, and the relative positions of the designed AONs. [Figure 3A] RNA analysis of homozygous c.768G>T patient-derived fibroblasts after AON delivery. (A) Analysis of ABCA4 transcripts by RT-PCR and nested PCR in patient-derived fibroblasts treated with AON2-AON8 or untreated (NT). Wild-type fibroblasts (WT) were used as control. ACTB was used as loading control. MQ represents the negative control for the first and second (nested) PCR reactions. AON1 produced similar results as AON2 (data not shown). [Figure 3B] RNA analysis of fibroblasts from a homozygous c.768G>T patient after AON delivery. (B) Chromatogram of the bands highlighted in gels 1-3. The band with an asterisk was found to be a contaminant since the sequence showed only a peak representing G as in band 1 and the patient carried the c.768G>T mutation homozygously. AON8 was able to correct the splicing as shown by the sequence in band 2. [Figure 4] AON position compared to the target region. AON position relative to the target region. AONs 10-34 contained 2'-OMe modifications with a phosphorothioate (PS) backbone, whereas AONs 35-40 contained 2'-O-MOE modifications with a phosphorothioate (PS) backbone. Exonic portions are depicted by grey boxes. The c.768G>T mutation is indicated by an arrow and in bold. The 35 nucleotide expansion found in the STGD1 case is listed in italics. [Diagram 5]AON screening in HEK293T cells using midigene carrying the c.768T variant. RT-PCR analysis of HEK293T cells co-transfected with midigene carrying the c.768T variant (MUT) and either AON (A#) or sense oligo (SON) with 2'-OMe modification with phosphorothioate (PS) backbone. Wild-type midigene carrying c.768G (WT) was used as control. Non-transfected HEK293T cells (HEK) were used as negative control. MQ indicates negative control of PCR. In the lower panel, semi-quantification of the ratio between correct (WT) and aberrant (MUT) transcripts (expressed as percentage) per condition is presented. [Figure 6] Screening of 2'-O-MOE AONs in HEK293T cells. RT-PCR analysis of HEK293T cells co-transfected with mutant midigene (MUT) and several AONs (A#) or sense oligos (SON) with 2'-MOE chemical modification and PS backbone. Wild-type midigene (WT) was used as positive control, whereas non-transfected cells (HEK) were used as negative control. MQ lane corresponds to negative control of PCR. The lower panel represents semi-quantification of bands expressed as percentage of correct (WT) and aberrant (MUT) transcripts. AON concentration: 1 μM. [Figure 7] AON efficacy analysis in patient-derived fibroblasts. Analysis of the efficacy of several AONs (A#) chemically modified with either 2'-OMe or 2'-MOE with a PS backbone in a patient-derived fibroblast cell line (HOM) homozygously carrying the c.768G>T variant. Fibroblasts from healthy individuals were used as control (CON). As aberrant transcripts are subject to nonsense-mediated decay (NMD), both CON and HOM cells were cultured in the absence (-) or presence (+) of cycloheximide (CHX). All conditions involving AON delivery are performed in the presence of CHX. MQ indicates the negative control for PCR. ACTB was used as loading control. Semi-quantitative analysis of the ratio of correct (WT) and aberrant (MUT) transcripts expressed as a percentage per condition. AON concentration: 1 μM.

[0117] [Array description] [Table 1-1] [Table 1-2] [Table 1-3] EXAMPLES

[0118] The recurrent mutation in ABCA4 underlying Stargardt disease (STGD1) is c.768G>T. This mutation changes the last nucleotide of exon 6 but does not alter the amino acid sequence, i.e., the valine residue encoded by the corresponding codon remains valine (p.Val256Val). Instead, the strength of the splice donor site of exon 6 is reduced by the presence of T instead of G (Figure 1A, Figure 4). As a consequence, an alternative splice site 35 nt downstream in intron 6 is used by the splicing machinery, resulting in a 35-nt expansion of exon 6, as demonstrated in patient-derived fibroblasts (Figure 1B). This abnormal transcript is most likely degraded in cells, since it was not detected in the absence of cycloheximide (an inhibitor of nonsense-mediated decay). Alternatively, if the mutant RNA is translated, the 35-nt expansion causes a frameshift predicted to result in premature termination of protein synthesis.

[0119] We designed AONs that would block the alternative splice donor site in intron 6, thereby evaluating whether AON administration could restore the splice defect associated with the c.768G>T mutation in ABCA4 by targeting control of the splicing machinery back to the original splice donor site.

[0120] Example 1 Experimental details are first described in the Materials and Methods section, while the results are described and explained further below in the Results section.

[0121] Materials and Methods AON Design and Inspection A smaller 257 bp region of interest was selected to design the AONs (SEQ ID NO: 5). Oligonucleotides of approximately 20 nucleotides in length were then designed. All oligonucleotides were subjected to in silico RNA structure prediction. Eight AONs that showed differences in predicted structure and / or had the best accessibility were designed and ordered in different chemistries (see Table 2 and Figure 2).

[0122] [Table 2]

[0123] AON Inspection Skin biopsies from STGD1 patients carrying the c.768G>T in homozygous form were obtained and fibroblast cell lines were generated. The fibroblast cell lines were then transfected with eight different AONs (listed in Table 2) in the presence of cycloheximide. The transfected cells were then subjected to RT-PCR analysis.

[0124] RT-PCR analysis Total RNA was isolated by using the NucleoSpin RNA Clean-up kit (catalog no. 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the iScript cDNA synthesis kit (catalog no. 1708891; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Finally, the efficacy of the AON was evaluated by performing nested PCR using the following ABCA4 primers: PCR1 (exon4_Fw: 5'-CACCCGGAGAGAATTGCAG-3' (SEQ ID NO: 38) and exon8_Rv: 5'-CCTGCATACTCGGCCGATG-3' (SEQ ID NO: 39)); PCR2 or nested PCR (exon5_Fw: 5'-GGAATACGAATAAGGGATATCTTG-3' (SEQ ID NO: 40) and exon7_Rv: 5'-CTTGAATTCTTGGTGACATATCAG-3' (SEQ ID NO: 41)).

[0125] result AON8 was able to block the alternative splice site in intron 6 and control the splice site back to the original splice donor site, which is weakened by the c.768G>T mutation but can still be used (Figures 3A and 3B), thereby restoring ABCA4 pre-mRNA splicing.

[0126] Example 2 Next, we report an expansion of Example 1 using many more AONs (different in terms of sequence and chemistry) and with multiple cell lines (HEK293T cells and patient-derived fibroblasts).

[0127] Materials and Methods AON design To design the AONs, a small ABCA4 region of 235 bp was selected; ABCA4 c.669-c.768+135. A total of 26 AONs (each 21 nucleotides long) were then designed, designated AON10-AON35. AON10-AON34 were designed with 2'-O-methyl (2'-OMe) chemical modification, and AON35 was designed with 2'O-methoxyethyl (2'-O-MOE) chemistry. After the first results, five more AONs with 2'-O-MOE chemistry were ordered, designated AON36-AON40, which correspond to the sequences of AON14, 15, 16, 19, and 23, respectively. For each chemistry, a sense oligonucleotide was ordered with sequences complementary to AON22 and 35, respectively. All AONs (and SONs) are listed in Table 3. All of the listed AONs have a phosphorothioate backbone.

[0128] [Table 3-1] [Table 3-2]

[0129] AON walk in HEK293T cells HEK293T cell transfection All experiments were performed in duplicate. HEK293T cells were seeded in 6-well plates and transfected with 6 μg of a midigene containing wild-type ABCA4 exon 6 and intron 6 (BA4_WT) and the same midigene but containing the c.768G>T mutation (BA4_MUT). After overnight incubation, HEK293T cells were trypsinized and plated in 12-well plates. Once the cells had become attached, they were transfected with AON or SON at a concentration of 1 μM using FuGENE (catalog no. E2311; Promega) and incubated for 48 h. Transfected cells were analyzed at the RNA level by RT-PCR.

[0130] RT-PCR analysis Total RNA was isolated by using the Nucleospin RNA Cleanup Kit (Cat. No. 740955250; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the SuperScript VILO cDNA Synthesis Kit (Cat. No. 11755050; Invitrogen) according to the manufacturer's instructions, then diluted in H2O to a working concentration of 20 ng / μl. AON efficacy was evaluated by performing reverse transcription PCR using the following BA4 midigene specific primers: ABCA4 ex6_Fw: 5'-CTTCAGCCAGAGACGCGGGGC-3' (SEQ ID NO: 81) and pCI-Neo-Rho ex5_Rev: 5'-AGGTGTAGGGGATGGGAGAC-3' (SEQ ID NO: 82). RT-PCR using primers targeting RHO exon 5 was used as a control to evaluate midigene transfection efficiency (pCI-Neo-Rho ex5_Fw: 5'-ATCTGCTGCGGCAAGAAC-3' (SEQ ID NO: 83) and pCI-Neo-Rho ex5_Rev: 5'-AGGTGTAGGGGATGGGAGAC-3' (SEQ ID NO: 84)). RT-PCR analysis was performed with 10 μM of each primer, 2 μM dNTPs, 2.5 mM MgCl2, 1 U Taq polymerase (Roche, Basel, Switzerland) and 40 ng of cDNA in a total reaction of 25 μl using the following PCR conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 2 min, with a final extension at 72°C for 3 min. PCR products were separated on 2.5% agarose gels and selected bands were confirmed by Sanger sequencing. Non-specific bands observed were not considered in the analysis. Semi-quantitative analysis of heteroduplex, mutant and wild-type bands was performed using Fiji software. Heteroduplex bands were equally distributed between correct and aberrant transcripts for graphical representation.

[0131] Comparison of AON efficacy in fibroblasts Fibroblast transfection Skin biopsies from a healthy individual and an STGD1 patient homozygous for ABCA4 c.768G>T were obtained to generate two fibroblast cell lines. Fibroblasts were plated in 6-well plates and transfected with AONs at a concentration of 1 μM using Hugein (catalog no. E2311; Promega) in the presence of cycloheximide. Transfected cells were analyzed at the RNA level by RT-PCR.

[0132] RT-PCR analysis Total RNA was isolated by using the Nucleospin RNA Cleanup Kit (catalog no. 740955250; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the Superscript VILO cDNA Synthesis Kit (catalog no. 11755050; Invitrogen) according to the manufacturer's instructions and then diluted in H2O to a working concentration of 20 ng / μl. Efficacy of the AONs was evaluated by performing RT-PCR in fibroblast control cell lines using the following ABCA4 primers: Exon5_Fw: 5'GGAATACGAATAAGGGATATCTTG-3' (SEQ ID NO: 85) and Exon7_Rev: 5'-CTTGAATTCTTGGTGACATATCAG-3' (SEQ ID NO: 86), and nested PCR in fibroblasts harboring ABCA4 c.768G>T using the following ABCA4 primers: PCR1 Exon4_Fw: 5'-CACCCGGAGAGAATTGCAG-3' (SEQ ID NO: 87) and Exon8_Rev: 5'-CCTGCATACTCGGCCGATG-3' (SEQ ID NO: 88) and PCR2 Exon5_Fw: 5'GGAATACGAATAAGGGATATCTTG-3' (SEQ ID NO: 89) and Exon7_Rev: 5'-CTTGAATTCTTGGTGACATATCAG-3' (SEQ ID NO: 90). RT-PCR in fibroblast control cell lines was performed with 10 μM exon 5_Fw and exon 7_Rw primers, 2 μM dNTPs, 2.5 mM MgCl2, 1 U Taq polymerase (Roche, Basel, Switzerland) and 80 ng of cDNA in a total reaction of 25 μl using the following PCR conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 2 min, with a final extension at 72°C for 3 min.RT-PCR in fibroblasts carrying the ABCA4 c.768G>T mutation was performed with 10 μM exon4_Fw and exon8_Rw primers, 2 μM dNTPs, 2.5 mM MgCl2, 1 U Taq polymerase (Roche, Basel, Switzerland) and 80 ng cDNA in a total reaction of 25 μl using the following PCR conditions: 94° C. for 3 min, followed by 40 cycles of 94° C. for 30 s, 58° C. for 30 s and 72° C. for 2 min, with a final extension of 3 min at 72° C. Nested PCR was performed as described above and 0.5 μl of PCR1 product was used as PCR template for nested PCR using the following conditions: 94° C. for 3 min, followed by 35 cycles of 94° C. for 30 s, 58° C. for 30 s and 72° C. for 2 min, with a final extension of 3 min at 72° C. All PCR products were resolved on 2.5% agarose gels and selected bands were confirmed by Sanger sequencing. Semi-quantitative analysis of mutant and wild type bands was performed using Fiji software. Actin (ACTB) was amplified as a loading control. Primers: P49750_Fw_ACTB Exon 3: 5'-ACTGGGACGACATGGAGAAG-3' (SEQ ID NO: 91) Primer: P49751_Rev_ACTB Exon 4: 5'-TCTCAGCTGTGGTGGTGAAG-3' (SEQ ID NO: 92).

[0133] Results and Discussion AON rescue in HEK293T cells To identify the most potent AONs able to rescue the splice defect associated with the c.768G>T mutation (35-nt expansion), an "oligo-walk" was performed, whereby AON sequences targeting the region around the alternative splice site (at position c.768+35) differed from one another by only one or two nucleotides (Figure 4). A midigene carrying a genomic fragment of ABCA4 with either a G (WT) or a T (MUT) at position c.768 was transfected into HEK293T cells and then cotransfected without or with the different AONs. In the first series, the majority of the AONs contained the previously used chemistry with phosphorothioate backbone and 2'-O-methyl modification (2'-OMe), only AON35 differed, with 2'-O-methoxyethyl (2'-O-MOE) instead of 2'-OMe and with the same sequence as AON22. As can be observed from FIG. 5, all AONs were able to some extent to control the splicing defects observed when transfecting the MUT midigene. AONs 14, 15, 16, 19, 22 and 23 appeared to be the most potent with 2'-OMe chemistry. Interestingly, the recently "published" AON 35 with 2'-O-MOE chemistry was the most able to control splicing towards the normal ABCA4 transcript, as confirmed by quantification and Sanger sequencing. All experiments were performed in duplicate. The results of AON administration to HEK293T cells carrying the WT midigene did not show any splicing defects (data not shown).

[0134] Considering the promising results with 2'-O-MOE chemistry, the applicants decided to order AONs with the sequences of AON14, 15, 16, 19 and 23 (AON35 already corresponded to AON22), also with 2'-O-MOE chemistry. These AONs were made AON36-40 and their potential efficacy was evaluated in the same manner in HEK293T cells. As can be observed in Figure 6, the previously effective AON35, but also AON36, 38 and 40, were highly capable of correcting the aberrant splicing process. AON39 was somewhat less effective, while AON37 barely showed rescue. AON rescue in fibroblasts

[0135] Next, for the six most effective AONs per chemistry in HEK293T cells, we aimed to evaluate their splice control ability in patient-derived fibroblasts, which carry the c.768G>T mutation in their genomic DNA, thus allowing the evaluation of the associated splice defect due to endogenous ABCA4 expression. Fibroblasts from STGD1 individuals carrying the c.768G>T mutation in the homozygous state were transfected with the most potent AONs with either 2'-OMe or 2'-O-MOE chemistry. As shown in Figure 7, some degree of splicing correction was measured, but with less efficacy compared to the results obtained in HEK293T cells. Of all AONs, AONs 35 and 40, both with 2'-O-MOE chemistry, appeared to have the strongest effect. The reason for the lower efficacy in fibroblasts compared to HEK293T cells is most likely explained by the fact that in HEK293T cells only a small portion of the ABCA4 transcript is expressed, whereas in fibroblasts the entire mRNA is present. Thus, folding of the pre-mRNA molecule may be completely different, affecting AON accessibility. In addition, the stability as well as transfection efficacy of ABCA4 pre-mRNA may vary from cell to cell.

[0136] conclusion Expanding the number of AONs that could potentially control aberrant ABCA4 splicing due to the c.768G>T mutation provided a number of interesting insights. First, using the ABCA4 midigene system in combination with the "oligo-walk", we found that nearly all AONs were able to control splicing to some degree, but significant differences were observed between AONs. AONs 14, 15, 16, 19, 22 and 23 all had 2'-OMe chemistry and were the most effective. Interestingly, AON 35, which had 2'-O-MOE chemistry, outperformed all 2'-OMe chemistries. Similar observations were made with a number of additional AONs (AON 36-AON 40) that all had 2'-O-MOE chemistry, but again with differing efficacy between AON sequences. In fibroblasts, AONs were less effective compared to HEK293T cells, suggesting either less efficient uptake of AONs by these cells and / or less accessibility of the target transcript to the AONs, e.g., due to some folding of the ABCA4 pre-mRNA in the endogenous environment that is not an issue in the HEK293T midigene system.

[0137] In summary, the applicants identified the most potent AON sequences for targeting the splice defect caused by c.768G>T, and the applicants showed that AONs with 2'-O-MOE chemistry appear to be more effective compared to AONs with 2'-OMe chemistry.

[0138] References Albert S, Garanto A, Sangermano R, Khan M, Bax NM, Hoyng CB, Zernant J, Lee W, Allikmets R, Collin RWJ, Cremers FPM (2018). Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease. Am J Hum Genet, 102: 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, BP & De Baere, EBCA. noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutated. 36, 39-42 (2015), doi:10.1002 / humu.22716. 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. Braun, T. A., Mullins, R. F., Wagner, A. H., Andorf, J. L., Johnston, R. M., Bakall, B. B., Deluca, A. P., Fishman, G. A., Lam, B. L., Weleber, R. G., Cideciyan, A. V., Jacobson, S. G., Sheffield, V. C., Tucker, B. A. & Stone, E. M. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367. Collin RWJ, den Hollander AI, van der Velde-Visser S, Bennicelli J, Bennett J, Cremers FPM (2012). Antisense oligonucleotide (AON)-based therapy for Leber Congenital Amaurosis caused by a frequent mutation in CEP290. Mol Ther Nucl Acids, e14. Cremers, FPM, van de Pol, DJ, van Driel, M., den Hollander, AI, van Haren, FJ, Knoers, NV, Tijmes, N., Bergen, AA, Rohrschneider, K., Blankenagel, A., Pinckers, AJ, Deutman, AF & Hoyng, CB autosomal recessive and recessive pigment dystrophy. caused by splice site mutations in the Stargardt's disease gene ABCR. Hum. Mole. Gen. 7, 355-362 (1998). Fujinami, K., Zernant, J., Chana, RK, Wright, GA, Tsunoda, K., Ozawa, Y., Tsubota, K., Webster, AR, Moore, AT, Allikmets, R. & Michaelides, M. ABCA4 gene screening by next-generation sequencing in a British cohort. Invest. Ophthalmol. Screw. 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. Garanto A & Collin RWJ (2018). Design and in vitro use of antisense oligonucleotides to correct pre-mRNA splicing defects in inherited retinal dystrophies. Methods Mol Biol, 1715: 61-78. Garanto, A., et al., In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet, 2016. 25(12): p. 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. The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am. J. Hum. Genet. 64, 1024-1035 (1999). Maugeri, A., Klevering, B. J., Rohrschneider, K., Blankenagel, A., Brunner, H. G., Deutman, A. F., Hoyng, C. B. & Cremers, F. P. M. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am. J. Hum. Genet. 67, 960-966 (2000), doi:10.1086 / 303079. Martinez-Mir, A., Paloma, E., Allikmets, R., Ayuso, C., del Rio, T., Dean, M., Vilageliu, L., Gonzalez-Duarte, R. & Balcells, S. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR. Nat. Genet. 18, 11-12 (1998), doi:10.1038 / ng0198-11. Rivera, A., White, K., Stohr, H., Steiner, K., Hemmrich, N., Grimm, T., Jurklies, B., Lorenz, B., Scholl, H. P., Apfelstedt-Sylla, E. & Weber, B. H. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am. J. Hum. Genet. 67, 800-813 (2000), doi:10.1086 / 303090. Sangermano R, Bax NM, Bauwens M, van den Born LI, de Baere E, Garanto A, Collin RWJ, Goercharn-Ramlal AS, den Engelsman-van Dijk AH, Rohrschneide K, Hoyng CB, Cremers FPM, Albert S (2016). Photoreceptor progenitor mRNA analysis reveals exon skipping resulting from the ABCA4 c.5461-10T>C mutation in Stargardt disease. Ophthalmology, 123: 1375-1385. Sangermano R, Khan M, Cornelis SS, Richelle V, Albert S, Garanto A, Elmelik D, Qamar R, Lugtenberg D, van den Born LI, Collin RWJ, Cremers FPM (2018). ABCA4 midigenes reveal the full splice spectrum of all reported noncanonical splice site variants in Stargardt disease. Genome Res, 28: 100-110. Schulz, H. L., Grassmann, F., Kellner, U., Spital, G., Ruther, K., Jagle, H., Hufendiek, K., Rating, P., Huchzermeyer, C., Baier, M. J., Weber, B. H. & Stohr, H. Mutation spectrum of the ABCA4 gene in 335 Stargardt disease patients from a multicenter German cohort-impact of selected deep intronic variants and common SNPs. Invest. Ophthalmol. Vis. Sci. 58, 394-403 (2017), doi:10.1167 / iovs.16-19936. van Driel, M. A., Maugeri, A., Klevering, B. J., Hoyng, C. B. & Cremers, F. P. M. ABCR unites what ophthalmologists divide(s). Ophthalmic Genet. 19, 117-122 (1998). Webster, A. R., Heon, E., Lotery, A. J., Vandenburgh, K., Casavant, T. L., Oh, K. T., Beck, G., Fishman, G. A., Lam, B. L., Levin, A., Heckenlively, J. R., Jacobson, S. G., Weleber, R. G., Sheffield, V. C. & Stone, E. M. An analysis of allelic variation in the ABCA4 gene. Invest. Ophthal. Vis. Sci. 42, 1179-1189 (2001). Zernant, J., Lee, W., Collison, F. T., Fishman, G. A., Sergeev, Y. V., Schuerch, K., Sparrow, J. R., Tsang, S. H. & Allikmets, R. Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration. J. Med. Genet. 54, 404-412 (2017), doi:10.1136 / jmedgenet-2017-104540. Zernant, J., Schubert, C., Im, K. M., Burke, T., Brown, C. M., Fishman, G. A., Tsang, S. H., Gouras, P., Dean, M. & Allikmets, R. Analysis of the ABCA4 gene by next-generation sequencing. Invest. Ophthalmol. Vis. Sci. 52, 8479-8487 (2011), doi:10.1167 / iovs.11-8182. Zhou, S., et al., Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling. Development, 2015. 142(19): p. 3294-306.

Claims

1. An antisense oligonucleotide for controlling splicing which 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 which binds to a polynucleotide having SEQ ID NO:5 or is complementary to a polynucleotide having SEQ ID NO:5, more preferably an antisense oligonucleotide which binds to a polynucleotide having SEQ ID NO:80 or is complementary to a polynucleotide having SEQ ID NO:80, even more preferably an antisense oligonucleotide which binds to a polynucleotide selected from the group consisting of SEQ ID NOs:7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21 or is complementary to a polynucleotide selected from the group consisting of SEQ ID NOs:7, 8, 9; 11, 12, 13; 15, 16, 17; 19, 20 and 21.

2. 2. The antisense oligonucleotide for controlling splicing according to claim 1, wherein the nucleotide in the antisense oligonucleotide may 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 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

4. An antisense oligonucleotide for controlling splicing according to any one of claims 1 to 3, comprising SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77, or consisting of SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77.

5. 4. An antisense oligonucleotide for controlling splicing according to any one of claims 1 to 3, comprising a 2'-O alkyl phosphorothioate antisense oligonucleotide, such as a 2'-O-methyl modified ribose (RNA), a 2'-O-ethyl modified ribose, a 2'-O-methoxyethyl modified ribose, a 2'-O-propyl modified ribose and / or substituted derivatives, such as halogenated derivatives, having these modifications.

6. The antisense oligonucleotide for controlling splicing according to any one of claims 1 to 5, comprising or consisting of SEQ ID NO: 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 73, 75 or 77, and comprising a 2'-O-methyl modified ribose (RNA) or a 2'-O-methoxyethyl modified ribose and a phosphorothioate backbone.

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

8. A pharmaceutical composition comprising an antisense oligonucleotide for controlling splicing according to any one of claims 1 to 4 or a viral vector according to claim 7, and a pharma- ceutically 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 ranging from 0.1 to 1 mg of total antisense oligonucleotide for controlling splicing per eye, 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 of total antisense oligonucleotide for controlling splicing per eye.

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

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

14. 14. An 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.

Images