Field of the invention The invention relates to the fields of medicine and immunology. In particular, it relates to novel antisense oligonucleotides that may be used in the treatment, prevention and/or delay of an ABCA4- associated condition.

Background of the invention Autosomal recessive mutations in ABCA4 cause Stargardt disease, a progressive disorder characterized by central vision loss and often leading to complete blindness. A typical hallmark of Stargardt disease is the presence of many yellow spots (flecks) distributed throughout the fundus of the patients. The ABCA4 gene is comprised of 50 exons and encodes a protein consisting of 2273 amino acids. This protein is expressed in the outer segments of cone and rod photoreceptor cells and plays an important role in the removal of waste products following phototransduction.

Besides STGD1 , variants in ABCA4 can also lead to other subtypes of retinal disease ranging from bull’s eye maculopathy to autosomal recessive cone-rod dystrophy (arCRD; Cremers et al, 1998; Maugeri et al, 2000) and pan-retinal dystrophies (Cremers et al, 1998; Martinez-Mir et al, 1998), depending on the severity of the alleles. Biallelic ABCA4 variants can be identified in approximately 80% of the cases with STGD1 (Allikmets et al, 1997; Fujinami et al, 2013; Lewis et al, 1999; Maugeri et al, 1999; Rivera et al, 2000; Schulz et al, 2017; Webster et al, 2001 ; Zernant et al, 2011 ; Zernant et al, 2017), and 30% of cases with arCRD (Maugeri et al, 2000, supra), after sequencing coding regions and flanking splice sites. In general, individuals with arCRD or pan-retinal dystrophy carry two severe ABCA4 alleles, whereas individuals with STGD1 carry two moderately severe variants or a combination of a mild and a severe variant (Maugeri et al, 1999; van Driel et al, 1998). It has been hypothesized that the majority of the missing ABCA4 variants in STGD1 patients reside in intronic regions of the gene, and indeed, over the last few years, several groups have demonstrated the existence of such deep-intronic variants (Bauwens et al, 2015; Bax et al, 2015; Braun et al, 2013; Lee et al, 2016; Schulz et al, 2017; Albert et al, 2018, Sangermano et al 2019 and Bauwens et al., 2019). Many of these deep- intronic mutations activate cryptic splice acceptor or donor sites, or change exonic splice enhancer or silencer motifs, all resulting in the inclusion of pseudoexons (PEs) to a significant proportion of ABCA4 transcripts. The degree of PE insertion can differ per ABCA4 variant, but also depends on which tissue is studied, i.e. the degree of PE inclusion can be proportionally higher in ‘retina-like’ tissues. Whilst most of the ABCA4 mutations are spread all over the ABCA4 gene, there are some mutations that tend to cluster, including a series of variants in intron 36 of this gene. Specifically, we identified four intron 36 variants (c.5196+10134>G; c.5196+10564>G; c.5196+1137G>A; c.5196+12164>G) that lead to the inclusion of pseudoexons to ABCA4 pre-mRNA, and consequently, predicted loss of ABCA4 protein function. The fact that a considerable amount of the mutations in ABCA4 affect pre-mRNA splicing of ABCA4, renders it an attractive target for antisense oligonucleotide (AON)-based splice modulation therapy. Accordingly, there is an urge to develop AONs for splice modulation of the ABCA4 gene to enable expression of a functional ABCA4 protein in subjects suffering from Stargardt disease.

Summary of the invention

In a first aspect, the invention relates to an antisense oligonucleotide for redirecting splicing that binds to and/or is complementary to a polynucleotide with the nucleotide sequence as shown in SEQ ID NO: 4. Preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 8, 9 and 10. More preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 5, 6, and 7. Even more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 12, 13, 14, 16, 17, 18, 20, 21 , 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 , and 42. In a second aspect, the invention relates to a viral vector expressing antisense oligonucleotide for redirecting splicing as defined herein when placed under conditions conducive to expression of the molecule.

In a third aspect, the invention relates to a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to the invention or a viral vector according to the invention and a pharmaceutically acceptable excipient.

In a fourth aspect, the invention relates to an antisense oligonucleotide for redirecting splicing according to the invention, a vector according the invention or the pharmaceutical composition according the invention for use as a medicament. Preferably for use as a medicament for treating an ABCA4-related disease or a condition requiring modulating splicing of ABCA4. Preferably, the ABCA4-related disease or condition is Stargardt disease.

In a fifth aspect, the invention relates to an antisense oligonucleotide for redirecting splicing according the invention, the vector according invention or the pharmaceutical composition according the invention for treating an ABCA4-related disease or a condition requiring modulating splicing of ABCA4. In a sixth aspect, the invention relates to a method for modulating splicing of ABCA4 in a cell, said method comprising contacting said cell with an antisense oligonucleotide for redirecting splicing as defined herein, the vector according to the invention or the pharmaceutical composition according to the invention. Detailed Description of the invention

By definition, antisense oligonucleotides (AONs) are substantially complementary (antisense) to their target, allowing them to bind to the corresponding pre-mRNA molecule, thereby, without wishing to be being bound by theory, preventing the binding of proteins essential for splicing. Usually, this lack of binding results in the skipping of the targeted exon, as the present inventors have previously shown for several mutations in ABCA4 (WQ2018/109011). In addition, AONs may redirect the splicing machinery towards adjacent splice acceptor or donor sites. This has led the inventors to select ABCA4 mutations that may also be amenable for AON- based splice modulation therapy. These mutations are all deep-intronic variants that create novel splice acceptor, splice donor or exonic splice enhancer binding sites, and result in the inclusion of pseudoexons to the mRNA of the corresponding gene. AONs will be employed to block the recognition of (and thereby induce skipping of) the pseudoexon, thereby fully restoring the wild-type transcript and corresponding protein function.

The following mutations have been selected:

- c.5196+1013A>G. This mutation results in the inclusion of a 129-nt pseudoexon in between exons 36 and 37 of ABCA4.

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

- c.5196+1137G>A. This mutation results in the inclusion of a 73-nt pseudoexon in between exons 36 and 37 of ABCA4. - c.5196+1216A>G. This mutation results in the inclusion of a 73-nt pseudoexon in between exons

36 and 37 of ABCA4.

These four ABCA4 variants in intron 36 have been identified in several patients, in particular the c.5196+1137G>A variant, which was found heterozygously in 15 reported STGD1 cases and in 19 unpublished STGD1 cases. Two of the four variants generate similar pseudoexons, thus the same AON molecules are able to correct the defect caused by two different mutations. The inventors herein show that specific AONs can restore aberrant ABCA4 splicing caused by intron 36 variants. The inventors have provided AONs to modulate splicing for the mutation classes depicted here above; the terms “modulate splicing” and “redirect splicing” are used herein interchangeably and encompass AON-based splice modulation therapy forthe mutations depicted here above. The term “redirecting splicing” is herein defined as redirecting the ABCA4 pre-mRNA splicing to yield the original transcript.

Accordingly, the present invention provides for an antisense oligonucleotide for redirecting splicing that binds to and/or is complementary to a polynucleotide with the nucleotide sequence as shown in SEQ ID NO: 4, preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 8, 9 and 10, more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 5, 6, and 7, even more preferably the antisense oligonucleotide binds to or is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 12, 13, 14, 16, 17, 18, 20, 21 , 22, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 40, 41 , and 42. The terms "antisense oligonucleotide" or “AON” are used interchangeably herein and are understood to refer to an oligonucleotide molecule comprising a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogeneous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. Binding of an AON to its target can easily be assessed by the person skilled in the art using techniques that are known in the field such as the gel mobility shift assay as described in EP1619249.

The term "complementary" used in the context of the invention indicates that some mismatches in the antisense sequence are allowed as long as the functionality, i.e. redirecting splicing is achieved. 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. Optionally, said AON may further be tested by transfection into retina-like cells of patients. The complementary regions are preferably designed such that, when combined, they are specific for the pseudoexon 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 will also 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 system.

The AON according to the invention preferably does not contain a stretch of CpG, more preferably does not contain any CpG. The presence of a CpG or a stretch of CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (Dorn and Kippenberger, 2008). 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 according to 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 AON according to the invention furthermore preferably has 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 oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (www. unc. edu/-cail/biotool/oligo/index) for single stranded RNA using the basic Tm and the nearest neighbor model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). 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.

In all embodiments, a nucleotide in the antisense oligonucleotide according to the invention may be an RNA residue, a DNA residue, or a nucleotide analogue or equivalent, or a combination thereof. A preferred AON for redirecting splicing according to the invention, has a length of from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 23 nucleotides, such as 16, 17, 18, 19, 20, 21 , 22, or 23 nucleotides. In an embodiment, the antisense oligonucleotide for redirecting splicing according the invention comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 11 , 15, 19, 23, 27, 31 , 35 and 39.

In a preferred embodiment, an AON for redirecting the aberrant splicing of ABCA4 that is caused by the c.5196+1013A>G mutation comprises or consists of SEQ ID NO: 27 or SEQ ID NO: 31 . In another preferred embodiment, an AON for redirecting the aberrant splicing of ABCA4 that is caused by the c.5196+1056A>G mutation comprises or consists of an AON selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 31 , SEQ ID NO: 35 and SEQ ID NO; 39. More preferably, the AON for redirecting the aberrant splicing of ABCA4 that is caused by the c.5196+1056A>G mutation is selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 31 and SEQ ID NO: 35

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

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

It is preferred that an AON for redirecting splicing according to the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense oligonucleotide for the target sequence. Therefore, in a preferred embodiment, the AON 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.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided 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. A recent report, demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

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). PNA-based molecules are true mimics of DNA molecules in terms of base-pair 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 (Govindaraju and Kumar, 2005). 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). A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent according to the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H- phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent according to the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a -OH; - F; substituted or unsubstituted, linear or branched lower (CI-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; 0-, S-, or N- allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. 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 of. 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'-0, 4'-C- ethylene-bridged nucleic acid (Morita et al., 2001). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA. In another embodiment, a nucleotide analogue or equivalent according to the invention comprises one or more base modifications or substitutions. 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. 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 aforementioned analogues or equivalents may be incorporated in a single AON or even at a single position within an AON. In certain embodiments, an AON according to the invention has at least two different types of analogues or equivalents. Accordingly, in a preferred embodiment an antisense oligonucleotide for redirecting splicing according to the invention comprises at least one 2'-0 alkyl phosphorothioate antisense oligonucleotide, such as a 2'-0-methyl modified ribose, a 2'-0-ethyl modified ribose, a 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives, preferably the antisense oligonucleotide comprises at least one 2'-0-propyl modified ribose In another preferred embodiment, an antisense oligonucleotide for redirecting splicing according to the invention, comprises a 2'-0 alkyl phosphorothioate antisense oligonucleotide, such as 2'-0- methyl modified ribose, 2'-0-ethyl modified ribose, 2'-0-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives, preferably the antisense oligonucleotide comprises a 2'-0-propyl modified ribose. In a preferred embodiment an antisense oligonucleotide for redirecting splicing according to the invention, comprises a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 11 , and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone. In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 15 and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 19 and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 23 , and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone. In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 27, and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 31 , and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 35 , and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

In an embodiment, an AON for redirecting splicing according to the invention, comprises or consists of SEQ ID NO: 39, and comprises a 2'-0-methyl modified ribose (RNA) and a phosphorothioate backbone.

It will also be understood by a skilled person that different antisense oligonucleotides can be combined for efficient redirection of splicing. In a preferred embodiment the invention comprises a set of AONs for redirecting splicing of ABCA4 according to the invention, preferably such set comprises at least two AONs for redirecting splicing according to the invention. Preferably such set comprises at least two, at least three, at least four antisense oligonucleotides selected from the group consisting of SEQ ID NO: 11 , 15, 19, 23, 27, 31 , 35 and 39.

An AON for redirecting splicing 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 as such, as a so-called ‘naked’ AON. It may also be administered in the form of an expression vector wherein the expression vector encodes an RNA transcript comprising the sequence of said AON according to the invention. The expression vector is preferably 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 for redirecting splicing according to the invention. Accordingly, the invention provides for a viral vector expressing antisense oligonucleotide for redirecting splicing according to the invention when placed under conditions conducive to expression of the molecule.

A cell can be provided with an AON for redirecting splicing according to the invention by plasmid- derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno- associated virus-based vectors. Expression may be driven by an RNA polymerase II promoter (Pol II) such as a U7 RNA promoter or an RNA 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 AON for redirecting splicing according to the invention. Preferred for the invention are those vectors wherein transcription is driven from Poll II 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 Pollll driven transcripts, preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such fusions may be generated as previously described (Gorman et al., 1998).

A preferred expression system for an AON for redirecting splicing according to the invention is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of antisense nucleotide sequences for highly efficient redirection of splicing. A preferred AAV-based vector, for instance, comprises an expression cassette that is driven by an RNA polymerase Ill-promoter (Pol III) or an RNA polymerase II promoter (Pol II). A preferred RNA promoter is, for example, a Pol III U6 RNA promoter, or a Pol II U7 RNA promoter. The invention accordingly provides for a viral-based vector, comprising a Pol II or a Pol III promoter driven expression cassette for expression of an AON for redirecting splicing according to the invention.

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 encoded AON for redirecting splicing 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, AAV8, AAV9 and others. A protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1 , 2, 3, 4, 5, 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 for redirecting splicing 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, see e.g. US 5,139,941 , incorporated herein by reference. 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 according to 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 method according to Garanto et al., 2016 which is herein incorporated by reference.

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

Improvements in means for providing an individual or a cell, tissue, organ of said individual with an AON for redirecting splicing according to the invention, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method according to the invention. An AON for redirecting splicing according to the invention can be delivered as such as a ‘naked’ AON to an individual, a cell, tissue or organ of said individual. When administering an AON for redirecting splicing according to the invention, it is preferred that the molecule is dissolved in a solution that is compatible with the delivery method. Retina cells can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution.

Alternatively, a preferred delivery method for an AON for redirecting splicing or a plasmid for expression of such AON is a viral vector or are nanoparticles. Preferably, viral vectors or nanoparticles are delivered to retina or other relevant cells. Such delivery to retina cells or other relevant cells may be in vivo, in vitro or ex vivo see e.g. Garanto et al., 2016, which is herein incorporated by reference.

Alternatively, a plasmid can be provided by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient or transfection agents 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 agents 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 agentia 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 constitutent as defined herein to a cell, preferably a retina cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as AONs 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 dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.

Polycations such as diethylaminoethylaminoethyl (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 each constituent as defined herein, preferably an AON according to the invention, 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 oligonucleotide. 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 retention molecule for use in the current invention to deliver it for the prevention, treatment or delay of ABCA4-related disease or condition. "Prevention, treatment or delay of an 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 that is caused by a genetic defect in the ABCA4 gene.

In addition, an AON for redirecting splicing according to the invention could be covalently or non- covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognizing cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, an AON for redirecting splicing according to the invention is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery.

It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be suitably formulated in one single combination or composition or preparation. Depending on their identity and specific features, 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 for redirecting splicing according to the invention and a further adjunct compound as later defined herein. If required and/or if desired, an AON for redirecting splicing according to the invention or a vector, preferably a viral vector, according to the invention, expressing an AON for redirecting splicing according to the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.

Accordingly, the invention also provides for a composition, preferably a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to the invention or a viral vector according to the invention and a pharmaceutically acceptable excipient Such composition may comprise a single AON for redirecting splicing or viral vector according to the invention, but may also comprise multiple, distinct AONs for redirecting splicing 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, 2000. Each feature of said composition has earlier been defined herein.

A preferred route of administration is through intravitreal injection of an aqueous solution or specially adapted formulation for intraocular administration. EP2425 814 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. Therefor in one embodiment, there is provided for a pharmaceutical composition suitable for intravitreal administration and dosed in an amount ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg of total antisense oligonucleotides per eye. A suitable intravitreal dose is provided and comprises between 0.05 mg and 5mg, preferably between 0.1 and 1mg of total antisense oligonucleotides per eye, such as about per eye: 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg.

A preferred AON for redirecting splicing according to the invention, is forthe treatment of an ABCA4- related disease or condition of an individual. In all embodiments of the invention, the term "treatment" is understood to include the prevention and/or delay of the ABCA4-related disease or condition. An individual, which may be treated using an AON for redirecting splicing according to the invention may already have been diagnosed as having an ABCA4-related disease or condition. Alternatively, an individual which may be treated using an AON for redirecting splicing 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 being. In all embodiments of the invention, the ABCA4- related disease or condition preferably is Stargardt disease. Accordingly, the invention further provides for an antisense oligonucleotide for redirecting splicing according to the invention, or a viral vector according to the invention, or a (pharmaceutical) composition according to the invention for use as a medicament, preferably as a medicament for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4 and for use as a medicament for the prevention, treatment or delay of an ABCA4-related disease or condition. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for treating an ABCA4-related disease or condition requiring modulating splicing of ABCA4. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for, a method of treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4, comprising said method comprising contacting a cell of said individual with an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for the preparation of a medicament for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for an antisense oligonucleotide for redirecting splicing according to the invention, the use according the invention or the method according to the invention, wherein the ABCA4-related disease or condition is Stargardt disease.

Treatment in a use or in a method according to the invention is preferably at least once, and preferably lasts at least one week, one month, several months, one year, 2, 3, 4, 5, 6 years or longer, such as life-long. Each AON for redirecting splicing according to the invention 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 an 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 according to the invention may depend on several parameters such as the severity of the disease, the age of the patient, the mutation of the patient, the number of AON for redirecting splicing according to the invention (i.e. dose), the formulation of the AON, composition, compound or adjunct compound according to the invention, the route of administration and so forth. The frequency of administration may vary between daily, weekly, at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.

Dose ranges of an AON, composition, compound or adjunct compound according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. An AON according to the invention may be used at a dose which is ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg. A suitable intravitreal dose would be between 0.05 mg and 5mg, preferably between 0.1 and 1 mg per eye, such as about per eye: 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 .0 mg. In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged from 0.1 nM and 1 pM is used. Preferably, this range is for in vitro use in a cellular model such as retina cells or retinal tissue. More preferably, the concentration used is ranged from 1 to 400 nM, even more preferably from 10 to 200 nM, even more preferably from 50 to 100 nM. If multiple distinct AONs are used, this concentration or dose may refer to the total concentration or dose of the AONs or the concentration or the dose of each AON added.

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 1x1 ⁰⁹ — 1x10 ¹⁷ virus particles per injection, more preferably from 1x10 ¹⁰ — 1x10 ¹² virus particles per injection. The ranges of concentration or dose of AONs as depicted 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. An AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention for use according to the invention may be administered to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing a ABCA4-related disease or condition, and may be administered in vivo, ex vivo or in vitro. An AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual already affected by or at risk of developing a ABCA4-related disease or condition, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Stargardt disease has a pronounced phenotype in retina cells, it is preferred that said targeted cells are retina cells, it is further preferred that said tissue is the retina and it is further preferred that said organ comprises or consists of the eye.

The invention further provides for a method for a method for modulating splicing of ABCA4 in a cell, said method comprising contacting the cell, preferably a retina cell, with an antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention or the pharmaceutical composition according to the invention The features of this aspect are preferably those defined earlier herein. Contacting the cell with an AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention may be performed by any method known by the person skilled in the art. Use of the methods for delivery of AONs for redirecting splicing, viral vectors and compositions as described earlier herein is included. Contacting may be directly or indirectly and may be in vivo, ex vivo or in vitro.

Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

Definitions In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting 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 element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the polypeptide obtainable by expression of the gene present in SEQ ID NO: 1 containing the nucleic acid sequence coding for the polypeptide should prevail.

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

Description of the figures

Figure 1: Schematic drawing of intron 36 variants in ABCA4 that underlie STGD1 , and the PE insertions they result in. The c.5196+1013A>G and the c.5196+1056A>G variants result in an overlapping PE with the same splice acceptor but a different splice donor site.

Figure 2: Overview of the PE insertions induced by the different ABCA4 mutations, as well as the relative positions of the eight AONs that were designed. Note that AON7 contains a mismatch towards the 129-nt PE inserted by the c.5196+1013A>G variant, as it was designed specific for the 177-nt PE inserted by the c.5196+1056A>G mutation.

Figure 3: RT-PCR analysis of HEK293T cells transfected with either wild-type or mutant ABCA4 midigenes, as well as with or without AONs or a sense oligonucleotide (SON, negative control). RHO was amplified to demonstrate equal transfection efficiencies of the midigenes. MQ: MilliQ water. A) C.5196+1216A , B) C.5196+1056G, C) C.5196+1013G. Figure 4: RT-PCR analysis of photoreceptor precursor cells (PPCs) transfected with or without AONs or SON. The STGD1 patient was compound heterozygous for the c.5196+1137G>A variant in conjunction with a deletion and missense mutation on the second allele. Actin was amplified to reveal equal cDNA input. CHX: cycloheximide. MQ: MilliQ water.

Description of the sequences Table 1: Sequences

Examples

We have assessed the in vitro efficacy of a number of AONs to redirect splicing of ABCA4 in cells. For this we used midigene constructs, i.e. plasmids that harbour the sequence of a part of the ABCA4 gene, usually the region of interest with or without the mutation, and flanked by at least 100 bp of wild-type ABCA4 sequence on each side. In addition to the midigene assays, for mutation c.5196+1137G>A, we also used iPSC technology to assess the efficacy of the AONs targeting the effects of that mutation.

Materials and Methods

AON design

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

Table 2: AONs

AON testing in HEK293T

Following the design of AONs, HEK293T cells were transfected with midigenes that contain either a part of the wild-type ABCA4 gene (SEQ ID NO: 44), or mutant forms harboring the different target intron 36 mutations described in Table 2, i.e. c.5196+1013A>G, c.5196+1056A>G and c.5196+1216C>A (SEQ ID NO: 45, 46 and 47). For each mutation, wild-type or mutant midigene- transfected cells were treated either without AON (NT), with the AONs designed for the specific PE (AON 1-4 for c.5196+1216C>A, and AON5-8 for c.5196+1013A>G and c.5196+1056A>G), or with a sense oligonucleotide (SON) that acts as a negative control (SEQ ID NO: 43). Forty-eight hours after transfection, cells were harvested and RNA was isolated. Subsequently, RT-PCR analysis was performed to determine which AONs were capable of correcting the splice defects.

RT-PCR analysis

Total RNA was isolated by using the NucleoSpin RNA Clean-up Kit (catalog no., 740955-50; Macherey-Nagel, Diiren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 pg RNA by using the iScript cDNA synthesis kit (catalog no., 1708891 ; Bio-Rad, Hercules, CA) following the manufacturer's instructions. Finally, the efficacy of the AONs was assessed by performing a nested PCR using the ABCA4 primers represented by SEQ ID NO: 48 and SEQ ID NO: 49. To assess transfection efficiency, exon 5 of rhodopsin was amplified using the following primer pair SEQ ID NO: 50 and SEQ ID NO: 51 .

Results

As shown in Figure 3, for each PE insertion, AONs can fully or at least partially restore the splicing defect. More specifically, for the c.5196+1216C>A variant, both AON1 and AON2 fully converted the aberrantly spliced midigene to the correctly spliced version, whereas AON3 and AON4 substantially restored the splicing defect-(Figure 3A). For the c.5196+1056A>G change, AON5 appeared to be the most potent one. For AON7 and to a little lesser extent for AON6, also the majority of PE-containing transcripts were converted. AON8 was partially able to restore the splice defect (Figure 3B). Finally, for the c.5196_1013A>G change, AON5 and AON6 appeared to be the most potent ones. AON7, despite the one nucleotide mismatch between the AON (designed for the other variant) and the PE, was still able to at least partially redirect splicing. For the c.5196_1013A>G change AON8 did not show any efficacy, which given its position outside the 129- nt PE inserted by this variant was not totally unexpected (Figure 3C).

AON testing

Because the resulting PE variant of the c.5196+1137G>A mutation was hardly detectable in the midigene assay iPSC technology was employed to assess the efficacy of the AONs targeting this PE. Blood cells derived from a patient with STGD1 harboring the c.5196+1137G>A variant in conjunction with a partial ABCA4 deletion and a missense mutation (c.[2918+775_3328+640del;4462T>Cj p.[Ser974Glnfs*64;Cys1488Argj) on the other allele were reprogrammed to iPS cells (Sangermano et al, 2016) , and subsequently differentiated to photoreceptor precursor cells (PPCs) for thirty days. At day 28, AONs 1-4 (or SON) were added to the cells at a final concentration of 1 pM. At day 29, cycloheximide (CHX) was added to block nonsense-mediated decay of transcripts harboring premature stop codons. Finally, at day 30, cells were collected and subjected to RNA analysis (as described above). In this case, actin ( ACTB ) was amplified to normalize samples using a forward primer in exon 3 as represented by SEQ ID NO: 52 and a reverse primer in exon 4 as represented by SEQ ID NO: 53. Results

As it can be observed in Figure 4, the PE insertion was more readily detected in the PPCs. In addition, two out of the four AONs that were tested (AON2 and -3) successfully converted PE- containing ABCA4 transcripts into correctly spliced products. Conclusion

For all the different PEs (77-nt, 129-nt and 177-nt AON could effectively restore the splice defect were identified. For c.5196+1013A>G, AON5 and AON6 appear to be the most efficacious ones whereas for c.5196+1056A>G, the PE insertion seems to be best prevented by AON5, AON6 or AON7. For c.5196+1137G>A, AON2 and AON3 seemed to be most potent, at least in the PPCs. Finally for c.5196+1216C>A, AON1 and AON2 fully restored the splice defect, but also AON3 and AON4 displayed splicing correction. References

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