Antisense oligonucleotides for the treatment of Stargardt disease - Patent Application 20070223333

Abstract

The present invention relates to the field of medicine. In particular, the present invention relates to novel antisense oligonucleotides that can be used in the treatment, prevention and / or delay of Stargardt disease.

Description

[Technical field]

[0001] The present invention relates to the field of medicine. In particular, the present invention relates to novel antisense oligonucleotides that can be used in the treatment, prevention and / or delay of Stargardt's disease. [Background technology]

[0002] Autosomal recessive mutations in ABCA4 are responsible for Stargardt disease, a progressive disorder characterized by loss of central vision, often resulting in total blindness. A typical feature of Stargardt disease is the presence of many macula (spots) distributed throughout the fundus of the patient's eye. The ABCA4 gene is composed of 50 exons and encodes a protein of 2273 amino acids. This protein is expressed in the outer segments of cone and rod photoreceptor cells and plays an important role in the clearance of waste products after phototransduction.

[0003] In the case of STGD1, the ABCA4 transcript is bound to the high-frequency transcription factor Free Radiation Cotton Cancer (arCRD;Cremers, FPM, van de Pol, DJ, van Driel, M, den Hollander, AI, van Haren, FJ, Knoers, NV, Tijmes, N, Bergen, AA, Rohrschneider, K, Blankenagel, A, Pinckers, AJ, Deutman, A; [ PubMed ] Brunner , HG , Deutman , AF , Hoyng , CB & Cremers , The major cause of autosomal recessive cone-rod dystrophy , J. Genet , 960–966 . doi:10.1086 / 303079) in the manufacture of skeletal muscle (Cremers, FPM, van de Pol, DJ, van Driel, M., den Hollander, AI, van Haren, FJ, Knoers, NV, Tijmes, N., Bergen, AA, Rohrschneider, K. , Blankenagel , A. , Pinckers , A. , Deutman , A. ;Martinez-Mir , A. , Paloma , E. , Allikmets , R. , Ayuso , C. , del Rio , T. , Dean , M. , Vilageliu , L. , Gonzalez-Duarte , R. & Balcells , S. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR Nat.18, 11-12 (1998), doi:10.1038 / ng0198-11;Shroyer et al, 2001;Duncker et al, 2014).

[0004] Biallelic ABCA4 variants account for approximately 80% of cases involving STGD1 after sequencing of the coding region and adjacent splice sites (Allikmets, R., Singh, N., Sun, H., Shroyer, NF, Hutchinson, A., Chidambaram, A., Gerrard, B., Baird, L., Stauffer, D., Peiffer, A., Rattner, A., Smallwood, P., Li, Y., Anderson, KL, Lewis, RA, Nathans, J., Leppert, M., Dean, M. & Lupski, J. R. A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat. Genet. 15, 236-246 (1997), doi:10.1038 / ng0397-236;Fujinami, K., Zernant, J., Chana, RK, Wright, GA, Tsunoda, K., Ozawa, Y., Tsubota, K., Webster, AR, Moore, AT, Allikmets, R. & Michaelides, M. ABCA4 gene screening by next-generation sequencing in a British cohort. Invest. Ophthalmol. Vis. Sci. 54, 6662-6674 (2013), doi:10.1167 / iovs.13-12570;Lewis, RA, Shroyer, NF, Singh, N., Allikmets, R., Hutchinson, A., Li, Y., Lupski, JR, Leppert, M. & Dean, M.Genotype / Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am. J. Hum. Genet. 64, 422-434 (1999), doi:10.1086 / 302251;Maugeri, A., van Driel, M. A., van de Pol, D. J., Klevering, B. J., van Haren, F. J., Tijmes, N., Bergen, A. A., Rohrschneider, K., Blankenagel, A., Pinckers, A. J., Dahl, N., Brunner, H. G., Deutman, A. F., Hoyng, C. B. & Cremers, F. P. M. The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am. J. Hum. Genet. 64, 1024-1035 (1999);Rivera, A., White, K., Stohr, H., Steiner, K., Hemmrich, N., Grimm, T., Jurklies, B., Lorenz, B., Scholl, H. P., Apfelstedt-Sylla, E. & Weber, B. H. A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration. Am. J. Hum. Genet. 67, 800-813 (2000), doi:10.1086 / 303090;Schulz, H. L., Grassmann, F., Kellner, U., Spital, G., Ruther, K., Jagle, H., Hufendiek, K., Rating, P., Huchzermeyer, C., Baier, M. J., Weber, B. H. & Stohr, H. Mutation spectrum of the ABCA4 gene in 335 Stargardt disease patients from a multicenter German cohort-impact of selected deep intronic variants and common SNPs. Invest. Ophthalmol. Vis. Sci. 58, 394-403 (2017), doi:10.1167 / iovs.16-19936;Webster, A. R., Heon, E., Lotery, A. J., Vandenburgh, K., Casavant, T. L., Oh, K. T., Beck, G., Fishman, G. A., Lam, B. L., Levin, A., Heckenlively, J. R., Jacobson, S. G., Weleber, R. G., Sheffield, V. C. & Stone, E. M. An analysis of allelic variation in the ABCA4 gene. Invest. Ophthal. Vis. Sci. 42, 1179-1189 (2001);Zernant, J., Schubert, C., Im, K. M., Burke, T., Brown, C. M., Fishman, G. A., Tsang, S. H., Gouras, P., Dean, M. & Allikmets, R. Analysis of the ABCA4 gene by next-generation sequencing. Invest. Ophthalmol. Vis. Sci. 52, 8479-8487 (2011), doi:10.1167 / iovs.11-8182;Zernant, J., Lee, W., Collison, FT, Fishman, GA, Sergeev, YV, Schuerch, K., Sparrow, JR, Tsang, SH & Allikmets, R. Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration. J. Med. Genet. 54, 404-412 (2017), doi:10.1136 / jmedgenet-2017-104540) and arCRD (Maugeri, A., Klevering, BJ, Rohrschneider, K., Blankenagel, A., Brunner, HG, Deutman, AF, Hoyng, CB & Cremers, FPM Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am. J. Hum. Genet. 67, 960-966 (2000), doi:10.1086 / 303079). Typically, individuals with arCRD or panretinal dystrophy carry two severe ABCA4 alleles, while individuals with STGD1 carry two moderate variants or a combination of a mild and a severe variant (Maugeri, A., van Driel, MA, van de Pol, DJ, Klevering, BJ, van Haren, FJ, Tijmes, N., Bergen, AA, Rohrschneider, K., Blankenagel, A., Pinckers, AJ, Dahl, N., Brunner, HG, Deutman, AF, Hoyng, CB& Cremers, FPM The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease. Am. J. Hum. Genet. 64, 1024-1035 (1999);van Driel, MA, Maugeri, A., Klevering, BJ, Hoyng, CB & Cremers, FPM ABCR unites what ophthalmologists divide(s). Ophthalmic Genet. 19, 117-122 (1998)). It has been hypothesized that the majority of ABCA4 deletion 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, M., De Zaeytijd, J., Weisschuh, N., Kohl, S., Meire, F., Dahan, K., Depasse, F., De Jaegere, S., De Ravel, T., De Rademaeker, M., Loeys, B., Coppieters, F., Leroy, BP & De Baere, E. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutat. 36, 39-42 (2015), doi:10.1002 / humu.22716;Bax, NM, Sangermano, R., Roosing, S., Thiadens, AA, Hoefsloot, LH, van den Born, LI, Phan, M., Klevering, B. J., Westeneng-van Haaften, C., Braun, T. A., Zonneveld-Vrieling, M. N., de Wijs, I., Mutlu, M., Stone, E. M., den Hollander, A. I., Klaver, C. C., Hoyng, C. B. & Cremers, F. P. M. Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum. Mutat. 36, 43-47 (2015), doi:10.1002 / humu.22717;Braun, T. A., Mullins, R. F., Wagner, A. H., Andorf, J. L., Johnston, R. M., Bakall, B. B., Deluca, A. P., Fishman, G. A., Lam, B. L., Weleber, R. G., Cideciyan, A. V., Jacobson, S. G., Sheffield, V. C., Tucker, B. A. & Stone, E. M. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367;Lee, W., Xie, Y., Zernant, J., Yuan, B., Bearelly, S., Tsang, S. H., Lupski, J. R. & Allikmets, R. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet.135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y;Schulz, HL, Grassmann, F., Kellner, U., Spital, G., Ruther, K., Jagle, H., Hufendiek, K., Rating, P., Huchzermeyer, C., Baier, MJ, Weber, BH & Stohr, H. Mutation spectrum of the ABCA4 gene in 335 Stargardt disease patients from a multicenter German cohort-impact of selected deep intronic variants and common SNPs. Invest. Ophthalmol. Vis. Sci. 58, 394-403 (2017), doi:10.1167 / iovs.16-19936;Zernant et al, 2014). In 2013, Braun and coworkers (Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367) identified two variants in intron 30 (c.4539+2001G>A and c.4539+2028C>T, respectively, are denoted as M1 and M2 below.) has been described, but no experimental evidence has been provided yet. So far, M2 has been identified in 13 cases (Bauwens, M., De Zaeytijd, J., Weisschuh, N., Kohl, S., Meire, F., Dahan, K., Depasse, F., De Jaegere, S., De Ravel, T., De Rademaeker, M., Loeys, B., Coppieters, F., Leroy, B. P. & De Baere, E. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutat. 36, 39-42 (2015), doi:10.1002 / humu.22716;Bax, N. M., Sangermano, R., Roosing, S., Thiadens, A. A., Hoefsloot, L. H., van den Born, L. I., Phan, M., Klevering, B. J., Westeneng-van Haaften, C., Braun, T. A., Zonneveld-Vrieling, M. N., de Wijs, I., Mutlu, M., Stone, E. M., den Hollander, A. I., Klaver, C. C., Hoyng, C. B. & Cremers, F. P. M. Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum. Mutat. 36, 43-47 (2015), doi:10.1002 / humu.22717;Braun, T. A., Mullins, R. F., Wagner, A. H., Andorf, J. L., Johnston, R. M., Bakall, B. B., Deluca, A. P., Fishman, G. A., Lam, B. L., Weleber, R. G., Cideciyan, A. V., Jacobson, S. G., Sheffield, V. C., Tucker, B. A. & Stone, E. M. Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367;Lee, W., Xie, Y., Zernant, J., Yuan, B., Bearelly, S., Tsang, S. H., Lupski, J. R. & Allikmets, R. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet. 135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y;Schulz, H. L., Grassmann, F., Kellner, U., Spital, G., Ruther, K., Jagle, H., Hufendiek, K., Rating, P., Huchzermeyer, C., Baier, M. J., Weber, B. H. & Stohr, H. Mutation spectrum of the ABCA4 gene in 335 Stargardt disease patients from a multicenter German cohort-impact of selected deep intronic variants and common SNPs. Invest. Ophthalmol. Vis. Sci. 58, 394-403 (2017), doi:10.1167 / iovs.16-19936; Zernant et al., 2014). M1 was found in 31 cases and, interestingly, was particularly frequent in the Dutch and Belgian populations (Bauwens, M., De Zaeytijd, J., Weisschuh, N., Kohl, S., Meire, F., Dahan, K., Depasse, F., De Jaegere, S., De Ravel, T., De Rademaeker, M., Loeys, B., Coppieters, F., Leroy, B. P. & De Baere, E. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutat. 36, 39-42 (2015), doi:10.1002 / humu.22716; Bax, N. M., Sangermano, R., Roosing, S., Thiadens, A. A., Hoefsloot, L. H., van den Born, L. I., Phan, M., Klevering, B. J., Westeneng-van Haaften, C., Braun, T. A., Zonneveld-Vrieling, M. N., de Wijs, I., Mutlu, M., Stone, E. M., den Hollander, A. I., Klaver, C. C., Hoyng, C. B. & Cremers, F. P. M. Heterozygous deep-intronic variants and deletions. in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum. Mutat. 36, 43-47 (2015), doi:10.1002 / humu.22717;Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367;Lee, W., Xie, Y., Zernant, J., Yuan, B., Bearelly, S., Tsang, SH, Lupski, JR & Allikmets, R. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet. 135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y;Zernant et al, 2014). Furthermore, we identified several additional deep intronic ABCA4 mutations that all lead to the insertion of pseudoexons by activating cryptic acceptor or splice donor sites or by reinforcing ESE motifs located within the pseudoexon.These additional mutations include c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T, and c.5197-557G>T.

[0005] Currently, several clinical trials on STGD1 are being conducted using various therapeutic strategies (http: / / www.clinicaltrials.gov): i) gene replacement therapy by delivering the complete ABCA4 cDNA (approximately 6.8 kb) by lentiviral vector (NCT01367444 and NCT01736592); ii) subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) (NCT02445612 and NCT02941991) and iii) administration of C20-D3-retinyl acetate (NCT02402660). Each of these approaches has its limitations, and to date, no valid data has been reported from these clinical trials.

[0006] Because a significant amount of mutations in ABCA4 affect pre-mRNA splicing of ABCA4, they represent attractive targets for antisense oligonucleotide (AON)-based splice modulation therapy. Thus, there is a need to develop AONs for splice modulation of the ABCA4 gene that allow expression of functional ABCA4 protein in subjects suffering from Stargardt disease. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cremers, FPM, van de Pol, DJ, van Driel, M., den Hollander, AI, van Haren, FJ, Knoers, NV, Tijmes, N., Bergen, AA, Rohrschneider, K., Blankenagel, A., Pinckers, AJ, Deutman, A.

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[0008] The present invention relates to - is complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof; - preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 11 or SEQ ID NO: 31, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 12 or SEQ ID NO: 32, or a part thereof; - more preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 16, 19, 163, 166, 169, 33, 36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143, 146, 149, 263, 266 and SEQ ID NO: 269, and portions thereof; more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 164, 167, 170, 34, 37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144, 147, 150, 264 and SEQ ID NO: 270, and parts thereof, Antisense oligonucleotides are provided to redirect splicing.

[0009] The present invention further provides an antisense oligonucleotide for redirecting splicing according to any of the preceding claims, comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 128, 131, 145, 148, 151, 265, 268 and SEQ ID NO: 271.

[0010] The present invention further provides viral vectors which, when placed under conditions conducive to expression of the exon skipping antisense oligonucleotide, express an antisense oligonucleotide for redirecting splicing according to the present invention.

[0011] The present invention further provides a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to the present invention or a viral vector according to the present invention and a pharma- ceutically acceptable excipient.

[0012] The present invention further provides an antisense oligonucleotide for redirecting splicing according to the present invention, a vector according to the present invention and a composition according to the present invention for use as a medicament.

[0013] The present invention further provides antisense oligonucleotides for redirecting splicing according to the present invention, vectors according to the present invention and compositions according to the present invention for use in the treatment of an ABCA4-related disease or condition which requires modulating the splicing of ABCA4.

[0014] The present invention further provides the use of an antisense oligonucleotide for redirecting splicing according to the present invention, a vector according to the present invention and a composition according to the present invention for the preparation of a medicament.

[0015] The present invention further provides the use of the antisense oligonucleotides for redirecting splicing according to the present invention, the vectors according to the present invention and the compositions according to the present invention for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4.

[0016] The present invention further provides a method for modulating splicing of ABCA4 in a cell, comprising contacting said cell with an antisense oligonucleotide for redirecting splicing according to the present invention, a vector according to the present invention and a composition according to the present invention.

[0017] The present invention further provides a method for the treatment of an ABCA4-related disease or condition requiring modulating the splicing of ABCA4 in an individual in need thereof, comprising contacting cells of said individual with an antisense oligonucleotide for redirecting splicing according to the present invention, a vector according to the present invention and a composition according to the present invention. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the results of Example A, in which rescue of the splice defects caused by the ABCA4 mutations c.4539+1100A>G and c.4539+1106C>T was achieved by delivery of AONs in a minigene assay. [Diagram 2] FIG. 1 shows the results of Example C, in which rescue of the splice defect caused by the ABCA4 mutation c.4539+2001G>A by AONs was achieved by delivery of AONs to cultured patient-derived photoreceptor progenitor cells. [Diagram 3] FIG. 1 shows the results of Example B, in which rescue of the splice defect caused by the ABCA4 mutation c.4539+2001G>A by AONs was achieved by delivery of AONs in a minigene assay. [Figure 4A-4B] (A) Results from Example C, in which splice defects caused by ABCA4 mutations c.4539+2001G>A (M1) and c.4539+2028C>T (M2) are identified. Aberrantly spliced ​​bands were detected, especially after cycloheximide (CHX) treatment (+). Actin (ACTB) RT-PCR was used as a control. (B) Quantification of the ratio of correctly spliced ​​to aberrantly spliced ​​ABCA4 transcripts for cell lines with and without CHX, respectively. [Diagram 5] Figure 2: In silico characterization of the effect caused by deep intronic variants M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T). The boundaries of the 345 bp pseudoexon with the positions of M1 and M2, the genomic positions of the splice sites, the splicing events detected, and the splice site predictions for both acceptor and donor are represented diagrammatically. The dashed line represents the splicing from the cryptic splice donor site in exon 30 at position g.94,495,074 (GRCh37 / hg19) to the normal splice acceptor site in exon 31 (r.4467_4539del, p.Cys1490Glufs*12). The predicted values ​​of the splice acceptor and donor sites in the control and mutant context did not show any differences. In the middle panel, the effect of variants that enhance or create new ESE motifs is shown. Splice Site Finder-like (SSFL) and Human Splicing Finder (HSF). [Figures 6A-6D](A) Schematic of variant location, SC35 motif with highest score and pseudoexon showing antisense oligonucleotide (AON). (B) RNA analysis for cells treated with AON. RT-PCR of ABCA4 exon 30 to exon 31 in control, M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T) containing photoreceptor progenitor cells (PPCs) for AON delivery. Samples were normalized using actin (ACTB) mRNA amplification. NT-: untreated and cycloheximide (CHX); NT+: untreated in the presence of CHX; A1: AON1; A2: AON2; A3: AON3; A4: AON4; S: SON and MQ: PCR negative controls. (C) Semiquantification of the ratio of correctly spliced ​​versus aberrantly spliced ​​transcripts in all M1 and M2 samples. (D) Percentage of correction for each AON compared to NT+ based on the ratios observed in Figure 3C. Statistically significant differences in efficacy of AONs against M1 and M2 are indicated with an asterisk (*: p ≤ 0.05 using Mann-Whitney test). [Figure 7A-7B] (A) Gene expression profiles of one control and M1 / M2 derived induced pluripotent stem cells (iPSCs) compared to their respective parental fibroblast cell lines. (B) Gene expression profiles of one control and M1 / M2 derived photoreceptor progenitor cells (PPCs) after 1 month of differentiation compared to iPSCs. The emergence of PPCs can be inferred by increased CRX expression. Differentiation towards photoreceptor-like cells is indicated by increased expression of OPN1SW, OPN1M / LW, RCV1 and ABCA4 compared to the pluripotency genes Oct3 / 4. Results are shown as mean ± SD. All data were plotted against expression of ACTB. [Figure 8] Figure 2: RT-PCR analysis of ABCA4 exon 2 to exon 5 in control (CON), M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T) photoreceptor progenitor cells in the absence (-) and presence (+) of CHX. Human adult retina RNA was used as a control, while MQ was the negative control for the reaction. [Figure 9] Screening of a total of 26 AON sequences for their ability to correct the splice defect caused by the c.4539+2001G>A mutation. (A) Representative electrophoretic picture of RT-PCR to detect exon 30 to exon 31 of ABCA4 in patient-derived PPC. The lower band represents the correct transcript, whereas the upper band represents the aberrant transcript. The aberrant bands were detected after cycloheximide (CHX) treatment, indicating that these transcripts undergo nonsense-mediated decay (NMD). 26 different AON molecules were delivered together with two SONs ​​(negative controls designated SON1 and SON2) into cells. Results were compared to untreated cells (NT) in the presence of CHX (+CHX). Actin was used as a loading control. MQ describes the negative control of PCR. Representation of the percentage of (B) correct (corrected) and (C) aberrant transcripts after semi-quantification of two independent replicates. Based on this percentage, AON molecules were classified as effective (solid grey), moderately effective (polka dots), poorly effective (stripes) and ineffective (crosses). Solid, dotted and dashed lines indicate the thresholds that determine the effectiveness of different AONs. Controls showing basal levels of aberrant transcripts are in white. In black are samples that were not treated with AONs and were not subjected to CHX treatment. [Figure 10] Figure 1 shows the screening of AONs for seven mutations in the ABCA4 gene that cause the inclusion of a pseudoexon. To insert this mutation found in humans, the genomic region containing the midi gene was mutagenized. These midi genes were then transfected into HEK293T cells, and after 24 hours, different AONs were delivered to these cells. Analysis was performed by RT-PCR. For every variant, three AONs were designed and SON was delivered as a negative control. NT describes untreated and represents transfected cells that were not subjected to AON treatment. HEK lane is an extra negative control that is made for untransfected HEK293T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Array Description Sequence number Name: 1 Genomic DNA ABCA4 2 cDNA ABCA4 3. Protein ABCA4 10 Pseudoexon 30-31(68) RNA 11 Pseudoexon 30-31(68) RNA; smaller target 12 Pseudoexon 30-31 (68) RNA; smaller target (AON area +10) 13 AON-1 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 14 AON-1 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 15 AON-1 pseudoexon 30-31 (68) 16 AON-2 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 17 AON-2 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 18 AON-2 pseudoexon 30-31 (68) 19 AON-3 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 20 AON-3 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 21 AON-3 pseudoexon 30-31 (68) 30 Pseudoexon 30-31 (345) RNA 31 Pseudoexon 30-31 (345) RNA; smaller target 32 Pseudoexon 30-31 (345) RNA; smaller target (AON area +10) 33 AON-1 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 34 AON-1 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 35 AON-1 pseudoexon 30-31 (345) 36 AON-2 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 37 AON-2 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 38 AON-2 pseudoexon 30-31 (345) 39 AON-3 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 40 AON-3 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 41 AON-3 pseudoexon 30-31 (345) 42 AON-4 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 43 AON-4 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 44 AON-4 pseudoexon 30-31 (345) 45 SON-1 pseudoexon 30-31 (345) Sense version of SEQ ID NO:35 50 pCI-Neo-Rho-ABCA4-30-31 wild type 51 pCI-Neo-Rho-ABCA4-30-31 c.4539+1100G 52 pCI-Neo-Rho-ABCA4-30-31 c.4539+1106T 53 pCI-Neo-Rho-ABCA4-30-31 c.4539+2001A 54 ABCA4_ex2 Fw 55 ACTB_ex3 Fw 56 ABCA4_ex30 Fw 57 ABCA4_ex20 / 21 Fw 58 CRX Fw 59 LIN28 Fw 60 NANOG Fw 61 OCT4 Fw 62 OPN1M / LW Fw 63 OPN1SW Fw 64 RCV1 Fw 65 SOX2 Fw 66 ABCA4_ex5 Rv 67 ACTB_ex4 Rv 68 ABCA4_ex31 Rv 69 ABCA4_ex21 Rv 70 CRX Rv 71 LIN28 Rv 72 NANOG Rv 73 OCT4 Rv 74 OPN1M / LW Rv 75 OPN1SW Rv 76 RCV1 Rv 77 SOX2 Rv 80 Pseudoexon 6-7 (162) 81 Pseudoexon 6-7 (162) Larger target + adjacent sequence (+50nt) 82 pseudoexons 6-7 (162) larger target + flanking sequence (+20nt) 83 AON-1 pseudoexon 6-7 (162) target site and adjacent sequence (+10nt) 84 AON-1 pseudoexon 6-7 (162) target site and adjacent sequence (+5nt) 85 AON-1 pseudoexon 6-7 (162) 86 AON-2 pseudoexon 6-7 (162) target site and adjacent sequence (+10nt) 87 AON-2 pseudoexon 6-7 (162) target site and adjacent sequence (+5nt) 88 AON-2 pseudoexon 6-7 (162) 89 AON-3 Pseudoexon 6-7 (162) Target site and adjacent sequence (+10nt) 90 AON-3 pseudoexon 6-7 (162) target site and adjacent sequence (+5nt) 91 AON-3 pseudoexon 6-7 (162) 100 Pseudoexon 7-8 (141) 101 Pseudoexon 7-8 (141) Larger target + adjacent sequence (+50nt) 102 Pseudoexon 7-8 (141) Larger target + adjacent sequence (+20nt) 103 AON-1 pseudoexon 7-8 (141) target site and adjacent sequence (+10nt) 104 AON-1 pseudoexon 7-8 (141) target site and adjacent sequence (+5nt) 105 AON-1 pseudoexon 7-8 (141) 106 AON-2 pseudoexon 7-8 (141) target site and adjacent sequence (+10nt) 107 AON-2 pseudoexon 7-8 (141) target site and adjacent sequence (+5nt) 108 AON-2 pseudoexon 7-8 (141) 109 AON-3 Pseudoexon 7-8 (141) Target site and adjacent sequence (+10nt) 110 AON-3 pseudoexon 7-8 (141) target site and adjacent sequence (+5nt) 111 AON-3 pseudoexon 7-8 (141) 120 Pseudoexon 7-8 (56) 121 Pseudoexon 7-8 (56) Larger target + adjacent sequence (+50nt) 122 Pseudoexon 7-8 (56) Larger target + adjacent sequence (+20nt) 123 AON-1 pseudoexon 7-8 (56) target site and adjacent sequence (+10nt) 124 AON-1 pseudoexon 7-8 (56) target site and adjacent sequence (+5nt) 125 AON-1 pseudoexon 7-8 (56) 126 AON-2 pseudoexon 7-8 (56) target site and adjacent sequence (+10nt) 127 AON-2 pseudoexon 7-8 (56) target site and adjacent sequence (+5nt) 128 AON-2 pseudoexon 7-8 (56) 129 AON-3 pseudoexon 7-8 (56) target site and adjacent sequence (+10nt) 130 AON-3 pseudoexon 7-8 (56) target site and adjacent sequence (+5nt) 131 AON-3 pseudoexon 7-8 (56) 140 Pseudoexon 13-14 (134) 141 Pseudoexon 13-14 (134) Larger target + adjacent sequence (+50nt) 142 Pseudoexon 13-14 (134) Larger target + adjacent sequence (+20nt) 143 AON-1 pseudoexon 13-14 (134) target site and adjacent sequence (+10nt) 144 AON-1 pseudoexon 13-14 (134) target site and adjacent sequence (+5nt) 145 AON-1 pseudoexon 13-14 (134) 146 AON-2 pseudoexon 13-14 (134) target site and adjacent sequence (+10nt) 147 AON-2 pseudoexon 13-14 (134) target site and adjacent sequence (+5nt) 148 AON-2 pseudoexon 13-14 (134) 149 AON-3 pseudoexon 13-14 (134) target site and adjacent sequence (+10nt) 150 AON-3 pseudoexon 13-14 (134) target site and adjacent sequence (+5nt) 151 AON-3 pseudoexon 13-14 (134) 160 Pseudoexon 30-31 (68) 161 Pseudoexon 30-31 (68) Larger target + adjacent sequence (+50nt) 162 pseudoexon 30-31 (68) larger target + adjacent sequence (+20nt) 163 AON-1 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 164 AON-1 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 165 AON-1 pseudoexon 30-31 (68) 166 AON-2 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 167 AON-2 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 168 AON-2 pseudoexon 30-31 (68) 169 AON-3 pseudoexon 30-31 (68) target site and adjacent sequence (+10nt) 170 AON-3 pseudoexon 30-31 (68) target site and adjacent sequence (+5nt) 171 AON-3 pseudoexon 30-31 (68) 180 Pseudoexon 30-31 (345) 181 Pseudoexon 30-31 (345) Larger target + adjacent sequence (+20nt) 182 AON-1 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 183 AON-1 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 184 AON-1 pseudoexon 30-31 (345) 185 AON-2 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 186 AON-2 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 187 AON-2 pseudoexon 30-31 (345) 188 AON-3 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 189 AON-3 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 190 AON-3 pseudoexon 30-31 (345) 191 AON-4 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 192 AON-4 pseudoexon 30-31 (345) target site and adjacent sequence (+5nt) 193 AON-4 pseudoexon 30-31 (345) 194 AON-5 pseudoexon 30-31 (345) target site and adjacent sequence (+10nt) 195 AON-5 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 196 AON-5 pseudoexon 30-31 (345) 197 AON-6 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 198 AON-6 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 199 AON-6 pseudoexon 30-31 (345) 200 AON-7 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 201 AON-7 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 202 AON-7 pseudoexon 30-31 (345) 203 AON-8 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 204 AON-8 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 205 AON-8 pseudoexon 30-31 (345) 206 AON-9 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 207 AON-9 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 208 AON-9 pseudoexon 30-31 (345) 209 AON-10 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 210 AON-10 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 211 AON-10 pseudoexon 30-31 (345) 212 AON-11 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 213 AON-11 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 214 AON-11 pseudoexon 30-31 (345) 215 AON-12 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 216 AON-12 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 217 AON-12 pseudoexon 30-31 (345) 218 AON-13 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 219 AON-13 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 220 AON-13 pseudoexon 30-31 (345) 221 AON-14 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 222 AON-14 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 223 AON-14 pseudoexon 30-31 (345) 224 AON-15 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 225 AON-15 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 226 AON-15 pseudoexon 30-31 (345) 227 AON-16 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 228 AON-16 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 229 AON-16 pseudoexon 30-31 (345) 230 AON-17 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 231 AON-17 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 232 AON-17 pseudoexon 30-31 (345) 233 AON-18 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 234 AON-18 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 235 AON-18 pseudoexon 30-31 (345) 236 AON-19 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 237 AON-19 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 238 AON-19 pseudoexon 30-31 (345) 239 AON-20 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 240 AON-20 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 241 AON-20 pseudoexon 30-31 (345) 242 AON-21 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 243 AON-21 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 244 AON-21 pseudoexon 30-31 (345) 245 AON-22 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 246 AON-22 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 247 AON-22 pseudoexon 30-31 (345) 248 AON-23 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 249 AON-23 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 250 AON-23 pseudoexon 30-31 (345) 251 AON-24 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 252 AON-24 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 253 AON-24 pseudoexon 30-31 (345) 254 AON-25 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 255 AON-25 Pseudoexon 30-31 (345) Target site and adjacent sequences (+5nt) 256 AON-25 pseudoexon 30-31 (345) 257 AON-26 Pseudoexon 30-31 (345) Target site and adjacent sequence (+10nt) 258 AON-26 Pseudoexon 30-31 (345) Target site and adjacent sequence (+5nt) 259 AON-26 pseudoexon 30-31 (345) 260 Pseudoexon 36-37 (188) 261 Pseudoexon 36-37 (188) Larger target + adjacent sequence (+50nt) 262 Pseudoexon 36-37 (188) Larger target + adjacent sequence (+20nt) 263 AON-1 pseudoexon 36-37 (188) target site and adjacent sequence (+10nt) 264 AON-1 pseudoexon 36-37 (188) target site and adjacent sequence (+5nt) 265 AON-1 pseudoexon 36-37 (188) 266 AON-2 pseudoexon 36-37 (188) target site and adjacent sequence (+10nt) 267 AON-2 pseudoexon 36-37 (188) target site and adjacent sequence (+5nt) 268 AON-2 pseudoexon 36-37 (188) 269 ​​AON-3 pseudoexon 36-37 (188) target site and adjacent sequence (+10nt) 270 AON-3 pseudoexon 36-37 (188) target site and adjacent sequence (+5nt) 271 AON-3 pseudoexon 36-37 (188) 280 SON-1 (c.4539+2001G>A, sense version of AON1 30-31 (345)) 281 SON-2 (c.4539+2001G>A, sense version of AON4 30-31 (345)) 282 SON-3 (c.1937+435C>G, sense version of AON2 13-14 (134) 290 pCI-Neo-Rho-ABCA4-intron6-intron7 wild type 291 pCI-Neo-Rho-ABCA4-intron6-intron7 c.769-784T 292 pCI-Neo-Rho-ABCA4-intron6-intron11 wild type 293 pCI-Neo-Rho-ABCA4-intron6-intron11 c.859-540G 294 pCI-Neo-Rho-ABCA4-intron6-intron11 c.859-506C 295 pCI-Neo-Rho-ABCA4-intron 11-intron 15 wild type 296 pCI-Neo-Rho-ABCA4-intron 11-intron 15 c.1937+435G 297 pCI-Neo-Rho-ABCA4-intron29-intron32 wild type 298 pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539+1100G 299 pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539+1106T 300 pCI-Neo-Rho-ABCA4-intron 31-intron 37 wild type 301 pCI-Neo-Rho-ABCA4-intron31-intron37 c.5197-557T 302 RHO_ex3 fw 303 ABCA4_ex7 rev 304 ABCA4_ex7 fw 305 ABCA4_ex8 rev 306 ABCA4_ex13 fw 307 ABCA4_ex14 rev 308 ABCA4_ex30 fw 309 ABCA4_ex32 rev 310 ABCA4_ex32 fw 311 ABCA4_ex37 rev

[0020] [Table 1] JPEG0007659291000002.jpg245161JPEG0007659291000003.jpg166161

[0021] Some mutations are located within pseudoexons (e.g., if the mutation creates an ESE that in turn creates a pseudoexon, the mutation is part of the pseudoexon). AONs designed to redirect splicing will have a mismatch with respect to the wild-type sequence at the site of the mutation. This is the case for AONs with SEQ ID NOs: 35 / 184, 131 and 226 and SONs ​​with SEQ ID NOs: 45 / 280, where the mutations with respect to the wild-type sequence are shown in bold and underlined.

[0022] [Table 2]

[0023] Table 2 describes the characteristics of 26 AONs tested for their efficacy in redirecting PE inclusion with the c.4539+2001G>A change. AONs are listed from the 5' to 3' end of the pseudoexon. Column 2 lists the location relative to the PE. Columns 3-6 list the number of predicted exonic splice enhancer motifs, i.e., SF2, SC35, SRp40, and SRp55, that overlap with the corresponding AON. Column 7 lists the RNA configuration at the location of the AON, i.e., open, closed, or mixed configuration.

[0024] Detailed Description of the Invention By definition, AONs are substantially complementary to their targets (antisense) and are able to bind to the corresponding pre-mRNA molecules, thereby preventing the binding of proteins essential for splicing. As we previously showed for the c.2991+1655A>G mutation in CEP290 (Collin, RW, den Hollander, AI, van der Velde-Visser, SD, Bennicelli, J., Bennett, J., and Cremers, FP (2012). Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290. Molecular therapy Nucleic acids 1, e14;Garanto, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular Genetics 25, 2552-2563), and the lack of this binding usually leads to skipping of the target exon. Furthermore, AONs can redirect the splicing mechanism to adjacent splice acceptor or donor sites. This led us to select ABCA4 mutations that may also be suitable for AON-based splice modulation therapy. All of these mutations are deep intronic variants that create novel splice acceptor, splice donor or exon splice enhancer binding sites, resulting in the inclusion of pseudoexons in the mRNA of the corresponding gene.AONs are used to block the recognition of the pseudoexon (thereby inducing the skipping of the pseudoexon), thereby fully restoring the wild-type transcript and the corresponding protein function. The following mutations have been selected: - c.769-784C>T. This mutation results in a 162 nt pseudoexon insertion between exons 6 and 7 of ABCA4. - c.859-540C>G. This mutation results in a 141 nt pseudoexon insertion between exons 7 and 8 of ABCA4. - c.859-506G>C. This mutation results in a 56 nt pseudoexon insertion between exons 7 and 8 of ABCA4. - c.1937+435C>G. This mutation results in a 134 nt pseudoexon insertion between exons 13 and 14 of ABCA4. - c.4539+1100A>G and c.4539+1106C>T. These mutations result in the same insertion of a 68 nt pseudoexon between exons 30 and 31 of ABCA4 and can therefore be processed by the same AON. - c.4539+2001G>A and c.4539+2028C>T. These mutations result in the same insertion of a 345 nt pseudoexon between exons 30 and 31 of ABCA4 and can therefore be processed by the same AON. - c.5197-557G>T. This mutation results in a 188 nt pseudoexon insertion between exons 36 and 37 of ABCA4.

[0025] The inventors have provided AONs that modulate splicing for the mutation classes set forth herein above; the terms "modulate splicing" and "redirect splicing" are used interchangeably herein and encompass AON-based splice modulation therapy for the mutations set forth herein above. Thus, the present invention provides: - is complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof; - preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 11 or SEQ ID NO: 31, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 12 or SEQ ID NO: 32, or a part thereof; - more preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 16, 19, 163, 166, 169, 33, 36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143, 146, 149, 263, 266 and SEQ ID NO: 269, and portions thereof; more preferably, is complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 164, 167, 170, 34, 37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144, 147, 150, 264, 268 and SEQ ID NO: 270, and parts thereof, Antisense oligonucleotides for redirecting splicing are provided.

[0026] References made herein to "SEQ ID NOs: 10, 161, 30, 81, 101, 121, 141 and SEQ ID NO: 261, or portions thereof." In the context of the present invention: SEQ ID NOs: 11, 12, 13, 14, 16, 17, 19, 20, 160, 162, 163, 164, 166, 167, 169 and 170 or portions thereof are preferred portions of SEQ ID NOs: 10 and 161, respectively; SEQ ID NOs: 181, 180, 31, 32, 33, 34, 36, 37, 39, 40, 42, 43, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 2 54, 257, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, and 258 or portions thereof are each preferred portions of SEQ ID NO: 30; SEQ ID NOs: 82, 80, 83, 86, 89, 84, 87 and 90 or portions thereof are each preferred portions of SEQ ID NO: 81; SEQ ID NOs: 102, 100, 103, 106, 109, 104, 107 and 110 or portions thereof are each preferred portions of SEQ ID NO: 10; SEQ ID NOs: 122, 120, 123, 126, 129, 124, 127 and 130 or portions thereof are each preferred portions of SEQ ID NO: 121; SEQ ID NOs: 142, 140, 143, 146, 149, 144, 147 and 150 or portions thereof are each preferred portions of SEQ ID NO: 141; SEQ ID NOs:262, 260, 263, 266, 269, 264, 267 and 270 or portions thereof are each preferred portions of SEQ ID NO:261.

[0027] The term exon skipping is defined herein as inducing, producing or increasing the production in a cell of a mature mRNA that does not contain a particular exon present in the mature mRNA without exon skipping. Exon skipping is achieved by providing a cell expressing a pre-mRNA of said mature mRNA for a molecule capable of interfering with sequences such as (cryptic) splice donor or (cryptic) splice acceptor sequences necessary to allow the enzymatic process of splicing, or with exon inclusion signals necessary for the recognition of a stretch of nucleotides as an exon to be included in the mature mRNA, such molecules being referred to herein as exon skipping molecules. The term pre-mRNA refers to an unprocessed or partially processed precursor mRNA that is synthesized from a DNA template of a cell by transcription, for example in the cell nucleus.

[0028] The term exon retention is defined herein as inducing, producing or increasing the production in a cell of a mature mRNA that does not retain a particular exon that would be present in the mature mRNA without (abnormal) exon skipping. Exon retention is achieved by providing a cell expressing a pre-mRNA of said mature mRNA with an AON molecule that can interfere with a sequence, such as an alternative splice site upstream or downstream of a normal splice site. The term "antisense oligonucleotide" or "AON" is understood to refer to an oligonucleotide molecule that comprises a nucleotide sequence that is substantially complementary to a target nucleotide sequence of a pre-mRNA molecule, hnRNA (heterogeneous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that the molecule comprising the antisense sequence is capable of forming a stable hybrid with the target nucleotide sequence of an RNA molecule under physiological conditions.

[0029] The terms "antisense oligonucleotide", "AON" and "oligonucleotide" are used interchangeably herein and are understood to refer to an oligonucleotide comprising an antisense sequence. Binding of an AON to a target can be readily assessed by the skilled artisan using techniques known in the art, such as the gel mobility shift assay described in EP 1619249. The term "substantial complementarity" as used in the context of the present invention indicates that some mismatches in the antisense sequence are tolerated as long as they are functional, i.e., induce exon skipping or exon retention. Preferably, the complementarity is 90% to 100%. Generally, this allows for 1 or 2 mismatches in a 20 nucleotide AON, or 1, 2, 3, 4, 5 or 6 mismatches in a 60 nucleotide AON, etc. The AON may be further tested by transfection into retinal cells of a patient. Exon skipping or exon retention may be assessed by RT-PCR (e.g. as described in EP 1619249). The complementary regions are preferably designed to be specific for the exons of the pre-mRNA when combined. Such specificity can be made with complementary regions of different lengths, since it depends on the actual sequence of the other (pre-)mRNA molecules in the system. The risk that the AON can also hybridize to one or more other pre-mRNA molecules decreases with increasing size of the AON. It is clear that AONs that contain mismatches in the region of complementarity but retain the ability to hybridize and / or bind to the target region of the pre-mRNA can be used in the present invention. However, it is preferred that at least the complementary portion does not contain such mismatches, since AONs without mismatches in the complementary portion typically have higher efficacy and higher specificity than AONs with such mismatches in one or two complementary regions.It is believed that a higher strength of hybridization (ie, an increased number of interactions with opposing strands) is preferred in order to increase the effectiveness of the process in interfering with the splicing machinery of the system.

[0030] The AON according to the invention preferably does not contain a stretch of CpG, more preferably does not contain any CpG. The presence of CpG or a stretch of CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (Dorn, A., and Kippenberger, S. (2008). Clinical application of CpG-, non-CpG-, and antisense oligodeoxynucleotides as immunomodulators. Current opinion in molecular therapeutics 10, 10-20). This increased immunogenicity is undesirable as it may induce damage to the treated tissue, i.e. the eye. Immunogenicity can be evaluated in animal models by assessing the presence of CD4+ and / or CD8+ cells and / or inflammatory mononuclear cell infiltration. Immunogenicity may be evaluated in the blood of animals or humans treated with the AON according to the invention by detecting the presence of neutralizing antibodies and / or antibodies recognizing said AON, using standard immunoassays known to those skilled in the art. Inflammatory response, type I-like interferon production, IL-12 production and / or increased immunogenicity may be assessed by detecting the presence or increased amount of neutralizing antibodies or antibodies recognizing said AON using standard immunoassays. Even more preferably, the AON according to the present invention has acceptable RNA binding kinetics and / or thermodynamic properties. The RNA binding kinetics and / or thermodynamic properties are at least partially determined by the melting temperature (Tm; calculated by the Oligonucleotide Properties Calculator for single-stranded RNA using the model closest to the basic Tm (www.unc.edu / -cail / biotool / oligo / index)) of the oligonucleotide and / or the free energy of the AON-target-exon complex (using RNA Structure version 4.5). If the Tm is too high, the AON is expected to have low specificity. Acceptable Tm and free energy vary depending on the sequence of the AON. It is therefore difficult to give a preferred range for each of these parameters.Acceptable Tm can range between 35 and 70° C., and acceptable free energies can range between 15 and 45 kcal / mol. Thus, the skilled artisan can first select an AON as an effective therapeutic compound that binds to and / or is complementary to SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof, as defined herein below. The skilled artisan can check that said AON is capable of binding to said sequence, as defined herein above. In a second step, the skilled artisan can use the present invention to further optimize said AON by checking for the absence of CpG and / or optimizing the Tm and / or free energy of the AON-target complex. One skilled in the art will appreciate that by selecting a distinct sequence of ABCA4 to which the AON is complementary (including SEQ ID NOs: 10, 161, 30, 81, 101, 121, 141 and SEQ ID NO: 261, or portions thereof), few or preferably no CpGs and / or a more acceptable Tm and / or free energy are obtained. Alternatively, if an AON complementary to a given stretch within SEQ ID NO: 10 or 30 does not contain CpGs and / or has an acceptable Tm and / or free energy, one may improve any of these parameters by decreasing the length of the AON and / or by selecting a distinct stretch within any of SEQ ID NOs: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261 to which the AON is complementary and / or by altering the chemistry of the AON.

[0031] An AON according to the present invention is said to induce exon skipping if the percentage of skipping as measured by real-time quantitative RT-PCR analysis is at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%.

[0032] An AON according to the present invention is said to induce exon retention if the percentage of retention as measured by real-time quantitative RT-PCR analysis is at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%.

[0033] Preferably, an AON according to the invention comprising a portion that is (substantially) complementary to SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof, is an AON in which the (substantially) complementary portion is at least 50%, 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 at least 98% or even more preferably at least 99%, or even more preferably 100% of the length of the AON according to the invention. Preferably, an AON according to the invention comprises or consists of a sequence that is complementary or substantially complementary to a portion of SEQ ID NO: 10 or 30. By way of example, an AON comprises a sequence that is complementary or substantially complementary to a portion of SEQ ID NO: 10 or 30, and may comprise further flanking sequences.

[0034] Preferably, the AON according to the present invention is an AON whose nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID NO: 30, or a part thereof, comprises at least one ESE (exon splice enhancer) motif, preferably 2, 3, 4 or more ESE motifs. ESE motifs are known to those skilled in the art. Identification and determination of ESEs is preferably carried out as in the examples herein. In an embodiment, the AON according to the present invention does not comprise an ESE motif.

[0035] Preferably, the AON described in the present invention is an AON in which the portion that is (substantially) complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, has a length of about 8 to about 40 nucleotides, for example, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to about 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, the AON described in the present invention is an AON in which the portion that is (substantially) complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, has a length of 8 to 40 nucleotides, for example, preferably 10 to 40 nucleotides, more preferably 14 to 30 nucleotides, more preferably 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, the AON according to the invention comprises a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, or a part (substantially) complementary to a part thereof, the polynucleotide having a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. The AON has a length of up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length, and the portion (substantially) complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof, is a polynucleotide having a length of up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0036] The additional sequence, the (substantially) complementary portion, may be used to modify the binding of a protein, e.g., a splice promoter, to the AON or to modify the thermodynamic properties of the AON, e.g., to modify the target RNA binding affinity.

[0037] Preferred AONs for redirecting splicing according to the present invention have a length of about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. More preferred AONs for redirecting splicing according to the present invention have a length of 8 to 100 nucleotides, preferably 10 to 40 nucleotides, more preferably 14 to 30 nucleotides, more preferably 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, the AON according to the invention has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. Preferably, the AON according to the invention has a maximum of 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, 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 or 100 nucleotides in length.

[0038] In embodiments, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 128, 131, 145, 148, 151, 265, 268, and SEQ ID NO:271.

[0039] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 15. Preferred AONs comprising SEQ ID NO: 15 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0040] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 18. Preferred AONs comprising SEQ ID NO: 18 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0041] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 21. A preferred AON comprising SEQ ID NO: 21 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0042] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 35. Preferred AONs comprising SEQ ID NO: 35 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0043] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 38. Preferred AONs comprising SEQ ID NO: 38 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0044] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 41. Preferred AONs comprising SEQ ID NO: 41 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0045] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 44. Preferred AONs comprising SEQ ID NO: 44 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0046] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 165. Preferred AONs comprising SEQ ID NO: 165 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0047] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 168. Preferred AONs comprising SEQ ID NO: 168 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0048] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 171. Preferred AONs comprising SEQ ID NO: 171 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0049] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 184. Preferred AONs comprising SEQ ID NO: 184 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0050] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 187. Preferred AONs comprising SEQ ID NO: 187 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0051] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 190. Preferred AONs comprising SEQ ID NO: 190 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0052] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 193. Preferred AONs comprising SEQ ID NO: 193 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0053] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 196. Preferred AONs comprising SEQ ID NO: 196 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0054] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 199. Preferred AONs comprising SEQ ID NO: 199 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0055] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 202. Preferred AONs comprising SEQ ID NO: 202 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0056] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 205. Preferred AONs comprising SEQ ID NO: 205 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0057] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 208. Preferred AONs comprising SEQ ID NO: 208 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0058] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 211. Preferred AONs comprising SEQ ID NO: 211 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0059] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 214. Preferred AONs comprising SEQ ID NO: 214 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0060] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 217. Preferred AONs comprising SEQ ID NO: 217 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0061] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 220. Preferred AONs comprising SEQ ID NO: 220 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0062] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 223. Preferred AONs comprising SEQ ID NO: 223 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0063] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 226. Preferred AONs comprising SEQ ID NO: 226 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0064] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 229. Preferred AONs comprising SEQ ID NO: 229 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0065] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 232. Preferred AONs comprising SEQ ID NO: 232 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0066] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 235. Preferred AONs comprising SEQ ID NO: 235 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0067] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 238. Preferred AONs comprising SEQ ID NO: 238 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0068] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 241. Preferred AONs comprising SEQ ID NO: 241 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0069] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 244. Preferred AONs comprising SEQ ID NO: 244 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0070] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 247. Preferred AONs comprising SEQ ID NO: 247 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0071] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 250. Preferred AONs comprising SEQ ID NO: 250 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0072] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 253. Preferred AONs comprising SEQ ID NO: 253 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0073] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 256. Preferred AONs comprising SEQ ID NO: 256 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0074] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 259. Preferred AONs comprising SEQ ID NO: 259 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0075] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 85. Preferred AONs comprising SEQ ID NO: 85 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0076] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 88. Preferred AONs comprising SEQ ID NO: 88 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0077] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 91. Preferred AONs comprising SEQ ID NO: 91 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0078] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 105. Preferred AONs comprising SEQ ID NO: 105 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0079] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 108. Preferred AONs comprising SEQ ID NO: 108 preferably comprise from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0080] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 111. A preferred AON comprising SEQ ID NO: 111 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0081] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 125. Preferred AONs comprising SEQ ID NO: 125 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0082] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 128. Preferred AONs comprising SEQ ID NO: 128 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0083] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 131. A preferred AON comprising SEQ ID NO: 131 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0084] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 145. Preferred AONs comprising SEQ ID NO: 145 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0085] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 148. Preferred AONs comprising SEQ ID NO: 148 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0086] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 151. A preferred AON comprising SEQ ID NO: 151 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0087] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 265. Preferred AONs comprising SEQ ID NO: 265 preferably comprise about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0088] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 268. A preferred AON comprising SEQ ID NO: 268 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0089] In a preferred embodiment, an AON is provided that comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 271. A preferred AON comprising SEQ ID NO: 271 preferably comprises about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, or preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0090] AONs for redirecting splicing according to the present invention may comprise one or more RNA residues (ribonucleotides), or one or more DNA residues (deoxyribonucleotides), and / or one or more nucleotide analogs or equivalents, as further detailed herein below.

[0091] AONs for redirecting splicing according to the present invention preferably comprise one or more residues which are modified to increase nuclease resistance and / or to increase the affinity of the antisense oligonucleotide for a target sequence. Thus, in a preferred embodiment, the AON comprises at least one nucleotide analogue or equivalent, where a nucleotide analogue or equivalent is defined as a residue having a modified base, and / or a modified backbone, and / or a non-natural internucleoside linkage, or a combination of these modifications.

[0092] In a preferred embodiment, nucleotide analog or equivalent comprises modified backbone.Examples of such backbone are provided by morpholino backbone, carbamate backbone, siloxane backbone, sulfide, sulfoxide and sulfone backbone, formacetyl and thioformacetyl backbone, methyleneformacetyl backbone, riboacetyl backbone, alkene-containing backbone, sulfamate, sulfonate and sulfonamide backbone, methyleneimino and methylenehydrazino backbone, and amide backbone.Phosphorodiamidate morpholino oligomer is a modified backbone oligonucleotide that has been previously investigated as antisense agent.

[0093] Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six-membered ring and the phosphodiester bond is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by blocking 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, although 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 into cells. Recent reports have demonstrated triplex formation by morpholino oligonucleotides, and due to the non-ionic backbone, these studies have shown that morpholino oligonucleotides can form triplexes in the absence of magnesium.

[0094] It is further preferred that the bonds between residues in the backbone, e.g., short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or linkages formed by one or more short chain heteroatom or heterocyclic internucleoside linkages, do not contain a phosphorus atom.

[0095] Preferred nucleotide analogs or equivalents include peptide nucleic acids (PNAs) with modified polyamide backbones (Nielsen, PE, Egholm, M., Berg, RH, and Buchardt, O. (1991). Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500). PNA-based molecules are true mimics of DNA molecules in terms of base pair recognition. The backbone of PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, with the nucleobases attached to the backbone by methylene carbonyl bonds. Alternative backbones include one-carbon extended pyrrolidine PNA monomers (Govindaraju, T., and Kumar, VA (2005). Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): design, synthesis and DNA / RNA binding studies. Chemical communications, 495-497). Because the backbone of the PNA molecule contains uncharged phosphate groups, PNA-RNA hybrids are generally more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, SM, Driver, DA, Berg, RH, Kim, SK, Norden, B., and Nielsen, PE (1993). PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568). Further preferred backbones include morpholino nucleotide analogs or equivalents in which the ribose or deoxyribose sugar is replaced with a six-membered morpholino ring.The most preferred nucleotide analogs or equivalents include phosphorodiamidate morpholino oligomers (PMOs), in which the ribose or deoxyribose sugar is replaced with a six-membered morpholino ring and the anionic phosphodiester bond between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate bond.

[0096] In yet another embodiment, the nucleotide analog or equivalent according to the present invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester bond. This modification slightly destabilizes base pairing, but adds significant resistance to nuclease degradation.Preferred nucleotide analog or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonates including 3'-alkylene phosphonate, 5'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

[0097] Further preferred nucleotide analogs or equivalents according to the invention include one or more sugar moieties that are mono- or di-substituted at the 2', 3' and / or 5' positions, such as -OH; -F; substituted or unsubstituted, linear or branched lower (Cl-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, optionally interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety may be a pyranose or a derivative thereof, or a deoxypyranose or a derivative thereof, preferably a ribose or a derivative thereof, or a deoxyribose or a derivative thereof. Preferred derivatized sugar moieties include Locked Nucleic Acids (LNAs), in which the 2' carbon atom is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. Preferred LNAs include 2'-O,4'-C-ethylene-bridged nucleic acids (Morita, K., Hasegawa, C., Kaneko, M., Tsutsumi, S., Sone, J., Ishikawa, T., Imanishi, T., and Koizumi, M. (2001). 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) with nuclease-resistance and high affinity for RNA. Nucleic acids research Supplement, 241-242). These substitutions render the nucleotide analogs or equivalents RNase H and nuclease resistant and increase affinity for target RNA. In another embodiment, the nucleotide analogs or equivalents according to the invention contain one or more base modifications or substitutions.Modified bases include synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will become known in the art.

[0098] It will be understood by those skilled in the art that not all positions of an AON need to be uniformly modified. Moreover, more than one of the above analogs or equivalents may be incorporated into a single AON or even at a single position within an AON. In certain embodiments, the AONs described in the present invention have at least two different types of analogs or equivalents.

[0099] Thus, preferred AONs for redirecting splicing according to the present invention include 2'-O alkyl phosphorothioate antisense oligonucleotides, e.g., 2'-O-methyl modified ribose (RNA), 2'-O-ethyl modified ribose, 2'-O-propyl modified ribose, and / or substituted derivatives of these modifications, such as halogenated derivatives.

[0100] It will also be understood by those skilled in the art that different AONs described in the present invention can be combined for efficient therapy. In embodiments, a combination of at least two AONs described in the present invention is used, such as two different AONs described in the present invention, three different AONs described in the present invention, four different AONs described in the present invention, or five AONs described in the present invention.

[0101] The AON for redirecting splicing according to the present invention can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably retinal cells. Examples of such moieties are cholesterol, carbohydrates, vitamins, biotin, lipids, phospholipids, cell penetrating peptides including, but not limited to, antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, polyarginine, oligolysine or polylysine, antigen binding domains such as those provided by antibodies, Fab fragments of antibodies, or single chain antigen binding domains such as camelid-like single domain antigen binding domains.

[0102] The AON for redirecting splicing according to the invention may be administered indirectly using suitable means known in the art. The AON may be provided to an individual or to a cell, tissue or organ of said individual itself, for example as a so-called "naked" AON. The AON may also be administered in the form of an expression vector encoding 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 by a gene delivery vehicle. In a preferred embodiment, a viral-based expression vector is provided that comprises an expression cassette or transcription cassette that drives the expression or transcription of the AON for redirecting splicing according to the invention. Thus, the invention provides a viral vector that expresses an antisense oligonucleotide for redirecting splicing according to the invention when placed under conditions that promote the expression of the antisense oligonucleotide for redirecting splicing. Cells may be provided with the AON for redirecting splicing according to the invention by plasmid-derived antisense oligonucleotide expression or viral expression provided by an adenovirus or adeno-associated virus-based vector. Expression can be driven by an RNA polymerase II promoter (Pol II), such as the U7 RNA promoter, or an RNA polymerase III (Pol III) promoter, such as the U6 RNA promoter. Preferred delivery vehicles are viral vectors, such as adeno-associated viral vectors (AAV), or retroviral vectors, such as lentiviral vectors. Plasmids, artificial chromosomes, plasmids useful for targeted homologous recombination and integration in the human genome of cells may also be suitably applied for the delivery of AONs to redirect splicing according to the present invention. Preferred for the present invention are those vectors in which the transcription is driven from a PolIII promoter and / or the transcript is in the form of a fusion with an U1 or U7 transcript, which gives good results for delivering small transcripts. It is within the skill of the person skilled in the art to design suitable transcripts. Preferred are transcripts driven by PolIII, preferably in the form of a fusion transcript with an U1 or U7 transcript.Such fusions can be obtained as previously described (Gorman, L., Suter, D., Emerick, V., Schumperli, D., and Kole, R. (1998). Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proceedings of the National Academy of Sciences of the United States of America 95, 4929-4934).

[0103] A preferred expression system for AONs for redirecting splicing according to the present invention is an adenovirus-associated virus (AAV)-based vector. Short and double stranded AAV-based vectors have been developed that can be used for extended expression of antisense nucleotide sequences for highly efficient redirection of splicing. Preferred AAV-based vectors include, for example, expression cassettes driven by RNA polymerase III promoter (Pol III) or RNA polymerase II promoter (Pol II). Preferred RNA promoters are, for example, Pol III U6 RNA promoter or Pol II U7 RNA promoter.

[0104] Thus, the present invention provides viral-based vectors comprising a Pol II or Pol III promoter-driven expression cassette for expression of an AON to redirect splicing according to the present invention.

[0105] The AAV vector according to the present invention is a recombinant AAV vector, which refers to an AAV vector that comprises a portion of the AAV genome that includes the AON encoded for redirecting splicing according to the present invention, encapsidated in a protein shell of capsid proteins derived from an AAV serotype as indicated elsewhere herein. The portion of the AAV genome may contain inverted terminal repeats (ITRs) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, etc. The protein shell composed of capsid proteins may be derived from an AAV serotype, such as AAV1, 2, 3, 4, 5, 8, 9, etc. The protein shell may also be referred to as a capsid protein shell. The AAV vector may be deleted of one or preferably all wild-type AAV genes, but still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for replication, rescue and packaging of AAV virions. The ITR sequences may be wild-type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with the wild-type sequences, or may be modified, for example, by insertion, mutation, deletion, or substitution of nucleotides, so long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into a capsid shell and then allow expression in the infected host or target cell. In the context of the present invention, the capsid protein shell may be of a different serotype than the genomic ITRs of the AAV vector. Thus, the AAV vector according to the present invention may be composed of a capsid protein shell, i.e., an icosahedral capsid, comprising the capsid proteins (VP1, VP2, and / or VP3) of one AAV serotype, for example, AAV serotype 2, while the ITR sequences contained in the AAV5 vector may be of any of the AAV serotypes mentioned above, including the AAV2 vector. Thus, an "AAV2 vector" comprises a capsid protein shell of AAV serotype 2, while, for example, an "AAV5 vector" comprises a capsid protein shell of AAV serotype 5, thereby either may encapsidate any of the AAV vector genome ITRs described in the present invention.

[0106] Preferably, a recombinant AAV vector according to the invention comprises an AAV serotype 2, 5, 8 or AAV serotype 9 capsid protein shell, wherein the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9, and such AAV vectors are referred to as AAV2 / 2, AAV2 / 5, AAV2 / 8, AAV2 / 9, AAV5 / 2, AAV5 / 5, AAV5 / 8, AAV5 / 9, AAV8 / 2, AAV8 / 5, AAV8 / 8, AAV8 / 9, AAV9 / 2, AAV9 / 5, AAV9 / 8 or AAV9 / 9 vectors.

[0107] 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 a vector is referred to as an AAV2 / 5 vector.

[0108] 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 a vector is referred to as an AAV2 / 8 vector.

[0109] 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 a vector is referred to as an AAV2 / 9 vector.

[0110] More preferably, a recombinant AAV vector according to the invention comprises an AAV serotype 2 capsid protein shell, and the AAV genome or ITRs present in said vector are derived from AAV serotype 2; such vectors are referred to as AAV2 / 2 vectors.

[0111] A nucleic acid molecule encoding an AON for redirecting splicing according to the present invention represented by a selected nucleic acid sequence, e.g., an expression construct comprising an expression regulatory element operably linked to the coding sequence and a 3'-terminal sequence, is preferably inserted between the AAV genome or ITR sequences identified above. "AAV helper functions" generally refer to the corresponding AAV functions required for AAV replication and packaging that are provided in trans to the AAV vector. AAV helper functions complement AAV functions missing in the AAV vector but lacking the AAV ITRs (provided by the genome of the AAV vector). AAV helper functions include the two major ORFs of AAV, i.e., the rep and cap coding regions or sequences substantially functionally identical thereto. Rep and Cap regions are well known in the art and see, for example, (Chiorini, JA, Kim, F., Yang, L., and Kotin, RM (1999). Cloning and characterization of adeno-associated virus type 5. Journal of virology 73, 1309-1319), which are incorporated herein by reference, or U.S. Pat. No. 5,139,941. AAV helper functions can be provided in an AAV helper construct, which may be a plasmid. Introduction of the helper construct into the host cell can occur, for example, by transformation, transfection, or transduction, prior to or simultaneously with introduction of the AAV genome present in the AAV vectors specified herein. Thus, the AAV helper constructs according to the invention can be selected to produce the desired combination of serotypes for the capsid protein shell of the AAV vector on the one hand and the AAV genome present in the replication and packaging of said AAV vector on the other hand.

[0112] "AAV helper virus" provides additional functions necessary for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV type 1 and type 2) and vaccinia virus. The additional functions provided by the helper virus can be introduced into the host cell by a vector, as described in U.S. Patent No. 6,531,456, which is incorporated herein by reference.

[0113] Preferably, the AAV genome as present in the recombinant AAV vector described in the present invention does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or capsid genes. The AAV genome may further comprise marker or reporter genes, such as, for example, antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products (e.g., lacZ, aph, etc.) known in the art.

[0114] Preferably, the AAV vector according to the present invention is constructed and produced according to the method described in (Garanto, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular genetics 25, 2552-2563), which is incorporated herein by reference.

[0115] A preferred AAV vector according to the invention is - is complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a portion thereof; - preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 11 or SEQ ID NO: 31, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 12 or SEQ ID NO: 32, or a part thereof; - more preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 16, 19, 163, 166, 169, 33, 36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143, 146, 149, 263, 266 and SEQ ID NO: 269, and portions thereof; - more preferably, is complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20, 164, 167, 170, 34, 37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144, 147, 150, 264, 268 and SEQ ID NO: 270, and portions thereof. The AAV vector expressing an AON for redirecting splicing according to the present invention is an AON comprising, or preferably consisting of, a sequence, preferably an AAV2 / 5, AAV2 / 8, AAV2 / 9 or AAV2 / 2 vector.

[0116] Further preferred AAV vectors according to the invention are those represented by SEQ ID NOs: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 1 The present invention relates to an AAV vector, preferably an AAV2 / 5, AAV2 / 8, AAV2 / 9 or AAV2 / 2 vector, expressing an AON for redirecting splicing according to the present invention, comprising, or preferably consisting of, a sequence selected from the group consisting of SEQ ID NO: 28, 131, 145, 148, 151, 265, 268 and SEQ ID NO: 271.

[0117] Improvements in the means for providing an individual or a cell, tissue or organ of said individual with an AON for redirecting splicing according to the invention are expected to take into account the progress already achieved so far. Such future improvements may of course be incorporated to achieve the effects mentioned for reconstructing mRNA using the method according to the invention. The AON for redirecting splicing according to the invention may be delivered as such to an individual, a cell, tissue or organ of said individual as a "naked" AON. When administering the AON for redirecting splicing according to the invention, it is preferred that the molecule is dissolved in a solution compatible with the delivery method. Retinal cells may be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution.

[0118] Alternatively, a preferred delivery method for AONs or plasmids for the expression of such AONs to redirect splicing is a viral vector or a nanoparticle. Preferably, the viral vector or nanoparticle is delivered to the retina or other relevant cells. Such delivery to retinal cells or other relevant cells may be in vivo, in vitro or ex vivo; see, for example, (Garanto, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular genetics 25, 2552-2563), which is incorporated herein by reference.

[0119] Alternatively, the plasmids can be provided by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal and / or intracerebrovascular administration, the solution is preferably saline. Particularly preferred in the present invention is the use of excipients or transfection agents that aid in the delivery of each of the components defined herein to and / or in cells, preferably retinal cells. Preferred are excipients or transfection agents that can form complexes, nanoparticles, micelles, vesicles and / or liposomes that deliver each of the components defined herein complexed or entrapped in the vesicles or liposomes through the cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agents include polyethyleneimine (PEI, polyethyleneimine; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers including polypropyleneimine or polyethyleneimine copolymers (PEC, polyethyleneimine copolymers) and derivatives, synthetic amphiphiles (SAINT-18), lipofectin™, DOTAP and / or viral capsid proteins capable of self-assembly into particles capable of delivering each of the components defined herein to cells, preferably retinal cells. Such excipients have been shown to efficiently deliver oligonucleotides such as AONs to a wide variety of cultured cells, including retinal cells. They combine their high transfection capacity with an expected low to moderate toxicity in terms of total cell survival. The ease of structural modification can be used to allow further modifications and further analysis of (in vivo) nucleic acid transfer characteristics and toxicity.

[0120] Lipofectin represents an example of a liposomal transfection agent. Lipofectin consists of two lipid components, the cationic lipid N-[1-(2,3 dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (compare DOTAP, the methyl sulfate salt) and the neutral lipid dioleylphosphatidylethanolamine (DOPE). The neutral component mediates intracellular release. Another group of delivery systems are polymeric nanoparticles.

[0121] Polycations such as diethylaminoethylaminoethyl (DEAE)-dextran, which is well known as a DNA transfection agent, can be combined with butyl cyanoacrylate (PBCA) and hexyl cyanoacrylate (PHCA) to formulate cationic nanoparticles capable of delivering each component defined herein, preferably the AONs described in the present invention, through the cell membrane and into cells.

[0122] In addition to these common nanoparticle materials, the cationic peptide protamine provides another approach to formulate oligonucleotides with colloids. This colloidal nanoparticle system can form so-called proticles, which can be prepared by a simple self-assembly process for packaging and mediating intracellular release of oligonucleotides. A person skilled in the art can select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver exon skipping molecules for use in the present invention to deliver exon skipping molecules for the prevention, treatment or delay of ABCA4-related diseases or conditions. "Prevention, treatment or delay of ABCA4-related diseases or conditions" is defined herein as preferably preventing, arresting, halting the progression of, or reversing partial or complete vision impairment or blindness caused by genetic defects in the ABCA4 gene.

[0123] Furthermore, the AONs for redirecting splicing according to the present invention can be covalently or non-covalently linked to targeting ligands specifically designed to promote uptake into the cell, cytoplasm and / or its nucleus. Such ligands can include (i) compounds (including but not limited to peptide(-like) structures) that recognize cell-, tissue- or organ-specific elements that promote intracellular uptake and / or (ii) chemical compounds that can promote intracellular uptake and / or intracellular release of oligonucleotides from vesicles, e.g., endosomes or lysosomes.

[0124] Thus, in a preferred embodiment, the AON for redirecting splicing according to the present invention is provided together with at least one excipient and / or targeting ligand for delivery and / or a delivery device thereof to a cell and / or is formulated in a composition or pharmaceutical or composition facilitating its intracellular delivery.

[0125] It should be understood that when the composition comprises further components such as auxiliary compounds as defined later herein, each component of the composition cannot be appropriately formulated in one simple combination or composition or preparation. Depending on their identity and specific characteristics, the skilled artisan will know which type of formulation is most suitable for each component as defined herein. In a preferred embodiment, the present invention provides a composition or preparation in the form of a kit of parts, which comprises the AON for redirecting splicing according to the present invention and further auxiliary compounds as defined later herein.

[0126] If necessary and / or desired, the AON for redirecting splicing according to the present invention or a vector according to the present invention, preferably a viral vector, expressing an AON for redirecting splicing according to the present invention, can be placed into a pharma- ceutically active mixture by adding a pharma- ceutically acceptable carrier.

[0127] Thus, the present invention also provides a composition, preferably a pharmaceutical composition, comprising an AON for redirecting splicing according to the present invention, or a viral vector according to the present invention, and a pharma- ceutically acceptable excipient. Such a composition may comprise a single AON for redirecting splicing according to the present invention, or a viral vector according to the present invention, but may also comprise a plurality of separate AONs for redirecting splicing according to the present invention, or a viral vector according to the present invention. Such a pharmaceutical composition may comprise any pharma- ceutically acceptable excipient, including carriers, fillers, preservatives, adjuvants, solubilizers and / or diluents. Such pharma- ceutically acceptable carriers, fillers, preservatives, adjuvants, solubilizers and / or diluents can be found, for example, in Remington, 2000. Each feature of said composition has been defined herein above.

[0128] The preferred route of administration is by intravitreal injection of an aqueous solution or formulation specifically adapted for intraocular administration. EP 2425 814 discloses an oil-in-water emulsion specifically adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. This emulsion has a lower density than the vitreous humor, so that the emulsion floats on top of the vitreous humor, avoiding the injected drug from impairing vision.

[0129] When multiple separate AONs for redirecting splicing according to the invention are used, the concentration or dose defined herein may refer to the total concentration or dose of all oligonucleotides used, or the concentration or dose of each exon skipping molecule used or added. Thus, in embodiments, compositions are provided in which each or the total amount of AONs for redirecting splicing according to the invention used is administered in an amount ranging from 0.01 to 20 mg / kg per eye, preferably 0.05 to 20 mg / kg per eye. Suitable intravitreal doses are provided, including between 0.05 mg and 5 mg per eye, preferably 0.1 to 1 mg, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg per eye.

[0130] A preferred AON for redirecting splicing according to the invention is for the treatment of an ABCA4-related disease or condition in an individual. In all embodiments of the invention, the term "treatment" is understood to include the prevention and / or delay of an ABCA4-related disease or condition. An individual who may be treated using an AON for redirecting splicing according to the invention may already have been diagnosed with an ABCA4-related disease or condition. Alternatively, an individual who may be treated using an AON for redirecting splicing according to the invention may not yet have been diagnosed with an ABCA4-related disease or condition, but may have an increased risk of developing an ABCA4-related disease or condition in the future, given the individual's genetic background. A preferred individual is a human. In all embodiments of the invention, the ABCA4-related disease or condition is preferably Stargardt's disease.

[0131] Thus, the present invention further provides an AON for redirecting splicing according to the present invention, or a viral vector according to the present invention, or a (pharmaceutical) composition according to the present invention, for use as a medicament, preferably for the treatment of an ABCA4-related disease or condition that requires modulating the splicing of ABCA4, and for use as a medicament for the prevention, treatment or delay of an ABCA4-related disease or condition. Each feature of all medical use embodiments herein is as defined herein above, preferably as defined herein above.

[0132] The present invention further provides a method for treating an ABCA4-related disease or condition that requires modulating the splicing of ABCA4, comprising contacting a cell of said individual with an AON for redirecting splicing as described in the present invention, a vector as described in the present invention or a (pharmaceutical) composition as described in the present invention. Each feature of all medical use embodiments herein is as hereinbefore defined, preferably as hereinbefore defined.

[0133] The present invention further provides the use of an AON for redirecting splicing according to the present invention, a vector according to the present invention or a (pharmaceutical) composition according to the present invention for the preparation of a medicinal product. Each feature of all medical use embodiments herein is as hereinbefore defined, preferably as hereinbefore defined.

[0134] The present invention further provides the use of an AON for redirecting splicing according to the present invention, a vector according to the present invention or a (pharmaceutical) composition according to the present invention for the preparation of a medicament for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. Each feature of all medical use embodiments herein is as hereinbefore defined, preferably as hereinbefore defined.

[0135] The present invention further provides the use of an AON for redirecting splicing according to the present invention, a vector according to the present invention or a (pharmaceutical) composition according to the present invention for treating an ABCA4-related disease or condition that requires modulating the splicing of ABCA4. Each feature of all medical use embodiments herein is as hereinbefore defined, preferably as hereinbefore defined.

[0136] The treatment in the use or method according to the invention is preferably at least once and lasts preferably at least one week, one month, several months, one year, two, three, four, five, six years or longer, e.g. lifelong. Each AON for redirecting splicing according to the invention or its equivalent as defined herein for the use according to the invention may be suitable for direct administration in vivo to cells, tissues and / or organs of an individual already suffering from an ABCA4-related disease or condition or at risk of developing an ABCA4-related disease or condition, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of the AON, composition, compound or auxiliary compound according to the invention may depend on several parameters, such as, for example, the severity of the disease, the age of the patient, the mutation of the patient, the number of AONs for redirecting splicing according to the invention (i.e., dose), the formulation of the AON, composition, compound or auxiliary compound according to the invention, the route of administration, etc. The frequency of administration may vary between daily, weekly, biweekly, or at least once every three or four or five weeks or longer.

[0137] The dose range of the AON, composition, compound or auxiliary compound described in the present invention is preferably designed based on an ascending dose study of a clinical trial (in vivo use) where there are strict protocol requirements. The AON described in the present invention can be used at a dose ranging from 0.01 to 20 mg / kg, preferably 0.05 to 20 mg / kg. A suitable intravitreal dose would be 0.05 mg to 5 mg per eye, preferably 0.1 to 1 mg, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg per eye.

[0138] In a preferred embodiment, a concentration of oligonucleotide as defined herein in the range of 0.1 nM to 1 μM is used. Preferably, this range is for in vitro use in cell models such as retinal cells or tissues. More preferably, the concentration used is in the range of 1 to 400 nM, even more preferably 10 to 200 nM, even more preferably 50 to 100 nM. When multiple separate AONs are used, this concentration or dose may refer to the total concentration or dose of the AONs or the concentration or dose of each AON added.

[0139] In a preferred embodiment, the viral vectors described herein above, preferably AAV vectors, as delivery vehicles for the molecules described in the present invention, are administered at a dose of 1×10 per injection. 9 ~1×10 17 1 x 10 viral particles per injection, more preferably 1 x 10 10 ~1×10 12 The vaccine is administered at a dose ranging from 10 to 15 viral particles.

[0140] The above-mentioned ranges of AON concentrations or dosages are preferred concentrations or dosages for in vivo, in vitro or ex vivo use. Those skilled in the art will understand that depending on the AON used, the target cells to be treated, the gene target and its expression level, the medium used, and the transfection and incubation conditions, the AON concentration or dosage used may further vary and may need to be further optimized.

[0141] The AON for redirecting splicing according to the invention, or the viral vector according to the invention, or the composition according to the invention for use according to the invention may be administered in vivo, or in vivo, ex vivo or in vitro to cells, tissues and / or organs of an individual already suffering from an ABCA4-related disease or condition or at risk of developing an ABCA4-related disease or condition. The AON for redirecting splicing according to the invention, or the viral vector according to the invention, or the composition according to the invention may be administered in vivo directly or indirectly, or in vivo, ex vivo or in vitro to cells, tissues and / or organs of an individual already suffering from an ABCA4-related disease or condition or at risk of developing an ABCA4-related disease or condition. As Stargardt's disease has a prominent phenotype in retinal cells, it is preferred that said target cells are retinal cells, more preferably that said tissue is the retina, and more preferably that said organ comprises or consists of the eye.

[0142] The present invention further provides a method for modulating the splicing of ABCA4 in a cell, comprising contacting a cell, preferably a retinal cell, with an AON for redirecting splicing according to the present invention, or a viral vector for redirecting splicing according to the present invention, or a (pharmaceutical) composition for redirecting splicing according to the present invention. The features of this embodiment are preferably as defined herein above. Contacting a cell with an AON for redirecting splicing according to the present invention, or a viral vector for redirecting splicing according to the present invention, or a composition for redirecting splicing according to the present invention can be performed by any method known to the skilled person. The use of the methods for delivery of AONs, viral vectors and compositions for redirecting splicing as described herein above is included. The contacting can be direct or indirect, and can be in vivo, ex vivo or in vitro.

[0143] Unless otherwise specified, each embodiment described herein may be combined with any other embodiment described herein.

[0144] definition In this document and in the claims, the verb "to comprise" and its conjugations are used in an open-ended sense, meaning that items following this word are included, but items not specifically mentioned are not excluded. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element is present. Thus, the indefinite article "a" or "an" usually means "at least one."

[0145] The words "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that a value may be 5% more or less than the given value (10).

[0146] The sequence information provided herein should not be narrowly interpreted to require the inclusion of erroneously specified bases.Those skilled in the art can identify such erroneously specified bases and know how to correct such errors.In the case of sequence errors, the sequence of the polypeptide that can be obtained by expression of the gene present in SEQ ID NO:1 that contains the nucleic acid sequence that codes for the polypeptide should prevail.

[0147] All patent and literature references cited herein are incorporated by reference in their entirety.

[0148] EMBODIMENTS OF THE PRESENT DISCLOSURE 1. - is complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID NO: 30, or a part thereof; - preferably complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 11 or SEQ ID NO: 31, or a part thereof; - more preferably, complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 12 or SEQ ID NO: 32, or a part thereof; - more preferably, is complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:42, and portions thereof; - more preferably an antisense oligonucleotide for redirecting splicing which is complementary or substantially complementary to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40 and SEQ ID NO:43, and parts thereof. 2. The antisense oligonucleotide for redirecting splicing according to embodiment 1, wherein the portion complementary or substantially complementary to a polynucleotide having a nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID NO: 30, or a part thereof, has a length of about 8 to about 40 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to about 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. 3. The antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments, having a length of about 8 to about 100 nucleotides, preferably about 10 to about 40 nucleotides, more preferably about 14 to about 30 nucleotides, more preferably about 16 to about 24 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. 4. The antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41 and SEQ ID NO:44. 5. 2. The antisense oligonucleotide for redirecting splicing of any of the preceding embodiments, comprising at least one ribonucleotide. 6. 2. The antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments, comprising at least one ESE (exon splice enhancer) motif. 7. An antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments, comprising a 2'-O alkyl phosphorothioate antisense oligonucleotide, e.g., a 2'-O-methyl modified ribose (RNA), a 2'-O-ethyl modified ribose, a 2'-O-propyl modified ribose, and / or substituted derivatives of these modifications, e.g., halogenated derivatives. 8. A viral vector that expresses an antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments when placed under conditions conducive to expression of the exon skipping antisense oligonucleotide. 9. A pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to any one of embodiments 1 to 7 or a viral vector according to embodiment 7, and a pharma- ceutically acceptable excipient. 10. The pharmaceutical composition of embodiment 9, wherein the composition is for intravitreal administration and the total amount of antisense oligonucleotide for redirecting splicing per eye is administered in an amount ranging from 0.05 mg to 5 mg. 11. The pharmaceutical composition of embodiment 10, which is for intravitreal administration and is administered in an amount ranging from 0.1 to 1 mg of the total antisense oligonucleotide for redirecting splicing per eye, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg of the total antisense oligonucleotide for redirecting splicing per eye. 12. An antisense oligonucleotide for redirecting splicing according to any one of embodiments 1 to 7, a vector according to embodiment 8 or a composition according to any one of embodiments 9 to 11, for use as a pharmaceutical. 13. An antisense oligonucleotide for redirecting splicing according to any of embodiments 1 to 7, a vector according to embodiment 9 or a composition according to any of embodiments 9 to 11 for use in the treatment of an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. 14. Use of an antisense oligonucleotide for redirecting splicing according to any of embodiments 1 to 7, a vector according to embodiment 8 or a composition according to any of embodiments 9 to 11 for the preparation of a medicament. 15. Use of an antisense oligonucleotide for redirecting splicing according to any of embodiments 1 to 6, a vector according to embodiment 7 or a composition according to any of embodiments 8 to 10 for the preparation of a medicament for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. 16. Use of an antisense oligonucleotide for redirecting splicing according to any of embodiments 1 to 7, a vector according to embodiment 7 or a composition according to any of embodiments 9 to 11 for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4. 17. A method for modulating splicing of ABCA4 in a cell, comprising contacting said cell with an antisense oligonucleotide for redirecting splicing as defined in any of embodiments 1 to 7, a vector according to embodiment 7 or a composition according to any of embodiments 9 to 11. 18. A method for the treatment of an ABCA4-related disease or condition requiring modulation of splicing of ABCA4 in an individual in need thereof, comprising contacting a cell of said individual with an antisense oligonucleotide for redirecting splicing as defined in any of embodiments 1-7, a vector according to embodiment 7 or a composition according to any of embodiments 9-11. 19. The antisense oligonucleotide for redirecting splicing for the use according to embodiment 12 or 13, the use according to embodiment 15 or 16, or the method according to embodiment 18, wherein the ABCA4 related disease or condition is Stargardt disease. [Example]

[0149] First, we evaluated the in vitro efficacy of a number of AONs to redirect the splice defects due to the c.4539+1100A>G, c.4539+1106C>T and c.4539+2001G>A mutations of ABCA4 in human embryonic kidney (HEK293T) cells. For this, we used minigene constructs, i.e. plasmids carrying the sequence of a portion of the ABCA4 gene, usually the desired region with or without a mutation, flanked on each side by at least 500 bp of the wild-type ABCA4 sequence. The plasmids also contain exon sequences and intron-exon boundaries of exons 3 and 5 of the RHO gene, respectively, on each side of the ABCA4 sequence. In this way, the effect of ABCA4 variants on the splicing of the corresponding exon or pseudoexon can be easily measured. Later, we used longer constructs (generated midigenes) to evaluate the properties of other deep intron variants discovered, including c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T. The generation of these midigenes is described in (Sangermano R, Khan M, Cornelis SS, Richelle V, Albert S, Elmelik D, Garanto A, Qamar R, Lugtenberg D, van den Born LI, Collin RWJ, Cremers FPM. Genome Res (2018), epub ahead of print, doi: 10.1101 / gr.226621.117). Finally, in addition to the minigene assay, we also used photoreceptor progenitor cells (PPCs) from a patient carrying a compound heterozygous ABCA4 mutation, i.e., c.4539+2001G>A mutation together with c.4892T>C (p.Leu1631Pro) on the other allele, to evaluate the ability of AONs to rescue the splice defect.PPC was also used to evaluate the ability of AONs to rescue a splice defect from a patient with a compound allele containing c.302+68C>T and c.4539+2028C>T (M2) and the deletion c.6148-698_6670delinsTGTGCACCTCCCTAG on the other allele (Lee, W., Xie, Y., Zernant, J., Yuan, B., Bearelly, S., Tsang, SH, Lupski, JR & Allikmets, R. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet. 135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y). Experimental details are described first in the Materials and Methods section, while the results are further described and illustrated in the Results section below.

[0150] Materials and Methods A) Mutations: c.4539+1100A>G and c.4539+1106C>T - minigene Generation of minigenes for each mutation A minigene was generated containing part of intron 29, the complete exon 30, intron 30 and exon 31, and part of intron 31. This genomic region was cloned into the pCI-Neo-Rhodopsin vector using the Gateway System. The resulting vector (generated pCI-Neo-Rho-ABCA4-30-31 wild type, SEQ ID NO: 50) was used to introduce the c.4539+1100A>G and c.4539+1106C>T mutations by site-directed mutagenesis (generated new vectors pCI-Neo-Rho-ABCA4-c.4539+1100G, SEQ ID NO: 51 and pCI-Neo-Rho-ABCA4-c.4539+1106T, SEQ ID NO: 52). Control and mutant vectors were confirmed by Sanger sequencing. The minigenes were then transfected into HEK293T cells, which were harvested 48 h posttransfection and subjected to RT-PCR to detect splicing defects.

[0151] Design and Testing of AON RNA analysis of HEK293T cells transfected with the minigene showed a splicing defect in the pre-mRNA consisting of the insertion of a pseudoexon. Several AONs were designed using the sequence of this pseudoexon. The AONs were then transfected into HEK293T with the minigene. To confirm the efficacy of the AONs, the cells were subjected to RT-PCR analysis. The efficacy of each of the AONs was evaluated by delivering the same amount of minigene and various concentrations of the AONs, followed by RT-PCR analysis.

[0152] RT-PCR analysis Total RNA was isolated using the NucleoSpin RNA Clean-up kit (catalog no. 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg of RNA using the iScript cDNA synthesis kit (catalog no. 1708891; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Finally, the efficacy of the AONs was evaluated by performing PCR from exon 30 to exon 31 or PCR spanning exons 29 to 34.

[0153] B) Mutation: c.4539+2001G>A - minigene Generation of minigenes A minigene was generated that contained a portion of intron 29, the complete exon 30, intron 30 and exon 31, and a portion of intron 31. This genomic region was cloned into the pCI-Neo-Rhodopsin vector using the Gateway System. The resulting vector (generated pCI-Neo-Rho-ABCA4-30-31 wild type, SEQ ID NO: 50) was used to introduce the c.4539+2001G>A mutation by site-directed mutagenesis (generated a new vector pCI-Neo-Rho-ABCA4-c.4539+2001A, SEQ ID NO: 53). Both the control and mutant vectors were confirmed by Sanger sequencing. The minigene was then transfected into HEK293T cells, which were harvested 48 hours after transfection and subjected to RT-PCR analysis to detect splicing defects.

[0154] Design and Testing of AON Transfection of the minigene pCI-Neo-Rho-ABCA4-c.4539+2001A in HEK293T cells showed the insertion of a pseudoexon. Using the sequence of this pseudoexon, several AONs were designed. The AONs were delivered together with the minigene into HEK293T cells. The transfected cells were subjected to RNA analysis.

[0155] RNA analysis Total RNA was isolated using the NucleoSpin RNA Clean-up kit (catalog no. 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg of RNA using the iScript cDNA synthesis kit (catalog no. 1708891; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Finally, the efficacy of the AONs was evaluated by performing PCR from exon 30 to exon 31 or PCR spanning exons 29 to 34.

[0156] C) Mutations: c.4539+2001G>A and c.4539+2028C>T - PPC evaluation Generation of photoreceptor precursor cells (PPCs) Skin biopsies were obtained from a patient carrying c.4539+2001G>A (M1) in a heterozygous fashion and from a patient carrying c.4539+2028C>T (M2) in a heterozygous state, and fibroblast cell lines were generated.Induced pluripotent stem cells (iPSCs) were then reprogrammed as previously described (Sangermano, R., Bax, NM, Bauwens, M., van den Born, LI, De Baere, E., Garanto, A., Collin, RW, Goercharn-Ramlal, AS, den Engelsman-van Dijk, AH, Rohrschneider, K., et al. (2016). Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T-->C Mutation in Stargardt Disease. Ophthalmology 123, 1375-1385) and then expressed in vitro. The cells were differentiated into photoreceptor progenitor cells (PPCs) using a method adapted from Goercharn-Ramlal, AS, den Engelsman-van Dijk, AH, Rohrschneider, K., et al. (2016). Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T-->C Mutation in Stargardt Disease. Ophthalmology 123, 1375-1385) or (Flamier, A., Barabino, A. & Bernier, G. Differentiation of human embryonic stem cells into cone photoreceptors. Bio-protocol 6, e1870 (2016), doi:10.21769 / BioProtoc.1870). Differentiated cells were subjected to RT-PCR analysis.

[0157] Analysis of ABCA4 transcripts After 30 days of differentiation, control and patient-derived PPCs were harvested. Reverse transcription-PCR (RT-PCR) analysis was performed using primers located in exon 2 (forward) and exon 5 (reverse) or exon 30 (forward) and exon 31 (reverse) of the ABCA4 gene. Actin (ACTB) primers were used as controls. The sequences of the primers are represented by SEQ ID NOs: 54-77. The total reaction mixture (50 μl) contained 10 μM of each primer pair, Taq DNA polymerase, 1 U / μl (catalog number 11647679001, Roche, Basel, Switzerland), 10× PCR buffer without MgCl2, 25 mM MgCl2, 10 mM dNTPs, and 50 ng of cDNA. PCR conditions were: a first denaturation step at 94°C for 5 min, followed by 35 cycles of melting (94°C for 30 s), annealing (58°C for 30 s), and extension (72°C for 1 min) steps, with a final extension step at 72°C for 5 min. PCR products were separated on a 1% (w / v) agarose gel, and the resulting bands were excised and purified using the NucleoSpin® Gel & PCR cleanup kit (cat. no. 740609.250, Macherey-Nagel) according to the manufacturer's protocol. Finally, 100 ng of purified PCR products were analyzed by Sanger sequencing on a 3100 DNA Analyzer or a 3730 DNA Analyzer (Thermo Fisher Scientific).

[0158] Antisense oligonucleotide (AON) design The sequences of the PE plus 50 base pairs flanking both sides were analyzed as previously described (Aartsma-Rus, A. Overview on AON design. Methods Mol. Biol. 867, 117-129 (2012), doi:10.1007 / 978-1-61779-767-5_8). Briefly, the total RNA structure of the desired region was analyzed using mfold software (http: / / unafold.rna.albany.edu / ?q=mfold / RNA-Folding-Form, last accessed July 23, 2017) to identify partially open and closed regions. Splice enhancer motifs were determined using ESE finder 3.0 (http: / / krainer01.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi?process=home, last accessed July 23, 2017). Particular attention was paid to the SC35 region, since it was demonstrated that a positive correlation exists between the presence of such motifs and the efficacy of AONs (Aartsma-Rus, A. Overview on AON design. Methods Mol. Biol. 867, 117-129 (2012), doi:10.1007 / 978-1-61779-767-5_8). Initially, this analysis led to the design of four AONs, two overlapping with the highest scoring SC35 motifs (AON2 and AON3), one at the 5' end of PE (AON4) and one overlapping with the c.4539+2001G>A mutation (AON1). At a later stage, 22 further AONs were designed to find the correlation between the efficacy of AONs and their position relative to the pseudoexon, their overlap with certain ESE motifs, and their specificity (i.e. whether a single nucleotide mismatch abolishes their efficacy). The final AON sequences were also evaluated for the free energy of the molecule alone, the potential to form dimers, and interactions with desired regions.For this, we used the RNA Secondary Structure Tool (http: / / rna.urmc.rochester.edu / RNAstructureWeb, last accessed July 23, 2017), which uses RNA secondary structure and bifold prediction tools. We ensured that all AONs had free energy values ​​above -4 on their own, above -14 as dimers, and between 21 and 28 for binding of the AON region. This was calculated using the estimated energy of the desired region minus the energy of the AON bound to the region. All AON sequences had a length of 19 nucleotides, a Tm above 46 °C, and a GC content between 40% and 65%. The sequences and characteristics of the AONs are listed in Table 1; further characteristics of the AONs relative to the pseudoexon 30-31 (345) are listed in Table 2. AONs were chemically modified by adding a phosphorothioate backbone and 2-O-methyl sugar modification 2OMe / PS to each nucleotide and were purchased from Eurogentec (Liege, Belgium). AONs were dissolved in PBS 1x (autoclaved twice) to a final concentration of 100 μM. Two sense oligonucleotides (SON-1 [SEQ ID NO: 280] and SON-2 [SEQ ID NO: 281]) were ordered with the same chemistry to be used as negative controls.

[0159] Processing by AON After differentiation, PPCs were treated with AONs (0.5 and 1 μM) by mixing naked AONs directly with the culture medium. After 24 hours, cycloheximide (CHX, Cat. No. C4859, Sigma Aldrich) was added at a final concentration of 0.1 mg / ml and cells were incubated for another 24 hours. 48 hours after AON delivery, cells were harvested, rinsed in PBS, and RNA was isolated. cDNA synthesis was performed using 1 μg of RNA as described above. All reactions were diluted to 20 ng / μl by adding 30 μl of distilled water. For RT-PCR analysis, 80 ng of cDNA was used for all ABCA4 reactions, while 40 ng of cDNA was used for ACTB analysis. The total reaction mixture (25 μl) contained 10 μM of each primer pair, 1 U / μl Taq DNA polymerase (catalog no. 11647679001, Roche), 10× PCR buffer with MgCl2 supplemented with 1 mM MgCl2, 2 μM dNTPs, and 80 or 40 ng of cDNA. PCR conditions for the ABCA4 fragment from exon 30 to 31 were as follows: 94°C for 2 min, 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 90 s, followed by a final step of 72°C for 2 min. For actin amplification, PCR was performed under the same conditions, except that the extension time was 30 s. The total volume of the ABCA4 PCR product and 10 μl of the actin amplicon were resolved on a 2% (w / v) agarose gel. The resulting bands were analyzed using Sanger sequencing.The ratio between correctly and aberrantly spliced ​​variants was assessed using Fiji software (Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, JY, White, DJ, Hartenstein, V., Eliceiri, K., Tomancak, P. & Cardona, A. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682 (2012), doi:10.1038 / nmeth.2019).

[0160] D) Mutations: c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T - midi gene Generation of midigenes for each mutation Midigenes were generated for each mutation (Sangermano R, Khan M, Cornelis SS, Richelle V, Albert S, Elmelik D, Garanto A, Qamar R, Lugtenberg D, van den Born LI, Collin RWJ, Cremers FPM. Genome Res (2018), epub ahead of print, doi: 10.1101 / gr.226621.117). These midigenes contain substantial fragments of ABCA4 genomic DNA on each side of the corresponding mutation, often encompassing one or more flanking exons. The genomic regions were cloned into the pCI-Neo-Rhodopsin vector using the Gateway System.The resulting vectors (pCI-Neo-Rho-ABCA4-intron 6-intron 7 wild type (SEQ ID NO: 290), pCI-Neo-Rho-ABCA4-intron 6-intron 11 wild type (SEQ ID NO: 292), pCI-Neo-Rho-ABCA4-intron 11-intron 15 wild type (SEQ ID NO: 295), pCI-Neo-Rho-ABCA4-intron 29-intron 32 wild type (SEQ ID NO: 297), pCI-Neo-Rho-ABCA Using pCI-Neo-Rho-ABCA4-intron6-intron7 wild type (SEQ ID NO: 300), the c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T mutations were introduced into the corresponding vectors by site-directed mutagenesis (pCI-Neo-Rho-ABCA4-intron6-intron7 wild type (SEQ ID NO: 300)) to generate the new vectors. c.769-784T (SEQ ID NO: 291), pCI-Neo-Rho-ABCA4-intron 6-intron 11 c.859-540G (SEQ ID NO: 293), pCI-Neo-Rho-ABCA4-intron 6-intron 11 c.859-506C (SEQ ID NO: 294), pCI-Neo-Rho-ABCA4-intron 11-intron 15 c.1937+435G (SEQ ID NO: 296), pCI-Neo-Rho-ABCA4-intron 29-intron 32 c.4539+1100G (SEQ ID NO: 298), pCI-Neo-Rho-ABCA4-intron 29-intron 32 c.4539+1106T (SEQ ID NO: 299) and pCI-Neo-Rho-ABCA4-intron 31-intron 37 c.5197-557T (SEQ ID NO: 301). The control and mutant vectors were confirmed by Sanger sequencing. The midi genes were then transfected into HEK293T cells, harvested 48 hours after transfection, and subjected to RT-PCR analysis to detect splicing defects.

[0161] Design and Testing of AON RNA analysis of HEK293T cells transfected with midigene showed splicing defects of pre-mRNA consisting of insertion of pseudoexon. Several AONs were designed using the sequence of this pseudoexon. AONs were then transfected into HEK293T with midigene. To confirm the efficacy of AONs, cells were subjected to RT-PCR analysis. The efficacy of each of the AONs was evaluated by delivering the same amount of minigene and various concentrations of AONs and then performing RT-PCR analysis. In each experiment, one SON was included as a negative control. During the final check, we found that AON1 designed against the c.859-540C>G mutation was not correctly ordered, and instead, the sequence of AON3 against the c.5197-557G>T mutation was invaded and provided. This also affects the interpretation of the results.

[0162] RT-PCR analysis Total RNA was isolated using the NucleoSpin RNA Clean-up kit (catalog no. 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol. RNA was quantified and cDNA was synthesized from 1 μg of RNA using the iScript cDNA synthesis kit (catalog no. 1708891; Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Finally, the efficacy of the AONs was assessed by performing PCR using the corresponding ABCA4 primers (SEQ ID NO:302, Rhodopsin ex3 fw; SEQ ID NO:303, ABCA4 ex7 rev; SEQ ID NO:304, ABCA4 ex7 fw; SEQ ID NO:305, ABCA4 ex8 rev; SEQ ID NO:306, ABCA4 ex13 fw; SEQ ID NO:307, ABCA4 ex14 rev; SEQ ID NO:308, ABCA4 ex30 fw; SEQ ID NO:309, ABCA4 ex32 rev; SEQ ID NO:310, ABCA4 ex32 tw; SEQ ID NO:311, ABCA4 ex37 rev).

[0163] result A) Minigene constructs carrying the c.4539+1100A>G or c.4539+1106C>T mutations were transfected into HEK293T cells together with a construct carrying the wild-type ABCA4 sequence. As shown in Figure 1, both mutations resulted in the insertion of an 86 bp pseudoexon into the transcript (lane marked NT), although some remaining wild-type transcripts were also detected. Transfection of three different AONs showed that for both mutations, the insertion of the pseudoexon was completely abolished in the presence of AON1 (AON-1 pseudoexon 30-31(68), SEQ ID NO: 15) and AON2 (AON-2 pseudoexon 30-31(68), SEQ ID NO: 18), whereas AON3 (AON-3 pseudoexon 30-31(68), SEQ ID NO: 21) resulted in a partial redirection of the splicing event (Figure 1). These data demonstrate the ability of AONs to redirect aberrant splicing events due to the c.4539+1100A>G or c.4539+1106C>T mutations.

[0164] B) A minigene construct carrying the c.4539+2001A>G mutation (A) was co-transfected with a construct carrying the wild-type ABCA4 sequence (G) into HEK293T cells. A minigene construct carrying the c.4539+2001G>A mutation was co-transfected with a construct carrying the wild-type ABCA4 sequence into HEK293T cells, and RT-PCR analysis using RNA from these cells revealed inclusion of a pseudoexon corresponding to a 345-bp sequence in intron 30 except when cells were cultured in the presence of cycloheximide (+CHX), a drug commonly used to inhibit nonsense-mediated decay of aberrant transcripts. As shown in FIG. 3, all four AONs (AON1=AON-1 pseudoexon 30-31(345), SEQ ID NO: 35; AON2=AON-2 pseudoexon 30-31(345), SEQ ID NO: 38; AON3=AON-3 pseudoexon 30-31(345), SEQ ID NO: 41; AON4=AON-4 pseudoexon 30-31(345), SEQ ID NO: 44) completely redirected ABCA4 splicing, unlike SONs. Using the WT construct (left lane), only normal, intact products without pseudoexons were detected, as expected. Here, AONs are interchangeably denoted as AON-n and AONn (where n is an integer), and AONs may be denoted with or without "-".

[0165] C) In photoreceptor precursor cells derived from a patient heterozygous for the c.4539+2001G>A(M1) mutation in ABCA4, RT-PCR analysis using RNA from these cells revealed inclusion of a pseudoexon corresponding to a 345-bp sequence in intron 30 except when the cells were cultured in the presence of cycloheximide (+CHX), a drug commonly used to inhibit nonsense-mediated decay of aberrant transcripts. As illustrated in Figure 2, upon transfection of four different AONs targeting this pseudoexon (AON1 = AON-1 pseudoexon 30-31(345), SEQ ID NO: 35; AON2 = AON-2 pseudoexon 30-31(345), SEQ ID NO: 38; AON3 = AON-3 pseudoexon 30-31(345), SEQ ID NO: 41; AON4 = AON-4 pseudoexon 30-31(345), SEQ ID NO: 44), the pseudoexon insertion was completely abolished after administration of AON1 and AON4. This was not the case for the negative control oligo SON (SEQ ID NO: 45) with the complementary sequence of AON1, ​​demonstrating that AON1 and AON4 effectively and specifically redirect the aberrant splicing events caused by the c.4539+2001G>A mutation.

[0166] To determine whether the variants c.4539+2001G>A (M1) and c.4539+2028C>T (M2) result in aberrant splicing of ABCA4 pre-mRNA, we generated fibroblast cell lines from two unrelated Stargardt disease (STGD1) patients. STGD1 patients with M1 had the missense variant c.4892T>C (p.Leu1631Pro) in trans (Webster, AR, Heon, E., Lotery, AJ, Vandenburgh, K., Casavant, TL, Oh, KT, Beck, G., Fishman, GA, Lam, BL, Levin, A., Heckenlively, JR, Jacobson, SG, Weleber, RG, Sheffield, VC & Stone, EM An analysis of allelic variation in the ABCA4 gene. Invest. Ophthal. Vis. Sci. 42, 1179-1189 (2001)). STGD1 patients with M2 harbored a deep intronic variant c.302+68C>T in cis, while the other allele had a deletion c.6148-698_6670delinsTGTGCACCTCCCTAG(p.?). In addition, fibroblast cell lines derived from healthy controls were generated. All cells were cultured in the absence and presence of cycloheximide (CHX), a compound used to suppress nonsense-mediated decay of RNA products carrying protein-truncating mutations. RT-PCR analysis using primers located in exons 30 and 31 revealed only one clear product corresponding to the expected product encompassing exons 30 and 31 (Figure 4). No aberrantly spliced ​​products were detected in fibroblasts derived from STGD1 patients.

[0167] To investigate the potentially retina-specific splicing defects caused by the two deep intronic ABCA4 mutations, control and patient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) by lentiviral transduction of Yamanaka factors (Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006), doi:10.1016 / j.cell.2006.07.024). Quantitative PCR (q-PCR) (Figure 7) and immunofluorescence analysis (not shown) confirmed the pluripotency of the iPSCs. These iPSCs were then differentiated into photoreceptor progenitor cells (PPCs) for one month. Because the cells primarily affected by STGD1 are cone photoreceptor cells, we obtained a relatively homogenous population of cone cells using a protocol previously described by Flamier and coworkers (Flamier, A., Barabino, A. & Bernier, G. Differentiation of human embryonic stem cells into cone photoreceptors. Bio-protocol 6, e1870 (2016), doi:10.21769 / BioProtoc.1870). Characterization of control and patient-derived PPCs revealed a significant increase in expression of ABCA4, approximately 40-fold higher in control PPCs than in control iPSCs, but only approximately 3-fold higher in M1-PPCs and M2-PPCs compared to M1-iPSCs and M2-iPSCs. Further characterization of PPCs revealed that control PPCs expressed higher amounts of CRX and OPN1SW compared with M1-PPCs and M2-PPCs, but all three cell lines differentiated into S cones ( Fig. 7B ).

[0168] As ABCA4 was strongly expressed in PPCs, we performed RT-PCR analysis of exon 30 to exon 31, which showed aberrant transcripts in both M1- and M2-derived PPCs upon CHX treatment, but not in control PPCs (Figure 4A). Semi-quantification of the ratio between correctly and aberrantly spliced ​​variants in CHX-treated samples revealed that approximately 25% of ABCA4 transcripts in patients with M1 and approximately 15% of ABCA4 transcripts in patients with M2 were aberrant (Figure 4B). More detailed analysis of all bands by Sanger sequencing revealed that the truncated protein product p.Arg1514Leufs * A 345nt PE containing a premature stop codon predicted to give rise to 36 was revealed (Figure 5). Interestingly, both variants contained identical PEs in the mRNA transcript upon CHX treatment. Once the sequences were identified, we studied the effect of both variants on splicing. According to all prediction software, neither M1 nor M2 changed the strength of the splice acceptor and donor sites (Figure 5). The splice donor site of the 345nt PE contains "GC" as the canonical splice site sequence recognized only by the Splice-Site-Finder-Like (SSFL) software. Furthermore, in silico predictions showed that M1 increases the strength of the exon splicing enhancer SF2 site and creates a new SRp55 motif, while M2 creates one SC35 and two SRp40 motifs (Figure 5).

[0169] Subsequent in-depth analysis of all bands observed by RT-PCR revealed that one band contained a heteroduplex of a correctly spliced ​​transcript together with a PE-containing transcript (Figure 4). In addition, an extra faint band missing the last 73 bp of exon 30 was seen in all samples treated with CHX, including the control. A relatively weak splice donor site (Human Splicing Finder (HSF) score: 75.9) explains this alternative transcript that was also detected in the heteroduplex band (Figure 4). This splice product (r.4467_4539del, p.Cys1490Glufs *12) was also identified as a result of a non-canonical splice site variant at the "natural" splice donor site in exon 30 (Sangermano, R., Bax, NM, Bauwens, M., van den Born, LI, De Baere, E., Garanto, A., Collin, RW, Goercharn-Ramlal, AS, den Engelsman-van Dijk, AH, Rohrschneider, K., et al. (2016). Photoreceptor Progenitor mRNA Analysis Reveals Exon Skipping Resulting from the ABCA4 c.5461-10T-->C Mutation in Stargardt Disease. Ophthalmology 123, 1375-1385). Interestingly, this new donor site was previously reported as a splice acceptor site (HSF score: 89.6) generating an isoform lacking the first 114 bp of exon 30 (Gerber, S., Rozet, JM, van de Pol, TJ, Hoyng, CB, Munnich, A., Blankenagel, A., Kaplan, J. & Cremers, FPM Complete exon-intron structure of the retina-specific ATP binding transporter gene (ABCR) allows the identification of novel mutations underlying Stargardt disease. Genomics 48, 139-142 (1998), doi:10.1006 / geno.1997.5164).

[0170] In the seven STGD1 cases with M2 examined, c.302+68C>T was found in cis (R. Allikmets, unpublished data; Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367; Lee, W., Xie, Y., Zernant, J., Yuan, B., Bearelly, S., Tsang, SH, Lupski, JR & Allikmets, R. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet. 135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y and Zernant et al., 2014). To study the contribution of this variant to STGD1 pathology, we performed RT-PCR of mRNA from control PPCs, M1-PPCs and M2-PPCs, treated and untreated with CHX, as well as from mRNA from adult retina. As shown in Figure 8, PCR primers located in exons 2 and 5 generated a 459 nt cone splice product in all PPCs and human retina, along with a fragment shorter than 317 nt. Confirmation of the band by Sanger sequencing revealed that the 317 nt fragment lacked exon 3 (size: 142 bp).No other splice products were observed, indicating that the c.302+68C>T variant does not result in activation of cryptic splice sites and / or exonic splice enhancers.

[0171] Having explained the molecular mechanisms associated with M1 and M2 variants, we aimed to design a therapeutic approach for skipping PE based on splicing modulation. An attractive and efficient method is the use of AONs, which are small RNA molecules that can enter cells, bind to pre-mRNA, and modify the splicing pattern. To increase its binding affinity and avoid RNaseH activation (and thus transcript degradation), we used a previously reported AON (2OMe / PS) backbone of 2-O-methyl modified RNA with phosphorothioates (Collin, RW, den Hollander, AI, van der Velde-Visser, SD, Bennicelli, J., Bennett, J., and Cremers, FP (2012). Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290. Molecular therapy Nucleic acids 1, e14;Garanto, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant Splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular genetics 25, 2552-2563;Gerard, X., Perrault, I., Hanein, S., Silva, E., Bigot, K., Defoort-Delhemmes, S., Rio, M., Munnich, A., Scherman, D., Kaplan, J., Kichler, A. & Rozet, J.M.AON-mediated exon skipping restores ciliation in fibroblasts harboring the common Leber congenital amaurosis CEP290 mutation. Mol. Ther. Nucleic Acids 1, e29 (2012), doi:10.1038 / mtna.2012.21 and Slijkerman et al., 2016). In total, we designed four AONs: two that block the highest scoring SC35 motif located at the 3' end of PE (AON2, AON3), one that blocks the second highest scoring SC35 at the 5' end of PE (AON4), and one that blocks the SRp55 motif newly created by M1 (AON1; Figure 6A). In addition, we designed a sense oligonucleotide (SON) in the same region that is complementary to AON1 and contains the same chemical modifications as the other AONs but cannot bind to pre-mRNA. AONs and SONs ​​were delivered to PPCs differentiated for about one month, and RNA was analyzed 48 hours later. As expected, CHX treatment increased the presence of aberrantly spliced ​​transcripts in untreated cells (Figures 6B and 6C). Furthermore, there was no difference between untreated and SON-treated cells. We demonstrated that AONs are efficient in exon skipping. We found that AON4 could efficiently produce up to about 75% PE skipping in both cell lines at two different concentrations (Figure 6D), while AON1 was highly efficient in M1 cell lines. AON2 showed variable efficacy, whereas AON3 was able to redirect splicing at both 0.5 μM and 1 μM (Figures 6B, 6C, and 6D). One explanation for AON2 and AON3 behaving differently despite targeting the same region could be the properties of AONs (Tables 1, 2). Compared to AON2, AON3 has a lower GC content and Tm, which may affect its stability and binding ability, thus explaining its lower efficiency.

[0172] To further expand our search for the most effective AONs to redirect the splice defect caused by the c.4539+2001G>A mutation, we designed and tested 22 additional AONs (AON5 to AON26, SEQ ID NOs: 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, and 256, respectively) to assess their ability to redirect splicing of ABCA4 by preventing the inclusion of the 345 nt pseudoexon. Previously tested AON1-4 (SEQ ID NO: 35 / 184, 38 / 187, 41 / 190 and 44 / 193, respectively) were treated with two sense oligonucleotides (SON1 [SEQ ID NO: 280] and SON2 [SEQ ID NO: 281]). The results are shown in Figure 9. Besides AON1 and 4, other effective AONs included AON9, AON10, AON14, AON17, AON18, AON22, AON23 and AON24. Moderately effective AONs included AON2, AON8, AON11, AON13, AON16, AON20 and AON21. Little or no effective AONs included AON3, AON5, AON6, AON7, AON12, AON15, AON19, AON25 and AON26. When comparing the properties of these AONs, several things become clear: i) AONs targeting regions outside the pseudoexon (AON5, AON6, AON7, AON25 and AON26) are unable to redirect ABCA4 splicing. ii) AONs with a single mismatch with the target are not effective, i.e., AON1 is mutation-specific for the c.4539+2001G>A and does not redirect splicing in patients with the c.4539+2028C>T mutation (Figure 6D). Similarly, AON15 is mutation-specific for the pseudoexon with the c.4539+2028C>T change and is not effective in correcting the splicing defect due to the c.4539+2001G>A mutation (Figure 9). iii) AONs effective in redirecting splicing more frequently possessed SC35 motifs (both effective and moderately effective possessed on average 1.8-fold and 1.45-fold more SC35 motifs compared to poorly and ineffective AONs). No significant differences were observed for SF2 and SRp40 motifs. In moderately effective AONs, we detected 4-fold and 2.6-fold more abundant SRp55 motifs, respectively, when compared to effective, poorly and ineffective AON groups, respectively. iv) On average, there was no difference in the length of AONs that redirected splicing compared to those that did not redirect splicing, however, we observed that the melting temperature (Tm) was on average 2-3 degrees higher for effective AONs compared to the moderately effective group and the group composed of poorly effective and ineffective AONs. v) Also, on average, both effective and moderately effective AONs exhibited a percentage of GC content higher than 54%, while poorly or ineffective AONs averaged less than 48%. vi) We did not observe clear differences between AONs that bind to either predicted closed or open regions versus those that bind to predicted mixed regions with partially open and partially closed regions.

[0173] D) Midigene constructs carrying the c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T or c.5197-557G>T mutations were transfected into HEK293T cells together with constructs carrying the corresponding wild-type ABCA4 sequence. As shown in Figure 10, all mutations result in the insertion of a pseudoexon with variable length to a variable extent (lanes marked with NT). Transfection of three different AONs showed that for all mutations, at least one AON was effective in rescuing the pseudoexon insertion associated with this mutation, as well as one common SON for each mutation. Specifically, for c.769-784C>T, the addition of AON1 and AON2 results in a reduction of the pseudoexon, while AON3 partially rescues the splice defect. For c.859-540C>G, AON1 is ineffective and AON2 is very effective, while AON3 partially rescues the splice defect. However, during final checks, the inventors discovered that the AON1 designed for the c.859-540C>G mutation was not correctly ordered, and instead, the sequence of AON3 for the c.5197-557G>T mutation was invaded and provided. This also affects the interpretation of the results. Therefore, the negative results seen for AON1 were expected and should not have been studied, since the actual AON used is not specific for the corresponding pseudoexon. For c.859-506G>C, AON1 and AON3 result in a reduction of transcripts containing the pseudoexon, while AON2 is not effective. For c.1937+435C>G, all three AONs result in a reduction of ABCA4 transcripts carrying the pseudoexon. For the c.4539+1100A>G and c.4539+1106C>T mutations, AON1 and AON2 appear to be effective, whereas AON3 is clearly not. Finally, for the c.5197-557G>T mutation, all three AONs result in a reduction of transcripts carrying the pseudoexon.Overall, these data demonstrate the ability of AONs to redirect aberrant splicing events due to all deep intronic ABCA4 mutations tested, with at least one AON active for each pseudoexon.

[0174] Consideration In this study, we show that two nearby deep intronic variants in ABCA4, c.4539+2001G>A and c.4539+2028C>T, result in the retina-specific inclusion of a 345-nt pseudoexon (PE) in a population of ABCA4 transcripts. *36), was found as a low abundance alternative splice form of ABCA4 when performing deep RNA sequencing of human macula retina RNA (Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367). Quantification of RT-PCR products revealed more PE insertions in M1 than in M2. Based on the ocular phenotype of STGD1 patients with M1 and the nature of the variants observed in trans in these patients, it was proposed that M1 acts as a severe variant (Bauwens, M., De Zaeytijd, J., Weisschuh, N., Kohl, S., Meire, F., Dahan, K., Depasse, F., De Jaegere, S., De Ravel, T., De Rademaeker, M., Loeys, B., Coppieters, F., Leroy, BP & De Baere, E. An augmented ABCA4 screen targeting noncoding regions reveals a deep intronic founder variant in Belgian Stargardt patients. Hum. Mutat. 36, 39-42 (2015), doi:10.1002 / humu.22716;Bax, NM, Sangermano, R., Roosing , S. , Thiaden , AA , Hoefsloot , LH , van den Born , LI , Phan , M. , Klevering , BJ , Westeneng-van Haaften , C. , Braun , TA , Zonneveld-Vrieling , MN , de Wijs , I. , Mutlu , M. , Stone , EM , den Hollander , AI , Piano , CC , Hoyng , CB & . Cremers, FPM Heterozygous deep-intronic variants and deletions in ABCA4 in persons with retinal dystrophies and one exonic ABCA4 variant. Hum. Mutat. 36, 43-47 (2015), doi:10.1002 / humu.22717;Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, 2015; SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136–5145 (2013), doi:10.1093 / hmg / ddt367). For example, the M2 is based on the specific STGD1 cycle with a smooth surface (Lee, W. , Xie , Y. , Zernant , J. , Yuan , B. , Bearelly , S. , Tsang , SH , Lupski , J.R.Based on the results of the previous study (Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes. Hum. Genet. 135, 9-19 (2016), doi:10.1007 / s00439-015-1605-y), we hypothesize that M2 acts as a mild to moderately severe variant. Therefore, we expect that the amount of mutant mRNA in patients with M1, which has a missense variant in trans, should be equal to the amount of the correct product. This is not the case, but this comparison is difficult, since smaller products may amplify more effectively and NMD suppression may be incomplete. PE insertions with M2 are less pronounced than those for M1, which is consistent with its less severe characteristics. However, we cannot exclude the possibility that other cis-acting variants lost during positional sequencing (Zernant et al, 2014) may act in concert with these intron 30 variants. Moreover, both patient-derived PPC lines differentiated less well than control PPC lines, indicating a possible role of cell type-specific mechanisms and delayed differentiation. This has a significant impact on the amount of PE insertion. A clear example of the importance of retinal differentiation for PE recognition was described for the deep intronic c.2991+1655A>G variant of CEP290. Meanwhile, in lymphoblastomas and fibroblasts of patients carrying this heterozygous mutation, the ratio between correctly and aberrantly spliced ​​CEP290 was approximately 1:1 (Collin, RW, den Hollander, AI, van der Velde-Visser, SD, Bennicelli, J., Bennett, J., and Cremers, FP (2012). Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290.Molecular therapy Nucleic acids 1, e14;Guarante, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular genetics 25, 2552-2563;den Hollander, AI, Koenekoop, RK, Yzer, S., Lopez, I., Arends, ML, Voesenek, KE, Zonneveld, MN, Strom, TM, Meitinger, T., Brunner, HG, Hoyng, CB, van den Born, LI, Rohrschneider, K. & Cremers, 2011; FP Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet. 79, 556–561 (2006)) and the structural characteristics of the iPSC genes were obtained by CEP The 290's are smooth and smooth (fraction 1:4; Parfitt, DA , Lane , A , Ramsden , CM , Carr , AJ , Munro , PM , Jovanovic , K , Schwarz , N , Kanuga , N , Muthiah , MN , Hull , S , Gallo , JM , da Cruz , L , Moore AT , Hardcastle , AJ , Coffey , PJ & Cheetham , MEIdentification and correction of mechanisms underlying inherited blindness in human iPSC-derived optic cups. Cell stem Cell 18, 769-781 (2016), doi:10.1016 / j.stem.2016.03.021). This study not only reveals insight into why this mutation results in a nonsyndromic retinal phenotype despite ubiquitous expression of CEP290, but also demonstrates the enormous power of using patient-derived iPSC-derived retinal cells to study splice defects in relevant cell lines.

[0175] Previously, intronic variants associated with inherited retinal disease (IRD) created new splice acceptor or donor sites that allowed the insertion of PE (Braun, TA, Mullins, RF, Wagner, AH, Andorf, JL, Johnston, RM, Bakall, BB, Deluca, AP, Fishman, GA, Lam, BL, Weleber, RG, Cideciyan, AV, Jacobson, SG, Sheffield, VC, Tucker, BA & Stone, EM Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum. Mol. Genet. 22, 5136-5145 (2013), doi:10.1093 / hmg / ddt367; Bonifert, T., Gonzalez Menendez, I., Battke, F., Theurer, Y., Synofzik, M., Schols, L. & Wissinger, B. Antisense oligonucleotide mediated splice correction of a deep intronic mutation in OPA1. Mol. Ther. Nucleic Acids 5, e390 (2016), doi:10.1038 / mtna.2016.93;Webb, TR, Parfitt, DA, Gardner, JC, Martinez, A., Bevilacqua, D., Davidson, AE, Zito, I., Thiselton, DL, Ressa, JH, Apergi, M., Schwarz, N., Kanuga, N., Michaelides, M., Cheetham, ME, Gorin, MB & Hardcastle, AJDeep intronic mutation in OFD1, identified by targeted genomic next-generation sequencing, causes a severe form of X-linked retinitis pigmentosa (RP23). Hum. Mol. Genet. 21, 3647-3654 (2012), doi:10.1093 / hmg / dds194;van den Hurk, J. A., van de Pol, D. J., Wissinger, B., van Driel, M. A., Hoefsloot, L. H., de Wijs, I. J., van den Born, L. I., Heckenlively, J. R., Brunner, H. G., Zrenner, E., Ropers, H. H. & Cremers, F. P. M. Novel types of mutation in the choroideremia ( CHM) gene: a full-length L1 insertion and an intronic mutation activating a cryptic exon. Hum. Genet. 113, 268-275 (2003), doi:10.1007 / s00439-003-0970-0;Vache, C., Besnard, T., le Berre, P., Garcia-Garcia, G., Baux, D., Larrieu, L., Abadie, C., Blanchet, C., Bolz, H. J., Millan, J., Hamel, C., Malcolm, S., Claustres, M. & Roux, A. F. Usher syndrome type 2 caused by activation of an USH2A pseudoexon: implications for diagnosis and therapy. Hum. Mutat. 33, 104-108 (2012), doi:10.1002 / humu.21634;Rio Frio, T., McGee, T. L., Wade, N. M., Iseli, C., Beckmann, J. S., Berson, E. L. & Rivolta, C. A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance. Hum. Mutat. 30, 1340-1347 (2009), doi:10.1002 / humu.21071;Naruto, T., Okamoto, N., Masuda, K., Endo, T., Hatsukawa, Y., Kohmoto, T. & Imoto, I. Deep intronic GPR143 mutation in a Japanese family with ocular albinism. Sci. Rep. 5, 11334 (2015), doi:10.1038 / srep11334;Mayer et al、2016;Liquori et al、2016;den Hollander, A. I., Koenekoop, R. K., Yzer, S., Lopez, I., Arends, M. L., Voesenek, K. E., Zonneveld, M. N., Strom, T. M., Meitinger, T., Brunner, H. G., Hoyng, C. B., van den Born, L. I., Rohrschneider, K. & Cremers, F. P. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet.79, 556-561 (2006); Carss et al, 2017). To our knowledge, we are the first to report on the insertion of PEs not by this mechanism, but presumably by the creation of new ESE motifs in the IRD. The intronic region is filled with predicted splice acceptor and donor site pairs that could theoretically flank the PE. With regard to the identification of further PEs that are not activated by the creation of splice sites, it will be possible to determine the sequence motifs that make a cryptic PE a real PE.

[0176] M1 and M2-associated PE insertions were successfully blocked by several AONs. M1-specific AONs were only effective in M1 cell lines, and even at a double AON concentration, AON1 still failed to correct the splice defect in M2 cell lines. Furthermore, M2-specific AONs with a single mismatch to the PE sequence were not effective in patients with M1. These results highlight the sequence specificity and the fact that a single nucleotide mismatch is enough to alter the efficiency of AONs. The newly created SRp55 motif may play a crucial role in the detection of PE. If both variants activate the same PE and AON4 is able to skip PE in both cases, this should be further explained. One of the limitations of AONs is that they bind to specific sequences and therefore it is not possible to test the same AONs in animal models in the absence of conserved DNA / RNA regions unless a model is created in which a part of the human sequence is inserted at an orthologous position in the animal genome.However, it is already known that 2OMe / PS chemistry and 2MOE (2-O-methoxyethyl) / PS are not ocularly toxic, as shown in several animal models (Garanto, A., Chung, DC, Duijkers, L., Corral-Serrano, JC, Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, LH, and Collin, RW (2016). In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Human molecular genetics 25, 2552-2563;Gerard, X., Perrault, I., Munnich, A., Kaplan, J. & Rozet, JM Intravitreal injection of splice-switching oligonucleotides to manipulate splicing in retinal cells. Mol. Ther. Nucleic Acids 4, e250 (2015), doi:10.1038 / mtna.2015.24;Murray, SF, Jazayeri, A., Matthes, MT, Yasumura, D., Yang, H., Peralta, R., Watt, A., Freier, S., Hung, G., Adamson, PS, Guo, S., Monia, BP, LaVail, MM & McCaleb, ML Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration. Invest. Ophthalmol. Vis. Sci. 56, 6362-6375 (2015), doi:10.1167 / iovs.15-16400).Furthermore, the first commercialized AON was used to treat the eye condition CMV retinitis (Fomivirsen approved for CMV retinitis: first antisense drug. AIDS treatment news, 7 (1998)). Thus, AON technology appears to be a safe and promising approach to treat eye disorders. Although it remains to be explained whether the function of ABCA4 protein can be restored after treatment of these cells due to the lack of an animal model, the use of iPSC-derived photoreceptors seems to be a suitable option.

[0177] In conclusion, by using patient-derived iPSCs differentiated into S-cones, we were able to identify molecular defects due to two recurrent, nearby deep intronic variants underlying STGD1. The splice defect appears to be tissue-specific and consists of a 345-nt PE insertion, probably caused by the presence of a newly created exonic splice enhancer, rather than by the creation of a novel splice site. Furthermore, an AON-based therapeutic approach was designed and tested, showing that one AON was able to redirect the splice defect in both mutant cell lines. Moreover, variant-specific AONs were highly effective against M1 but not against M2, indicating that one single nucleotide mismatch can dramatically change the efficiency of AONs. For several other deep intronic mutations in ABCA4 (i.e., c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T or c.5197-557G>T), we showed that all result in the insertion of pseudoexons. AONs were designed to block the inclusion of these pseudoexons, and for each pseudoexon, at least one AON was able to significantly reduce the amount of aberrant ABCA4 transcript. Overall, these results highlight the potential of AONs as a therapeutic tool for Stargardt disease.

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Claims

1. An antisense oligonucleotide for redirecting splicing, the antisense oligonucleotide comprising: comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 15, 18, 165, 168, 35, 38, 44, 184, 187, 193, 205, 208, 211, 214, 220, 223, 229, 232, 235, 241, 244, 247, 250, and SEQ ID NO: 253; The antisense oligonucleotide.

2. The antisense oligonucleotide for redirecting splicing according to claim 1, having a length of 8 to 100 nucleotides.

3. 3. An antisense oligonucleotide for redirecting splicing according to claim 1 or 2, comprising at least one ribonucleotide.

4. An antisense oligonucleotide for redirecting splicing according to any one of claims 1 to 3, comprising at least one ESE (exon splice enhancer) motif.

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

6. A viral vector expressing an antisense oligonucleotide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 15, 18, 165, 168, 35, 38, 44, 184, 187, 193, 205, 208, 211, 214, 220, 223, 229, 232, 235, 241, 244, 247, 250, and SEQ ID NO: 253, for redirecting splicing according to any one of claims 1 to 5, when placed under conditions promoting expression of the exon skipping antisense oligonucleotide.

7. A pharmaceutical composition comprising an antisense oligonucleotide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 15, 18, 165, 168, 35, 38, 44, 184, 187, 193, 205, 208, 211, 214, 220, 223, 229, 232, 235, 241, 244, 247, 250, and SEQ ID NO: 253, or a viral vector according to claim 6, for redirecting splicing, and a pharma- ceutically acceptable excipient.

8. 8. The pharmaceutical composition of claim 7, which is for intravitreal administration and is administered in an amount ranging from 0.05 mg to 5 mg of the total amount of antisense oligonucleotide for redirecting splicing per eye.

9. The pharmaceutical composition of claim 8, wherein the composition is for intravitreal administration and is administered in an amount ranging from 0.1 to 1 mg of total antisense oligonucleotide for redirecting splicing per eye, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg of total antisense oligonucleotide for redirecting splicing per eye.

10. An antisense oligonucleotide for redirecting splicing according to any one of claims 1 to 5, a vector according to claim 6, or a composition according to any one of claims 7 to 9, for use as a medicament.

11. An antisense oligonucleotide for redirecting splicing according to any one of claims 1 to 5, a vector according to claim 6, or a composition according to any one of claims 7 to 9, for use in the treatment of an ABCA4-related disease or condition which requires modulating the splicing of ABCA4.

12. Use of an antisense oligonucleotide for redirecting splicing according to any one of claims 1 to 5, a vector according to claim 6, or a composition according to any one of claims 7 to 9 for the preparation of a medicament.

13. Use of an antisense oligonucleotide for redirecting splicing according to any one of claims 1 to 5, a vector according to claim 6, or a composition according to any one of claims 7 to 9 for the preparation of a medicament for treating an ABCA4-related disease or condition which requires modulating the splicing of ABCA4.

14. 10. An ex vivo method for modulating splicing of ABCA4 in a cell, comprising contacting the cell with an antisense oligonucleotide for redirecting splicing as defined in any one of claims 1 to 5, a vector according to claim 6, or a composition according to any one of claims 7 to 9.

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