Zebrafish disease model for cornea dystrophies

Abstract

The invention relates to in vivo zebrafish disease model of corneal dystrophy diseases and potential use of these models for drug or therapeutic development and / or potens tests as a preclinical in vivo test platform.

Description

[0001] ZEBRAFISH DISEASE MODEL FOR CORNEA DYSTROPHIES

[0002] Technical Field of the Invention

[0003] This patent relates to first in vivo disease model of corneal dystrophy diseases via mutagenesis of zebrafish chst6 gene, and its use as a drug test platform and preclinical disease model.

[0004] State of the Art of the Invention (Prior Art)

[0005] Macular corneal dystrophy (MCD) is linked to autosomal recessive carbohydrate sulfotransferase 6 (chst6) gene mutations, and leads to vision loss due to accumulation of unsulfated or low-sulfated keratan sulfate proteoglycans in the cornea. MCD usually becomes symptomatic in childhood, progressively leads to complete bilateral vision loss by age 30-40. No medical cure has been yet developed for MCD, while penetrating keratoplasty is applied in the advanced stage of the disease. However, disease relapses within 5 years of cornea transplantation due to invasion of healthy cornea by the patient’s keratocytes.

[0006] Macular corneal dystrophy is an autosomal recessively transmitted rare disease, and mutagenesis of chst6 gene is needed to develop a disease model. Mutagenesis of chst6 gene was not reported in any genetic animal model, and no in vivo MCD model has been reported. The disease causing chst6 gene is conserved in zebrafish, but not conserved in mice. The gene most similar to chst6 is chst5 in mouse, mutation of which does not induce MCD phenotype in mice. The structure of zebrafish cornea, the presence of different corneal layers and the composition and ratio of keratansulfate proteoglycans to the total proteoglycan content of cornea are highly similar to the human cornea. However, there is no model of the disease in zebrafish.

[0007] Patent and literature search related to in vivo model of macular corneal dystrophy revealed no in vivo model of MCD. An in vitro model that reflects MCD phenotypes completely has not yet been developed either. In 2016, Murab et al published an article titled “Establishment of an in vitro monolayer model of macular corneal dystrophy” in which a chemical inhibitor of 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS), sodium chlorate was applied on cells isolated from goat cornea, decrease of sulfated glycosaminoglycans was indicated by alcian blue staining, formation of aggregates in some cells was proposed by atomic force microscopy (AFM) analysis. However, inhibition of CHST6 enzyme was not directly proven with this method. This in vitro model contain one cell type, lacks tissue integrity and genetic modification, hence differs from this invention.

[0008] While no cure is known for macular corneal dystrophy, due to its autosomal recessive hereditary nature disease the symptoms may recur after corneal transplantation. Development of a medical cure or treatment for MCD has become an important need. Enzyme replacement therapies may be developed but no article or study or any method has been published yet..

[0009] Brief Description and Objects of the Invention

[0010] This invention relates to the development of macular corneal dystrophy disease model in zebrafish model organism, its use for in vivo and preclinical testing of therapeutics that are / will be developed. The model developed in this invention is the first in vivo model of the MCD. The zebrafish MCD model presents the potential to apply and test the effects of chemical, biotechnological, natural or synthetic therapeutic agents and reagents, as well as the potential to use for drug repositioning. This invention differs from the other studies in that the opaque aggregate formation in the eyes were shown phenotypically. The mutants were analyzed for changes in DNA and protein sequence of the chst6 gene, loss of enzyme function anlyses and histopathological analyses can be performed.

[0011] The main aim of the invention is to provide a live platform, that shows similar symptoms to human MCD, to test therapeutic methods on. This invention also allows for testing of therapeutics to be developed on live organism and because the disease is modelled in model organism, it will be possible to test the treatment response at the preclinical stage. Preclinical models contribute to acceleration of treatment development. Broad aim of this invention to help improve life quality of corneal dystrophy patients without the need for corneal transplantation or until a transplantation is needed. The chst6 mutant zebrafish lines generated in this invention also provides a model to research mechanisms of MCD further. With this invention zebrafish chst6 gene was targeted with the CRISPR / Cas9 technology, the first 3'-fosfoadenosin-5'-fosfosulfat (PAPS) domain which was determined to be the enzyme active site was targeted to be mutated. Two different guide RNA (gRNA)s were used. gRNAl that binds to the DNA sequence encoding for 37.-43. residues of the CHST6 protein, was used to generate f24 and f42 alleles of the chst6 gene. gRNA2, that binds to the DNA sequence encoding for 58.-63. residues of the CHST6 protein, was used together with gRNAl to induce PAPS1 deletion mutants and pd4, pd5, pd57 and pd66 mutant alleles of the chst6 gene were generated. The sequences of each mutant allele were shown. Sulfated corneal keratan sulfate amount was quantified with ELISA. Loss of sulfated corneal keraten sulfates in the eyes of mutant larvae and adults were shown.

[0012] In the MCD model, opaque aggregate formation in the eyes of adult zebrafish were shown. Histopathological analysis and alcian blue staining showed aggregate formation and deformation of the corneal structure.

[0013] This invention proves that zebrafish model of macular corneal dystrophy is generated via inactivation of chst6 gene, this model mimics the clinical symptoms and can be used as a preclinical disease model.

[0014] Definitions of Figures Describing the Invention

[0015] Figure 1: Immunofluorescence staining with the 5D4 antibody that recognizes the sulfated keratan sulfate (cKS) epitope that has been sufated by CHST6 enzyme, in the wild type and mutant larvae. Scale bar: 100 pm. cKS: the antibody that recognizes sulfated KS epitope, DAPL nucleus stain.

[0016] Figure 2: Change of KS levels in mutants is shown with ELISA. (A) The graph showing the standard curve analysed by 4PL method, using the standards in the KS ELISA test kit. Formula: y=-2.666855 + (-0.003460337- (-2.666855)) / (l+(x / 0.6290704)A0.9516325),

[0017] R2=0,9993. (B) The amount of cKS in wild type and mutant larvae (n=50). Figure 3: Opaque aggregates in the adult zebrafish cornea in the Zebrafish MCD Model. Eye images of (A) wild type, (B) pd4, (C) pd5, (D) pd57, (E) f42 homozygous chst6 mutant alleles, and (F) f24 heterozygous chst6 mutant allele adult zebrafish. Scale bar: 500 pm.

[0018] Figure 4: Alcian blue staining of corneal section of zebrafish MCD model. Zebrafish corneal tissue section images of (A) wildtype, (B) pd4, (C) pd5, (D) f42 homozygous chst6 mutant alleles, and (E) f24 heterozygous chst6 mutants. Scale bar: 50 pm, E: epithelium, S: stroma.

[0019] Figure 5: Zebrafish MCD model, cKS staining of the cornea tissue. A) wildtype, (B) pd4, (C) pd5, (D) f42 homozygous chst6 mutant alleles, and (E) f24 heterozygous mutants. Scale bar: 20 pm. cKS: the antibody that recognizes sulfated KS epitope, DAPI: nucleus stain.

[0020] Detailed Description of the Invention

[0021] In this invention Macular Corneal Dystrophy disease model is developed by mutagenesis of zebrafish chst6 gene. Different mutant alleles are confirmed with DNA sequence analysis, loss of CHST6 enzyme function is shown with immunofluorescence staining and ELISA, MCD phenotype is shown in the eyes of mutant zebrafish microscopically and hi stopathologically .

[0022] This invention includes mutagenesis of chst6 gene and generation of loss of function mutants. This invention includes induction of MCD phenotype in the eye via deletions generated in the chst6 gene and reduction or loss of the activity of the enzyme N-acetilglucosamine-6-O- sulfotransferase (GlcNAc6ST). Targeted gene editing was used to induce mutagenesis. gRNAs were designed with respect to coding region for the sulfate binding region (49.-55. aa) which is required for the activity of the GlcNAc6ST enzyme. gRNAl and gRNA2 were designed to target coding sequences for 37.-43. and 58.-63. aa, respectively. Indel mutations were generated by single use of gRNAl and alleles named f42 and f24 were generated. Small deletion mutations were generated by dual use of gRNAl and gRNA2 and alleles named pd4, pd5 and pd57 were generated. Mutant alleles were sequenced with sanger sequencing. The DNA sequences of the alleles and determined protein sequences are listed in appendix and at the end of the detailed description of the invention section. f24 mutant allele has deletion of 125.-130. nucleotides in the coding sequence. f42 mutant allele has deletion of 123.-126. nucleotides in the coding sequence. pd4 mutant allele has deletion of 124.-180. nucleotides (57 nt) and insertion of T instead in the coding sequence. pd5 mutant allele has deletion of 120.-181. nucleotides (62 nt) in the coding sequence. pd57 mutant allele has deletion of 123.- 284. nucleotides (162 nt) and insertion of AA instead in the coding sequence. The mutation f24 leads to deletion of 42nd and 43rd aminoacids. The mutation f42 leads to frameshift that starts at 42nd aminoacid. The mutation pd57 leads to deletion and frameshift that starts at 42nd aminoacid.

[0023] In this invention, zebrafish chst6 gene was targeted with the CRISPR / Cas9 technology, l.PAPS region which was determined to be the active site of the sulfotransferase enzyme encoded by the chst6 gene was targeted with mutagenesis . Two different guide RNAs (gRNAs) were used, small indel mutations before 1. PAPS was induced with gRNAl that binds to the DNA sequence encoding the 37.-43. residues and f24 and f42 mutant alleles were generated. PAPS deletion mutants were generated with simultaneous use of gRNAl and gRNA2 that binds to the DNA sequence that endoces for 58.-63. residues, and pd4, pd5, pd57 and pd66 mutant alleles were generated. The sequence analysis of mutants and the quantitation of sulfated corneal keratan sulfate (cKS) amount with ELISA was shown. Loss of cKS in mutant larvae and the cornea of the adult mutants were shown. The adult MCD zebrafish developed opaque aggreagates in the eye, and histopathological examination thereof showed alcian blue positive aggregates and disruption of tissue structure. This invention shows generation of the zebrafish MCD disease model by mutagenesis of chst6 gene and that the clinical symptoms of the model is phenocopies, and can be used as a preclinical model.

[0024] Loss of function in the developed mutants were proven with analyses that were conducted with the antibody that recognizes fully sulfates KS epitopes that were sulfated by the GlcNAc6ST enzyme. 4 days post fertilization wild type and mutant larvae were fixated and incubated with the anti-cKS primary antibody, which was stained with fluorescently labeled secondary antibody. Figure 1 shows presence of cKS epitope in the cornea, brain and otic vesicle (ear) of the wildtype larvae. The homozygous mutants carrying the pd4 and f42 alleles do not have cKS in the cornea and the otic vesicle (ear) and reduced cKS in the brain tissues (Figure 1). The amount of cKS in the whole-body lysates were measured with ELISA method. The larvae were snap frozen and lysed in PBS, total protein and cKS concentrations in wild type and mutant lysates were measured (Figure 2). The amount of total cKS was 35pg in wt, 4.9 pg in chst6pd4 heterozygous and 3.4 pg in pd4 homozygous and 14.2 pg in heterozygous mutant larvae (Table 1). The loss of function in chst6 mutants were shown.

[0025] Table 1. Total protein, total cKS amount and cKS concentration in lysates of 50 wt or mutant larvae of indicated genotypes

[0026] The aggregates observed in the human MCD patient corneas were shown to develop in the zebrafish MCD model, causing opacity as observed with brightfield microscopy (Figure 3). Paraffin sections of adult MCD zebrafish and wildtype fish stained with alcian blue showed increase of blue staining in the stroma and epithelium of the cornea, indicating and increase in glycosaminoglycan aggregation as seen in human MCD patients (Figure 4). Deformations in the cornea structure, presence of blue stained aggregates and change in tissue thickness was observed.

[0027] Cyrosections of the eyes of adult WT, chst6 pd4, pd5 and f42 homozygous mutants and f24 heterozygous mutant. Homozygous mutants lost cKS signal and f24 heterozygous mutant has cKS in the epithelium whereas no stromal expression was detected (Figure 5).

[0028] This invention and reported data showed that the phenotypes of the human MCD cornea is formed in the zebrafish MCD model at the molecular, macroscopic and histopathological levels.

[0029] Sequence No:l represents Danio rerio carbohydrate sulfotransferase 6 wild-type cds. Sequence No:2 represents f24 mutant allele of Danio rerio carbohydrate sulfotransferase 6 cds. Sequence No:3 represents f42 mutant allele of Danio rerio carbohydrate sulfotransferase 6 cds. Sequence No:4 represents pd4 mutant allele of Danio rerio carbohydrate sulfotransferase 6 cds. Sequence No: 5 represents pd5 mutant allele of Danio rerio carbohydrate sulfotransferase 6 cds. Sequence No:6 represents pd57 mutant allele of Danio rerio carbohydrate sulfotransferase 6 cds. Sequence No:7 represents protein sequence, wildtype Danio rerio carbohydrate sulfotransferase 6. Sequence No: 8 represents protein sequence, pd4 mutant allele Danio rerio carbohydrate sulfotransferase 6. Sequence No:9 represents the protein sequence, pd5 mutant allele Danio rerio carbohydrate sulfotransferase 6. Sequence No: 10 represents the protein sequence, f24 mutant allele Danio rerio carbohydrate sulfotransferase 6. Sequence No: 11 represents the protein sequence, f44 mutant allele Danio rerio carbohydrate sulfotransferase 6. Sequence No: 12 represents the protein sequence, pd57 mutant allele Danio rerio carbohydrate sulfotransferase 6

[0030] >Danio rerio carbohydrate sulfotransferase 6 (chst6), cds (WT)

[0031] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCTGAGGGAAAGGTGCACGTGCTGCTTCTGTCC TCCTGGAGGTCCGGCTCATCCTTCCTGGGCCAGGTATTCAGCCAGCATCCTGACGTCTTTTATTTGATGGAGCCG GCCTGGCACGTGTGGATGACCATTAACCAATCCGGCGCACGTAGCCTGAGAATGGCGGTACGAGACACGATTCGC AGCATCTTTCAGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAACATCTCCAACCTGTTCATG TGGAGCCACAGTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGACCAGATTAGCATCGAACAA GACTGCAAAAAGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCAATCCTACAGCCATGTTGTT TTAAAAGAAGTTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCCGACTTTGAATGTGCGAATC ATCCATTTAGTTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAAAGCCTTGGTGAAAGACAGC AATATCGTGCTCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTTGCAGGAGATTTGCCGCAGT CACGTGCGCATTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACGCTACAAAATGATTCGCTAC GAAGATCTCGTGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGGTCTGGAGATGACCGAAACC CTGCAGGAGTGGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGAAGCATTCGATATCACTTCG CGAAACGCCGAGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGTACAGCGTATCCAGGATGTT TGTAAAGGAGCCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCAAAAGATGATGGATTTAGAC TTGATGAAGCCACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGACGGCTGCTAAATTATAG

[0032] Nucleotides deleted in chst6r24: GAAAGG

[0033] Nucleotides deleted in chst6pd4:

[0034] GGAAAGGTGCACGTGCTGCTTCTGTCCTCCTGGAGGTCCGGCTCATCCTTCCTGGGC

[0035] >F24 mutant allele_Danio rerio carbohydrate sulfotransferase 6 (chst6), cds (deletion of nucleotides 125-130.)

[0036] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCTGAGGTGCACGTGCTGCTTCTGTCCTCCTGG AGGTCCGGCTCATCCTTCCTGGGCCAGGTATTCAGCCAGCATCCTGACGTCTTTTATTTGATGGAGCCGGCCTGG CACGTGTGGATGACCATTAACCAATCCGGCGCACGTAGCCTGAGAATGGCGGTACGAGACACGATTCGCAGCATC TTTCAGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAACATCTCCAACCTGTTCATGTGGAGC

[0037] CACAGTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGACCAGATTAGCATCGAACAAGACTGC

[0038] AAAAAGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCAATCCTACAGCCATGTTGTTTTAAAA

[0039] GAAGTTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCCGACTTTGAATGTGCGAATCATCCAT

[0040] TTAGTTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAAAGCCTTGGTGAAAGACAGCAATATC

[0041] GTGCTCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTTGCAGGAGATTTGCCGCAGTCACGTG

[0042] CGCATTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACGCTACAAAATGATTCGCTACGAAGAT

[0043] CTCGTGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGGTCTGGAGATGACCGAAACCCTGCAG

[0044] GAGTGGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGAAGCATTCGATATCACTTCGCGAAAC

[0045] GCCGAGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGTACAGCGTATCCAGGATGTTTGTAAA

[0046] GGAGCCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCAAAAGATGATGGATTTAGACTTGATG

[0047] AAGCCACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGACGGCTGCTAAATTATAG

[0048] >F42 mutant allele_Danio rerio carbohydrate sulfotransferase 6 (chst6), cds (deletion of nucleotides 123-126.)

[0049] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCTGAAAGGTGCACGTGCTGCTTCTGTCCTCCT GGAGGTCCGGCTCATCCTTCCTGGGCCAGGTATTCAGCCAGCATCCTGACGTCTTTTATTTGATGGAGCCGGCCT GGCACGTGTGGATGACCATTAACCAATCCGGCGCACGTAGCCTGAGAATGGCGGTACGAGACACGATTCGCAGCA TCTTTCAGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAACATCTCCAACCTGTTCATGTGGA GCCACAGTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGACCAGATTAGCATCGAACAAGACT GCAAAAAGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCAATCCTACAGCCATGTTGTTTTAA AAGAAGTTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCCGACTTTGAATGTGCGAATCATCC ATTTAGTTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAAAGCCTTGGTGAAAGACAGCAATA TCGTGCTCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTTGCAGGAGATTTGCCGCAGTCACG TGCGCATTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACGCTACAAAATGATTCGCTACGAAG ATCTCGTGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGGTCTGGAGATGACCGAAACCCTGC AGGAGTGGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGAAGCATTCGATATCACTTCGCGAA ACGCCGAGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGTACAGCGTATCCAGGATGTTTGTA AAGGAGCCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCAAAAGATGATGGATTTAGACTTGA TGAAGCCACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGACGGCTGCTAAATTATAG

[0050] >pd4 mutant allele_Danio rerio carbohydrate sulfotransferase 6 (chst6), cds

[0051] (deletion of nucleotides 124-180., insertion of T)

[0052] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG

[0053] TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCTGAGTCAGGTATTCAGCCAGCATCCTGACGT

[0054] CTTTTATTTGATGGAGCCGGCCTGGCACGTGTGGATGACCATTAACCAATCCGGCGCACGTAGCCTGAGAATGGC

[0055] GGTACGAGACACGATTCGCAGCATCTTTCAGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAA

[0056] CATCTCCAACCTGTTCATGTGGAGCCACAGTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGA

[0057] CCAGATTAGCATCGAACAAGACTGCAAAAAGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCA

[0058] ATCCTACAGCCATGTTGTTTTAAAAGAAGTTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCC

[0059] GACTTTGAATGTGCGAATCATCCATTTAGTTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAA

[0060] AGCCTTGGTGAAAGACAGCAATATCGTGCTCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTT

[0061] GCAGGAGATTTGCCGCAGTCACGTGCGCATTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACG

[0062] CTACAAAATGATTCGCTACGAAGATCTCGTGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGG

[0063] TCTGGAGATGACCGAAACCCTGCAGGAGTGGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGA AGCATTCGATATCACTTCGCGAAACGCCGAGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGT

[0064] ACAGCGTATCCAGGATGTTTGTAAAGGAGCCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCA

[0065] AAAGATGATGGATTTAGACTTGATGAAGCCACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGAC

[0066] GGCTGCTAAATTATAG

[0067] >pd5 mutant allele Danio rerio carbohydrate sulfotransferase 6 (chst6), cds (deletion of nucleotides 120-181.)

[0068] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCAGGTATTCAGCCAGCATCCTGACGTCTTTTA TTTGATGGAGCCGGCCTGGCACGTGTGGATGACCATTAACCAATCCGGCGCACGTAGCCTGAGAATGGCGGTACG AGACACGATTCGCAGCATCTTTCAGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAACATCTC CAACCTGTTCATGTGGAGCCACAGTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGACCAGAT TAGCATCGAACAAGACTGCAAAAAGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCAATCCTA CAGCCATGTTGTTTTAAAAGAAGTTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCCGACTTT GAATGTGCGAATCATCCATTTAGTTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAAAGCCTT GGTGAAAGACAGCAATATCGTGCTCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTTGCAGGA GATTTGCCGCAGTCACGTGCGCATTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACGCTACAA AATGATTCGCTACGAAGATCTCGTGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGGTCTGGA GATGACCGAAACCCTGCAGGAGTGGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGAAGCATT CGATATCACTTCGCGAAACGCCGAGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGTACAGCG TATCCAGGATGTTTGTAAAGGAGCCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCAAAAGAT GATGGATTTAGACTTGATGAAGCCACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGACGGCTGC TAAATTATAG

[0069] >pd57 mutant allele_Danio rerio carbohydrate sulfotransferase 6 (chst6), cds

[0070] (deletion of nucleotides 123-284., insertion of AA)

[0071] ATGCTGCGCTGGAGAGTGTCGAAGGCGGCTGTGTTGAGCGTGTTGTTCGCTCAGGCTGTGACCGTGGGGCTCCTG

[0072] TATGGCTGGTACAGTCGACCGAATATTCAGAACGTGACTCAGCCTGAAAACGAGACACGATTCGCAGCATCTTTC

[0073] AGTGCGACATGTCCGTCATGGATTCATACATCCGCCAGCCCCAGAACATCTCCAACCTGTTCATGTGGAGCCACA

[0074] GTCGTGCGCTCTGCTCTCCGCCTGCGTGCCTCCAAACGCCGCGCGACCAGATTAGCATCGAACAAGACTGCAAAA

[0075] AGCACTGCGGGAAAAGCAATTTGAAGCTCGCTGAGTCGGCGTGTCAATCCTACAGCCATGTTGTTTTAAAAGAAG

[0076] TTCGCTTCTTCGAATTGGAGTCGCTTTATTCGCTACTTCAAGACCCGACTTTGAATGTGCGAATCATCCATTTAG

[0077] TTCGAGACCCACGAGCGGTGTTTCGCTCCAGAGATCGTTCCTACAAAGCCTTGGTGAAAGACAGCAATATCGTGC

[0078] TCGAGATGGCAAACATTCCTGAGAAGGATAAACCTTATCGCGTCTTGCAGGAGATTTGCCGCAGTCACGTGCGCA

[0079] TTTACGAGACCGCCATGCTTAAAGCGCCGAGTTTCCTTAAAGGACGCTACAAAATGATTCGCTACGAAGATCTCG

[0080] TGCACAACACGCAGGCTGAAATCGAGGCCATGTATGAATTCATCGGTCTGGAGATGACCGAAACCCTGCAGGAGT

[0081] GGATCTACCGCATCACTCACGGTAAAGGCAAGGGAACCAAGAAGGAAGCATTCGATATCACTTCGCGAAACGCCG

[0082] AGGATGTTTCGATGGCTTGGCGAACCACGTTACCGTTCGAGAAAGTACAGCGTATCCAGGATGTTTGTAAAGGAG

[0083] CCATGTCCCTGCTGGGATATTCGACGGTGGACAGTGAGAAGGAGCAAAAGATGATGGATTTAGACTTGATGAAGC

[0084] CACGCGAGCGCTATAAATTCAAATGGCTGCCTCCCAAAAGCACGACGGCTGCTAAATTATAG

[0085] Protein Sequence of WT CHST6 (I P APS sequence is underlined: LSSWRSG)

[0086] MLRWRVSKAAVLSVLFAQAVTVGLLYGWYSRPNIQNVTQPEGKVHVLLLSSWRSGSSFLGQVFSQHPDVFYL 70 M EPAWHVWMTINQSGARSLRMAVRDTI RSIFQCDMSVM DSYIRQPQNISNLFMWSHSRALCSPPACLQ.TP 140

[0087] RDQISIEQDCKKHCG KSNLKLAESACQSYSHVVLKEVRFFELESLYSLLQDPTLNVRI IHLVRDPRAVFRSRDRSYK 217

[0088] ALVKDSNIVLEMANIPEKDKPYRVLQEICRSHVRIYETAM LKAPSFLKGRYKMIRYEDLVHNTQAEI EAMYEFI 291

[0089] G LEMTETLQEWIYRITHGKGKGTKKEAFDITSRNAEDVSMAWRTTLPFEKVQRIQDVCKGAMSLLGYSTVDSE 364

[0090] KEQKM MDLDLM KPRERYKFKWLPPKSTTAAKL* 399

[0091] Protein Sequence of CHST6 PD4 mutant allele (residues resulting from frameshift are underlined) MLRWRVSKAAVLSVLFAQAVTVGLLYGWYSRPNIQNVTQPESGIQPAS*

[0092] Protein Sequence of CHST6 PD5 mutant allele (residues resulting from frameshift are underlined) M L R WR VS KA AV LS V LF AQ.AVTVG L LYG WYS R P N I QNVTQPGIQPAS*

[0093] Protein Sequence of CHST6 F24 mutant allele (l.PAPS sequence is underlined: LSSWRSG)

[0094] M LRWRVSKAAVLSVLFAQAVTVG LLYGWYSRPNIQNVTQPE- - VHVLLLSSWRSGSSFLGQVFSQHPDVFYL 68

[0095] M EPAWHVWMTINQSGARSLRMAVRDTI RSIFQCDMSVM DSYIRQPQNISNLFMWSHSRALCSPPACLQ.TP 138

[0096] RDQISIEQDCKKHCG KSNLKLAESACQSYSHVVLKEVRFFELESLYSLLQDPTLNVRI IHLVRDPRAVFRSRDRSYK 215

[0097] ALVKDSNIVLEMANIPEKDKPYRVLQEICRSHVRIYETAM LKAPSFLKGRYKMIRYEDLVHNTQAEI EAMYEFI 289

[0098] G LEMTETLQEWIYRITHGKGKGTKKEAFDITSRNAEDVSMAWRTTLPFEKVQRIQDVCKGAMSLLGYSTVDSE 362

[0099] KEQKM MDLDLMKPRERYKFKWLPPKSTTAAKL* 397

[0100] Protein Sequence of CHST6 F42 mutant allele (frameshift beginning in 42. residue. Residues resulting from frameshift are underlined)

[0101] M LRWRVSKAAVLSVLFAQAVTVG LLYGWYSRPNIQNVTQPERCTCCFCPPGGPAHPSWARYSASI LTSFI*

[0102] Protein Sequence of CHST6 PD57 mutant allele (frameshift beginning in 42. residue. Residues resulting from frameshift are underlined)

[0103] M LRWRVSKAAVLSVLFAQAVTVG LLYGWYSRPNIQNVTQPENETRFAASFSATCPSWIHTSASPRTSPTCSCGATVVRSALRLR ASKRRATRLASNKTAKSTAGKAI*

Claims

CLAIMS1. A mutant zebrafish model of corneal dystrophies, characterized in that it comprises at least one mutation in the sequence of the disease-causing gene.

2. The mutant zebrafish model according to Claim 1, characterized in that it comprises at least one mutation between l.-l 197 bp of the sequence of chst6 gene.

3. The mutant zebrafish model according to Claim 2, characterized in that the mutation is one or a combination of an indel, a deletion and / or a frameshift mutation.

4. The mutant zebrafish model according to Claim 2, characterized in that it comprises a deletion mutation between nucleotides 125.-130. of the chst6 coding sequence (Sequence No:2).

5. The mutant zebrafish model according to Claim 2, characterized in that it comprises a deletion mutation between nucleotides 123.-126. of the chst6 coding sequence (Sequence No:3).

6. The mutant zebrafish model according to Claim 2, characterized in that it comprises an indel mutation between nucleotides 124.-180. of the chst6 coding sequence (Sequence No:4).

7. The mutant zebrafish model according to Claim 2, characterized in that it comprises a deletion mutation of nucleotides 120.-181. of the chst6 coding sequence (Sequence No:5).

8. The mutant zebrafish model according to Claim 2, characterized in that it comprises an indel mutation between nucleotides 123.-284. of the chst6 coding sequence (Sequence No:6).

9. A mutant zebrafish model according to any one of Claims 1-8, wherein corneal dystrophy is a macular cornea dystrophy.

10. A mutant zebrafish development method according to Claim 9, characterized in that it comprises steps of; i. Analysis of zebrafish chst6 gene to determine active region, ii. Targeting the sequence that encodes for GlcNAc6ST enzyme active region(s), iii. Design of at least one guide RNA to inactivate GlcNAc6ST enzyme function, iv. Use of one or two guide RNA’s alone or simultaneously to bind and induce mutagenesis of targeted gene.

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