Method for treating retinal dystrophy by exon skipping strategy

Abstract

The present invention relates to skipping of CEP290 exon 36 in individuals suffering from retinal dystrophies caused by premature stop codons due to nonsense mutations or frameshift mutations in exon 36 or an upstream exon, including the c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A mutations, to bypass protein cleavage and reduce retinal damage. Here, we study fibroblasts from control individuals and two patients with the CEP290 c.4723A>T nonsense mutation, demonstrating low levels of spontaneous exon 36 skipping resulting from both endogenous basal skipping and mutagenic skipping. Minimal truncation and mutation-free CEP290 mRNA resulting from exon 36 skipping in the two patient fibroblasts is translated into a centrosome-localized protein isoform, allowing the formation of primary cilia but with elongated axonemes. Using an AON targeting the mRNA, consisting of the sequence shown in SEQ ID NO:1, which is complementary to the nucleic acid sequence of the CEP290 pre-mRNA and encodes the donor splice site (H36D), we were able to alter splicing by blocking recognition of exon 36, bypassing proteolytic cleavage while maintaining the open reading frame, resulting in the production of a nearly full-length CEP290 protein. They were able to increase the abundance of alternatively spliced ​​mRNA and truncated protein in patient cells and shorten axoneme length.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is in the field of gene therapy, in particular exon skipping strategies are used to treat retinal dystrophies.

[0002] Background of the Invention Leber congenital amaurosis (LCA, MIM204000) is a group of neonatal-onset, severe retinal dystrophies and a leading cause of intractable childhood blindness (prevalence 1:30,000; 20% of children attending schools for the blind in Western Europe) [1]. Retinal dystrophies typically occur as nonsyndromic disorders exhibiting large genetic, allelic, and physiopathological heterogeneity, making therapeutic development challenging [2]. Mutations in CEP290 (MIM610142), encoding a widely expressed centrosomal protein involved in ciliary formation and maintenance [3], are the primary cause of this disorder, termed LCA type 10 (LCA10; MIM611755) [4, 5]. Despite early-onset visual loss, LCA10 individuals show long-term (>30 years) preservation of central photoreceptors with intact visual brain pathways, creating the conditions for developing therapeutic approaches incorporating correction of the genetic lesion [6]. The very well-known c.2991+1655A>G (p.Cys998) is involved in 10% and 2.5% of all LCA cases, respectively. * ) and c.4723A>T (p.Lys1575 *Numerous LCA10 mutations have been reported, including the c.2991+1655A>G mutation [5, 7]. The c.2991+1655A>G change activates a deep intronic cryptic splice site, introducing a frameshift pseudoexon in the mRNA [8-11]. Antisense oligonucleotides (AONs) have proven effective in redirecting the splicing machinery to the consensus splice site and bypassing protein cleavage in primary fibroblasts, IPSC-derived 3D retinal organoids, and humanized mice harboring the mutation [8, 10, 12]. Subsequently, a phase II / III clinical trial (NCT03140969) has been initiated, demonstrating the safety and clinical significance (improved visual acuity) of intravitreal injection of a splice-modulating oligonucleotide (sepofarsen)

[11] . A Phase II / III clinical trial is underway (PQ-110-003, NCT03913143; a multiple-dose, double-blind, randomized, sham-controlled clinical trial of sepofarsen in patients with retinal disease). The c.4723A>T variant, like the majority of LCA10 mutations, is predicted to truncate the protein and is amenable to gene augmentation therapy. However, this approach is challenging due to both the CEP290 cDNA size (7.4 Kb), which exceeds the cargo capacity (<5 Kb) of AAV vectors preferred in the field of retinal disease [13-15], and the risk of overexpression toxicity [16, 17]. Interestingly, consistent with a critical role in ciliary metabolism, CEP290 mutations are associated with additional human phenotypes, including oculo-renal Senior-Loken syndrome (SLSN6, MIM610189), oculo-cerebro-renal Jaubert syndrome (JBTS5, MIM610188), and embryonic lethal Meckel syndrome type 4 (MKS4; MIM611134)

[18] . The observation of endogenous basal exon skipping, which produces low levels of alternatively spliced ​​coding CEP290 mRNA, suggests a pathogenetic model in which disease severity is a function of CEP290 dosage and cells may arise from mutant alleles

[19] .Consistently, low levels of PTC-free CEP290 mRNA, produced by endogenous basal alternative splicing and / or nonsense-associated altered splicing, have been identified in fibroblasts from individuals with biallelic CEP290 truncating mutations but a mild retinal phenotype [20-22]. Mitigating disease via a somatic frame-restoration mechanism implies genetic reversion in dystrophin-positive myofibers from patients with Duchenne muscular dystrophy, stimulating AON-mediated exon skipping to bypass dystrophin truncation and convert the disease to attenuated Becker muscular dystrophy.

[0003] Summary of the Invention The present invention relates to an antisense oligonucleotide consisting of a sequence complementary to the nucleic acid sequence of the CEP290 gene, wherein said antisense oligonucleotide targeting the donor splice site of exon 36 (H36D) has the following sequence: SEQ ID NO: 1, and is capable of altering splicing by blocking recognition of exon 36 by the spliceosome, allowing for bypassing the proteolytic cleavage associated with any mutation that introduces a premature stop codon in exon 36 while maintaining the open reading frame, resulting in the production of a nearly full-length CEP290 protein. In particular, the present invention is defined by the claims.

[0004] Detailed Description of the Invention In an attempt to better understand the relationship between CEP290 and retinal dystrophies, such as Leber congenital amaurosis (LCA), the inventors have studied different regions of the CEP290 gene and investigated the c.4723A>T mutation in exon 36 of the CEP290 gene. In particular, this mutation leads to the appearance of a premature stop codon in the portion of the CEP290 mRNA encoded by exon 36, resulting in degradation of the mutant mRNA by nonsense-mediated mRNA decay (NMD) and / or the translation of a non-functional truncated protein.

[0005] We have used antisense oligonucleotides complementary to sequences containing the donor splice site to mask consensus splice sites (donor and acceptor splice sites) from the spliceosome (splicing machinery). The present inventors hypothesized that AON-mediated skipping of CEP290 exon 36 could result from any CEP290 mutation, particularly at least one selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A, thereby bypassing protein truncation that would otherwise introduce a premature stop codon in exon 36. Here, we studied fibroblasts from control individuals and demonstrated that exon 36 undergoes low levels of endogenous skipping, producing minimally truncated CEP290 mRNA and very low levels of protein that preserves wild-type centrosomal localization. We also studied fibroblasts from two unrelated individuals homozygous for the c.4723A>T mutation, demonstrating that exon 36 skipping, combined with endogenous and mutagenic skipping, was more pronounced in controls than in controls. The minimally truncated, mutation-free CEP290 mRNA resulting from exon 36 skipping is translated into a protein isoform that localizes to the centrosome, enabling primary cilia to form but with elongated axonemes compared with matched controls. Using AONs specific for the donor consensus splice site of exon 36 in patient and control fibroblasts, we were able to increase the abundance of alternatively spliced ​​mRNA and truncated protein, thereby shortening axoneme length in patient cells.

[0006] Using AONs specific for the donor consensus splice site of exon 36 in patient and control fibroblasts, the AONs were able to increase the abundance of alternatively spliced ​​mRNA and truncated protein, thereby shortening axoneme length in patient cells.

[0007] We have shown that administration of AON induces the skipping of exon 36, allowing the production of a minimally truncated CEP290 protein that maintains the open reading frame and is surprisingly functional.

[0008] Here, we report data supporting the feasibility of an antisense oligonucleotide-mediated exon skipping strategy to bypass protein truncation and alleviate retinal damage caused by mutations that introduce premature stop codons in exon 36. Thus, the present invention provides for the use of such exon skipping strategies for the treatment of retinal dystrophies.

[0009] Antisense oligonucleotides consisting of sequences complementary to the nucleic acid sequence of the CEP290 gene In a first aspect, the present invention relates to an antisense oligonucleotide consisting of a sequence complementary to the nucleic acid sequence of the CEP290 gene, wherein said antisense oligonucleotide targeting the donor splice site of exon 36 (H36D) has the following sequence: SEQ ID NO: 1, and is capable of altering splicing by blocking the recognition of exon 36 by the splicing machinery, allowing for bypassing the protein cleavage associated with any mutation that introduces a premature stop codon in exon 36 while maintaining the open reading frame, resulting in the production of an almost full-length CEP290 protein.

[0010] In a particular embodiment, the CEP290 is a CEP290 pre-mRNA.

[0011] In a specific embodiment, the antisense oligonucleotides of the invention target the donor splice site of exon 36 of the CEP290 gene, removing this exon from the mature mRNA and thereby allowing bypassing of any truncating mutations in this exon.

[0012] In a further embodiment, in the antisense oligonucleotide of the invention, exon 36 of the CEP290 gene has at least one mutation selected from the group consisting of c.4723A>T, c.4732G>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4714G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A.

[0013] As used herein, the term "CEP290" has its general meaning in the art and refers to the protein encoded by the CEP290 gene. CEP290 is an essential component of the ciliary gate, which bridges the transition zone between the cilium and the cytoplasm. This protein plays an important role in maintaining the structural integrity of this gate and, therefore, in maintaining ciliary function (Craige, B et al. The Journal of Cell Biology. 190, 927-40 (2010)). The term includes naturally occurring "CEP290" and its mutants and variants. CEP290 can be derived from any source but is typically mammalian (e.g., human and non-human primate) CEP290, particularly human CEP290. An exemplary native human CEP290 amino acid sequence is provided in the GenPept database under accession number [EAW97414.1], and an exemplary native human nucleotide sequence encoding CEP290 is provided in the GenBank database under accession number [NM_025114.3].

[0014] The term "antisense oligonucleotide" or AON refers to a single strand of DNA, RNA, or modified nucleic acid that is complementary to a selected sequence. Antisense RNA can be used to modulate splicing or bind to a specific mRNA strand, thereby preventing protein translation of that mRNA strand. Antisense DNA can be used to target a specific complementary (coding or non-coding) RNA. In certain embodiments, the antisense oligonucleotide is antisense RNA. In other embodiments, the antisense oligonucleotide is antisense DNA.

[0015] Oligonucleotides are designed against appropriate complementary sequences, typically RNA sequences within a pre-mRNA molecule required for correct splicing of the target exon, thereby eliminating the mutant exon without blocking the splicing reaction or disrupting the reading frame that would incorporate the target exon into the mature mRNA. AONs typically bind to sequences that are complementary to them and sterically mask the splicing reaction. The sequences are selected to be specific; i.e., the AON is complementary only to the pre-mRNA sequence and not to other nucleic acid sequences. AONs used in the practice of the present invention can be of any suitable type, such as oligodeoxyribonucleotides, oligoribonucleotides, morpholinos, tricyclo-DNA-antisense oligonucleotides, U7- or U1-mediated AONs, or their conjugated products, such as peptide conjugates or nanoparticle-complexed AONs. AONs utilized in the practice of the present invention are generally about 10 to 50 nucleotides in length, and can be, for example, about 10 or less, or about 15, about 20, or about 30 or more nucleotides in length. The optimal length of an AON for a targeted complementary sequence generally ranges from about 15 to about 30 nucleotides in length, depending on the chemical backbone used and the target sequence. Typically, morpholino-AONs are about 25 nucleotides in length, 2'PMO-AONs are about 20 nucleotides in length, and tricyclo-AONs are about 15 nucleotides in length.

[0016] The AONs of the present invention can be synthesized de novo using any of a number of techniques well known in the art, such as the b-cyanoethyl phosphoramidite method (Beaucage et al., 1981); the nucleoside H-phosphonate method (Garegg et al., 1986; Froehler et al., 1986; Garegg et al., 1986; Gaffney et al., 1988). These chemistries can be performed using a variety of commercially available automated nucleic acid synthesizers. These nucleic acids can be referred to as synthetic nucleic acids. Alternatively, AONs can be produced on a large scale in plasmids (see Sambrook, et al., 1989). AONs can be prepared from existing nucleic acid sequences using known techniques, such as those utilizing restriction enzymes, exonucleases, or endonucleases. AONs prepared in this manner can be referred to as isolated nucleic acids.

[0017] AONs can be stabilized or are stabilized. A "stabilized" AON refers to an AON that is relatively resistant to in vivo degradation (e.g., via exonucleases or endonucleases). Stabilization can be a function of length or secondary structure. Alternatively, AON stabilization can be achieved by phosphate backbone modifications. Preferred stabilized AONs of the present invention have modified backbones, e.g., phosphorothioate linkages, to provide maximal activity and protect the AON from degradation by intracellular exonucleases and endonucleases. Other potential stabilizing modifications include phosphodiester modifications, combinations of phosphodiester and phosphorothioate modifications, methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. Chemically stabilized and modified versions of AONs, including "morpholinos" (phosphorodiamidate morpholino oligomers, PMOs), 2'-O-Met oligomers, 2'-fluoro (2'-F) oligomers, tricyclo(tc)-DNA, U7 small nuclear (sn)RNA, tricyclo-DNA-oligoantisense molecules (U.S. Ser. No. 61 / 212,384, filed April 10, 2009, entitled "Tricyclo-DNA Antisense Oligonucleotides, Compositions and Methods for the Treatment of Disease," the entire contents of which are incorporated herein by reference), unlocked nucleic acids (UNA), peptide nucleic acids (PNA), serinol nucleic acids (SNA), staggered intercalating nucleic acids (TINA), anhydrohexitol nucleic acids (HNA), cyclohexenyl nucleic acids (CeNA), D-altritol nucleic acids (ANA), and morpholino nucleic acids (MNA), have also been investigated for splice modulation.Recently, it has been reported that nucleobase-modified antisense oligonucleotides containing 2-thioribothymidine and 5-(phenyltriazole)-2-deoxyuridine nucleotides induce exon skipping (Chen S, Le BT, Chakravarthy M, Kosbar TR, Veedu RN. Systematic evaluation of 2'-Fluoro-modified chimeric antisense oligonucleotide-mediated exon skipping in vitro. Sci Rep. 2019 Apr 15;9(1):6078.).

[0018] In a specific embodiment, the antisense oligonucleotide of the present invention can be a 2'-O-Me RNA / ENA chimeric oligonucleotide (Takagi M, Yagi M, Ishibashi K, Takeshima Y, Surono A, Matsuo M, Koizumi M. Design of 2'-O-Me RNA / ENA chimera oligonucleotides to induce exon skipping in dystrophin pre-mRNA. Nucleic Acids Symp Ser (Oxf). 2004;(48):297-8).

[0019] Other forms of AONs that can be used to this effect are AON sequences coupled to small nuclear RNA molecules, such as U1 or U7, in combination with viral transfer methods based on, but not limited to, lentivirus or adeno-associated virus (Denti, MA, et al, 2008; Goyenvalle, A, et al, 2004).

[0020] In another specific embodiment, the antisense oligonucleotides of the invention are 2'-O-methyl-phosphorothioate oligonucleotides.

[0021] In certain embodiments, the antisense oligonucleotides of the invention are complementary to a sequence comprising a donor splice site (H36D) having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A.

[0022] In a particular embodiment, an antisense oligonucleotide complementary to the nucleic acid sequence of CEP290 exon 36 is required for skipping of exon 36 inserted in the mutant CEP290 mRNA.

[0023] In a specific embodiment, the antisense oligonucleotide of the invention comprises the nucleic acid sequence of SEQ ID NO:1.

[0024] In a more particular embodiment, the antisense oligonucleotide of the invention consists of the nucleic acid sequence of SEQ ID NO:1.

[0025] In a specific embodiment, the antisense oligonucleotides of the invention are complementary to a nucleic acid sequence essential for enhancing splicing of exon 36 of a CEP290 mRNA that contains a premature stop codon resulting from a mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon that introduces a premature stop codon into exon 36.

[0026] In a specific embodiment, the antisense oligonucleotide of the invention is complementary to a nucleic acid sequence essential for enhancing splicing of exon 36 of CEP290 mRNA containing at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A.

[0027] In a further embodiment, the antisense oligonucleotides of the invention are complementary to a nucleic acid sequence essential for enhancing splicing of exon 36 of CEP290 mRNA containing a nonsense mutation, eg, c.4723A>T.

[0028] As used herein, the term "complementary" includes "fully complementary" and "substantially complementary," which generally means that there will be greater than 80%, preferably greater than 85%, even more preferably greater than 90%, and most preferably greater than 95% complementarity between an oligonucleotide and its corresponding target sequence. For example, for a 20-nucleotide-long oligonucleotide with one mismatch between its sequence and its target sequence, the degree of complementarity is 95%. In particular, the present invention relates to antisense oligonucleotides complementary to the nucleic acid sequence of CEP290 exon 36, which is essential for skipping exon 36 inserted into a mutant mRNA, that share at least 25% sequence identity with the target sequence.

[0029] According to the present invention, a first amino acid sequence having at least 25% identity to a second amino acid sequence is defined as a sequence in which the first amino acid sequence has at least 25% identity to the second amino acid sequence; 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%; "Sequence identity" means having 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% identity. Sequence identity is often measured in terms of % identity (or similarity or homology); the higher the % the more similar the two sequences are. Methods of aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992, and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994. Altschul et al., Nat. Genet., 6:119-129, 1994 provides a detailed discussion of sequence alignment methods and homology calculations.For example, sequence comparisons can be performed using the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) (Internet Program® 1996, W.R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences to each other, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet, for example, at the NCSA website. Alternatively, for comparison of amino acid sequences of more than about 30 amino acids, the Blast 2 alignment function can be utilized using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and per-residue gap cost of 1). When aligning short peptides (less than about 30 amino acids), alignment should be performed using the Blast 2 sequence function, utilizing the PAM30 matrix set to default parameters (start gap of 9, extension gap penalty of 1). The BLAST sequence comparison system is available, for example, from the NCBI website. See also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997, and Zhang & Madden, Genome Res., 7:649-656, 1997.

[0030] In some embodiments, the antisense oligonucleotides of the invention comprise a sequence complementary to the nucleic acid sequence of CEP290 exon 36 that is essential for skipping exon 36 inserted in the mutant CEP290 mRNA.

[0031] In some embodiments, the antisense oligonucleotide of the present invention comprises a sequence complementary to the nucleic acid sequence of CEP290 exon 36, which is essential for skipping exon 36 inserted in a mutant CEP290 mRNA, wherein the antisense oligonucleotide comprises the sequence set forth in SEQ ID NO:1.

[0032] Combinations of said antisense oligonucleotides can also be used according to the invention to modulate the splicing of exon 36 of CEP290 mRNA containing at least one mutation.

[0033] In a specific embodiment, the antisense oligonucleotide of the present invention is capable of inducing exon skipping and consists of a sequence complementary to a nucleic acid sequence of the CEP290 gene essential for modulating the splicing of exon 36 of CEP290 containing at least one mutation.

[0034] In a particular embodiment, the antisense oligonucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0035] Typically, the antisense oligonucleotides have a length of at least 15 nucleotides.

[0036] In certain embodiments, in antisense oligonucleotides for use in accordance with the present invention, the antisense oligonucleotides comprise at least 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108 nucleotides in length.

[0037] [Table 1]

[0038] Table 1: Oligonucleotide sequences containing 2'-O-methyl RNA bases and full-length phosphorothioate backbones. H_sense is a sense oligonucleotide used as a control. H36D(+98-11) is an antisense oligonucleotide (AON) designed to target the donor splice site at the CEP290 exon 36 / intron 36 junction, and H36ESE(+63+84) is directed against the ESE sequence within CEP290 exon 36.

[0039] Within an intron, a donor site (at the 5' end of the intron), a branch site (near the 3' end of the intron), and an acceptor site (at the 3' end of the intron) are required for splicing. The donor splice site contains the nearly invariant sequence GU within a larger, less conserved region at the 5' end of the intron. The acceptor splice site at the 3' end of the intron terminates the intron with the nearly invariant sequence AG.

[0040] In a specific embodiment, the present invention relates to an antisense oligonucleotide for masking a splicing regulatory motif (ESE sequence). In a specific embodiment, the antisense oligonucleotide comprises the sequence shown in SEQ ID NO: 5 (H36ESE). In a specific embodiment, the antisense oligonucleotide consists of the sequence shown in SEQ ID NO: 5 (H36ESE).

[0041] In some embodiments, the antisense oligonucleotide of the present invention comprises the sequence set forth in SEQ ID NO: 5, which is complementary to the nucleic acid sequence of CEP290 exon 36, which is essential for skipping exon 36 inserted into the mutant CEP290 mRNA.

[0042] Antisense oligonucleotides are used as splice-switching oligonucleotides (SSOs) to bypass protein cleavage caused by mutations in exon 36 of CEP290. Typically, administration of an SSO designed to skip exon 36 bypasses the mutation, allowing the production of a minimally truncated mRNA with an open reading frame and a functional, minimally truncated CEP290 protein. Splice-switching oligonucleotides direct pre-mRNA splicing by binding to sequence elements and blocking access to the transcript by spliceosomes and other splicing factors. In the context of the present invention, the inventors used databases (http: / / mfold.rna.albany.edu / and http: / / rulai.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi) to identify targetable sequences in the CEP290 pre-mRNA surrounding the donor splice site in exon 36.

[0043] In a particular embodiment, the inventors used databases to identify targetable sequences in the CEP290 pre-mRNA surrounding the H36ESE and / or H36D sites.

[0044] Typically, such methods are referred to as exon skipping strategies.

[0045] As used herein, the term "exon" refers to a nucleic acid sequence that is represented in the mature form of an RNA molecule after a defined portion of a protein-coding nucleic acid or any portion of a pre-processing (or precursor) RNA has been removed by splicing. The mature RNA molecule can be messenger RNA (mRNA) or a functional form of non-coding RNA, such as rRNA or tRNA.

[0046] As used herein, the term "exon skipping" generally refers to a process in which an entire exon or a portion thereof is removed from a given pre-processed RNA, thereby excluding it from being present in the mature RNA, e.g., the mature mRNA that is translated into a protein. In the context of the present invention, exon skipping is used to bypass (hide) a mutation in exon 36 from the spliceosome machinery, increasing the abundance of alternatively spliced ​​mRNA and truncated protein and shortening the axoneme length. In this way, this strategy makes it possible to obtain a functional shortened CEP290 protein. In this way, this strategy prevents the occurrence of a premature stop codon in exon 36.

[0047] Thus, the portion of the protein that is somehow encoded by the skipped exon is not present in the expressed form of the protein, typically resulting in an altered, but still functional, form of the protein. In certain embodiments, the skipped exon is a mutant exon originally present in the human CEP290 gene, which may contain a mutation or other modification in its sequence that somehow results in a premature stop codon that leads to the formation of a truncated CEP290 protein.

[0048] As used herein, the terms "preventing the appearance of a premature stop codon in exon 36," "blocking the recognition of exon 36," or "removing a premature stop codon in exon 36" refer to the removal of said exon in mature mRNA by modifying splicing using an exon-skipping strategy (see FIG. 1). Therefore, the present invention provides a method for obtaining a functional protein using exon-skipping technology. This method involves blocking or preventing the incorporation of a targeted exon 36, encoding an amino acid sequence involved in protein dysfunction, into mature mRNA. The AON binds to a complementary required sequence in pre-mRNA and causes splicing modification. Thus, the targeted exon is not included in the mature mRNA that is translated into protein, and the amino acid sequence encoded by the targeted exon is missing from the translated protein. In this way, a short functional protein, which is nearly the full-length CEP290 protein, can be obtained by performing an exon-skipping strategy.

[0049] Thus, the exon skipping strategy restores the function and / or stability of the CEP290 protein by at least about 10%, preferably about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%, compared to the protein translated from mutant CEP290. Such protein restoration can be observed at a microscopic level. For example, restoration of protein expression and / or localization can be assessed by immunohistochemistry, immunofluorescence, Western blot analysis; restoration / improvement of protein functionality as assessed by improved cilia assembly and / or maintenance, restoration / improvement of cone function; or at a macroscopic level (i.e., improvement / restoration of clinical symptoms such as vision).

[0050] Those skilled in the art will recognize that there are many methods for determining or measuring the level of protein functionality, for example, to determine the level of increased or decreased functionality in response to a treatment protocol. Such methods include, but are not limited to, measuring or detecting the activity of the protein. Such measurements are typically performed in comparison to a standard, control, or "normal" sample. Additionally, if a lack of protein functionality is involved in a disease process, disease symptoms can be monitored and / or measured to indirectly detect the presence or absence of a properly functioning protein or to measure the success of a treatment protocol intended to ameliorate the lack of protein functionality. In particular, the functionality of CEP290 can be measured by several art-recognized methods. Generally, removal of exon 36 containing a premature stop codon resulting from a mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon, in certain embodiments, removal of exon 36 containing at least one mutation, is performed using at least one antisense oligonucleotide (AON).

[0051] In certain embodiments, the antisense oligonucleotides of the invention can be delivered in vivo either alone or in association with a vector.

[0052] In its broadest sense, a "vector" is any vehicle that facilitates the transfer of the antisense oligonucleotides of the present invention into cells. Preferably, the vector transports the nucleic acid into the cell with reduced degradation relative to the level of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, naked plasmids, non-viral delivery systems (electroporation, sonoporation, cationic transfection agents, liposomes, nanoparticles, etc.), phagemids, viruses, and other vehicles derived from viral or bacterial sources that have been engineered with the insertion or incorporation of antisense oligonucleotide nucleic acid sequences. Viral vectors are a preferred type of vector, and include, but are not limited to, nucleic acid sequences derived from the following viruses: RNA viruses, such as retroviruses (e.g., Moloney murine leukemia virus and lentivirus-derived vectors), hedgehog sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; and poliovirus. Other vectors not named but known in the art are readily available.

[0053] Typically, viral vectors of the present invention include adenoviruses and adeno-associated (AAV) viruses, which are DNA viruses already approved for human use in gene therapy. Currently, 12 AAV serotypes (AAV1-12) are known, each with different tissue tropism (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAVs are derived from the parasite-dependent parvovirus AAV (Choi, VW J Virol 2005; 79:6801-07). Adeno-associated viruses types 1-12 can be engineered to be replication-deficient and capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). Furthermore, they possess advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition, thereby enabling transduction of multiple lineages. In addition, wild-type adeno-associated virus infection has been tracked in tissue culture for over 100 passages in the absence of selective pressure, indicating that adeno-associated virus genome integration is a relatively stable event. Adeno-associated viruses can also function extrachromosomally.

[0054] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, e.g., Sambrook et al., 1989. For several years, plasmid vectors have been used as DNA vaccines to deliver antigen-encoding genes to cells in vivo. They are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. On the other hand, these plasmids, which have promoters compatible with the host cell, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids can be delivered via a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. DNA plasmids can also be administered via nasal sprays or drops, rectal suppositories, and orally. Preferably, the DNA plasmid is injected via an intraocular route (intravitreal, subretinal, suprachoroidal...). The DNA plasmid can also be administered to the epidermis or mucosal surfaces using a gene gun. The plasmid can be provided in an aqueous solution, dried on gold particles, or associated with another DNA delivery system (including, but not limited to, liposomes, dendrimers, cochleates, and microencapsulation).

[0055] In certain embodiments, the antisense oligonucleotide nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter, which can also be, for example, a viral promoter, e.g., a CMV promoter, or any synthetic promoter.

[0056] Uses of the Antisense Oligonucleotides of the Invention In a second aspect, the present invention relates to a method for performing antisense oligonucleotide-mediated exon skipping in a subject in need thereof, the method comprising the step of delivering an amount of an antisense oligonucleotide to target cells of the subject, wherein the subject is suffering from retinal dystrophy caused by a mutation that modifies splicing and / or a mutation that results in a premature stop codon due to a nonsense mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon in a gene that is important for functionality and / or survival of the target cell, and wherein the nucleic acid sequence of the CEP290 gene is selected from the group consisting of an ESE sequence of exon 36 and a sequence comprising a donor splice site near the exon 36 / intron 36 boundary, and wherein the antisense oligonucleotide effects antisense oligonucleotide-mediated exon skipping in pre-mRNA derived from a gene whose mutation causes retinal dystrophy in the target cells of the subject.

[0057] In certain embodiments, the present invention provides a method for performing antisense oligonucleotide-mediated exon skipping, wherein the subject suffers from a retinal dystrophy caused by at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A, which modify splicing and introduce a premature stop codon in exon 36 in a gene important for the functionality and / or survival of the target cell. wherein the nucleic acid sequence of the CEP290 gene is selected from the group consisting of sequences comprising the ESE sequence of exon 36 and a donor or acceptor splice site (around the exon 36 / intron 36 boundary), and wherein the antisense oligonucleotide causes antisense oligonucleotide-mediated exon skipping in pre-mRNA derived from a gene whose mutation causes retinal dystrophy in the target cell of the subject.

[0058] As used herein, the term "target cells" in a subject refers to one or more of the following types of ocular cells, including but not limited to ciliated cells, photoreceptors, cone cells, rod cells, retinal epithelial cells, retinal bipolar cells, retinal ganglion cells, RPE cells, horizontal cells, amacrine cells, and Müller cells.

[0059] Mutations in CEP290 (MIM610142), which encodes a widely expressed centrosomal protein involved in the formation and maintenance of cilia [3] and cell division (Valente, EM; Silhavy, JL; Brancati, F.; Barrano, G.; Krishnaswami, SR; Castori, M.; Lancaster, MA; Boltshauser, E.; Boccone, L.; Al-Gazali, L.; et al. Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat. Genet. 2006, 38, 623-625.), are the primary cause of a disorder called LCA10.

[0060] Thus, in certain embodiments, the target cell is a ciliated cell. As used herein, the term "cilia" encompasses vibrating / motile cilia, primary cilia, or flagella expressed by a cell.

[0061] Within the eye, the retina is the tissue that lines the wall at the base of the eye. It is composed of three cell layers, including the photoreceptor cell layer. Photoreceptors are divided into two parts: an inner segment and an outer segment, connected to each other via a connecting cilium. This primary cilium is an essential structure for transporting proteins from the inner segment, where they are synthesized, to the outer segment, where phototransduction occurs. The protein encoded by the CEP290 gene is localized at the base of the connecting cilium. Altered function can lead to abnormalities in intracellular transport, resulting in ciliary structural defects and / or loss or shortening of the outer segment within the photoreceptor cell.

[0062] As used herein, the term "retinal dystrophy" refers to chronic and progressive disorders that alter the anatomy and / or function of the retina. The retina is located at the back of the eye near the optic nerve and is composed of millions of light-sensitive cells called "photoreceptors." The purpose of the retina is to receive light focused by the lens, convert the light into neural signals, and send these signals to the brain for visual perception. When damage occurs to the photoreceptor cells, the retina is unable to function properly and has difficulty processing and transmitting visual information to the brain.

[0063] In certain embodiments, the methods of the invention, wherein the retinal dystrophies are selected from the group consisting of Leber's congenital amaurosis and other early-onset severe retinal dystrophies (LCA-like), rod-cone dystrophies (retinitis pigmentosa), cone-rod dystrophies, macular dystrophies including age-related macular degeneration, any ciliary-related disease associated with the retina including Joubert syndrome, Senior-Loken syndrome, Bardet-Biedl syndrome, Meckel and Meckel-like syndromes, Refsum syndrome, Stargardt disease, Usher syndrome, hereditary optic neuropathies, congenital stationary night blindness, color deficiency, and color vision deficiency.

[0064] In a particular embodiment, the method of the present invention, wherein the retinal dystrophy is Leber congenital amaurosis (LCA). The term "Leber congenital amaurosis (LCA)" is a common cause of childhood blindness (10%). Leber congenital amaurosis is the most severe inherited retinal dystrophy, resulting in blindness or severe visual impairment at birth or within the first few months of life. Leber congenital amaurosis can manifest as early-onset severe rod-cone dystrophy or early-onset severe cone-rod dystrophy.

[0065] In certain embodiments, the methods of the present invention comprise methods wherein the antisense oligonucleotide is selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, locked nucleic acid (LNA) oligonucleotides, morpholino oligonucleotides, tricycloDNA antisense oligonucleotides, U7- or U1-mediated antisense oligonucleotides, peptide-linked, nanoparticle-conjugated antisense oligonucleotides, 2'-O-MeRNA / ENA chimeric oligonucleotides, and 2'-O-methyl-phosphorothioate oligonucleotides.

[0066] In a third aspect, the present invention relates to an antisense oligonucleotide consisting of a sequence complementary to the nucleic acid sequence of the CEP290 gene, which alters splicing and is necessary to exclude an exon encoding a premature stop codon inserted into CEP290 mRNA due to a nonsense mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon in a subject having said mutation, wherein said nucleic acid sequence of the CEP290 gene is a sequence containing an ESE motif, and wherein said sequence is selected from the group consisting of an exon splicing enhancer (ESE) sequence of the mutated exon and a sequence containing a donor splice site for use in restoring CEP290 function in a cell having a nonsense mutation present in exon 36 of the CEP290 gene that introduces a premature stop codon in exon 36 or a frameshift mutation in exon 36 or an upstream exon.

[0067] Specifically, the present invention relates to an antisense oligonucleotide comprising a sequence complementary to the nucleic acid sequence of the CEP290 gene, which alters splicing and is necessary for excluding an exon encoding a premature stop codon inserted into CEP290 mRNA due to a nonsense mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon in a subject having said mutation, wherein the nucleic acid sequence of the CEP290 gene is a sequence comprising an ESE motif, and wherein the sequence is selected from the group consisting of an exon splicing enhancer (ESE) sequence of the mutated exon and a sequence comprising a donor splice site mutation suitable for restoring CEP290 function in cells having a nonsense mutation present in exon 36 of the CEP290 gene that introduces a premature stop codon into exon 36 or a frameshift mutation in exon 36 or an upstream exon.

[0068] In a specific embodiment, the antisense oligonucleotides of the invention target the splice donor site (H36D).

[0069] In a particular embodiment, an antisense oligonucleotide for use according to the invention is provided, wherein the premature stop codon inserted within exon 36 is due to at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A of CEP290 mRNA, wherein said nucleic acid sequence of the CEP290 gene is a sequence comprising an ESE motif, wherein said sequence is identical to that of the mutation present in the CEP290 gene in a subject having said mutation. and a sequence comprising an exon splicing enhancer (ESE) sequence of the exon and a donor splice site for use in restoring CEP290 function in cells having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A.

[0070] In a particular embodiment, an antisense oligonucleotide complementary to the nucleic acid sequence of the CEP290 gene for use according to the invention, wherein the cell having a nonsense mutation present in exon 36 that introduces a premature stop codon in exon 36 present in the CEP290 gene or a frameshift mutation in exon 36 or an upstream exon is a ciliated cell.

[0071] In a particular embodiment, an antisense oligonucleotide complementary to the nucleic acid sequence of the CEP290 gene for use according to the invention, wherein the cells having at least one mutation present in the CEP290 gene selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A are ciliated cells.

[0072] In a particular embodiment, an antisense oligonucleotide complementary to a nucleic acid sequence of the CEP290 gene for use according to the invention, wherein the antisense oligonucleotide consists of the nucleic acid sequence of SEQ ID NO:1.

[0073] In a particular embodiment, an antisense oligonucleotide complementary to a nucleic acid sequence of the CEP290 gene for use according to the invention, wherein said antisense oligonucleotide has a length of at least 15 nucleotides.

[0074] In a particular embodiment, an antisense oligonucleotide complementary to a nucleic acid sequence of the CEP290 gene for use in the present invention, wherein said antisense oligonucleotide is at least 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, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, , 102, 103, 104, 105, 106, 107 or 108 nucleotides in length.

[0075] In a particular embodiment, an antisense oligonucleotide complementary to the nucleic acid sequence of the CEP290 gene for use according to the invention is administered intravenously to a subject having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A.

[0076] In a fourth aspect, the present invention relates to a method for treating retinal dystrophy in a subject carrying a nonsense mutation located in exon 36 of the CEP290 gene, or a frameshift mutation in exon 36 or an upstream exon, which results in the appearance of a premature stop codon in exon 36 resulting in a truncated protein, comprising modulating the splicing of exon 36 containing the premature stop codon resulting from the nonsense mutation or the frameshift mutation in exon 36 or an upstream exon, wherein the method is performed by exposing a pre-mRNA comprising exon 36 encoding the CEP290 protein to an antisense oligonucleotide (AON) complementary to the sequence of the mutated exon with the premature stop codon.

[0077] In a particular embodiment, the method of the present invention for treating retinal dystrophy comprises modulating the splicing of exon 36 containing at least one mutation resulting in a truncated protein, said method being carried out by exposing a pre-mRNA containing exon 36 encoding the CEP290 protein to at least one antisense oligonucleotide (AON) of the present invention.

[0078] In a particular embodiment, the present invention provides a method for treating retinal dystrophy in a subject having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A located in exon 36 of the CEP290 gene, which results in the appearance of a premature stop codon resulting in a truncated protein, and modulating the splicing of exon 36 containing at least one mutation selected from the group consisting of c.4786_4790del, c.4791_4794del, c.4732G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A, wherein the method is carried out by exposing a pre-mRNA containing exon 36 encoding a CEP290 protein to an antisense oligonucleotide (AON) complementary to the sequence of the mutated exon having a premature stop codon.

[0079] In certain embodiments, methods of the invention comprise administering a therapeutically effective amount of an AON of the invention to a subject having a premature stop codon in exon 36 caused by a nonsense mutation in exon 36 containing the c.4723A>T mutation or a frameshift mutation in exon 36 or an upstream exon.

[0080] In certain embodiments, the methods of the invention comprise administering a therapeutically effective amount of an AON of the invention to a subject having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A.

[0081] In certain embodiments, the methods of the invention wherein the antisense oligonucleotide is complementary to a sequence comprising the donor splice site or acceptor splice site described above.

[0082] In a particular embodiment, the method of the present invention wherein the antisense oligonucleotide comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0083] In a particular embodiment, the method of the present invention wherein the antisense oligonucleotide consists of the nucleic acid sequence set forth in SEQ ID NO:1.

[0084] As used herein, the term "treat" or "treatment" refers to both prophylactic or preventive treatment and curative or disease-modifying treatment. This includes treatment of subjects at risk of or suspected of having a disease, as well as diseased subjects or subjects diagnosed with a disease or medical condition, including suppression of clinical recurrence. Treatment can be administered to subjects with a medical disorder or subjects who may ultimately acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder beyond what would be expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of disease treatment, e.g., an administration pattern used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The term "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of a drug to a subject during the initial period of the treatment regimen. The induction regimen may utilize (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than the physician would use during a maintenance regimen, administering the drug more frequently than the physician would use during a maintenance regimen, or both. The terms "maintenance regimen" or "maintenance period" refer to a treatment regimen (or part of a treatment regimen) used to maintain a subject during disease treatment, for example, to keep the subject in remission for an extended period of time (months or years). A maintenance regimen may utilize continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0085] As used herein, the term "subject" refers to any mammal, for example, a rodent, cat, dog, or primate. In particular, in the present invention, the subject is a human affected or susceptible to a mutation in CEP290 exon 36. In a particular embodiment, the subject is a human affected or susceptible to a premature stop codon in exon 36 caused by a nonsense mutation in exon 36, including the c.4723A>T mutation, or a frameshift mutation in exon 36 or an upstream exon of the CEP290 gene. In another embodiment, the subject is a human affected or susceptible to at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A in exon 36 of CEP290. In a particular embodiment, the subject is a human affected or susceptible to retinal dystrophy. In particular, the subject is affected or susceptible to LCA.

[0086] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an AON of the present invention, alone or with a vector) into a subject, particularly into the vitreous, aqueous humor, ciliary tissue or cells and / or extraocular muscles, the retina (e.g., after retinal detachment), or even the epithalamic space. Electroporation or sonoporation means may also be suitable for delivering the antisense oligonucleotides of the present invention (alone or with a vector of the present invention). When a disease or a symptom thereof is treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or a symptom thereof is prevented, administration of the substance typically occurs before the onset of the disease or its symptoms.

[0087] In certain embodiments, naked AONs are administered. In some embodiments, methods of the invention include those in which the AONs are delivered intraocularly, intravitreally, subretinally, parenterally, intravenously, intracerebroventricularly, or intrathecally. In certain embodiments, aqueous solutions (naked) are particularly suitable for intravenous, intramuscular, intravitreal, subretinal, subcutaneous, and intraperitoneal administration.

[0088] In a specific embodiment, an antisense oligonucleotide of the invention (naked or associated with a vector of the invention) is administered intravitreally to a subject having a premature stop codon in exon 36 caused by a nonsense mutation or a frameshift mutation within exon 36 or an upstream exon in the CEP290 gene.

[0089] In a specific embodiment, an antisense oligonucleotide of the invention (naked or associated with a vector of the invention) is administered intravitreally to a subject having at least the c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A mutation in the CEP290 gene.

[0090] The term "therapeutically effective amount" refers to the minimum amount of an active agent required to confer a therapeutic benefit on a subject. For example, a "therapeutically effective amount" for a subject is one that induces, ameliorates, or otherwise causes an improvement in the pathological symptoms, progression of a disease, or physiological condition associated with a disorder, or resistance to succumbing to the disorder. It will be understood that the total daily usage of the compounds of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound utilized; the specific composition utilized; the age, weight, general health, sex, and diet of the subject; the timing, route of administration, and excretion rate of the specific compound utilized; the duration of treatment; drugs used in combination with or concurrently with the specific compound utilized, as well as factors well known in the medical field. For example, it is well within the skill of one in the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, allowing for symptomatic adjustment of the dosage to the subject being treated. A pharmaceutical product typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg body weight per day.

[0091] Pharmaceutical Composition In a fifth aspect, the present invention relates to a pharmaceutical composition comprising an antisense oligonucleotide of the present invention (alone or in combination with a vector of the present invention) for use in treating retinal dystrophy in a subject carrying a premature stop codon in exon 36 caused by a nonsense mutation or a frameshift mutation in exon 36 or an upstream exon in the CEP290 gene.

[0092] In a particular embodiment, the present invention relates to a pharmaceutical product of the present invention (alone or together with a vector of the present invention) for use in treating retinal dystrophy in a subject with at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A.

[0093] The pharmaceutical compositions of the present invention can also include a pharmaceutically or physiologically acceptable carrier, such as saline, sodium phosphate, etc. The compositions will usually, but not always, be in liquid form. Suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propylhydroxybenzoates, mineral oil, etc. The formulation can also include lubricants, wetting agents, emulsifiers, preservatives, buffers, etc. Those skilled in the art will also recognize that nucleic acids are often delivered in combination with lipids (e.g., cationic lipids or neutral lipids or mixtures thereof), often in the form of liposomes or other suitable micro- or nanostructured materials (e.g., micelles, lipocomplexes, dendrimers, emulsions, cubic phases, nanoparticles, etc.).

[0094] Typically, the antisense oligonucleotides of the present invention (naked or with a vector of the present invention) can be delivered in a pharmaceutically acceptable ophthalmic vehicle so that they can penetrate the cornea and internal regions of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid / retina, and sclera. The pharmaceutically acceptable ophthalmic vehicle can be, for example, an ointment, vegetable oil, or an encapsulating material.

[0095] Alternatively, the antisense oligonucleotides of the invention (naked or with a vector of the invention) can be injected directly into the vitreous, aqueous humor, ciliary tissue or cells and / or extraocular muscles, the retina (e.g., after retinal detachment), or even into the epithalamic space. Electroporation or sonoporation procedures may also be suitable for delivering the antisense oligonucleotides of the invention (alone or with a vector of the invention).

[0096] In a specific embodiment, a pharmaceutical composition containing an antisense oligonucleotide of the invention (naked or associated with a vector of the invention) is administered intravitreally to a subject having a premature stop codon in exon 36 caused by a nonsense mutation or a frameshift mutation within exon 36 or an upstream exon in the CEP290 gene.

[0097] In a specific embodiment, a pharmaceutical composition containing an antisense oligonucleotide of the invention (naked or associated with a vector of the invention) is administered intravitreally to a subject having at least the c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A mutation in the CEP290 gene.

[0098] Those skilled in the art will recognize that the amount of AON administered will be sufficient to induce improvement in undesired disease symptoms. Such an amount can vary depending on factors such as the patient's gender, age, weight, overall physical condition, etc., among others, and can be determined on a case-by-case basis. This amount can also vary depending on the type of condition being treated and other components of the treatment protocol (e.g., administration of other drugs, such as steroids, etc.).

[0099] If viral-based delivery of AONs is chosen, the appropriate dose will depend on various factors, such as the viral strain utilized and the route of delivery (intramuscular, intravenous, intraarterial, etc.).

[0100] Those skilled in the art will recognize that such parameters are typically calculated during clinical trials. Even partial or intermittent relief of symptoms can be of significant benefit to the recipient. In addition, patient treatment is typically not a single event. Rather, the AON of the present invention may be administered on multiple occasions, which may be days, weeks, months, or even years apart, depending on the results obtained. This is particularly true for the treatment of Leber congenital amaurosis. The disease is not cured by this treatment, i.e., the gene encoding the protein will still be defective, and the encoded protein will still have undesirable destabilizing mechanisms, such as exposed proteolytic recognition sites, unless the AON of the present invention is administered.

[0101] The present invention will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the invention in any way. [Brief explanation of the drawings]

[0102] [Figure 1]Figure 1: AON-mediated splicing alteration of CEP290 pre-mRNA bypassing proteolytic cleavage. An exonic mutation (c.4723A>T, red) in the CEP290 pre-mRNA introduces a premature termination codon (PTC) within exon 36 into the CEP290 mRNA. Administration of an AON (black) targeting the splice donor site (H36D) is predicted to alter splicing by blocking the recognition of exon 36. Exclusion of exon 36 (CEP290Δ36) should allow bypassing proteolytic cleavage while maintaining the reading frame, resulting in the production of a nearly full-length CEP290 protein. [Figure 2A] Figure 2: Naturally occurring exclusion of CEP290 exon 36 encompassing a premature stop codon. Relative expression of (A) WT (gray bars) and mutant (black bars) full-length isoforms and (B) skipped (CEP290Δ36; hatched bars) CEP290 mRNA in control (C1-C3) and patient (P1 and P2) fibroblasts as measured by RT-qPCR using the GUSB and RPLP0 genes as standards. C corresponds to the C1, C2, and C3 pooled values. Values ​​are means ± SEM from 10 independent experiments. ****p ≤ 0.0001, ns = not significant. [Figure 2B] Figure 2: Naturally occurring exclusion of CEP290 exon 36 encompassing a premature stop codon. Relative expression of (A) WT (gray bars) and mutant (black bars) full-length isoforms and (B) skipped (CEP290Δ36; hatched bars) CEP290 mRNA in control (C1-C3) and patient (P1 and P2) fibroblasts as measured by RT-qPCR using the GUSB and RPLP0 genes as standards. C corresponds to the C1, C2, and C3 pooled values. Values ​​are means ± SEM from 10 independent experiments. ****p ≤ 0.0001, ns = not significant. [Figure 3]Figure 3: Effect of spontaneous elimination of PTC-encoding exon 36 on CEP290 protein production. Immunodetection of CEP290 protein in control cell lines (C1-C3) and mutant fibroblasts (P1 and P2). β-actin was used for normalization. Quantification of CEP290 protein abundance. C corresponds to the C1, C2, and C3 pooled value. Values ​​are the mean ± SEM from four independent experiments, determined by computerized densitometry analysis of CEP290 and β-actin expression in each sample. **p ≤ 0.01, ns = not significant. [Figure 4] Figure 4: Evaluation of CEP290 expression in quiescent cells. Quantification of CEP290 immunofluorescence intensity at basal bodies in each cell line (C represents the pooled value of C1, C2, and C3). Each dot represents the protein labeling intensity in an individual cell from six microscopic fields (scored automatically). The solid line indicates the mean. ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 5] Figure 5: Localization and abundance of CEP290 centriolar satellite partners. Quantification of RAB8A-positive cilia. Values ​​are means ± SEM (n ≥ 50 cilia for each group). Quantification of CP110 immunofluorescence intensity at centrosomes in quiescent fibroblasts. Values ​​are means ± SEM. Immunolabeling was performed from 90-100% confluent cells. Fully automated intensity measurements were recorded from six fields. C corresponds to the C1, C2, and C3 pooled value. ****p ≤ 0.0001, ns = not significant. AU = arbitrary units. [Figure 6A-B] Figure 6. Ciliogenesis and axonemal transport. (A) Percentage of ciliated cells and (B) length of ciliary axonemes in control and patient fibroblasts. A minimum of 90 ciliated cells were considered for each cell line. (C) Quantification of IFT88-positive cilia. Bars represent mean ± SEM (n ≥ 80 cilia for each group). C regroups values ​​for C1, C2, and C3. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant. [Figure 6C]Figure 6. Ciliogenesis and axonemal transport. (A) Percentage of ciliated cells and (B) length of ciliary axonemes in control and patient fibroblasts. A minimum of 90 ciliated cells were considered for each cell line. (C) Quantification of IFT88-positive cilia. Bars represent mean ± SEM (n ≥ 80 cilia for each group). C regroups values ​​for C1, C2, and C3. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns = not significant. [Figure 7A] Figure 7: Effect of AON-mediated exon 36 skipping on CEP290 mRNA. (A) RT-PCR analysis of CEP290 transcripts expressed in control (C3) and patient (P1 and P2) fibroblasts untreated or treated for 24 hours with lipofectamine alone or lipofectamine associated with H36D antisense oligonucleotide. The agarose gel image shows the amplicon produced using a primer pair encompassing the mutated exon 36. (B) Relative expression levels of full-length (black bars) and exon 36-skipped (gray bars) CEP290 mRNA in control (C1-C3) and patient (P1 and P2) fibroblasts as measured by RT-qPCR. Bars indicate the mean ± SEM from three independent experiments. C represents the pooled value for C1, C2, and C3. ****p ≤ 0.0001, ns not significant. [Figure 7B]Figure 7: Effect of AON-mediated exon 36 skipping on CEP290 mRNA. (A) RT-PCR analysis of CEP290 transcripts expressed in control (C3) and patient (P1 and P2) fibroblasts untreated or treated for 24 hours with lipofectamine alone or lipofectamine associated with H36D antisense oligonucleotide. The agarose gel image shows the amplicon produced using a primer pair encompassing the mutated exon 36. (B) Relative expression levels of full-length (black bars) and exon 36-skipped (gray bars) CEP290 mRNA in control (C1-C3) and patient (P1 and P2) fibroblasts as measured by RT-qPCR. Bars indicate the mean ± SEM from three independent experiments. C represents the pooled value for C1, C2, and C3. ****p ≤ 0.0001, ns not significant. [Figure 8A]Figure 8. Optimization of transfection conditions. (A) RT-qPCR analysis of reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts after transfection with untreated or increasing doses (20 nM–300 nM) of H36D oligonucleotide. The graph shows the mean amounts (±SEM) of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars) from three independent experiments. C regroups the values ​​obtained for C1, C2, and C3. ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant. (B) Reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts untreated or treated for increasing treatment times (4 h–72 h) with 75 nM H36D oligonucleotide. RT-qPCR analysis of mRNA. The graph shows the abundance of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars). C corresponds to the C1, C2, and C3 pool values. (C) CEP290 protein analysis in control (C3) and mutant (P1 and P2) cell lines untreated or treated with 75 nM H36D for 24 or 48 h. (D) Relative quantification of CEP290 protein abundance as a function of treatment time. β-actin was used for normalization. [Figure 8B]Figure 8. Optimization of transfection conditions. (A) RT-qPCR analysis of reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts after transfection with untreated or increasing doses (20 nM–300 nM) of H36D oligonucleotide. The graph shows the mean amounts (±SEM) of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars) from three independent experiments. C regroups the values ​​obtained for C1, C2, and C3. ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant. (B) Reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts untreated or treated for increasing treatment times (4 h–72 h) with 75 nM H36D oligonucleotide. RT-qPCR analysis of mRNA. The graph shows the abundance of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars). C corresponds to the C1, C2, and C3 pool values. (C) CEP290 protein analysis in control (C3) and mutant (P1 and P2) cell lines untreated or treated with 75 nM H36D for 24 or 48 h. (D) Relative quantification of CEP290 protein abundance as a function of treatment time. β-actin was used for normalization. [Figure 8C]Figure 8. Optimization of transfection conditions. (A) RT-qPCR analysis of reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts after transfection with untreated or increasing doses (20 nM–300 nM) of H36D oligonucleotide. The graph shows the mean amounts (±SEM) of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars) from three independent experiments. C regroups the values ​​obtained for C1, C2, and C3. ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant. (B) Reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts untreated or treated for increasing treatment times (4 h–72 h) with 75 nM H36D oligonucleotide. RT-qPCR analysis of mRNA. The graph shows the abundance of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars). C corresponds to the C1, C2, and C3 pool values. (C) CEP290 protein analysis in control (C3) and mutant (P1 and P2) cell lines untreated or treated with 75 nM H36D for 24 or 48 h. (D) Relative quantification of CEP290 protein abundance as a function of treatment time. β-actin was used for normalization. [Figure 8D]Figure 8. Optimization of transfection conditions. (A) RT-qPCR analysis of reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts after transfection with untreated or increasing doses (20 nM–300 nM) of H36D oligonucleotide. The graph shows the mean amounts (±SEM) of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars) from three independent experiments. C regroups the values ​​obtained for C1, C2, and C3. ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant. (B) Reverse-transcribed CEP290 mRNA extracted from control (C1–C3) and patient (P1 and P2) fibroblasts untreated or treated for increasing treatment times (4 h–72 h) with 75 nM H36D oligonucleotide. RT-qPCR analysis of mRNA. The graph shows the abundance of full-length (CEP290; black bars) and exon 36-skipping transcripts (CEP290Δ36; gray bars). C corresponds to the C1, C2, and C3 pool values. (C) CEP290 protein analysis in control (C3) and mutant (P1 and P2) cell lines untreated or treated with 75 nM H36D for 24 or 48 h. (D) Relative quantification of CEP290 protein abundance as a function of treatment time. β-actin was used for normalization. [Figure 9A-B]Figure 9: Effect of AON treatment at the protein level and its impact on cilia. (A) CEP290 protein analysis in control and mutant cell lines untreated or treated with 75 nM H36D for 48 h. (B) Quantification of CEP290 protein amount relative to β-actin. Bars correspond to the mean ± SEM from three independent experiments. = no significant difference. AU = arbitrary units. (C) Quantification of CEP290 immunofluorescence intensity at the basal body in each cell line. Each dot represents the protein labeling intensity in an individual cell, automatically recorded from six microscopic fields. The solid line represents the mean. (D) Quantification of CP110 immunofluorescence intensity at the centrosome in quiescent fibroblasts. Fully automated intensity measurements were recorded from six fields. (E) Percentage of ciliated cells in control and mutant fibroblasts and (F) length of ciliary axonemes. A minimum of 90 ciliated cells were considered for each cell line. C corresponds to the C1, C2, and C3 pooled values. *p≦0.05, *p≦0.01, ***p≦0.001, ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 9C] Figure 9: Effect of AON treatment at the protein level and its impact on cilia. (A) CEP290 protein analysis in control and mutant cell lines untreated or treated with 75 nM H36D for 48 h. (B) Quantification of CEP290 protein amount relative to β-actin. Bars correspond to the mean ± SEM from three independent experiments. = no significant difference. AU = arbitrary units. (C) Quantification of CEP290 immunofluorescence intensity at the basal body in each cell line. Each dot represents the protein labeling intensity in an individual cell, automatically recorded from six microscopic fields. The solid line represents the mean. (D) Quantification of CP110 immunofluorescence intensity at the centrosome in quiescent fibroblasts. Fully automated intensity measurements were recorded from six fields. (E) Percentage of ciliated cells in control and mutant fibroblasts and (F) length of ciliary axonemes. A minimum of 90 ciliated cells were considered for each cell line. C corresponds to the C1, C2, and C3 pooled values. *p≦0.05, *p≦0.01, ***p≦0.001, ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 9D]Figure 9: Effect of AON treatment at the protein level and its impact on cilia. (A) CEP290 protein analysis in control and mutant cell lines untreated or treated with 75 nM H36D for 48 h. (B) Quantification of CEP290 protein amount relative to β-actin. Bars correspond to the mean ± SEM from three independent experiments. = no significant difference. AU = arbitrary units. (C) Quantification of CEP290 immunofluorescence intensity at the basal body in each cell line. Each dot represents the protein labeling intensity in an individual cell, automatically recorded from six microscopic fields. The solid line represents the mean. (D) Quantification of CP110 immunofluorescence intensity at the centrosome in quiescent fibroblasts. Fully automated intensity measurements were recorded from six fields. (E) Percentage of ciliated cells in control and mutant fibroblasts and (F) length of ciliary axonemes. A minimum of 90 ciliated cells were considered for each cell line. C corresponds to the C1, C2, and C3 pooled values. *p≦0.05, *p≦0.01, ***p≦0.001, ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 9E] Figure 9: Effect of AON treatment at the protein level and its impact on cilia. (A) CEP290 protein analysis in control and mutant cell lines untreated or treated with 75 nM H36D for 48 h. (B) Quantification of CEP290 protein amount relative to β-actin. Bars correspond to the mean ± SEM from three independent experiments. = no significant difference. AU = arbitrary units. (C) Quantification of CEP290 immunofluorescence intensity at the basal body in each cell line. Each dot represents the protein labeling intensity in an individual cell, automatically recorded from six microscopic fields. The solid line represents the mean. (D) Quantification of CP110 immunofluorescence intensity at the centrosome in quiescent fibroblasts. Fully automated intensity measurements were recorded from six fields. (E) Percentage of ciliated cells in control and mutant fibroblasts and (F) length of ciliary axonemes. A minimum of 90 ciliated cells were considered for each cell line. C corresponds to the C1, C2, and C3 pooled values. *p≦0.05, *p≦0.01, ***p≦0.001, ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 9F]Figure 9: Effect of AON treatment at the protein level and its impact on cilia. (A) CEP290 protein analysis in control and mutant cell lines untreated or treated with 75 nM H36D for 48 h. (B) Quantification of CEP290 protein amount relative to β-actin. Bars correspond to the mean ± SEM from three independent experiments. = no significant difference. AU = arbitrary units. (C) Quantification of CEP290 immunofluorescence intensity at the basal body in each cell line. Each dot represents the protein labeling intensity in an individual cell, automatically recorded from six microscopic fields. The solid line represents the mean. (D) Quantification of CP110 immunofluorescence intensity at the centrosome in quiescent fibroblasts. Fully automated intensity measurements were recorded from six fields. (E) Percentage of ciliated cells in control and mutant fibroblasts and (F) length of ciliary axonemes. A minimum of 90 ciliated cells were considered for each cell line. C corresponds to the C1, C2, and C3 pooled values. *p≦0.05, *p≦0.01, ***p≦0.001, ****p≦0.0001, ns=not significant. AU=arbitrary units. [Figure 10] Figure 10. Effect of AON-mediated exon 36 skipping on CEP290 mRNA. Relative expression levels of full-length (black bars) and exon 36-skipped (gray bars) CEP290 mRNA in control (C1-C3) and patient (P1 and P2) fibroblasts as measured by RT-qPCR. Bars show mean ± SEM from three independent experiments. C represents the pooled value of C1-C3. ****p≦0.0001, ns not significant. [Figure 11]Figure 11. Biallelic elimination of Cep290 exon 35 in Cep290del / del mice accounts for a milder retinal phenotype compared with biallelic elimination in compound heterozygous Cep290ptc / del mice. (A) Dark-adapted (scotopic; left graph) and light-adapted (light-adapted; right graph) ERG recordings in Cep290del / del and Cep290ptc / del compared with C57BL / 6J mice at P30 (N≧9). (B) ONL thickness measured from control, Cep290del / del, and Cep290ptc / del retinal sections shows a more severe reduction in Cep290ptc / del retinas compared with corresponding Cep2del / del at P30 (N=3). All data were statistically analyzed using a two-way analysis of variance with post hoc Sidak's test and are presented as mean ± SEM.

[0103] Example 1 Materials and Methods Genetic analysis P1 and P2 are two unrelated unrelated cases born to apparently unrelated parents from the transnational region of Flanders. They were referred to our Molecular Diagnosis Unit of our Genetic Department for molecular diagnosis of early-onset, severe retinal dystrophy. Patient DNA was subjected to panel-based molecular testing of 199 genes involved in retinal dystrophy (Supplementary Material, Figure S1), and the variant dataset was filtered using Polydiag software from the in-house developed Polyweb series. Biallelicity for the apparently homozygous CEP290 c.4723A>T variant was assessed by Sanger sequencing of parental DNA using primers specific for CEP290 exon 36 (Supplementary Material, Table S1). Written informed consent was obtained from all participating individuals. The study was approved by the Comite de Protection des Personnes "Ile-De-France II" (2015-03-03 / DC 2014-2272).

[0104] Splicing nonsense c.4723A>T (p.Lys1575 * ) In silico analysis of mutations The consequences of the c.4723A>T substitution on splicing were assessed using several prediction software as previously described

[21] .

[0105] cell culture Fibroblast cell lines were obtained from skin biopsies of affected subjects (P1 and P2) and three healthy individuals (C1, C2, and C3). A table summarizing their genetic and clinical characteristics is shown in Supplementary Material, Table S2. Primary fibroblasts (<15 passages) were cultured as previously described

[21] .

[0106] AONs and transfection Antisense oligonucleotides (AONs) specific for the donor splice site and ESE motifs in CEP290 exon 36 were identified using software prediction tools (the m-fold and ESEfinder3.0 programs available online at http: / / mfold.rna.albany.edu / and http: / / rulai.cshl.edu / cgi-bin / tools / ESE3 / esefinder.cgi, respectively) according to general recommendations

[23] .

[0107] The locations of the H36D (+98-11) and H36ESE (+63+84) antisense oligonucleotides are shown in Figure 6. The sequence of H36D was as follows: 5'-UAGAAUCUUACCCAAGCCGUUU-3' (SEQ ID NO: 1). The sequence of H36ESE was as follows: 5'-UAGUGAACUAUCAGCCUGUAGU-3' (SEQ ID NO: 5).

[0108] These AONs were synthesized by Eurofins Genomics (St. Quentin Fallavier, France) and contain 2'-O-methyl RNA and a full-length phosphorothioate backbone. Eighty percent confluent cells were transfected with various concentrations of AONs ranging from 20 to 300 nmol / L in Opti-MEM supplemented with 10% fetal bovine serum using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested at 4 to 72 hours for mRNA or protein analysis.

[0109] RNA preparation and cDNA synthesis Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's protocol. All samples were DNase-treated with an RNase-free DNase kit (Qiagen). The concentration and purity of total RNA were measured using a Nanodrop-2000 spectrophotometer (Thermo Scientific, Illkirch, France) and then stored at -80°C. First-strand cDNA synthesis was performed from 500 ng of extracted total RNA using the Verso cDNA Kit (Thermo Scientific) containing random hexamer:anchor oligo(dT) primers at a 3:1 (vol:vol) ratio, according to the manufacturer's instructions. A non-reverse transcription reaction (no enzyme; RT-) for one sample was prepared to serve as a control for reverse transcription PCR (RT-PCR) and real-time quantitative PCR (RT-qPCR) experiments.

[0110] RT-PCR CEP290 splicing isoforms were amplified from reverse-transcribed mRNA (2 μl) in 20 μl of 1× Phusion HF buffer containing 4 mM dNTPs (Thermo Scientific), 0.4 units Phusion High-Fidelity DNA Polymerase (Thermo Scientific), and 10 μM specific primer pairs (Supplementary Material, Figure S1 and Table S1). A no template reaction was used as a negative control (NTC). PCR was performed in a 2720 Thermal Cycler (Applied Biosystems, Courtaboeuf, France) under the following conditions: initial denaturation at 98°C for 5 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. PCR products (5 μl) were separated by electrophoresis in a 3% low-melting-point agarose gel stained with ethidium bromide, visualized under UV light, and excised. The in-gel PCR products were further sequenced using the Big Dye Terminator Cycle Sequencing Kit v3.1 (ABI Prism™, Applied Biosystems, Foster City, USA) in a 3500 automated sequencer (Applied Biosystems, Foster City, USA).

[0111] RT-qPCR analysis The abundance of CEP290 mRNA isoforms was measured using primers specific for the non-skipped (referred to as "full-length") or skipped CEP290 versions. Primer sequences are listed in Supplementary Material, Figure S1 and Table S2. Data were normalized using GUSB (NM_000181.3) and RPLP0 (NM_001002.3) mRNAs. Each sample's cDNA (5 μl diluted 1:25 in RNAse-free HO) was subjected to real-time PCR amplification in 20 μl of buffer containing SYBR GREEN PCR Master Mix (Life Technologies) and 300 nM forward and reverse primers under the following conditions: Taq polymerase activation and denaturation at 95°C for 10 minutes, followed by 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. The specificity of the amplification products was determined after melting curve analysis at the end of each amplification using one cycle at 95°C for 15 seconds, followed by a stepwise heat increase from 60°C to 95°C for 20 minutes. Data analysis and methods were performed as previously described [8].

[0112] Protein analysis For Western blot analysis, total proteins from treated and untreated cells were extracted and quantified as previously described

[21] , and the relative abundance of CEP290 protein was estimated by densitometry using β-actin as a reference in each cell line.

[0113] For immunocytochemical analysis, cells were grown on glass coverslips in 12-well plates for 24 hours to reach 90%–100% confluence and either transfected with H36D(+98-11) AON or left untreated. After 24 hours, treated and untreated cells were serum-starved for 48–72 hours, followed by cold methanol fixation and immunolabeling for ARL13B, CEP290, CP110, IFT88, pericentrin, RAB8A, γ-tubulin, and / or acetylated α-tubulin. Immunofluorescence images were acquired and processed to analyze cilia abundance, axoneme length, subcellular localization, and / or staining intensity. All experimental and analytical methods are described in Barny et al. (2018).

[0114] Immunocytochemical analysis Cells were seeded on glass coverslips in 12-well plates and transfected with H36D(+98-11) AON 24 hours later using the conditions previously described. 24 hours after transfection, cells were incubated in serum-free medium for 48-72 hours. After serum deprivation, cells were fixed with cold methanol (-20°C for 7 minutes) and washed twice with PBS. Untreated fibroblasts were treated under the same conditions. Cells were permeabilized and nonspecific sites were saturated for 1 hour in a PBS solution containing 3% bovine serum albumin and 0.5% Triton X-100. The permeabilized cells were incubated overnight at 4°C with the following primary antibodies in PBS containing 3% bovine serum albumin and 0.1% Triton X-100: anti-pericentrin rabbit antibody (1:1000, Abcam), anti-γ-tubulin mouse antibody (1:500, Sigma-Aldrich), anti-CEP290 rabbit antibody (1:100, Novus Biologicals), anti-CP110 rabbit antibody (1:100, ProteinTech), anti-acetylated α-tubulin mouse antibody (1:1000; Sigma-Aldrich), anti-ARL13B rabbit antibody (1:200, ProteinTech), anti-IFT88 rabbit antibody (1:100, ProteinTech), and anti-RAB8A mouse antibody (1:50, Abnova). After washing three times with PBS, the cells were incubated with secondary antibodies: goat anti-rabbit IgG conjugated to Alexa-Fluor 488 and goat anti-mouse IgG conjugated to Alexa-Fluor 568 (1:1000; Life Technologies) in a PBS solution containing 3% bovine serum albumin and 0.1% Triton X-100 for 1 hour at room temperature. After three additional washes with PBS, the coverslips were mounted on slides using mounting medium containing DAPI (ProLong Gold antifade reagent with DAPI, Invitrogen) to stain cell nuclei. Immunofluorescence images were obtained using a Zeiss spinning disk microscope. Exposure times and settings for image processing were kept constant for all samples to allow for inter-sample comparisons.The number of Z-stacks collected varied between samples but was optimized to capture maximum fluorescence signal. Deconvolved images were projected into a single image using the ImageJ software Z-projection tool with maximum intensity settings. The total fluorescence contained within a 16-μm square centered on the centrosome, as determined by gamma-tubulin staining, was recorded to measure the intensity of CEP290 and CP110 labeling in the pericentriolar region. The integrated pixel density was quantified in each square using ImageJ software. Final images were generated using ImageJ software.

[0115] statistics All statistical analyses were performed using Prism 6 software, and significant differences were determined using one-way analysis of variance with post hoc Tukey's test. Data from C1, C2, and C3 were systematically pooled for immunolabeling analysis. Error bars reflect SEM.

[0116] result Panel-based molecular diagnostic testing identifies homozygosity for the CEP290 c.4723A>T founder mutation in two patients with congenital retinal dystrophies of different severity We studied two apparently unrelated, sporadic cases from Belgium and / or Flanders, France, addressing congenital retinal dystrophy without extraocular complications. The first individual (P1) presented at birth with nystagmus, light aversion, and hyperopia (+6 diopters LRE). He had no cone-derived electroretinogram but a severely reduced voltage rod-derived response. His visual acuity improved spontaneously over the first decade of life. At age 20, he presented with tubular fields with visual acuities (VA) of 20 / 67 (RE) and 20 / 50 (LE), thin retinal vessels, optic nerve atrophy, and peripheral pigmentation of the fundus. Initial symptoms suggested early-onset severe cone-rod dystrophy, but this outcome is consistent with a rod-dominant LCA-like disorder called early-onset severe rod-cone dystrophy. The second individual (P2) presented with typical LCA10-related disease, i.e., stationary congenital blindness with nystagmus, inability to follow lights or objects, and flat cone and rod electroretinogram responses. Panel-based molecular diagnosis (190 genes) and Sanger-based familial segregation analysis for inherited retinal dystrophies identified the Flemish founder gene CEP290 c.4723A>T (p.Lys1575) in two cases. * ) homozygosity for the mutation was identified.

[0117] In silico analysis suggests that the CEP290 c.4723A>T mutation induces a nonsense-associated splicing alteration The impact of the c.4723A>T mutation on splicing was analyzed using a prediction software solution that probes splice signals and ESS / ESE binding sites. Substitution of adenine at c.4723 by thymine (mutation) or guanine is predicted to increase the ESS / ESE ratio of exon 36 compared to the wild-type sequence, thus increasing its susceptibility to skipping (substitution by cytosine is not predicted to result in this) (Table 2).

[0118] [Table 2]

[0119] Effect of the nucleotide change at position c.4723 in the ESS and ESE motifs by the EX-SKIP and HOT-SKIP prediction programs. The WT allele and the mutant allele identified in this study are labeled in blue and red, respectively. ESS = exonic splicing silencer; ESE = exonic splicing enhancer.

[0120] mRNA analysis supports c.4723A>T-mediated nonsense-associated splicing alteration and basal endogenous alternative splicing of exon 36 Agarose gel analysis and Sanger sequencing (table not shown) of RT-PCR products generated from mRNA from P1 and P2 skin fibroblasts homozygous for the c.4723A>T variant using primers specific for CEP290 exons 35 and 37 detected the full-length mutant cDNA (CEP290) and an alternative splicing product lacking exon 36 (CEP290Δ36), which does not contain the PTC. Control fibroblasts expressed the full-length wild-type cDNA, but the CEP290Δ36 product was not detectable, in contrast to human retina, where both isoforms were identified. These observations indicate that CEP290 exon 36 undergoes endogenous basal skipping in the retina and, consistent with in silico analysis, that the c.4723A>T variant induces a nonsense-associated splicing change (data not shown). Interestingly, RT-qPCR analysis using primers specific for the CEP290Δ36 isoform (table not shown) detected the product in control fibroblasts (Figure 2B). This observation supports some contribution of endogenous basal exon skipping in the CEP290 frame restoration demonstrated in P1 and P2 mutant fibroblasts. RT-qPCR analysis using primers specific for the full-length mutant / wild-type cDNA (table not shown) demonstrated a decreased abundance of the mutant product in P1 and P2 cell lines compared with the corresponding wild-type in controls, supporting nonsense-mediated RNA decay (NMD) of mRNAs carrying the nonsense c.4723A>T mutation (Figure 2A).

[0121] Protein analysis detects low levels of centrosome-localized CEP290 protein in patient fibroblasts homozygous for the c.4723A>T nonsense mutation Western blot analysis of protein extracts from serum-starved P1 and P2 fibroblasts detected a minimal CEP290 product of approximately 290 kDa (Figures 3 and 4). This product was localized to the centrosome based on immunocytochemical analysis (Figure 4). These results indicate that the PTC-free alternative splicing product lacking exon 36 is translated into a stable protein that can be recruited to the centrosome similarly to the wild-type protein.

[0122] Cilia analysis of serum-starved patient fibroblasts reveals apparently normal RAB8A localization at the centrosome but no axoneme elongation CEP290 exon 36 encodes 36 amino acids that contribute to the CEP290 domain, which binds RAB8A. Recruitment of RAB8A to the centrosome results in the release of the ciliogenesis suppressor CP110, thereby initiating ciliogenesis during the cell transition from proliferation to quiescence [24-26]. Interestingly, RAB8A immunolabeling in quiescent fibroblasts demonstrated comparable localization in the basal bodies of patient and control fibroblasts, suggesting that the absence of the information encoded by exon 36 does not alter RAB8A recruitment to the centrosome (Figure 6). Furthermore, comparable CP110 abundance was observed at the centrosomes of control and P1 fibroblasts, indicating correct release upon RAB8A recruitment (Figure 6A). In contrast, CP110 abundance at the centrosomes of P2 cells was significantly higher than that of control and P1 cells (p ≤ 0.0001; Figure 6A). Accumulation of CP110 in P2 compared with P1 could be correlated with decreased abundance of CEP290Δ36aa protein. Consistent with normal and impaired CP110 release, cilia abundance was within the normal range and decreased in P1 and P2 fibroblasts, respectively (p ≤ 0.001; Figure 5B).

[0123] Another noteworthy point is that we measured cilia length and observed statistically significant axoneme elongation in both patient cell lines compared with controls (mean axoneme size 3.9 μm in controls vs. 4.4 μm in P1 and 4.9 μm in P2, p ≤ 0.0001; Figure 6C). Cilia from P2, which express less CEP290Δ36aa isoform in the centrosome, exhibited significantly longer axonemes than their P1 counterparts (p ≤ 0.01; Figure 6C). This further supports the correlation between the severity of the ciliary phenotype and the amount of minimally shortened CEP290 cells generated by the mutant allele.

[0124] As observed in control cells, IFT88 immunolabeling in patient fibroblasts revealed all of this IFT complex B protein along the axoneme (Fig. 6C), suggesting that the abnormal ciliary elongation in patient cells is not related to defects in IFT88-driven anterograde transport.

[0125] Targeting the consensus donor splice site allows dose- and time-dependent skipping of CEP290 exon 36 The RNA conformation around CEP290 exon 36 and splicing regulatory elements was predicted in silico using the m-fold and ESEfinder 3.0 programs to design splicing-switching AONs. We designed 2'-O-methyl-phosphorothioate (2'-OMePs) AONs targeting either the donor site (H36D(+98-11)) (Figure 1). AONs were delivered to patient and control fibroblasts at a final concentration of 150 nM for 24 h before mRNA analysis. Treatment with the H36D(+98-11) AON significantly increased the abundance of the product lacking exon 36 and halved the abundance of the full-length mutant and wild-type products in patient and control fibroblasts, respectively (Figure 7). NMD significantly reduced the abundance of the full-length mutant in patient cells compared to control cells (Figure 7B). Consistent with a switch from NMD-prone mRNA to a PTC-free isoform, the abundance of an alternative splicing product lacking exon 36 in patient fibroblasts was comparable to that in controls after treatment with H36D(+98-11) AON. Treatment with transfection reagent alone did not alter CEP290 expression in any cell line.

[0126] To assess dose-dependent skipping efficiency, patient and control fibroblasts were treated with increasing doses of H36D(+98-11) AON for 24 hours. This revealed that the amount of alternative splice products lacking exon 36 reached a maximum in almost all cell lines at an AON concentration of 75 nmol / L (Figure 8A). At this concentration, accumulation of alternative splice products and CEP290 protein was observed with treatment time (Figures 8B, 8C, and 8D).

[0127] The RNA conformation around CEP290 exon 36 and splicing regulatory elements was predicted in silico using the m-fold and ESEfinder 3.0 programs to design splice-switching AONs. 2'-O-methyl-phosphorothioate (2'-OMePs) AONs targeting the donor site (H36D(+98-11)) and the exon splicing enhancer region (H36ESE(+63+84)) were designed. AONs were delivered to patient and control fibroblasts at a final concentration of 150 nM, and transfected cells were maintained in culture for 24 h before mRNA analysis. Treatment with H36ESE(+63+84) did not affect the relative abundance of the transcript lacking exon 36, whereas transfection with H36D(+98-11) AON statistically significantly increased the abundance of the product lacking exon 36 and reduced the abundance of the full-length mutant and wild-type products by half in patient and control fibroblasts, respectively (Figure 10). NMD significantly reduced the abundance of the full-length mutant in patient fibroblasts compared with control cells (Figure 10). Consistent with a switch from NMD-prone mRNA to a PTC-free isoform, the abundance of the alternatively spliced ​​product lacking exon 36 in patient fibroblasts was comparable to that in controls after treatment with H36D(+98-11) AON. Treatment with transfection reagent alone did not alter CEP290 expression in any cell line (Figure 10).

[0128] AON-mediated skipping allows bypassing of protein cleavage but may not restore full CEP290 function The abundance of full-length CEP290 mRNA in control cells treated with 75 nM H36D(+98-11) AON for 48 h was approximately 60% of that in untreated control cells, as measured by RT-qPCR (Figure 8B). This indicates that approximately 40% of full-length CEP290 pre-mRNA underwent AON-mediated skipping of exon 36. The amount of CEP290 protein (full-length + Δ36aa) was comparable in treated and untreated cells (Figures 9A and 9B). This suggests that the CEP290 Δ36aa isoform is stable. However, treated cells showed a moderate decrease in CEP290 staining at centrosomes, with a statistically significant difference (p ≤ 0.05, Figure 9C), but minimal changes in CP110 centrosomal staining (p ≤ 0.05, Figure 9D). The abundance of ciliated cells tended to be slightly reduced (95.5% vs. 92.5%, Figure 9F), as did the mean axoneme length (3.9 µm vs. 3.6 µm, p ≤ 0.01, Figure 9E). Collectively, these results suggest that the CEP290Δ36aa isoform, as opposed to its wild-type counterpart, may impair ciliary movement via disorganization of centriolar satellites. The same treatment in P1 cells resulted in a highly significant increase in the abundance of CEP290Δ36aa protein, as determined by Western blot and immunocytochemical analysis (Figures 9A, 9B, and 9C). Interestingly, the intensity of CEP290 staining at the centrosomes reached that of treated control cells (Figure 9C). On the other hand, increased expression levels of the CEP290Δ36aa isoform in cells lacking wild-type CEP290 altered the dynamics of centriolar satellites, as evidenced by increased dispersion of CP110-specific centrosomal staining (p ≤ 0.001, Figure 9D). Consistently, the proportion of ciliated cells tended to decrease with AON treatment (89% vs. 79.8%) (Figure 9F), as did axoneme length (4.4 μm vs. 3.5 μm, p ≤ 0.0001, Figure 9E).

[0129] In summary, here we report PTC-free CEP290 mRNA expression resulting from endogenous and selective elimination of exon 36, encompassing the founder CEP290 c.4723A>T nonsense mutation, in two apparently unrelated individuals. Upon serum deprivation, centrosome-localized CEP290 isoforms are produced, and cilia are produced, but their production is inappropriate, as evidenced by significant axoneme elongation.

[0130] Example 2 Previously, we demonstrated that targeting the donor splice site of CEP290 exon 36 using 2'OMePS antisense splice-switching oligonucleotides could improve ciliary metabolism in patient-derived fibroblasts homozygous for the c.4723A>T (p.1575X, exon 36) mutation. Furthermore, we demonstrated proof-of-concept AON-mediated skipping of the orthologous exon (exon 35) in mice using an intravitreal delivery route in wild-type C57BL / 6J mice (46). To evaluate the therapeutic efficacy of intravitreal injection of the previously reported m35ESE AON (46), we generated a mouse model with a homozygous endogenous deletion of exon 35 (Cep290del / del) and a mouse strain with a compound heterozygous deletion with a premature stop codon (Cep290del / PTC). Here, we report retinal degeneration in both Cep290del / del and Cep290del / PTC mouse strains.

[0131] Materials and Methods Development of the Cep290 mouse model Two Cep290 mouse models were generated at the Imagine Institute using the CRISPR / Cas9 system. Guide RNAs (sgRNAs) were designed using CRISPOR (http: / / crispor.tefor.net / ) to introduce a premature termination codon (PTC) into coding exon 35 of the Cep290 gene, respectively, skipping this exon (del35). Four-week-old C57BL / 6J female mice were superovulated by intraperitoneal injection of 5 IU PMSG (SYNCRO-PART® PMSG 600 UI, Ceva) followed by 5 IU hCG (Chorulon 1500 UI, Intervet) at 46-48 hour intervals and mated with C57BL / 6J male mice. The next day, zygotes were retrieved from the oviducts and exposed to hyaluronidase (H3884, Sigma-Aldrich) to remove cumulus cells. They were then placed in M2 medium (M7167, Sigma-Aldrich) in a CO2 incubator (5% CO2, 37°C). The sgRNA was hybridized with the cas9 (WT) protein and injected into the pronuclei of C57Bl / 6J zygotes. Surviving zygotes were placed in KSOM medium (MR-106-D, Merck-Millipore) and cultured overnight to the two-cell stage. They were then transferred into the oviducts of B6CBAF1 pseudopregnant females. Mice carrying either mutation were selected by genotyping genomic DNA by Sanger sequencing using specific primers flanking exon 35. Heterozygous mice were crossed with C57BL / 6J mice to remove potential off-target genes, and the offspring were bred to generate homozygous exon 35 deletion (Cep290del / del) and compound heterozygous exon 35 deletion with PTC, respectively. Cep290del / del and Cep290del / PTC mice were bred and maintained under a 12-hour dark / light cycle at the LEAT Facility of Imagine Institute. Electrophysiological and histological analyses were then performed at postnatal days (P) 30, 60, and 120 (P30, P60, and P120) and 18, 21, and 30 (P18, P21, and P30), respectively.Age-matched wild-type C57BL / 6J mice were used as controls in all analyses. All animal procedures were performed in compliance with French guidelines for animal experimentation and in accordance with ethical principles.

[0132] Electroretinography Electroretinograms (ERGs) were recorded using a rodent Celeris™ ERG (Diagnosys LLC, Cambridge, UK). Briefly, mice were dark-adapted overnight and anesthetized with an intramuscular injection of 120 mg / kg ketamine and 16 mg / kg xylazine. After pupil dilation with a drop of 0.5% tropicamide and a drop of 10% phenylephrine, sterile ophthalmic gel was applied to the corneal surface to ensure electrical contact and maintain corneal integrity. Animals were maintained on a Celeris™ warming support throughout the ERG procedure to maintain body temperature at 38°C. Stimulation and recording were generated with a Celeris™ electrode stimulator, and a ground electrode was inserted subcutaneously. The dark-adapted ERG protocol consisted of four steps in which the stimulus intensity increased from 0.01 to 3 cd.s / m². Light-adapted ERGs were recorded after 8 minutes of light adaptation. Photopic recordings consisted of two steps of increasing stimuli from 3 to 10 cd.s / m². Statistical analysis was performed using Prism 6 software, and significant differences in a-wave amplitude between the Cep290 mouse model and age-matched wild-type C57BL / 6J mice were determined using a two-way analysis of variance with a post hoc Sidak test.

[0133] histology Mice were euthanized by cervical dislocation. Eyes were enucleated and immediately fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 12 hours. At the Imagine Institute's histology platform, eyecups were dehydrated in a serial gradient of ethanol using an automated tissue processor (ASP300S, Leica), embedded in paraffin, and then microtomed (2xHM 340E, Microm France). Six-micron serial sections were cut longitudinally and stained with hematoxylin and eosin. Each slide was scanned using a commercially available imaging system (NanoZoomer S210, Hamamatsu) and analyzed using NDPview software. The thickness of the outer nuclear layer was plotted against the distance from the optic nerve (ON) (0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, and 2.25 mm). Three mice from each group were included in this analysis. The ONL thickness of the Cep290 mouse model was compared with that of age-matched wild-type C57BL / 6J mice by two-way ANOVA with post hoc Sidak test (Prism 6.0 software, San Diego, CA).

[0134] result Using CRISPR-Cas9 technology, a premature termination codon (c.4749del, p.His1583Glnfs6) was identified in exon 35 of mouse Cep290. *) were introduced and proved to be efficient in deleting entire exons, respectively. Very few Cep290PTC / PTC offspring were produced from Cep290PTC / + × Cep290PTC / + matings; the animals exhibited developmental delay, ataxia, hydrocephalus, defective cerebellar development, polycystic kidneys, severe retinal degeneration, and did not survive beyond P45 (data not shown). In contrast, Cep290PTC / + × Cep290del / + matings produced Cep290+ / +, Cep290+ / del, Cep290PTC / +, and Cep290del / PTC animals. These animals survived and developed normally. The ERG response of Cep290del / del photoreceptors decreased from P30 and was completely absent by P120 (Figure 11A). Histological analysis at P30 revealed a moderate reduction in the thickness of the outer nuclear layer (ONL; photoreceptor nuclei) compared with wild-type Cep290+l+ retinas (Figure 11B). ONL thickness was significantly reduced by P60 and reduced to one to two layers of nuclei by P120 (Figure 11B). Retinal degeneration in Cep290del / PTC animals occurred earlier and progressed more rapidly. At P14 (eye opening), ERG responses of both rod and cone photoreceptors from Cep290del / PTC mice were significantly reduced (Figure 11A). Although the histological structure appeared normal at this age, ONL thickness decreased very rapidly as eyes opened, reducing to only one row of nuclei at P30 (Figure 11B).

[0135] conclusion In humans, homozygosity and compound heterozygosity for truncating mutations in the 35th coding exon (exon 36) of the CEP290 gene, including the founder c.4723A>T mutation, result in congenital or early-onset, severe, nonsyndromic retinal degeneration (LCA10 and EOSRD, respectively). In mice, results indicate that homozygosity for a truncating mutation in the orthologous exon (c.4749del; Cep290PTC / PTC) results in a very severe ciliary pathway phenotype reminiscent of Meckel syndrome type 4 (MKS4). In contrast, mice homozygous for a deletion of exon 35 that does not alter the reading frame exhibited a moderate, isolated retinal phenotype. This supports the notion that CEP290 isoforms lacking residues encoded by exon 35 retain some function. Interestingly, mice carrying a compound heterozygous deletion for c.4749del had an intermediate phenotype consisting of severe retinal disease reminiscent of LCA with moderate extraocular abnormalities. Although the onset and severity of multiorgan pathology in Cep290PTC / PTC precludes in vivo manipulation of splicing, the Cep290del / PTC model is ideally suited to assess whether AON-mediated skipping of exon 35 carrying c.4749del can delay retinal degeneration from LCA-like disease to a Cep290del / del EOSRD phenotype (Figure ​(Figure11). 11).

[0136] References Throughout this application various references are made to describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure.

[0137] [Table 3] TIFF0007682808000004.tif241165 TIFF0007682808000005.tif248165 TIFF0007682808000006.tif220165

Claims

1. An antisense oligonucleotide consisting of the sequence shown by SEQ ID NO:

1.

2. The antisense oligonucleotide according to claim 1, which is complementary to the nucleic acid sequence of CEP290 pre-mRNA, wherein the antisense oligonucleotide targeting the donor splice site (H36D) can change splicing by blocking the recognition of exon 36, and bypasses protein cleavage associated with any mutation that introduces a premature stop codon into exon 36 while maintaining the open reading frame, resulting in the production of a nearly full-length CEP290 protein. Antisense oligonucleotide.

3. An antisense oligonucleotide for use in performing antisense oligonucleotide-mediated exon skipping in a subject suffering from retinal dystrophy caused by a mutation that modifies splicing and / or a nonsense mutation in exon 36 of the CEP290 gene or a frameshift mutation in exon 36 of the CEP290 gene that causes premature termination, wherein the antisense oligonucleotide comprises the nucleic acid sequence shown by SEQ ID NO: 1, the antisense oligonucleotide is complementary to the nucleic acid sequence of the CEP290 gene containing the donor splice site near the exon 36 / intron 36 boundary, and the antisense oligonucleotide performs antisense oligonucleotide-mediated exon skipping in the pre-mRNA derived from the CEP290 gene that causes retinal dystrophy in the target cells of the subject by the mutation. Antisense oligonucleotide.

4. The antisense oligonucleotide according to claim 3, wherein the subject suffers from retinal dystrophy caused by at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A.

5. The antisense oligonucleotide according to claim 3 or 4, wherein the retinal dystrophy is selected from the group consisting of Leber congenital amaurosis and other early-onset severe retinal dystrophies (LCA-like), rod-cone dystrophy (retinitis pigmentosa), cone-rod dystrophy, macular dystrophy including age-related macular degeneration, any cilia-related disease related to the retina including Joubert syndrome, Senior-Loken syndrome, Bardet-Biedl syndrome, Meckel and Meckel-like syndromes, Refsum syndrome, Stargardt disease, Ascher syndrome, hereditary optic neuropathy, congenital stationary night blindness, color weakness and color vision abnormality.

6. An antisense oligonucleotide consisting of a sequence complementary to the nucleic acid sequence of the CEP290 gene, which is necessary to change splicing and exclude the exon encoding the premature stop codon inserted into the CEP290 mRNA due to a nonsense mutation in exon 36 or a frameshift mutation in exon 36 or an upstream exon, wherein the antisense oligonucleotide contains the nucleic acid sequence shown in SEQ ID NO: 1, and the nucleic acid sequence of the CEP290 gene is a sequence containing the donor splice site of CEP290 exon 36. An antisense oligonucleotide for use in restoring the function of CEP290 in a cell having a nonsense mutation present in exon 36 of the CEP290 gene or a frameshift mutation in exon 36 in a subject having said mutation.

7. The antisense oligonucleotide according to claim 6, wherein the cell having at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A present in the CEP290 gene is a ciliated cell.

8. Retinal dystrophy in a subject having a nonsense mutation located in exon 36 of the CEP290 gene that results in the appearance of a premature stop codon in exon 36 that gives rise to a truncated protein, or a frameshift mutation in exon 36 or an upstream exon of the CEP290 gene, By exposing CEP290 pre-mRNA to an antisense oligonucleotide (AON) complementary to a sequence containing the donor splice site of CEP290 exon 36, modulating the splicing of exon 36 of the CEP290 gene containing a premature stop codon resulting from the nonsense mutation or the frameshift mutation within exon 36, An antisense oligonucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 1 for use in treating. **Claim 9** The antisense oligonucleotide according to claim 8, wherein the subject has at least one mutation selected from the group consisting of c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG(35), c.4792_4795del, c.4801C>T, c.4805C>T or c.4811G>A. **Claim 10** The antisense oligonucleotide according to any one of claims 3-9, wherein the antisense oligonucleotide is selected from the group consisting of an oligodeoxyribonucleotide, an oligoribonucleotide, a locked nucleic acid (LNA) oligonucleotide, a morpholino oligonucleotide, a tricyclo DNA antisense oligonucleotide, a U7 or U1-mediated antisense oligonucleotide, a peptide bond or nanoparticle conjugate antisense oligonucleotide, a 2'-O-MeRNA / ENA chimeric oligonucleotide and a 2'-O-methyl-phosphorothioate oligonucleotide. **Claim 11** The antisense oligonucleotide according to any one of claims 3-10, wherein the antisense oligonucleotide is complementary to a sequence containing the donor splice site of CEP290 exon 36. **Claim 12** The antisense oligonucleotide according to any one of claims 3-11, wherein the antisense oligonucleotide consists of the nucleic acid sequence set forth in SEQ ID NO:

1. **Claim 13** The antisense oligonucleotide according to claim 3, which is administered intravitreally to a subject having a nonsense mutation or an early termination codon in exon 36 of the CEP290 gene.

14. A pharmaceutical composition containing the antisense oligonucleotide according to any one of claims 6 to 13, alone or together with a vector, for use in the treatment of retinal dystrophy in a subject having a nonsense mutation or an early termination codon in exon 36 resulting from a frameshift mutation in exon 36 or an upstream exon of the CEP290 gene.

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