WO2022090256A1

WO2022090256A1 - Antisense oligonucleotides for the treatment of stargardt disease

Info

Publication number: WO2022090256A1
Application number: PCT/EP2021/079723
Authority: WO
Inventors: Jim SWILDENS; Melita KALTAK
Original assignee: Proqr Therapeutics Ii B.V.
Priority date: 2020-10-26
Filing date: 2021-10-26
Publication date: 2022-05-05

Abstract

The invention relates to the field of medicine and more in particular to single-stranded antisense oligonucleotides (AONs) that can induce the skip of exon 17 from human ABCA4 pre-mRNA. The AONs of the present invention target sequences within exon 17 and/or surrounding sequences in intron 16 and intron 17. The AONs of the present invention are applicable in treating a human subject suffering from a disorder caused by a mutation in the ABCA4 gene, preferably mutations affecting exon 17, more preferably the c.2588G>C mutation at the 5' terminus of exon 17.

Description

Antisense oligonucleotides for the treatment of Stargardt disease

Field of the invention

The invention relates to the field of medicine and biotechnology. It relates to antisense oligonucleotides that can modulate splicing of human ABCA4 pre-mRNA into mature mRNA in patients suffering from an ABCA4-related disorder or condition, such as Stargardt disease.

Background of the invention

Stargardt disease (STGD or STGD1 ) is the most common inherited macular dystrophy causing progressive impairment of central vision, with onset typically in childhood or young adulthood, and least frequently in later adulthood, with a better prognosis generally associated with a later onset. The disease has a prevalence of 1 in 8,000-10,000 and has an autosomal recessive mode of inheritance associated with disease-causing mutations in the gene coding for the photoreceptor cell-specific ATP- binding cassette, sub-family A, member 4 protein (ABCA4, sometimes referred to as ABCR). The protein contains 2273 amino acids, is predominantly expressed in the retina (photoreceptor cells and Retinal Pigment Epithelium (RPE)) and localizes to the rims and cone outer segments disks. Its mode of action is contemplated to relate to flipping N-retinyl-idene-phosphatidylethanolamine from the luminal to the cytosolic face of the photoreceptor disks. Stargardt disease links tightly with a massive deposition of lipofuscin content in the RPE, failure in toxic substance removal and significant loss in photoreceptor cells. A major component of lipofuscin, di-retinoid-pyridinium- ethanolamine is formed when the ABCA4 protein is missing or dysfunctional. Indeed, multiple reports have been published that confirmed that ABCA4 is the gene underlying Stargardt disease, showing a large number (-1000) of disease-causing variants, of which more than half have been described only once. ABCA4 variants have been observed in the vast majority (95%) of patients with autosomal recessive Stargardt disease, -30% of patients with autosomal recessive cone-rod dystrophy (CRD) and -5% of patients with autosomal recessive panretinal dystrophy (Cremers et al. 1998. Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt’s disease gene ABCR. Hum Mol Genet 7:291-297; Maugeri et al. 1999. 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; Maugeri et al. 2000. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am J Hum Genet 67:9060-966). Most mutations are missense, followed by nonsense mutations, small insertions/deletions, and mutations affecting RNA splicing. An unusually high proportion of Stargardt disease cases from northern Europe and the United States (-30%) is the result of a single ABCA4 variant. It is known that the third most frequent ABCA4 variant referred to as c.5461-10T>C present in intron 38, causes a severe form of Stargardt disease due to skipping of exon 39, or skipping of exon 39 + exon 40 in the mRNA of ABCA4. The skipping of exon 39 results in a frameshift deletion of 124 nucleotides, whereas the double skip of exon 39 and 40 results in a frameshift deletion of 254 nucleotides. It is estimated that approximately 7000 Stargardt disease patients in the Western world suffer from this mutation.

The three main routes of intervention to treat Stargardt disease are currently stem cell therapy, gene replacement therapy and different pharmaceutical approaches. A relatively new therapeutic development for treating inherited eye diseases is the use of antisense oligonucleotides (AONs), which target the pre-mRNA transcribed from the mutant gene. AONs are generally small polynucleotide molecules (16- to 25-mers) that can interfere with splicing as their sequence is complementary to that of the target pre- mRNA molecule. The envisioned mechanism is such that upon binding of the AON to a target sequence, with which it is complementary, the targeted region within the pre- mRNA interferes with splicing factors which in turn results in altered splicing. Therapeutically, this methodology can be used in two ways: a) to redirect normal splicing of genes in which mutations activate cryptic splice sites and b) to skip exons that carry (protein-truncating) mutations in such a way, that the reading frame of the mRNA remains intact, and a (partially or fully) functional protein is made. Both methods have already been successfully applied in human patients. With respect to eye diseases, AONs have been shown to be promising for the treatment of Leber’s Congenital Amaurosis, or LCA (WO 2012/168435; WO 2013/036105; WO 2016/034680; WO 2016/135334). Further, WO 2016/005514 discloses exon skipping AONs for targeting the USH2A pre-mRNA, directed at skipping of exon 13, exon 50 and PE40, and/or retaining exon 12, for the treatment, prevention, or delay of Usher Syndrome Type II.

In relation to Stargardt disease, splice modulation by using AONs has also been applied. In respect of the methodology under b) as indicated above, WO 2015/004133 discloses the intravitreal administration of a 28-mer AON in mouse eyes trying to skip exon 10 from the ABCA4 pre-mRNA. In respect of the methodology under a) as indicated above, WO 2018/109011 discloses a variety of AONs for preventing the inclusion of so-called ‘pseudo exons’ (PE) that are erroneously introduced into the mRNA due to several intronic mutations (c.4539-1100A>G causing the appearance of PE30-31 (68); c.4539-1106C>T causing the appearance of PE30-31 (345); c.769- 784C>T causing the appearance of PE6-7 (162); c.859-540C>G causing the appearance of PE7-8 (141 ); c.859-506G>C causing the appearance of PE7-8 (56); c.1937-435C>G causing the appearance of PE13-14 (134); and c.5197-557G>T causing the appearance of PE36-37 (188)). The methodology described in WO 2018/189376 is yet another way of manipulating splicing, in the sense that it discloses antisense oligonucleotides that inhibit skip of exon 39 and exon 40. It was found that in Stargardt patients carrying this mutation, exon 39 and sometimes the co-skip of exon 39 + exon 40 from the human ABCA4 pre-mRNA was caused by a mutation referred to as c.5461-10T>C. The AONs disclosed in WO 2018/189376 can prevent such aberrant exon skipping and thereby prevent the translation product to go out of frame. A similar approach was used in WO 2020/015959 that describes the use of AONs to block the skip of exon 28 (or co-skip of exon 28 + exon 29, or the co-skip of exon 27 + exon 28) from the human ABCA4 pre-mRNA caused by the c.4253+43G>A mutation. Like the approach disclosed in WO 2018/109011 (see above), WO 2020/1151106 discloses the use of AONs to prevent the skip of PE6-7 (35) caused by the c.768G>T mutation.

As indicated, when treatment is considered through splice modulation, the prevention of exon skipping, or the prevention of inclusion of a pseudo exon, the resulting mRNA should be in-frame such that the translated protein is functional, or at least partly functional, and not prematurely terminated. However, many potentially Stargardt disease-causing mutations exist in the human ABCA4 gene that cannot be targeted by splice modulation. For instance, when the resulting transcript will go out of frame, or the resulting protein will lack an essential part required for executing its function. Hence, although many therapeutic endeavors relate to treating Stargardt disease by introducing AONs that modulate splicing (see the examples above), many Stargardt patients will not benefit because of it, because they carry a different kind of mutation in their ABCA4 gene. The c.5882G>A mutation in exon 42 is one that comes to mind, and which is one of the more common mutations in ABCA4 in the western world, with an estimated prevalence of 10.000-15.000 patients (Lewis et al. 1999. Am J Hum Genet 64:422-434). This mutation leads to a substitution of the amino acid glycine (G; codon: GGA) at position 1961 of the ABCA4 protein to a glutamic acid (E, codon: GAA). This amino acid is localized in the Nucleotide Binding Domain 2 (NBD2), which is essential for providing the energy for binding of substrate for transport. Exon 42, even though it is in-frame when it would be skipped, encodes for a part of the NBD2 that cannot be disrupted for a properly functioning ABCA4 protein. Biochemical assessment showed that the ABCA4 protein carrying this substitution has reduced ATPase activity (Sun et al. 2000. Nature Genetics 26:242-246), which is also an indication that even the smallest alteration in the NBD2 may cause the protein to become dysfunctional. WO 2021/130313 describes the use of RNA editing oligonucleotides that use the cellular endogenous ADAR enzyme to site-specifically deaminate the target mutation (adenosine) into an inosine that is read by the translational machinery of the cell as a guanine, which then makes that the mutation is reversed to wild type. This RNA editing approach may also be used for mutations elsewhere in the ABCA4 pre-mRNA, especially where exon skipping (or the prevention thereof) would not provide a solution.

Despite all these efforts, Stargardt disease patients that carry a mutation in another exon (or intron) not targeted by any of the described AONs will not benefit. The c.2588G>C mutation is in the first nucleotide position of exon 17 in the human ABCA4 gene and therefore is part of its splice acceptor site. The nucleotide change results in a missense mutation, causing an exchange of an alanine for glycine at amino acid residue 863, but also affects the splicing at the 3’ splice site of exon 17. Maugeri et al. (1999) identified that a cryptic 3’ splice site, 3 bp downstream, was used causing a deletion of Gly863. Hence, the effect of the c.2588G>C mutation is two-fold: when splicing is normal it causes a mutation from a glycine (wt) to an alanine (mutant), and when splicing is affected, the first three nucleotides (CAG in the case of the mutation (underlined C)) of exon 17 are skipped. The mutation itself is relatively mild and present in 1 of every 35 western Europeans. When it is present in the absence of another ABCA4 mutation it does not lead to Stargardt disease. However, due to its abundance, it is present in the genome of many patients suffering from Stargardt disease and contributes significantly to the occurrence of the disease. It is envisioned that patients that carry two or more mutations in the ABCA4 gene, including this mild frequent founder mutation c.2588G>C will benefit from treatments using AONs that influence the splicing of exon 17 in the ABCA4 pre-mRNA.

Summary of the invention

The invention relates to an antisense oligonucleotide (AON) that can induce the skip of exon 17 from human ABCA4 pre-mRNA, wherein the AON comprises a sequence that is 90 to 100%, preferably 100% complementary to a consecutive stretch of nucleotides within SEQ ID NO: 41. Preferably, the AON comprises a sequence that is 90 to 100%, preferably 100% complementary to a consecutive stretch of nucleotides within the sequence of exon 17 (SEQ ID NO: 42) or the 5’ part of intron 17 (SEQ ID NO: 49). In yet another preferred embodiment, the AON comprises a sequence that is 90 to 100%, preferably 100% complementary to a consecutive stretch of nucleotides that comprises that exon17/intron17 boundary. Preferably, the AON consists of 14 to 25 nucleotides, more preferably 16, 17, 18, 19, 20, 21 or 22 nucleotides. Preferably, the AON comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 10, 11 , 12, 13, 14, 19, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, 40, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, and 109. In one aspect, the AON comprises at least one 2’-O- methoxyethyl (2’-MOE) modification, and preferably, all nucleotides of the AON are 2’- MOE modified. In another aspect, the AON comprises at least one non-naturally occurring internucleoside linkage, such as a phosphorothioate (PS) linkage, and preferably all sequential nucleosides are interconnected by PS linkages.

The invention also relates to a viral vector expressing an AON according to the invention, and to a pharmaceutical composition comprising an AON according to the invention and a pharmaceutically acceptable carrier.

The invention also relates to an AON according to the invention, the viral vector according to the invention, or the pharmaceutical composition according to the invention for use in the treatment, prevention or delay of an ABCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene.

The invention also relates to the use of an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment, prevention or delay of an ABCA4-related disease or a condition requiring modulating splicing of ABCA4 pre- mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene.

In yet another aspect, the invention relates to an in vitro, ex vivo or in vivo method for modulating splicing of ABCA4 pre-mRNA in a cell, comprising the steps of: administering to the cell an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention; allowing the hybridization of the AON to its complementary sequence in ABCA4 target RNA molecule in the cell; and allowing the skip of exon 17 from the target RNA molecule. In yet another aspect, the invention relates to a method for the treatment of a ABCA4-related disease or condition requiring modulating splicing of ABCA4 pre-mRNA of an individual in need thereof, said method comprising contacting a cell of said individual with an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention.

Brief description of the drawings

Figure 1 shows the 5’ to 3’ sequence of exon 17 (bold, upper case) of the human ABCA4 gene, and parts of its surrounding intron sequences (lower case) together with the sequences of 40 initially tested antisense oligonucleotides (AONs 1 to 40, given from 3’ to 5’; SEQ ID NO: 1 to 40) at the location of the target sequences. Shortened derived oligonucleotides of AON 14, 24, 25, 27, 39, and 40 that are at least 16 nucleotides in length are also given (AON 14-A, -B, -C, and -D are SEQ ID NO: 56 to 59, respectively; AON 14-E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -O, -P, -Q, and -R are SEQ ID NO: 92 to 105, respectively; AON 24-A, -B, -C, -D, and -E are SEQ ID NO: 60 to 64, respectively; AON 25-A, -B, -C, -D, -E, -F, -G, -H, and -I are SEQ ID NO: 65 to 73, respectively; AON 27- A, -B, -C, -D, and -E are SEQ ID NO: 74 to 78, respectively; AON 27-F, -G, -H, and -I are SEQ ID NO: 106 to 109, respectively; AON 39-A, -B, -C, -D, -E, -F, -G, -H, and -I are SEQ ID NO: 79 to 87, respectively; AON 40-A, -B, -C, and -D are SEQ ID NO: 88 to 91 , respectively). The c.2588G>C mutation at the first nucleotide of exon 17 is given by an asterisk. The sequence of exon 17 plus the depicted sequences of intron 16 and intron 17 is provided as SEQ ID NO: 41. The sequence of exon 17 only is SEQ ID NO: 42. The sequence of intron 17 that is depicted here is SEQ ID NO: 49. AON 1 to 6 all target a sequence that is completely within intron 16. AON 7 targets a sequence that includes the c.2588G>C mutation. AONs 7 to 39 all target a sequence that is partly or completely within exon 17. AONs 17, 18, 19, 35, 36, 37, 38, and 39 target a sequence that includes the boundary between exon 17 and intron 17. AON 40 targets a sequence that is completely within intron 17. It is to be understood that the genomic version of the intron/exon sequences is provided, but that the target molecule for the AONs is the corresponding pre-mRNA.

Figure 2 shows the average of three separate experiments in a graph depicting the percentage of exon 17 skip occurring in wild-type ABCA4 pre-mRNA in retinoblastoma WERI-Rb1 cells, after gymnotic uptake of AONs 1 to 19, in comparison to a non-treated sample. Figure 3 shows the average of three separate experiments assessing exon 17 skipping in WERI-Rb1 cells (wild-type ABCA4) after gymnotic uptake of AONs 20 to 40, in comparison to AON 14 from the initial screen and a mock transfection.

Figure 4 shows the rate of exon 17 skipping from wild type human ABCA4 pre- mRNA in human retinal organoid tissue after incubation (in the medium) with AON 14, 24, 25, 27, 29, 39, 40, and a scrambled control AON. Each AON was tested separately in six separate organoids. These six independent values are depicted by small circles, and the bar represents the average of these six values.

Detailed description

The invention relates to an antisense oligonucleotide (AON) that can induce the skip of exon 17 from human ABCA4 pre-mRNA, wherein the AON comprises a sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a consecutive stretch of nucleotides within SEO ID NO: 41. In a preferred embodiment, the AON is 100% complementary to a consecutive sequence within SEQ ID NO: 42, which represents exon 17 of the human ABCA4 gene carrying at the first nucleotide the c.2588G>C mutation. This mutation causes either a 3-nucleotide deletion at the start of exon 17, or a mutation from a glycine to an alanine in the ABCA4 protein. In another preferred embodiment, the AON is 90 to 100% complementary to a consecutive sequence within SEQ ID NO: 49, which represents the 5’ part of intron 17 (see Figure 1 ). In yet another preferred embodiment, the AON is 90 to 100% complementary to a consecutive sequence that comprises the boundary between exon 17 and intron 17, such as exemplified by AON 19 and 39 (see accompanying examples). The inventors of the present invention envisioned that removal of exon 17 from the ABCA4 mRNA would be beneficial and that such would be less detrimental than the short deletion and/or the mutation. Hence, the inventors of the present invention pursued the possibility of skipping exon 17 from the human ABCA4 pre-mRNA by using AONs that target specific sequences within exon 17 (SEQ ID NO: 42) and/or its surrounding intronic sequences (represented in full by SEQ ID NO: 41 , and wherein SEQ ID NO: 49 represents the 5’ part of intron 17). Indeed, as shown herein, the inventors were able to identify certain AONs that were able to induce the skip of exon 17 from the pre-mRNA. Preferably, an AON is 100% complementary to its target sequence for optimal interaction. Also preferably, the AON consists of 14 to 25 nucleotides, more preferably 16, 17, 18, 19, 20, 21 or 22 nucleotides. The most optimal length depends on efficiency, cell entry, intracellular trafficking, the capability to interfere with the splicing machinery in the target cell (here a photoreceptor cell), GC content, Tm, and specificity for the target sequence. The skilled person knows that the shorter the AON, the greater the possibility of non-specific interactions elsewhere in the transcribed genome. In general, it is held that 16-mer AONs are still specific enough, but such needs to be assessed on a case-by-case basis. Also, the longer the AON, the more difficult it gets to enter a cell without the help of a vector, another delivery vehicle, or any other means to increase cell entry or endosomal release. Moreover, when AONs get longer, they get more prone to nuclease-dependent breakdown. In a preferred aspect, the invention relates to an AON that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 1 to 40 and 56 to 109, preferably SEQ ID NO: 10, 11 , 12, 13, 14, 19, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, 40, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, or 109. The skilled person understands that the number of deviations in length and additional nucleotides at the 5’ and/or 3’ end of each of these AONs, or wherein one or more nucleotides are removed from the 5’ and/or 3’ end of each of these AONs is enormous, and that removal or addition of a single nucleotide may substantially influence the skipping efficiency of an AON. It is generally envisioned that shortening an AON increases its ability to enter a target cell, whereas in its shorter version it is also less prone to nuclease-dependent degradation. Hence, it is generally preferred to generate an AON that is as short as possible without losing specificity for the targeting sequence, while maintaining exon skipping force. In any case, the inventors have now for the first time identified multiple AONs that can be used to skip exon 17 from human ABCA4 pre- mRNA and have shown that such is applicable for the treatment of Stargardt disease since exon skipping was amongst others, shown in human retinal organoids that represent the closest model to the human eye in vitro, even though the content (or chemical modifications) of such an AON may be further optimized. It is an essential aspect of the invention that the AON is 90 to 100% complementary to a consecutive stretch of nucleotides within SEQ ID NO: 41 (or SEQ ID NO: 42, or SEQ ID NO: 49, or a sequence that includes the boundary between exon 17 and intron 17), and that the AON can induce skipping of exon 17 from human ABCA4 pre-mRNA as shown herein. In another preferred aspect, the AON of the present invention comprises at least one 2’- O-methyl (2’-OMe) or one 2’-O-methoxyethyl (2’-MOE) modification. In a particularly preferred aspect, all nucleotides of the AON of the invention are 2’-MOE modified. In another aspect, the AON comprises at least one non-naturally occurring internucleoside linkage, preferably a phosphorothioate (PS) linkage. In a further preferred aspect, all sequential nucleosides are interconnected by PS linkages. The invention also relates to a viral vector expressing an AON according to the invention. Preferred viral vectors that are used to deliver an AON of the present invention are Adenovirus-Associated Viruses (AAV) as further outlined below.

The invention also relates to a pharmaceutical composition comprising an AON according to the invention or a viral vector according to the invention, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well- known in the art.

In yet another aspect the invention relates to an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention for use in the treatment, prevention or delay of an AfiCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene. In a preferred aspect, the AON of the present invention is for intravitreal administration and is dosed in an amount ranging from 5 pg to 500 pg of total AON per eye, preferably from 10 pg to 100 pg, more preferably from 25 pg to 100 pg. In a further preferred aspect, the AON is dosed in an amount ranging from 25 pg to 100 pg of total AON per eye, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 pg total AON per eye.

In yet another aspect, the invention relates to a use of an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment, prevention or delay of an AfiCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene.

In yet another aspect, the invention relates to an in vitro, ex vivo or in vivo method for modulating splicing of ABCA4 pre-mRNA in a cell, comprising the steps of: administering to the cell an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention; allowing the hybridization of the AON to its complementary sequence in ABCA4 target RNA molecule in the cell; and allowing the skip of exon 17 from the target RNA molecule.

In yet another aspect, the invention relates to a method for the treatment of a AfiCA4-related disease or condition requiring modulating splicing of ABCA4 pre-mRNA of an individual in need thereof, said method comprising contacting a cell of said individual with an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention. In a preferred aspect the AON of the present invention is an oligoribonucleotide. In a further preferred aspect, the AON comprises a 2'-0 alkyl modification, such as a 2'- O-methyl (2’-OMe) or a 2’-methoxyethoxy (2’-O-methoxyethyl, or 2’-MOE) modification of the sugar moiety. In a more preferred embodiment, all nucleotides in the AON are 2’- OMe modified. In another preferred aspect, the invention relates to an AON comprising a 2’-MOE modification. In a more preferred aspect, all nucleotides of said AON carry a 2’-MOE modification. In yet another aspect the invention relates to an AON comprising at least one 2’-OMe and at least one 2’-MOE modification. In another preferred embodiment, the AON according to the present invention comprises at least one phosphorothioate (PS) modified linkage. In another preferred aspect, all sequential nucleotides are interconnected by PS linkages.

In yet another aspect, the invention relates to a viral vector expressing an AON according to the invention. The invention also relates to a pharmaceutical composition comprising an AON according to the invention or a viral vector according to the invention, and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention for use in the treatment, prevention, or delay of Stargardt disease or a condition requiring modulating splicing of a mutated ABCA4 pre-mRNA, such as Stargardt disease. A preferred ABCA4-related disease or condition that is being treated is one that is caused by a mutation in exon 17 of the human ABCA4 gene, more preferably the c.2588G>C mutation (see Figure 1 ) that causes a 3-nucleotide deletion affecting correct splicing of the ABCA4 pre-mRNA. In one aspect, the invention relates to an AON for use according to the invention, wherein the AON is for intravitreal administration and is dosed in an amount ranging from 5 pg to 500 pg of total AON per eye, preferably from 10 pg to 100 pg, more preferably from 25 pg to 100 pg. Preferably, the AON is administered in a naked form (as is, without being carried by a particle such as a nanoparticle or liposome), and preferably the administration to the vitreous is by direct injection of the naked oligonucleotide (generally held in a pharmaceutically acceptable composition). Preferably, the AON for use according to the invention is administered to the eye, wherein the AON is dosed in an amount ranging from 5 pg to 500 pg of total AON per eye, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, or 320 pg total AON per eye. In another embodiment the invention relates to a use of an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention for the preparation of a medicament for the treatment, prevention or delay of an ABCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, caused by the c.2588G>C mutation.

In another embodiment, the invention relates to an in vitro, ex vivo or in vivo method for modulating splicing of ABCA4 pre-mRNA in a cell, comprising the steps of: administering to the cell an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention; allowing the hybridization of the AON to its complementary sequence in ABCA4 target RNA molecule in the cell; and inducing the skip of exon 17 from the target RNA molecule to yield an inframe transcript from the mRNA. Optionally, the method further comprises the step of analyzing whether the skip of exon 17 from the ABCA4 target RNA molecule has been induced in comparison to a situation in which no AON is administered, which can be performed using methods as disclosed herein and/or by other methods generally known to the person skilled in the art. The invention also relates to a method for the treatment of a ABCA4-related disease or condition requiring modulating splicing of ABCA4 pre- mRNA of an individual in need thereof, said method comprising contacting a cell of said individual with an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention. Contacting the cell of the individual may be in vivo, by direct intravitreal administration of the AON to the patient in need thereof, or through ex vivo procedures, wherein treated cells, that have received the AON, viral vector, or pharmaceutical composition, are transplanted back to the patient, thereby to treat the disease.

In all embodiments of the invention, the terms ‘modulating splicing’, ‘inducing the exclusion of an exon’, ‘causing or inducing exon skipping’ are considered synonymous. In respect of ABCA4, ‘splice switching’, ‘modulating splicing’ or ‘inducing exon skipping’ are to be construed as the skip of exon 17 from the ABCA4 pre-mRNA. For the invention, the terms ‘aberrant exon 17’ or ‘aberrant ABCA4 exon 17’ are synonymous and mean the presence of a mutation in exon 17 of the human ABCA4 gene.

Unless otherwise indicated, the following terms have the following meaning. The term ‘pre-mRNA’ refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template of a cell by transcription, such as in the nucleus.

The terms ‘antisense oligonucleotide’, ‘oligonucleotide’, single-stranded antisense oligonucleotide’, the abbreviation ‘AON’, and varieties thereof are understood to refer to a molecule with a nucleotide sequence that is substantially (= sufficiently) complementary to a target nucleotide sequence in a pre-mRNA, hnRNA (heterogenous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial/sufficient complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable double stranded hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. The terms ‘antisense oligonucleotide’, ‘oligonucleotide’, ‘AON’ and ‘oligo’ are often used interchangeably herein and are understood to refer to an oligonucleotide comprising an antisense sequence in respect of the target RNA (or DNA) sequence.

The verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by 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 there be one and only one of the elements. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. The use of “including” as well as other forms, such as “includes” and “included” is not limiting. Terms such as “element” or “component” encompass both elements and component that comprise more than one subunit, unless specifically stated otherwise. All documents, or portions of documents, cited herein, are hereby expressly incorporated by reference for the portions of the documents discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to the sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.

“2’-0-methoxyethyl” (also 2’-MOE, 2’-methoxyethoxy, or 2’-O(CH2)2-OCH₃) refers to an O-methoxy-ethyl modification at the 2’ position of a sugar ring, e.g. a furanose ring. A 2’-O-methoxyethyl modified sugar is a modified sugar. A “2’-MOE nucleoside” (also 2’-O-methoxyethyl nucleoside, or 2’-methoxyethoxy nucleoside) means a nucleoside comprising a 2’-MOE modified sugar moiety. “2’-substituted nucleoside” means a nucleoside comprising a substituent at the 2’-position of the furanosyl ring other than H or OH. In certain embodiments, 2’ substituted nucleosides include nucleosides with bicyclic sugar modifications.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within ±10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition”, it is implied that levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing an antisense oligonucleotide provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes but is not limited to intravitreal administration. The intravitreal administration may be by direct injection, which means that the compound is injected straight into the vitreous of the subject’s eye. The compound itself may be “naked”, or “as such”, but it may also be held in a delivery vehicle. When it is naked, it is generally contained in a formulation that besides the compound also comprises suitable and allowable pharmaceutical carriers, that are well- known to the person skilled in the art.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of Stargardt disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense oligonucleotide to its target molecule. In certain embodiments, antisense activity is an increase in the percentage of exon 17 skip from human ABCA4 pre-mRNA.

“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid, preferably a consecutive stretch of nucleotides within SEQ ID NO: 41 , or a part thereof, through hydrogen bonding. A preferred antisense compound according to the invention is a single stranded antisense oligonucleotide (AON) and is understood to refer to a nucleotide sequence which is substantially complementary to, and hybridizes to, a (target) pre-mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable double stranded hybrid with the target nucleotide sequence in the target RNA molecule under physiological conditions. The AONs of the present invention are not double stranded and are therefore not siRNAs. The AON of the present invention is man-made, and is chemically synthesized, generally in a laboratory by solid-phase chemical synthesis, followed by purification. It is typically purified or isolated.

“Antisense inhibition” means decrease of human ABCA4 mRNA levels that still contains exon 17 in the presence of an antisense compound complementary to (a part of) SEQ ID NO: 41 compared to such levels in the absence of the antisense compound, or in the presence of a non-targeting control antisense compound.

“Antisense mechanism” are all those mechanisms involving hybridization of an antisense compound with a target nucleic acid, wherein the outcome or effect of the hybridization is increase of (wild type or wild type like) protein activity translated from the ABCA4 mRNA.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2’-carbon and the 4’-carbon of the furanosyl.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4’-carbon and the 2’-carbon, wherein the bridge has the formula: 4’- CH(CH₃)-O-2’. Constrained ethyl nucleoside (or cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH₃)-O-2’ bridge.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. The term includes “fully complementary” and “substantially complementary”, meaning there will usually be a degree of complementarity between the oligonucleotide and its corresponding target sequence of more than 80%, preferably more than 85%, still more preferably more than 90%, most preferably more than 95%. For example, for an oligonucleotide of 20 nucleotides in length with one mismatch between its sequence and its target sequence, the degree of complementarity is 95%.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2’ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

“Derivative of an AON” as herein used refers to an AON that is either shortened at the 5’-end or at the 3’-end to generate a 16-, 17-, 18-, 19-, or 20-mer AON with a sequence that is completely within the sequence of the AON from which it is derived. For example (see Figure 1 ): AON 27-A (18-mer), AON 27-B (17-mer), AON 27-0 (16- mer), AON 27-D (18-mer), AON 27-E (17-mer), AON 27-F (16-mer), AON 27-G (17- mer), AON 27-H (16-mer), and AON 27-I (16-mer) are all derivatives of AON 27, which itself is a 19-mer oligonucleotide. Another example is AON 29, that can be considered as a ‘derivative’ of AON 14 (a 21-mer), because the sequence of AON 29 is completely within that of AON 14. The same holds true for AON 30.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

“Mismatch” or “non-complementary nucleobase” refers to a case when a nucleobase of an antisense compound is not capable of pairing with the corresponding nucleobase of a target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Modified sugar” or “modified sugar moiety” means substitution and/or change from a natural sugar moiety.

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ, or organism. For example, modulating splicing can mean to increase the level (the amount; or number of copies) of ABCA4 mRNA that lack exon 17, or to increase/influence the functionality of the resulting protein in a cell, tissue, organ, or organism.

“Natural sugar moiety” means a sugar moiety found in DNA (2’-H) or RNA (2 - OH).

“Naturally occurring internucleoside linkage” means a 3’ to 5’ phosphodiester linkage.

“Nucleoside” means a nucleobase linked to a sugar. “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents (such as an oligonucleotide) and a sterile aqueous solution.

“Phosphorothioate linkage” (often abbreviated to PS linkage) means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

"Prevention, treatment or delay of a ABCA4 related disease or condition" is herein preferably defined as preventing, halting, ceasing the progression of, or reversing partial or complete visual impairment or blindness, as well as preventing, halting, ceasing the progression of or reversing partial or complete auditory impairment or deafness that is caused by a genetic defect in the ABCA4 gene.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2’ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“Substantially complementary” used in the context of the invention indicates that some mismatches in the antisense sequence are allowed if the functionality, i.e. inducing skipping of the ABCA4 exon 17 is still acceptable. Preferably, the complementarity is from 90% to 100%. In general, this allows for 1 or 2 mismatches in an AON of 20 nucleotides or 1 , 2, 3 or 4 mismatches in an AON of 40 nucleotides, or 1 , 2, 3, 4, 5, or 6 mismatches in an AON of 60 nucleotides, etc. The skilled person understands that an AON may be 100% complementary to a sequence harboring a mutation, which means that it is not 100% complementary to the corresponding wild type sequence, while it is still active in skipping exon 17 in both wild type and mutant settings.

In one embodiment, an exon 17 skipping molecule as defined herein is an AON that binds and/or is complementary to a specified target RNA sequence within a target RNA molecule, preferably a target pre-mRNA molecule. Binding to one of the specified target sequences, preferably in the context of a mutated ABCA4 exon 17 may be assessed via techniques known to the skilled person. A preferred technique is gel mobility shift assay as described in EP1619249. In a preferred embodiment, an exon 17 skipping AON is said to bind to one of the specified sequences as soon as a binding of said molecule to a labeled target sequence is detectable in a gel mobility shift assay. The invention provides a method for designing an AON that can skip exon 17 of the human ABCA4 pre-mRNA. First, the AON is selected to bind to and/or to be complementary to a part of SEQ ID NO: 41. Subsequently, in a preferred method at least one of the following aspects has to be considered for designing, improving said exon retainment AON further: the AON preferably does not contain a CpG or a stretch of CpG; and the AON has acceptable RNA binding kinetics and/or thermodynamic properties. The presence of a CpG or a stretch of CpG in an AON is usually associated with an increased immunogenicity of said AON (Dorn and Kippenberger (2008) Curr Opin Mol Ther 10(1 ) 10-20). This increased immunogenicity is undesired since it may induce damage of the tissue to be treated, i.e., the eye. Immunogenicity may be assessed in an animal model by assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration. Immunogenicity may also be assessed in blood of an animal or of a human being treated with an AON of the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said AON using a standard immunoassay known to the skilled person. An inflammatory reaction, type l-like interferon production, IL-12 production and/or an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said AON using a standard immunoassay. The invention allows designing an AON with acceptable RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an AON (Tm), and/or the free energy of the AON-target exon complex, applying methods known to the person skilled in the art. If a Tm is too high, the AON is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the AON. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 70°C and an acceptable free energy may be ranged between 15 and 45 kcal/mol.

An AON of the invention is preferably one that can exhibit an acceptable level of functional activity. A functional activity of said AON is preferably to provide skipping of the ABCA4 exon 17 as further outlined herein, to a certain acceptable level, to provide an individual with a functional protein resulting from the produced mRNA in which exon 17 is absent.

Preferably, an AON, which comprises a sequence that is complementary or substantially complementary to a nucleotide sequence within SEQ ID NO: 41 is such that the (substantially) complementary part is at least 50% of the length of the AON according to the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99%, or even more preferably 100%. Preferably, an AON according to the invention comprises or consists of a sequence that is complementary to a consecutive stretch of nucleotides within SEQ ID NO: 41. In another preferred embodiment, the length of the consecutive complementary part of said AON is 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, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58,

59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 141 , 142 or

143 nucleotides. Additional flanking sequences may be used to modify the binding of a protein to the AON, or to modify a thermodynamic property of the AON, more preferably to modify target RNA binding affinity.

It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. For instance, when designing the AON, one may want to incorporate for instance a residue that does not base pair with the base on the complementary strand. Mismatches may, to some extent, be allowed, if under the circumstances in the cell, the stretch of nucleotides is sufficiently capable of hybridizing to the complementary part.

Optionally, the AON o the invention may further be tested by ex vivo transfection into retina cells of patients, by delivering the AONs directly to so-called eye-cups, which are ex vivo generated eye models (generally generated from patient’s cells, often skin cells), directly to organoids, or by direct intravitreal injection in an animal model, or by direct intravitreal administration in human patients in the course of performing clinical trials. The skip of exon 17 may be assessed by RT-PCR or by ddPCR. The complementary regions are preferably designed such that, when combined, they are specific for the exon and/or intron in the pre-mRNA. Such specificity may be created with various lengths of complementary regions as this depends on the actual sequences in other (pre-) mRNA molecules in the system. The risk that the AON also will be able to hybridize to one or more other pre-mRNA molecules decreases with increasing size of the AON. It is clear that AONs comprising mismatches in the region of complementarity but that retain the capacity to hybridize and/or bind to the targeted region(s) in the pre-mRNA, can be used in the invention. However, preferably at least the complementary parts do not comprise such mismatches as AONs lacking mismatches in the complementary part typically have a higher efficiency and a higher specificity, than AONs having such mismatches in one or more complementary regions. It is thought that higher hybridization strengths (i.e. increasing number of interactions with the opposing strand) are favorable in increasing the efficiency of the process of interfering with the splicing machinery of the target cell. Preferably, the complementarity is from 90% to 100%.

It is to be understood that an exon 17 skipping AON does not have to be complementary to the mutation site in exon 17. It may be that the AON is complementary to a wild type sequence (located away from the mutation), while still being able to give exon 17 skipping in a mutated pre-mRNA. The aim is to induce the skip of exon 17, not to have an AON that specifically targets a region containing the mutation in exon 17, although such is not explicitly excluded. Hence, the invention also relates to AONs that may be fully complementary to the wild type target sequence but may also be adjusted in sequence to become 100% complementary to a mutant sequence, if the mutation (or a mutation that is yet to be identified) is in the region of AON complementarity. In that case the AON is substantially complementary to the mutant sequence and may then differ from the wild type sequences of the AONs that are generally referred to herein.

A preferred exon 17 skipping AON of the invention comprises or consists of from 8 to 143 nucleotides, more preferably from 10 to 40 nucleotides, more preferably from 12 to 30 nucleotides, more preferably from 14 to 30 nucleotides. More preferably, the exon 17 skipping AON of the invention consists of 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides, and even more preferably consists of 16, 17, 18, 19, or 20 nucleotides.

In certain embodiments, the invention provides an AON selected from the group consisting of SEQ ID NO: 1 to 40 and 56 to 109, preferably SEQ ID NO: 10, 11 , 12, 13, 14, 19, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, and 40.

It is preferred that an AON of the invention comprises one or more residues that are modified by non-naturally occurring modifications to increase nuclease resistance, and/or to increase the affinity of the AON for the target sequence. Therefore, in a preferred embodiment, the AON sequence comprises at least one nucleotide analogue or equivalent, wherein 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. Specific modifications that may be introduced are further outlined in detail below.

The skilled person knows that an oligonucleotide, such as an RNA oligonucleotide, generally consists of repeating monomers. Such a monomer is most often a nucleotide or a nucleotide analogue. The most common naturally occurring nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G), and uridine monophosphate (U). These consist of a pentose sugar, a ribose, a 5’-linked phosphate group which is linked via a phosphate ester, and a T-linked base. The sugar connects the base and the phosphate and is therefore often referred to as the “scaffold” of the nucleotide. A modification in the pentose sugar is therefore often referred to as a “scaffold modification”. For severe modifications, the original pentose sugar might be replaced in its entirety by another moiety that similarly connects the base and the phosphate. It is therefore understood that while a pentose sugar is often a scaffold, a scaffold is not necessarily a pentose sugar.

A base, sometimes called a nucleobase, is generally adenine, cytosine, guanine, thymine or uracil, or a derivative thereof. Cytosine, thymine, and uracil are pyrimidine bases, and are generally linked to the scaffold through their 1 -nitrogen. Adenine and guanine are purine bases and are generally linked to the scaffold through their 9- nitrogen.

A nucleotide is generally connected to neighboring nucleotides through condensation of its 5’-phosphate moiety to the 3’-hydroxyl moiety of the neighboring nucleotide monomer. Similarly, its 3’-hydroxyl moiety is generally connected to the 5’- phosphate of a neighboring nucleotide monomer. This forms phosphodiester bonds. The phosphodiesters and the scaffold form an alternating copolymer. The bases are grafted on this copolymer, namely to the scaffold moieties. Because of this characteristic, the alternating copolymer formed by linked monomers of an oligonucleotide is often called the “backbone” of the oligonucleotide. Because phosphodiester bonds connect neighboring monomers together, they are often referred to as “backbone linkages”. It is understood that when a phosphate group is modified so that it is instead an analogous moiety such as a phosphorothioate, such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a “backbone linkage modification”. In general terms, the backbone of an oligonucleotide comprises alternating scaffolds and backbone linkages.

In one aspect, the nucleobase in an AON of the present invention is adenine, cytosine, guanine, thymine, or uracil. In another aspect, the nucleobase is a modified form of adenine, cytosine, guanine, or uracil. In another aspect, the modified nucleobase is hypoxanthine (the nucleobase in inosine), pseudouracil, pseudocytosine, 1- methylpseudouracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2-thiothymine, 5- halouracil, 5-halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5- propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5- aminomethylcytosine, 5-formylcytosine, 5-hydroxymethylcytosine, 7-deazaguanine, 7- deazaadenine, 7-deaza-2,6-diaminopunne, 8-aza-7-deazaguanme, 8-aza-7- deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, pseudoisocytosine, N4- ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2- aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamp, Super A, Super T, Super G, amino-modified nucleobases or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene, or absent like abasic sites (e.g. 1 -deoxyribose, 1 ,2- dideoxyribose, 1-deoxy-2-O-methylribose, azaribose). The terms ‘adenine’, ‘guanine’, ‘cytosine’, ‘thymine’, ‘uracil’ and ‘hypoxanthine’ as used herein refer to the nucleobases as such. The terms ‘adenosine’, ‘guanosine’, ‘cytidine’, ‘thymidine’, ‘uridine’ and ‘inosine’ refer to the nucleobases linked to the (deoxy)ribosyl sugar. The term ‘nucleoside’ refers to the nucleobase linked to the (deoxy)ribosyl sugar. The term ‘nucleotide’ refers to the respective nucleobase-(deoxy)ribosyl-phospholinker, as well as any chemical modifications of the ribose moiety or the phospho group. Thus, the term would include a nucleotide including a locked ribosyl moiety (comprising a 2’-4’ bridge, comprising a methylene group or any other group, well known in the art), a nucleotide including a linker comprising a phosphodiester, phosphotriester, phosphoro(di)thioate, methylphosphonates, phosphoramidate linkers, and the like. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative thereof. A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), 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. A preferred LNA comprises 2'-O, 4'-C-ethylene-bridged nucleic acid (Morita et al. 2001 . Nucleic Acid Res Supplement No.1 :241-242).

Sometimes the terms adenosine and adenine, guanosine and guanine, cytosine and cytidine, uracil and uridine, thymine and thymidine, inosine, and hypoxanthine, are used interchangeably to refer to the corresponding nucleobase, nucleoside, or nucleotide. Sometimes the terms nucleobase, nucleoside and nucleotide are used interchangeably, unless the context clearly requires differently. Modified bases comprise 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 that are or will be known in the art.

In one aspect, an AON of the present invention comprises a 2’-substituted phosphorothioate monomer, preferably a 2’-substituted phosphorothioate RNA monomer, a 2’-substituted phosphate RNA monomer, or comprises 2’-substituted mixed phosphate/phosphorothioate monomers. It is noted that DNA is considered as an RNA derivative in respect of 2’ substitution. An AON of the present invention comprises at least one 2’-substituted RNA monomer connected through or linked by a phosphorothioate or phosphate backbone linkage, or a mixture thereof. The 2’- substituted RNA preferably is 2’-F, 2’-H (DNA), 2’-O-Methyl or 2’-0-(2-methoxyethyl). The 2’-O-Methyl is often abbreviated to “2’-OMe” and the 2’-0-(2-methoxyethyl) moiety is often abbreviated to “2’-MOE”. In a preferred embodiment of this aspect is provided an AON according to the invention, wherein the 2’-substituted monomer can be a 2’- substituted RNA monomer, such as a 2’-F monomer, a 2’-NH2 monomer, a 2’-H monomer (DNA), a 2’-O-substituted monomer, a 2’-OMe monomer or a 2’-MOE monomer or mixtures thereof. Preferably, any other 2’-substituted monomer within the AON is a 2’-substituted RNA monomer, such as a 2’-OMe RNA monomer or a 2’-MOE RNA monomer, which may also appear within the AON in combination.

Throughout the application, a 2’-OMe monomer within an AON of the present invention may be replaced by a 2’-OMe phosphorothioate RNA, a 2’-OMe phosphate RNA or a 2’-OMe phosphate/phosphorothioate RNA. Throughout the application, a 2’- MOE monomer may be replaced by a 2’-MOE phosphorothioate RNA, a 2’-MOE phosphate RNA or a 2’-MOE phosphate/phosphorothioate RNA. Throughout the application, an oligonucleotide consisting of 2’-OMe RNA monomers linked by or connected through phosphorothioate, phosphate or mixed phosphate/phosphorothioate backbone linkages may be replaced by an oligonucleotide consisting of 2’-OMe phosphorothioate RNA, 2’-OMe phosphate RNA or 2’-OMe phosphate/phosphorothioate RNA. Throughout the application, an oligonucleotide consisting of 2’-MOE RNA monomers linked by or connected through phosphorothioate, phosphate or mixed phosphate/phosphorothioate backbone linkages may be replaced by an oligonucleotide consisting of 2’-MOE phosphorothioate RNA, 2’-MOE phosphate RNA or 2’-MOE phosphate/phosphorothioate RNA.

In addition to the specific preferred chemical modifications at certain positions in compounds of the invention, compounds of the invention may comprise or consist of one or more (additional) modifications to the nucleobase, scaffold and/or backbone linkage, which may or may not be present in the same monomer, for instance at the 3’ and/or 5’ position. A scaffold modification indicates the presence of a modified version of the ribosyl moiety as naturally occurring in RNA (i.e. the pentose moiety), such as bicyclic sugars, tetrahydropyrans, hexoses, morpholines, 2’-modified sugars, 4’- modified sugar, 5’-modified sugars and 4’-substituted sugars. Examples of suitable modifications include, but are not limited to 2’-O-modified RNA monomers, such as 2’- O-alkyl or 2’-O-(substituted)alkyl such as 2’-O-methyl, 2’-O-(2-cyanoethyl), 2’-MOE, 2’- O-(2-thiomethyl)ethyl, 2’-0-butyryl, 2’-O-propargyl, 2’-O-allyl, 2’-0-(2-aminopropyl), 2’- O-(2-(dimethylamino)propyl), 2’-O-(2-ammo)ethyl, 2 -O-(2-(dimethylamino)ethyl); 2’- deoxy (DNA); 2’-O-(haloalkyl)methyl such as 2’-O-(2-chloroethoxy)methyl (MCEM), 2’- O-(2,2-dichloroethoxy)methyl (DCEM); 2’-O-alkoxycarbonyl such as 2’-O-[2- (methoxycarbonyl)ethyl] (MOCE), 2’-O-[2-A/-methylcarbamoyl)ethyl] (MCE), 2’-O-[2- (A/,A/-dimethylcarbamoyl)ethyl] (DCME); 2’-halo e.g. 2’-F, FANA; 2'-O-[2-(methylamino)- 2-oxoethyl] (NMA); a bicyclic or bridged nucleic acid (BNA) scaffold modification such as a conformationally restricted nucleotide (CRN) monomer, a locked nucleic acid (LNA) monomer, a xy/o-LNA monomer, an a-LNA monomer, an a-L-LNA monomer, a [3-D-LNA monomer, a 2’-amino-LNA monomer, a 2’-(alkylamino)-LNA monomer, a 2’-(acylamino)- LNA monomer, a 2’-A/-substituted 2’-amino-LNA monomer, a 2’-thio-LNA monomer, a (2’-O,4’-C) constrained ethyl (cEt) BNA monomer, a (2’-O,4’-C) constrained methoxyethyl (cMOE) BNA monomer, a 2’,4’-BNA^NC(NH) monomer, a 2’,4’-BNA^NC(NMe) monomer, a 2’,4’-BNA^NC(NBn) monomer, an ethylene-bridged nucleic acid (ENA) monomer, a carba-LNA (cLNA) monomer, a 3,4-dihydro-2/7-pyran nucleic acid (DpNA) monomer, a 2’-C-bridged bicyclic nucleotide (CBBN) monomer, an oxo-CBBN monomer, a heterocyclic-bridged BNA monomer (such as triazolyl or tetrazolyl-linked), an amido-bridged BNA monomer (such as AmNA), an urea-bridged BNA monomer, a sulfonamide-bridged BNA monomer, a bicyclic carbocyclic nucleotide monomer, a TriNA monomer, an a-L-TriNA monomer, a bicyclo DNA (bcDNA) monomer, an F-bcDNA monomer, a tricyclo DNA (tcDNA) monomer, an F-tcDNA monomer, an alpha anomeric bicyclo DNA (abcDNA) monomer, an oxetane nucleotide monomer, a locked PMO monomer derived from 2’-amino LNA, a guanidine-bridged nucleic acid (GuNA) monomer, a spirocyclopropylene-bridged nucleic acid (scpBNA) monomer, and derivatives thereof; cyclohexenyl nucleic acid (CeNA) monomer, altriol nucleic acid (ANA) monomer, hexitol nucleic acid (HNA) monomer, fluorinated HNA (F-HNA) monomer, pyranosyl-RNA (p-RNA) monomer, 3’-deoxypyranosyl DNA (p-DNA), unlocked nucleic acid UNA); an inverted version of any of the monomers above.

A “backbone modification” indicates the presence of a modified version of the ribosyl moiety (“scaffold modification”), as indicated above, and/or the presence of a modified version of the phosphodiester as naturally occurring in RNA (“backbone linkage modification”). Examples of internucleoside linkage modifications are phosphorothioate (PS), chirally pure phosphorothioate, Rp phosphorothioate, Sp phosphorothioate, phosphorodithioate (PS2), phosphonoacetate (PACE), thophosphonoacetate, phosphonacetamide (PACA), thiophosphonacetamide, phosphorothioate prodrug, S- alkylated phosphorothioate, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, phosphoryl guanidine (PGO), methylsulfonyl phosphoroamidate, phosphoramidite, phosphonamidite, N3’->P5’ phosphoramidate, N3’->P5’ thiophosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido (TANA); and their derivatives.

The present invention also relates to a chirally enriched population of modified AONs according to the invention, wherein the population is enriched for modified AONs comprising at least one particular phosphorothioate internucleoside linkage having a particular stereochemical configuration, preferably wherein the population is enriched for modified AONs comprising at least one particular phosphorothioate internucleoside linkage having the Sp configuration, or wherein the population is enriched for modified AONs comprising at least one particular phosphorothioate internucleoside linkage having the Rp configuration.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone, exemplified by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting 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 method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells.

It is further preferred that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.

A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991 ) Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of basepair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)- glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer. Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA- DNA hybrids, respectively (Egholm et al. (1993) Nature 365:566-568).

It is understood by a skilled person that it is not necessary for all positions in an AON to be modified uniformly. In addition, more than one of the analogues or equivalents may be incorporated in a single AON or even at a single position within an AON. In certain embodiments, an AON of the invention has at least two different types of analogues or equivalents. A preferred exon skipping AON according to the invention comprises a 2'-0 alkyl phosphorothioated antisense oligonucleotide, such as 2'-OMe modified ribose (RNA), 2'-0-ethyl modified ribose, 2'-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives. An effective AON according to the invention comprises a 2'-OMe ribose and/or a 2’-MOE ribose with a (preferably full) phosphorothioated backbone.

It will also be understood by a skilled person that different AONs can be combined for efficiently skipping exon 17 from the aberrant ABCA4 pre-mRNA. In a preferred embodiment, a combination of at least two AONs are used in a method of the invention, such as 2, 3, 4, or 5 different AONs. Hence, the invention also relates to a composition comprising a set of AONs comprising at least one AON according to the present invention, optionally further comprising AONs as disclosed herein.

An AON of the present invention can be linked to a moiety that enhances uptake of the AON in cells, preferably retina cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain. Particularly preferred in the invention is the use of an excipient or transfection reagents that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell (preferably a retina cell). Preferred are excipients or transfection reagents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection reagents comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a cell, preferably a retina cell. Such excipients have been shown to efficiently deliver an AON to a wide variety of cultured cells, including retina cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity. Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1- (2,3 dioleoyloxy)propyl]-N, N, N- trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidyl ethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery system are polymeric nanoparticles. Polycations such as diethylamino ethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver AONs across cell membranes into cells. In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an AON. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an exon skipping molecule for use in the current invention to deliver it for the prevention, treatment, or delay of a ABCA4 related disease or condition, such as Stargardt disease.

An AON according to the invention may be indirectly administrated using suitable means known in the art. It may for example be provided to an individual or a cell, tissue, or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector may be introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of an AON as identified herein. Accordingly, the invention provides a viral vector expressing an exon 17 skipping AON according to the invention when placed under conditions conducive to expression of the AON. A cell can be provided with an AON of the invention by plasmid-driven expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. Expression may be driven by a polymerase ll-promoter (Pol II) such as a U7 promoter or a polymerase III (Pol III) promoter, such as a 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 lentivirus vector and the like. Also, plasmids, artificial chromosomes, plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from Pol III promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are Pol III driven transcripts, preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such fusions may be generated as described (Gorman et al. 1998. Proc Natl Acad Sci U S A 95(9):4929-34; Suter et al. 1999. Hum Mol Genet 8(13):2415-23).

The AON of the present invention may be delivered as such (or ‘naked’). However, the AON may also be encoded by the viral vector, as mentioned above. Typically, this is in the form of an RNA transcript that comprises the sequence of an oligonucleotide according to the invention in a part of the transcript. An AAV vector according to the invention is a recombinant AAV vector and refers to an AAV vector comprising part of an AAV genome comprising an AON according to the invention encapsidated in a protein shell of capsid protein derived from an AAV serotype as depicted elsewhere herein. Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and others. Protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1 , 2, 3, 4, 5, 6, 7, 8, 9 and others. A protein shell may also be named a capsid protein shell. AAV vector may have one or preferably all wild type AAV genes deleted but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue, and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in insertion, mutation, deletion, or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell. In the context of the invention a capsid protein shell may be of a different serotype than the AAV vector genome ITR. An AAV vector according to present the invention may thus be composed of a capsid protein shell, i.e., the icosahedral capsid, which comprises capsid proteins (VP1 , VP2, and/or VP3) of one AAV serotype, e.g., AAV serotype 2, whereas the ITRs sequences contained in that AAV5 vector may be any of the AAV serotypes described above, including an AAV2 vector. An “AAV2 vector” thus comprises a capsid protein shell of AAV serotype 2, while e.g., an “AAV5 vector” comprises a capsid protein shell of AAV serotype 5, whereby either may encapsidate any AAV vector genome ITR according to the invention. Preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2, 5, 8 or AAV serotype 9 wherein the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9; such AAV vector is referred to as an AAV2/2, AAV 2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV 5/9, AAV8/2, AAV 8/5, AAV8/8, AAV8/9, AAV9/2, AAV9/5, AAV9/8, or an AAV9/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 5; such vector is referred to as an AAV 2/5 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 8; such vector is referred to as an AAV 2/8 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 9; such vector is referred to as an AAV 2/9 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 2; such vector is referred to as an AAV 2/2 vector. A nucleic acid molecule encoding an AON according to the invention represented by a nucleic acid sequence of choice is preferably inserted between the AAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3’ termination sequence. “AAV helper functions” generally refers to the corresponding AAV functions required for AAV replication and packaging supplied to the AAV vector in trans. AAV helper functions complement the AAV functions which are missing in the AAV vector, but they lack AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art. The AAV helper functions can be supplied on an AAV helper construct, which may be a plasmid.

Introduction of the helper construct into the host cell can occur e.g., by transformation, transfection, or transduction prior to or concurrently with the introduction of the AAV genome present in the AAV vector as identified herein. The AAV helper constructs of the invention may thus be chosen such that they produce the desired combination of serotypes for the AAV vector’s capsid protein shell on the one hand and for the AAV genome present in said AAV vector replication and packaging on the other hand. “AAV helper virus” provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as described in US 6,531 ,456 incorporated herein by reference. Preferably, an AAV genome as present in a recombinant AAV vector according to the invention does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. An AAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art. Preferably, an AAV vector according to the invention is constructed and produced according to the methods in the Examples herein. 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 according to the invention that comprises, or preferably consists of, a sequence that is 100% complementary or substantially complementary to a consecutive nucleotide sequence stretch within SEQ ID NO: 41 . A further 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 exon 17 skipping AON according to the invention that comprises, or preferably consists of the sequence of SEQ ID NO: 1 to 40 and 56 to 109, more preferably SEQ ID NO: 10, 11 , 12, 13, 14, 19, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, or 40.

It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an AON according to the invention and a further adjunct compound as defined herein. If required, an AON according to the invention or a vector, preferably a viral vector, expressing an AON according to the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier. Accordingly, the invention also provides a composition, preferably a pharmaceutical composition, comprising an AON according to the invention, or a viral vector according to the invention and a pharmaceutically acceptable excipient. Such composition may comprise a single AON or viral vector according to the invention, but may also comprise multiple, distinct AONs or viral vectors according to the invention. Such a pharmaceutical composition may comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer and/or diluent may for instance be found in Remington (Remington. 2000. The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams Wilkins). Each feature of said composition has earlier been defined herein.

A preferred route of administration is through direct intravitreal injection of an aqueous solution or specially adapted formulation for intraocular administration. EP2425814 discloses an oil in water emulsion especially adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. This emulsion is less dense than the vitreous fluid, so that the emulsion floats on top of the vitreous, avoiding that the injected drug impairs vision.

A preferred ABCA4 exon 17 skipping AON according to the invention is for the treatment of an ABCA4-related disease or condition of an individual. In all embodiments of the invention, the term ‘treatment’ is understood to also include the prevention and/or delay of the ABCA4-related disease or condition. An individual, which may be treated using an AON according to the invention may already have been diagnosed as having a ABCA4-related disease or condition. Alternatively, an individual which may be treated using an AON according to the invention may not have yet been diagnosed as having a ABCA4-related disease or condition but may be an individual having an increased risk of developing a ABCA4-related disease or condition in the future given his or her genetic background. A preferred individual is a human individual. A preferred ABCA4-related disease or condition is Stargardt disease. A treatment in a use or in a method according to the invention is at least once a week, once a one month, once every several months, once every 1 , 2, 3, 4, 5, 6 years or longer, such as lifelong. Each AON or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing ABCA4-related disease or condition, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an AON, composition, compound, or adjunct compound of 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 (i.e., dose), the formulation of said AON(s), the route of administration and so forth. The frequency may vary between daily, weekly, at least once in two weeks, or three weeks or four weeks or five weeks or a longer time. Dose ranges of an AON according to the invention are preferably designed based on rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. In a preferred embodiment, a viral vector, preferably an AAV vector as described earlier herein, as delivery vehicle for a molecule according to the invention, is administered in a dose ranging from 1x10⁹ to 1x10¹⁷ virus particles per injection, more preferably from 1x10¹⁰ to 1x10¹² virus particles per injection. The ranges of concentration or dose of AONs as given above are preferred concentrations or doses for in vivo, in vitro or ex vivo uses. The skilled person will understand that depending on the AONs used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of AONs used may further vary and may need to be optimized any further.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person can identify such erroneously identified bases and knows how to correct for such errors.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. EXAMPLES

Example 1. Design of antisense oligonucleotides targeting a consecutive stretch of nucleotides in the pre-mRNA of exon 17 of human ABCA4 and/or its surrounding sequences

Initially a set of nineteen single-stranded antisense oligonucleotides (AONs) were designed, considering the GC content and Tm, that were 100% complementary with a consecutive stretch of nucleotides in intron 16 of the human ABCA4 pre-mRNA, that were 100% complementary with a consecutive stretch of nucleotides that overlapped with a sequence of intron 16 and exon 17 of the human ABCA4 pre-mRNA, that were 100% complementary with a consecutive stretch of nucleotides within exon 17 of the human ABCA4 pre-mRNA, or that were 100% complementary with a consecutive stretch of nucleotides that overlapped with a sequence of exon 17 and intron 17 of the human ABCA4 pre-mRNA. The sequences of all AONs are given in Figure 1 . The sequence of exon 17 carrying the c.2588G>C mutation is also given in Figure 1. Part of the surrounding sequences of exon 17 (the 3’ part of intron 16 and the 5’ part of intron 17) are also given in Figure 1. AON 7 targets a sequence that comprises the c.2588G>C mutation, while all other AONs target a sequence away from the mutation. All nineteen AONs (AONs 1 to 19; SEQ ID NO: 1 to 19) were modified such that all nucleotides carried a 2’-O-methoxyethyl (2’-MOE) modification in the sugar moiety and all AONs contained a full phosphorothioate backbone. The nineteen AONs were 20, 21 , or 22 nucleotides in length. After the initial experiments using these nineteen AONs, an additional set of twenty-one AONs was designed (AONs 20 to 40; SEQ ID NO: 20 to 40) of which the sequences and their respective target locations are also provided in Figure 1. These additional AONs were also fully 2’-MOE modified and contained a full phosphorothioate backbone. The length of the AONs in the additional set ranged from 17 to 19 nucleotides.

Example 2. Exon 17 skip from human ABCA4 pre-mRNA using AONs 1 to 19 in a gymnotic uptake experiment

The initial set of nineteen AONs (AONs 1 to 19) were tested for the efficiency in exon 17 skipping from wt human ABCA4 pre-mRNA in cells in vitro. For this, human retinoblastoma WERI-Rb1 cells (ATCC HTB-169) were cultured in a 12-well dish in RPM 11640/10%FBS in a concentration of 5x10⁵ cells/well. The antisense oligonucleotides were dissolved in Nuclease-free water (ThermoFisher Scientific, Cat. No. AM9937) to a concentration of 1 mM, which was confirmed using Thermo Scientific™ NanoDrop™ 2000 Spectrophotometer (Fisher Scientific, Cat. No. ND2000). The cells were incubated with 100 pL AON (10000 nM) and Opti-MEM I Reduced Serum Media (ThermoFisher Scientific, Cat. No. 11058-021 ) mixture for 48 h at 37°C / 5% CO2.

The content of each well was transferred to a 1.5 mL Eppendorf tube and centrifuged at max speed (14000 rpm) for 1 min. The supernatant was vacuum-removed and the pellet containing cells was lysed with 350 mL RLT plus buffer (part of the RNeasy Plus Mini Kit, QIAGEN, Cat. No. 74136). The RNA was extracted with the mentioned kit according to the manufacturer’s instructions. The RNA was eluted in 35 pL of RNAse free water (part of the RNeasy Plus Mini Kit, QIAGEN, Cat. No. 74136). The tubes containing RNA were placed on ice, and the concentration was measured on a Thermo Scientific™ NanoDrop™ 2000 Spectrophotometer (Fisher Scientific, Cat. No. ND2000). The RNA was stored at -80°C.

11 pL of mixture containing 500 ng of RNA and Nuclease-free water was reverse transcripted using the Verso cDNA Synthesis Kit (ThermoFisher Scientific, Cat. No. AB1453B) as follows: 4 pL/reaction 5x cDNA synthesis buffer, 2 pL/reaction dNTP mix (5 mM each), 1 pL/reaction RT enhancer, 1 pL/reaction hxm (400 ng/pL) and 1 pL/reaction Verso Enzyme Mix (200 U/pL). The sample mixtures were briefly vortexed and centrifuged, and placed in the Bio-Rad T100 Thermal Cycler (Bio-Rad, Cat. No. 1861096). The following program was used: 30 min 42°C, 2 min 95°C and °° at 4°C. The cDNA was stored at -20°C.

The samples were analyzed with two digital droplet PCR (ddPCR) assays, where the first assay, ABCA4_exon17_skip, determines the number of skipped copies of endogenous ABCA4 exon 17, and the second, ABCA4_exon8-9 determines the number of total copies of endogenous ABCA4 in WERI-Rb-1 cells. The primers used for the total RNA ddPCR were: ABCA4_exon8_FW: 5’-CCCTCATGCAGAATGGTGGT-3’ (SEQ ID NO: 43); ABCA4_exon9_RV: 5’-CGCCCTCCAAGCGATTTTG-3’ (SEQ ID NO: 44); and ABCA4_exon8-9_probe: 5’-5HEX/AGAAGAACAACATCCTTTTG/3IABkFQ-3’ (SEQ ID NO: 45). The primers for the determination of exon 17 skipped RNA ddPCR were: ABCA4_ex.16_FW: 5’-GTGGAGCAACATCGGGAACA-3’ (SEQ ID NO: 46); ABCA4_ex.18_RV: 5'-TGTTAGGGGCTCGGTCTTTT-3' (SEQ ID NO: 47); and ABCA4_ex.17.skip_probe: 5'-5HEX/TGTTTCCAGGGTGTTCAACC/3IABkFQ-3' (SEQ ID NO: 48).

The PCR master mix for each assay was made as follows: 0.525 pL/reaction of 10 pM forward primer, 0.525 pL/reaction of 10 pM reverse primer, 0.525 pL/reaction of 10 pM ddPCR probe, 10.5 pL/reaction of ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad, Cat. No. 186-3025) and 4.925 pL Nuclease-free water. The master mix (without cDNA) was aliquoted in separate 8-strip tubes and 4 pL of cDNA was added. A ddPCR cartridge (Bio-Rad, Cat. No. #186-4008) was placed in the DG8 cartridge holder

(Bio-Rad). Each well of the middle row was filled with 21 pL of sample mixture using the Electronic Multichannel (Rainin, Cat. No. E8-50XLS+). Each well of the bottom row of the cartridge was filled with 70 pL of Droplet Generation Oil for Probes (Bio-Rad, Cat. No. 1863005). A rubber gasket (Bio-Rad, Cat. No. #186-3009) was placed over the cartridge, and the cartridge was inserted in the QX200™ Droplet Generator (Bio-Rad, Cat. No. #1864002). When the droplet generator was finished, the cartridge was taken out and the rubber gasket was removed. 40 pL of oil emulsion was carefully pipetted with Electronic Multichannel (Rainin, Cat. No. E8-50XLS+) and transferred to a semi skirted 96 wells plate (Eppendorf, Cat. No. #0030 128.591 ). The plate was sealed with a Pierceable Foil Heat Seal (Bio-Rad, #181-4040) in the PX1 PCR Plate Sealer (Bio¬

Rad, Cat. No. #1814000) at 170°C. The plate was placed in the Bio-Rad T100 Thermal Cycler (Bio-Rad, Cat. No. 1861096). The following PCR program was used: enzyme activation at 95°C for 10 min (1 cycle), denaturation at 95°C for 30 sec and annealing/extension at 57°C for 1 min (40 cycles), enzyme deactivation at 98°C for 10 min (1 cycle) and hold at 4°C. After the PCR, the plate was placed in the QX200™ Droplet Reader (Bio-Rad, Cat. No. #1864003) to measure the number of positive droplets. The data was analyzed using the Quantasoft software, as follows: the negative and the positive droplet populations were separated, and the positive droplets were used for the further calculations. The positive droplets detected with the assay

ABCA4_exon17_skip were taken along as the “skipped copies of the ABCA4 exon 17 region”, while the positive droplets measured with the assay ABCA4_exon8-9 were taken along as “total copies of ABCA4”. The following formula was used to calculate the percentage of ABCA4 exon 17 skip:

ABCA4 exonl7 skip

% ABCA4 exon 17 skip = ( - -) x 100 v ABCA4 exon8 - 9 ⁷

The experiment was performed three times in separate setups. The results (average) of these three gymnotic uptake experiments (= no transfection) in WERI-Rb1 cells are shown in Figure 2. The graph shows that no signal was determined when AON 5 was used. This was likely due to a manufacturing error of this oligonucleotide because no proper RNA was found after its production. It also shows that several AONs that target a sequence within exon 17 (and of which some overlap with the 5’ intron/exon or 3’ exon/intron boundary) performed well. Especially AON 10, 11 , 12, 13, 14, and 19 (SEQ ID NO: 10, 11 , 12, 13, 14, and 19, respectively) gave significant levels of exon 17 skip of the wild type ABCA4 pre-mRNA in the retinoblastoma cell line, in the absence of transfection reagents. Example 3. Exon 17 skip from human ABCA4 pre-mRNA using AONs 20 to 40 m a gymnotic uptake experiment

An identical gymnotic uptake assessment in WERI-Rb1 cells as described in Example 2 was performed with a new set of AONs (AONs 20 to 40; SEQ ID NO: 20 to 40), again in three separate experiments. These were compared to the best performer of the first experiment, AON 14.

The average result of the three experiments using AONs 20-40 in gymnotic uptake in WERI-Rb1 cells are given in Figure 3. Clearly, several AONs performed well and sometimes even better than the best performer from the first screen, AON 14. Especially AON 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, and 40 gave significant levels of exon 17 skip of the wild-type ABCA4 pre-mRNA in the retinoblastoma cell line, in the absence of transfection reagents.

Example 4. Exon 17 skip from human ABCA4 pre-mRNA using AONs in human retinal organoids

To further investigate the potential of the AONs that appeared capable of skipping exon 17 from the ABCA4 pre-mRNA in human WERI-Rb1 cells, as shown above, a system was used in which the AONs were incubated with human retinal organoids (also referred to as ‘eye cups’). Such retinal organoids represent the structure of the human retina in an in vitro situation.

Generation of retinal organoids

Human induced pluripotent stem cell line (iPSC) 771 -3G (ReproCell, Cat. No. RCRP005N) was cultured in 6-well plates coated with Corning® Matrigel® hESC- Qualified Matrix (Corning, Cat. No. 354277) containing 2 mL mTeSR™1 medium (Stemcell Technologies, Cat. No. 85857) enriched with 25% mTeSR™ 1 5X Supplement (Stemcell Technologies, Cat. No. 85852) and 1 % pen/strep. The cells were transferred every 5-7 days to new 6-well plates in 200-500 clumps/well and grown at 37°C and 5% CO2. For organoid differentiation, iPSCs were in the growth log phase but not differentiated. Cells were washed with 1 ml PBS and detached with 1 ml TrypLE™ Express Enzyme (Thermofisher Scientific, Cat. No. 12604013). The cells were collected with 3 mL of iPSC culture medium and centrifuged for 5 min at 1000 rpm. The supernatant was removed and the cells were resuspended in Day 0 medium - iPSC culture medium with 10 pM Y-27632 dihydrochloride (Sigma, Cat. No. Y0503-1 mg). 100 pL of cell suspension was added in a well of the ultra-low adhesion U-shaped 96 well plate using the P200 multichannel pipette or P1200 electronic multichannel pipette. The plate was centrifuged for 3 min at 100xg and incubated at 5% CO2 and 37°C. The following day, the aggregates were topped with 100 pL medium Day 0 medium per well. After two days, the medium was removed and ‘Day 2’ medium (42.5% Iscove's Modified Dulbecco's Medium (ThermoFisher), 42.5% Ham’s F12 (ThermoFisher), 15% KnockOut Replacement Serum (ThermoFisher), 1% Glutamaxx (ThermoFisher), 0.5% Chemically Define Lipid Concentrate (ThermoFisher), 1% pen/strep, and 0,1% 0.45M 1 -Thioglycerol (Sigma)) was added. After 4 days, the medium was removed and ‘Day 6’ medium (42.5% IMDM, 42.5% Ham’s F12, 15% KnockOut Replacement Serum, 1% Glutamaxx, 0.5% Chemically Define Lipid Concentrate, 1 % pen/strep, 2nM BMP-4 (R&D Systems), and 0,1 % 0.45M 1 -Thioglycerol) was added. At day 9, 12 and 15, 50% of the medium was removed and ‘Day 2’ medium was again added. At day 18 the medium was removed and NR-RPE medium (86.9% DMEM/F12 (ThermoFisher), 10% FBS, 1% Glutamaxx, 1 % N-2 supplement (ThermoFisher), 3 pM CHIR99021 (Sigma), 5 pM SU-5402 (Sigma), 1 % pen/strep) was added. At day 24, the medium was removed and NR maintenance medium (87% DMEM/F12, 10% FBS, 1 % N-2 supplement, 1% Glutamaxx and 1% pen/strep) was added. 50% of the medium was refreshed every 2 days.

A ON treatment

AONs were administered to 190-day-old retinal organoids at a 10 pM concentration (six separate organoids for each of the following oligonucleotides: AON 14, 24, 25, 27, 29, 39, 40, and a scrambled control AON); the culture medium was fully removed and fresh medium with AON was added. Every two days half of the culture medium was removed and replaced by fresh culture medium. Four weeks posttreatment the culture medium was removed completely, the retinal organoids were washed in PBS and 300 pl TRIreagent (Zymo Research) was added. The samples were snap frozen in liquid nitrogen and stored at -80°C. For examination, the organoids were lysed with a 25-gauge needle, and the RNA was extracted with the Direct-Zol RNA MicroPrep kit (Zymo Research) according to the manufacturer’s instructions. 100 ng of extracted RNA was reverse transcribed in cDNA as described above. ddPCR

Exon skipping was examined generally as described above. The cDNA samples were analysed with four digital droplet PCR (ddPCR) assays: skip of ABCA4 exon 17 was measured for the degree of ABCA4 exon 17 skip, whereas the ABCA4 exon 28 wild type and ABCA4 exon 39-40 wild type measured the total amount of full length ABCA4 transcript. The primers for the determination of exon 17 skipped RNA ddPCR were as described in Example 2. The primers used for the total RNA ddPCR were: ABCA4_exon28_FW: 5’-GCTGCTGGTCAAGAGATTCCA-3’ (SEQ ID NO: 50); ABCA4_exon28_RV: 5’-ACGTCTGCAAGTACCGTGAA-3’ (SEQ ID NO: 51 ); ABCA4_exon28_probe: 5’-56FAM/ CCCTGGATA/ ZEN /TATGGGCAGCAGTACAC/ 3IABkFQ-3’ (SEQ ID NO: 52); ABCA4_ex39-40_FW: 5’-

GCGGTCATTCCCATGATGTA-3’ (SEQ ID NO: 53); ABCA4_ex39-40_RV: 5'- AACAATGGGCTCCTTAGTGG-3' (SEQ ID NO: 54); and ABCA4_ex39-40_probe: 5’- 56-FAMZ CCCGGTTTG/ ZEN /GTGAGGAGCAC/ 3IABkFQ-3' (SEQ ID NO: 55).

The PCR master mix for each assay was made as follows: 0.5 pM forward primer, 0.5 pM reverse primer, 0.25 pM probe, 1 x QIAcuity Probe PCR Kit (5 x 5 ml master mix, 4x cone.) (QIAGEN) and 5 ng of cDNA. Nuclease-free water was added up to total volume of 12 pL. The digital PCR was run following the thermal cycling conditions generally as set out in Example 2.

Using the QIAcuity Software Suite, the negative and the positive populations were separated with manually set thresholds, and the positive population was considered for further calculations. The positive copies detected with the assay ABCA4 exon 17 skip were taken as the “skipped copies of the ABCA4 exon 17 region”, while the positive copies measured with the assays ABCA4 exon 39-40 wild type and ABCA4 exon 28 wild type were considered “reference amount of ABCA4 copies”. The following formulas were used to calculate the fold change of ABCA4 exon 17 skip in samples after AON treatment:

ABCA4 exon 17 skip

AON effect on ABCA4 exon 17 skip - — — - - > - >

‘ ^J Reference ABCA4

Results

The copies detected with the ABCA4 exon 17 skip assay were used to identify the effect of AON treatment on the skip of exon 17 in ABCA4. The total expression of ABCA4 was assessed with the average value detected with ABCA4 exon 39-40 wild type and ABCA4 exon 28 wild type assays. The amount of transcript lacking ABCA4 exon 17 was compared to the wild type ABCA4 transcript. The results are depicted in Figure 4. All the AONs that were tested displayed clear ability to induce exon 17 skip in ABCA4 after a 4-week treatment on retinal organoids, which is completely in line with what was found first in WERI-Rb1 cells (see above). The control retinal organoid group, treated with scrambled AON showed almost only full length ABCA4 isoforms and no transcripts lacking exon 17. From these results it appeared that at least any of these seven oligonucleotides could be selected as lead molecule for exon 17 skip and to serve as a model molecule for designing (preferably shorter) oligonucleotides which are also tested in additional organoid studies and preclinical in vitro and in vivo studies. AON 14, 24, 25, 27, 29, 39, and 40 are shown in Figure 1 . Since it is often preferred to use short (16- to 18-mer) oligonucleotides over long oligonucleotides (>25 nucleotides), the invention also relates to derivatives of these good performing AONs in which the size is shortened from the 5’ and/or 3’ side. These derivatives are also depicted in Figure 1 (AON 14-A, - B, -C, and -D [SEQ ID NO: 56 to 59, respectively]; AON 14-E, -F, -G, -H, -I, -J, -K, -L, - M, -N. -O, -P, -Q, and -R [SEQ ID NO: 92 to 105, respectively]; AON 24-A, -B, -C, -D, and -E [SEQ ID NO: 60 to 64, respectively]; AON 25-A, -B, -C, -D, -E, -F, -G, -H, and -I [SEQ ID NO: 65 to 73, respectively]; AON 27-A, -B, -C, -D, and -E [SEQ ID NO: 74 to 78, respectively]; AON 27-F, -G, -H, and -I [SEQ ID NO: 106 to 109, respectively]; AON 39-A, -B, -C, -D, -E, -F, -G, -H, and -I [SEQ ID NO: 79 to 87, respectively]; AON 40-A, -

B, -C, and -D [SEQ ID NO: 88 to 91 , respectively]). As can be seen in Figure 1 also, AON 29 and AON 30 can both in fact be regarded as a shortened derivatives of AON 14.

Claims

Claims An antisense oligonucleotide (AON) that can induce the skip of exon 17 from human ABCA4 pre-mRNA, wherein the AON comprises a sequence that is 90 to 100%, preferably 100% complementary to a consecutive stretch of nucleotides within SEO ID NO: 41. The AON according to claim 1 , wherein the AON comprises a sequence that is complementary to a consecutive stretch of nucleotides within SEQ ID NO: 42, SEQ ID NO: 49, or a consecutive stretch of nucleotides within SEQ ID NO: 41 that includes the boundary between exon 17 and intron 17. The AON according to claim 1 or 2, wherein the AON consists of 14 to 25 nucleotides, more preferably 16, 17, 18, 19, 20, 21 or 22 nucleotides. The AON according to any one of claims 1 to 3, wherein the AON comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 10, 11 , 12, 13, 14, 19, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 39, 40, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, and 109. The AON according to any one of claims 1 to 4, wherein the AON comprises at least one 2’-O-methoxyethyl (2’-MOE) modification. The AON according to claim 5, wherein all nucleotides of the AON are 2’-MOE modified. The AON according to any one of claims 1 to 6, wherein the AON comprises at least one non-naturally occurring internucleoside linkage, such as a phosphorothioate (PS) linkage.

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. The AON according to claim 7, wherein all sequential nucleosides are interconnected by PS linkages. . A viral vector expressing an AON according to any one of claims 1 to 3.

10. A pharmaceutical composition comprising an AON according to any one of claims 1 to 8 or a viral vector according to claim 9, and a pharmaceutically acceptable carrier.

11 . The AON according to any one of claims 1 to 8, the viral vector according to claim 9, or the pharmaceutical composition according to claim 10 for use in the treatment, prevention or delay of an ABCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene.

12. The AON for use according to claim 11 , wherein the AON is for intravitreal administration and is dosed in an amount ranging from 5 pg to 500 pg of total AON per eye, preferably from 10 pg to 100 pg, more preferably from 25 pg to 100 pg.

13. The AON for use according to claim 12, wherein the AON is dosed in an amount ranging from 25 pg to 100 pg of total AON per eye, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 pg total AON per eye.

14. Use of an AON according to any one of claims 1 to 8, a viral vector according to claim 9, or a pharmaceutical composition according to claim 10 for the preparation of a medicament for the treatment, prevention or delay of an ABCA4-related disease or a condition requiring modulating splicing of ABCA4 pre-mRNA, such as Stargardt disease, preferably Stargardt disease caused by the c.2588G>C mutation in exon 17 of the ABCA4 gene.

15. An in vitro, ex vivo or in vivo method for modulating splicing of ABCA4 pre-mRNA in a cell, comprising the steps of: administering to the cell an AON according to any one of claims 1 to 8, a viral vector according to claim 9, or a pharmaceutical composition according to claim 10; allowing the hybridization of the AON to its complementary sequence in ABCA4 target RNA molecule in the cell; and allowing the skip of exon 17 from the target RNA molecule.

40 A method for the treatment of a ABCA4-re\ated disease or condition requiring modulating splicing of ABCA4 pre-mRNA of an individual in need thereof, said method comprising contacting a cell of said individual with an AON according to any one of claims 1 to 8, a viral vector according to claim 9, or a pharmaceutical composition according to claim 10.

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