Gene manipulation for treatment of retinal dysfunction disorder

A method for treating a retinal dysfunction disease, the method comprising administration of a composition for gene manipulation into a subject to be treated,

• wherein the composition for gene manipulation comprises:
• a guide RNA or a nucleic acid encoding the same,
• wherein the guide RNA targets a target region present in a retinal pigment epithelium-specific 65 kDa protein (RPE65) gene,
• wherein the target region has two strands having a guide nucleic acid-binding sequence and a guide nucleic acid-non binding sequence, which are sequences of 15 to 25 contiguous nucleotides,
• wherein the guide RNA comprises
• a guide sequence complementary binding with the guide nucleic acid-binding sequence of the target region;
• and
• a guide RNA comprises a nucleotide sequence comprising SEQ ID NO:
• 225, SEQ ID NO:
• 228, SEQ ID NO:
• 231, and SEQ ID NO:
• 233 arranged in a direction of 5′ to 3′, or a nucleotide sequence having 80% or more homologous therewith
• wherein the guide sequence is a RNA sequence having homology with the guide nucleic acid-non binding sequence of the target region,
• wherein the guide nucleic acid-non binding sequence of the target region in the RPE65 gene is selected from the group consisting of SEQ ID NOs:
• 1 to 44,
• wherein the guide sequence includes 0 to 5 nucleotides mismatched with the guide nucleic acid-binding sequence of the target region,
• and
• a Cas9 protein or a nucleic acid encoding the same,
• wherein the Cas9 protein is a Streptococcus pyogenes-derived Cas9 protein,
• a donor comprising a nucleic acid encoding a part of RPE65, wherein the Cas9 protein is complexed with the guide RNA to manipulate the RPE65 gene,
• wherein the nucleic acid encoding the part of RPE65 comprises one or more synonymous mutations compared to wild type sequence thereof, so as to prevent a donor cleavage and a recleavage of a Rpe65 locus repaired after homology directed repair (HDR).