Zinc finger nuclease, vector, and method for genome editing and method for visual cell production using said nuclease and vector

Abstract

Provided is a technique useful for genome editing for rhodopsin (RHO) gene locus using a zinc finger nuclease (ZFN), which is a ZFN pair combination for genome editing for the RHO gene locus, the ZFN combination having a zinc finger array that recognizes a specific nucleic acid sequence.

Description

Zinc finger nuclease, vector, and genome editing method and photoreceptor cell production method using the same

[0001] The present disclosure relates to zinc finger nucleases, vectors, and genome editing methods and cell production methods using them, and more particularly to zinc finger nucleases for genome editing of the rhodopsin (RHO) gene locus.

[0002] Human rhodopsin protein is a light-sensitive G protein-coupled receptor (GPCR) specific to rod photoreceptors, consisting of approximately 348 amino acids. The rhodopsin (RHO) gene locus, which encodes rhodopsin protein, consists of five exons and is approximately 6.7 kb long (chr3:129,528,639-129,535,344) including intron regions. Mutations in the rhodopsin gene cause retinitis pigmentosa, and approximately 110 types of genetic mutations have been reported to date. Patent Document 1 proposes a technology for treating retinitis pigmentosa caused by mutations in the rhodopsin gene through genome editing using designer nucleases such as TALE nucleases (TALENs), zinc-finger nucleases (ZFNs), and Cas nucleases.

[0003] ZFNs consist of a nucleic acid cleavage domain, FokI-ND, a nucleic acid binding domain, a zinc finger protein (ZFP), and a linker connecting them. Patent Document 2 discloses ZFNs that contain nucleic acid cleavage domains called nuclease domain 1 (ND1) and nuclease domain 2 (ND2) instead of FokI-ND. ND1 is said to be a nuclease domain derived from Bacillus SGD-V-76, and ND2 is said to be a nuclease domain derived from Clostridium botulinum. ND1 and ND2 are said to be superior to conventional FokI-NDs in terms of functionality such as cleavage activity, specificity, and target sequence selectivity.

[0004] International Publication No. 2021 / 010303 International Publication No. 2020 / 045281

[0005] “Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly”, Wright et al., Nature Protocols, 2006, Vol.1, No.3, p.1637-52

[0006] The main object of the present disclosure is to provide a technique useful for genome editing of the RHO locus using ZFNs.

[0007] To solve the above problems, the present disclosure provides the following [1] to

[12] . [1] A combination of a pair of zinc finger nucleases (ZFNs) for genome editing of the rhodopsin (RHO) gene locus, the combination being selected from the following group: (1) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 1 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 2. (2) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 5 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 6. (3) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 7 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 8. (4) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 9 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 10. (5) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 12. (6) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 13 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 14. (7) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 15 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 16. (8) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 31 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 32. (9) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 35 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 36. (10) A combination of a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 37 and a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 6.(11) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 38 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 8. (12) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 7 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 10. (13) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 14. (14) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 15 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 18. (15) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 45 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 32. (16) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:47 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:36. [2] A combination of [1] selected from the group consisting of: (2) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:5 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:6. (4) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:9 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:10. (5) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:11 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:12. (10) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:37 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:6. (12) A combination of a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 7 and a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 10.(13) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 14. (14) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 15 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 18. [3] A combination of [1] or [2], in which the spacer between the nucleic acid sequences recognized by the two zinc finger arrays is 5 bps or 6 bps. [4] Any combination of [1] to [3], in which the nucleic acid cleavage domain of the ZFN is ND1. [5] The combination of [4], in which the nucleic acid cleavage domain of the ZFN comprises the amino acid sequence of positions 391 to 585 of SEQ ID NO: 52, the amino acid sequence of SEQ ID NO: 57, or the amino acid sequence of SEQ ID NO: 58. [6] Any combination of [1] to [4], in which the RHO locus is the human RHO locus.

[0008] [7] A vector comprising a nucleic acid sequence encoding a pair of ZFNs according to any one of [1] to [6]. [8] The vector of [7], which is a bidirectional expression vector, comprising a nucleic acid sequence encoding a sense strand ZFN and a promoter sequence controlling the transcription of the nucleic acid sequence, and a nucleic acid sequence encoding an antisense strand ZFN and a promoter sequence controlling the transcription of the nucleic acid sequence, wherein the two promoter sequences are arranged contiguously without a spacer sequence between them. [9] The vector of [8], wherein the two promoters are a CMV promoter and a human EF1a (Elongation Factor alpha-1) promoter.

[0009]

[10] A therapeutic agent for a disease caused by the RHO gene, comprising any one of the vectors [7] to [9].

[0010]

[11] A genome editing method for the RHO locus, comprising the step of introducing any of the vectors [7] to [9] into photoreceptor cells in vivo or in vitro, preferably in vitro.

[12] A method for producing photoreceptor cells in which the RHO gene has been genome-edited, comprising the step of introducing any of the vectors [7] to [9] into photoreceptor cells in vivo or in vitro, preferably in vitro.

[0011] The present disclosure provides techniques useful for genome editing of the RHO locus using ZFNs.

[0012] Figure 1 shows EGFP fluorescence images of HEK293 cells transfected with a ZF-ND1 expression plasmid and an EGFP fragment expression plasmid containing a candidate ZF binding target sequence. EGFP fluorescence indicates cleavage of the candidate ZF binding target sequence by ZF-ND1. The structures of the bicistronic, dual promoter, and bidirectional promoter plasmid vectors are shown. Figure 1 shows EGFP fluorescence images of HEK293 cells transfected with a bicistronic, dual promoter, or bidirectional promoter and an EGFP fragment expression plasmid containing a candidate ZF binding target sequence. EGFP fluorescence indicates cleavage of the candidate ZF binding target sequence by ZF-ND1. ZF-ND1 expression plasmids (5461S, 5562AS) were transfected as positive controls. The results of Cel-I analysis of the target RHO sequence in HEK293 cells transfected with the bicistronic or bidirectional promoter are shown. The 220-bp and 130-bp bands indicate insertion and deletion (InDel). The positive control used was a ZF-ND1 expression plasmid (5461S, 5562AS). The results of Tide (Tracking of Indels by DEcomposition) analysis of the target RHO sequence in HEK293 cells transfected with a bicistronic or bidirectional promoter are shown. The X-axis of the histogram indicates the same sequence as WT (no InDels), with the positive axis representing the number of inserted bases and the negative axis representing the number of deleted bases. The Y-axis represents the frequency (percentage). Total eff indicates the sum of InDel frequencies (%). The positive control used was a ZF-ND1 expression plasmid (5461S, 5562AS).

[0013] 1. Zinc Finger Nucleases (ZFNs) The ZFNs disclosed herein can be used for genome editing of the rhodopsin (RHO) gene locus and are a combination of ZFNs with a zinc finger array that recognizes a specific nucleic acid sequence. The zinc finger array is composed of multiple zinc fingers (ZFs), each of which recognizes a three-base sequence. The zinc finger array may contain two or more ZFs, for example, 3-9, preferably 4-8, more preferably 5-7, and typically 6 ZFs. The relationship between the amino acid sequence of a ZF and the three-base sequence recognized by the ZF is publicly known. For example, the amino acid sequences listed in Supplemental Table 1 of Non-Patent Document 1 can be used for the ZF amino acid sequence depending on the target three-base sequence. Since there are multiple amino acid sequences for ZFs that recognize the same three-base sequence, these ZFs that recognize the same three-base sequence can be used interchangeably.

[0014] The ZFNs disclosed herein have zinc finger arrays selected from the following combinations (see Table 1 below): (1) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 1 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 2. (2) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 5 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 6. (3) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 7 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 8. (4) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 9 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 10. (5) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 12. (6) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 13 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 14. (7) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 15 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 16. (8) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 31 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 32. (9) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 35 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 36. (10) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 37 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 6. (11) A combination of a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 38 and a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 8.(12) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 7 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 10. (13) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 14. (14) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 15 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 18. (15) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 45 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 32. (16) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 47 with a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 36.

[0015] The recognition sequence of the zinc finger array of each ZFN, its SEQ ID NO: 1, and the amino acid sequence of the zinc finger array are shown in Table 1. For example, the pair of ZFNs in (1) above is a combination of a sense strand ZFN4350S having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 1 (ATTCTTGGGTGGGAGCAG) and an antisense strand ZFN4350AS having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 2 (AAGGCCTGAGCTCAGCCA). ZFN4350S consists of the amino acid sequence of SEQ ID NO: 59, and ZFN4350AS consists of the amino acid sequence of SEQ ID NO: 60. For example, the zinc finger array of ZFN 4350S has an amino acid sequence in which ZF91, ZF62, ZF106, ZF58, ZF103, and ZF88 (see Non-Patent Document 1, Supplemental Table 1), which recognize CAG, GAG, TGG, GGG, CTT, and ATT, respectively, are linked in order from the N-terminus.

[0016] The ZFN according to the present disclosure is more preferably a combination of any of the above (2), (4), (5), (10), (12), (13), and (14) because of its high cleavage activity.

[0017] The ZFNs disclosed herein recognize and bind to a nucleic acid sequence upstream of the start codon of exon 1 of the RHO gene (5'UTR of the RHO locus) using a zinc finger array at the RHO locus, resulting in a DNA double-strand break (DSB) via the nucleic acid cleavage domain.

[0018] The length of the spacer between the nucleic acid sequences recognized by two zinc finger arrays of a ZFN according to the present disclosure is not particularly limited, but is, for example, 1-20 bp, preferably 3-8 bp, 5 bps, or 6 bps. The nucleic acid sequence of the spacer is uniquely defined as the nucleic acid sequence between the nucleic acid sequence recognized by one zinc finger array and the nucleic acid sequence recognized by the other zinc finger array. The ZFN cleaves the double-stranded DNA within the spacer sequence.

[0019] For example, the pair of ZFNs described in (1) above cleaves double-stranded DNA in the spacer sequence (CGCAGC) between the nucleic acid sequence (ATTCTTGGGTGGGAGCAG) of SEQ ID NO: 1, which is the binding sequence of the sense strand ZFN, and the nucleic acid sequence (AAGGCCTGAGCTCAGCCA) of SEQ ID NO: 2, which is the binding sequence of the antisense strand ZFN.

[0020] The nucleic acid cleavage domain of the ZFN according to the present disclosure is not particularly limited and may be FokI-ND, ND1 and ND2 described in Patent Document 2, or variants thereof. The amino acid sequence of a full-length protein containing ND1 (derived from Bacillus SGD-V-76) is shown in SEQ ID NO: 52, and its nucleotide sequence is shown in SEQ ID NO: 53. The amino acid sequence of a full-length protein containing ND2 (derived from Clostridium botulinum) is shown in SEQ ID NO: 54, and its nucleotide sequence is shown in SEQ ID NO: 55. ND1 is typically a partial peptide corresponding to positions 391 to 585 of SEQ ID NO: 52, and ND2 is typically a partial peptide corresponding to positions 389 to 579 of SEQ ID NO: 54. The nucleic acid cleavage domain is preferably ND1.

[0021] The ND1 and ND2 variants may be polypeptides having nuclease activity, comprising an amino acid sequence in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence shown at positions 391 to 585 of SEQ ID NO: 52 or positions 389 to 579 of SEQ ID NO: 54. Here, "one to several" means, for example, 1 to 10, preferably 1 to 6, e.g., 1, 2, 3, 4 or 5.

[0022] The ND1 and ND2 variants may be polypeptides having nuclease activity and comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence shown at positions 391 to 585 of SEQ ID NO: 52 or positions 389 to 579 of SEQ ID NO: 54. Here, the "sequence identity" of an amino acid sequence is determined by comparing two sequences aligned to maximize sequence identity. Methods for determining the numerical value (%) of sequence identity are known to those skilled in the art. Any algorithm known to those skilled in the art (e.g., the BLAST algorithm, the FASTA algorithm, etc.) may be used to obtain optimal alignment and sequence identity. The sequence identity of an amino acid sequence can be determined using sequence analysis software such as BLASTP or FASTA.

[0023] Specific examples of ND1 and ND2 variants include variants containing aspartic acid (D483, D487, and D496) at positions corresponding to positions 483, 487, and 496 in the amino acid sequence of FokI (DDD variants), and variants containing arginine (R483, R487, and R537) at positions corresponding to positions 483, 487, and 537 in the amino acid sequence of FokI (RRR variants). The ND1 DDD variant (ND1DDD) has an amino acid sequence corresponding to positions 391 to 585 of SEQ ID NO: 52, in which the amino acids at positions 103 and 113 are substituted with aspartic acid (SEQ ID NO: 57). The ND1 RRR variant (ND1RRR) has an amino acid sequence corresponding to positions 391 to 585 of SEQ ID NO: 52, in which the amino acids at positions 100 and 154 are substituted with arginine (SEQ ID NO: 58).

[0024] The nucleic acid cleavage domain of a ZFN according to the present disclosure may be a polypeptide containing modified amino acids and / or unnatural amino acids. Modified amino acids include, but are not limited to, methylation, esterification, amidation, acetylation, alkylation, halogenation, etc. The modified amino acids and unnatural amino acids can be introduced by known methods.

[0025] The nucleic acid cleaving domain and the zinc finger protein (ZFP) comprising the zinc finger array may be linked directly or via a linker. The linker may consist of, for example, two or more amino acid residues. The length is not particularly limited, but may be, for example, 2 to 20 amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, or 20 amino acids. The type of linker is also not particularly limited, but examples include TGGS (SEQ ID NO: 56). The presence or absence of a linker and the length and type of the linker can be appropriately selected by those skilled in the art, taking into account the type of nucleic acid binding domain, etc.

[0026] The ZFNs of the present disclosure may have other effector domains instead of a nucleic acid cleavage domain, such as cytosine base editors (UGI, APOBEC), adenine base editors (TadA), transcriptional activators (VP16) / repressors (SID), epigenomic editors (DNMT3A, TET1), imaging proteins (Dye, fluorescent proteins), transposases (PiggyBac), and recombinases (Flippase).

[0027] The ZFNs of the present disclosure can be produced in vitro or in vivo by methods known in the art, such as artificial synthesis based on amino acid sequence information, or artificial synthesis of nucleic acids encoding ZFNs, insertion of the nucleic acids into an appropriate expression vector, and subsequent introduction into an appropriate host cell to express the ZFNs in the cell.

[0028] 2. Vector The vector according to the present disclosure comprises a nucleic acid sequence encoding the pair of ZFNs described above. In one embodiment, the vector according to the present disclosure is a bidirectional expression vector, in which the sense strand ZFN and the antisense strand ZFN of the pair of ZFNs are expressed under the control of separate promoters, and the two promoters are arranged in opposite directions. More specifically, the bidirectional expression vector according to the present disclosure comprises a nucleic acid sequence encoding the sense strand ZFN and a promoter sequence controlling the transcription of the nucleic acid sequence, and a nucleic acid sequence encoding the antisense strand ZFN and a promoter sequence controlling the transcription of the nucleic acid sequence, and preferably the two promoter sequences are arranged contiguously without a spacer sequence between them.

[0029] In the vectors of the present disclosure, the two promoters can be conventionally known promoters, such as the CMV promoter, human EF1a (Elongation Factor alpha-1) promoter, PGK (phosphoglycerate kinase) promoter, etc. The two promoters are preferably the CMV promoter and human EF1a.

[0030] In addition to a nucleic acid sequence encoding a ZFN and a promoter sequence, the vectors of the present disclosure may also contain elements present in conventional gene expression vectors, such as polyA addition sequences (e.g., SV40 pA, HGH pA, rabbit globin pA).

[0031] The vector is not particularly limited and may be selected from vectors used in the relevant field. Specific examples of vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements, viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, avian poxviruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, etc.), and vectors derived from combinations thereof (cosmids, phagemids, etc.). Among the above, plasmid vectors are preferred from the viewpoint of their versatility. Furthermore, from the viewpoint of their advanced clinical applications, viral vectors are preferred, and adeno-associated viral vectors (AAV vectors) are more preferred.

[0032] 3. Genome Editing Methods, etc. The present disclosure also provides a genome editing method for the RHO locus in photoreceptor cells, including the step of introducing the above-mentioned vector into photoreceptor cells in vivo or in vitro, preferably in vitro, as well as a method for producing genome-edited photoreceptor cells. Unlike genome editing systems using Cas nucleases, which involve ubiquitous expression of gRNA, ZFN-based genome editing systems are composed solely of proteins. Therefore, ZFN-based genome editing systems can easily achieve tissue-specific genome editing of interest by using tissue-specific promoters, drug-responsive promoters, etc.

[0033] Introduction of a vector into photoreceptor cells can be carried out by conventionally known techniques, including, but not limited to, electroporation, particle gun, microinjection, lipofection, and protein transduction.

[0034] Depending on the purpose of genome editing, donor DNA may be introduced into cells together with the vector. The donor DNA may be, for example, donor DNA encoding a normal RHO gene. The donor DNA may be configured to be capable of introducing the normal RHO gene into the gene locus using repair mechanisms such as nonhomologous end-joining (NHEJ) or homologous recombination (HR) at the DNA double-strand break site induced by ZFN.

[0035] In genome editing without donor DNA, the breakage site is mainly repaired by NHEJ.Because NHEJ is error-prone, at least one nucleotide deletion, insertion, or substitution, or a combination thereof, may occur during the repair of the breakage.In this way, the RHO locus is modified at the breakage site.

[0036] The photoreceptor cells may be derived from humans or non-human animals, including ungulates (such as cattle, boars, pigs, sheep, and goats), perissodactyls (such as horses), rodents (such as mice, rats, hamsters, and squirrels), lagomorphs (such as rabbits), and carnivores (such as dogs, cats, and ferrets).

[0037] 4. Therapeutic Agent The present disclosure also provides a therapeutic agent for a disease caused by the RHO gene, comprising the vector described above. The therapeutic agent may contain donor DNA encoding the normal RHO gene described above. Furthermore, genome-edited photoreceptor cells obtained by the genome editing method described above may be formulated as a cell medicine. Such a cell medicine may also be used as a therapeutic agent for a disease caused by the RHO gene.

[0038] The therapeutic agent can be formulated according to conventional methods, specifically by compounding the vector and, optionally, the donor DNA with pharmaceutical additives, or by compounding the genome-edited photoreceptor cells with pharmaceutical additives.

[0039] Pharmaceutical additives refer to substances other than the active ingredient contained in a formulation. Pharmaceutical additives are included in formulations for purposes such as facilitating formulation, stabilizing quality, and enhancing usefulness. Examples of pharmaceutical additives include excipients, binders, disintegrants, lubricants, flow agents (solid-preventing agents), colorants, capsule coatings, plasticizers, flavoring agents, sweeteners, flavoring agents, solvents, solubilizers, emulsifiers, suspending agents (adhesives), thickeners, pH adjusters (acidifiers, alkalinizers, buffers), wetting agents (solubilizers), antibacterial preservatives, chelating agents, suppository bases, ointment bases, hardening agents, softeners, medical water, propellants, stabilizers, preservatives, etc. These pharmaceutical additives can be readily selected by those skilled in the art based on the intended dosage form and route of administration and standard pharmaceutical practice.

[0040] The therapeutic agent may take any dosage form, including eye drops, tablets, capsules, oral preparations, external preparations, suppositories, injections, and inhalants.

[0041] Therapeutic agents can be prescribed as appropriate based on the judgment of a doctor or medical professional, and the dosage and administration schedule of the therapeutic agent can also be determined as appropriate based on the judgment of a doctor or medical professional.

[0042] The administration route of the therapeutic agent is appropriately selected depending on factors such as the dosage form of the therapeutic agent, the type and severity of the disease to be treated, etc. Examples of administration routes that are preferably used include subretinal injection, vitreous injection, and suprachoroidal injection.

[0043] Example 1: Identification of ZF Target Sequences 1. Search for Candidate Sequences A 100-bp region located upstream of the start codon of exon 1 of the RHO gene was searched for regions where ZFNs could induce double-stranded DNA cleavage. The 100-bp region was selected because it exhibited low evolutionary sequence conservation. ZFFinger Tools (https: / / www.scripps.edu / barbas / zfdesign / zfdesignhome.php), available from the Barbas Laboratory at Scripps Research Institute, was used to search for candidate sequences to which ZFNs could bind. The binding target sequence of each zinc finger array in the ZFN dimer was 18 bp (composed of six ZFs). When the base length between the binding target sequences (spacer) was set to 6 bp or 5 bp, 34 pairs of candidate binding target sequences were identified (see Table 1).

[0044] The amino acid sequence of each ZF was as described in the literature ("Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly," Wright et al., Nature Protocols, 2006, Vol. 1, No. 3, pp. 1637-52, Supplemental Table 1). A zinc finger array was constructed by linking one ZF to every three bases on the 3' end of the target sequence (a total of six ZFs). For example, the sense strand ZFN 4350S, which has a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 1 (ATTCTTGGGTGGGAGCAG), has an amino acid sequence in which ZF91, ZF62, ZF106, ZF58, ZF103, and ZF88 (see Supplemental Table 1 in the literature), which recognize CAG, GAG, TGG, GGG, CTT, and ATT, respectively, are linked in order from the N-terminus.

[0045]

[0046] 2. Evaluation of ZFN cleavage activity against candidate binding target sequences. Using the ND1 DDD mutant (ND1DDD, SEQ ID NO: 57) and RRR mutant (ND1RRR, SEQ ID NO: 58) as nucleases, the cleavage activity against each candidate binding target sequence was evaluated by SSA (single strand annealing) analysis. SSA analysis quantitatively evaluates the cleavage activity of ZFN against the binding target sequence using homologous recombination and single strand annealing (SSA), which are gene repair mechanisms when DNA breaks occur in cells. HEK293 cells and the following expression vectors were used for the analysis.

[0047] (1) ZF-ND1 Expression Plasmids (pCMV-ZF-ND1DDD, pCMV-ZF-ND1RRR) These plasmids express "ZF-ND1" under the control of the CMV promoter. In the term "ZF-ND1," "ZF" refers to one of the sense and antisense ZFNs of the 34 ZFN pairs listed in Table 1, and "ND1" refers to one of the subunits of the ND1 heterodimer (ND1DDD and ND1RRR). ND1DDD was used as the sense strand ZFN, and ND1RRR was used as the antisense strand ZFN. A nuclear localization signal (NLS) was attached to the 5' end of "ZF," and "ZF" and "ND1" were linked by a linker sequence (Thr-Gly-Gly-Ser: SEQ ID NO: 56). A polyA sequence was added to the 3' end of "ND1." The polyA sequence used was that of rabbit beta-globin.

[0048] (2) EGFP Fragment Expression Plasmid (pCAG-EGxxFP) This plasmid expresses EGFP under the control of the CAG promoter. EGxxFP consists of a 1-600 bp fragment of the EGFP gene, a stop codon, a multicloning site, and a 120-720 bp fragment of the EGFP gene, into which a potential target sequence is inserted. If the potential target sequence is not cleaved with ZF-ND1, the pCAG-EGxxFP plasmid expresses incomplete GFP due to the stop codon immediately following the 1-600 bp fragment of the EGFP gene. On the other hand, if the potential target sequence is cleaved with ZF-ND1, homologous recombination and single-strand annealing (SSA) occur between the overlapping sequences of the 120-720 bp fragment of the EGFP gene, resulting in expression of the complete EGFP gene and EGFP fluorescence.

[0049] pCMV-ZF-ND1DDD, pCMV-ZF-ND1RRR, and pCAG-EGxxFP were transfected into HEK293 cells using transfection reagents (FuGene6 (Promega) or Viafect (Promega)) according to the accompanying protocol. Fluorescence images were taken 48 hours after transfection. The results are shown in Figure 1. Green EGFP fluorescence was observed in 16 pairs (1, 3-8, 16, 18-23, 30, and 32) out of 34 pairs of candidate binding target sequences, confirming that cleavage by ZF-ND1 had occurred in the binding target sequences. High fluorescence intensity was observed in pairs 3, 5, 6, 19, and 21-23, confirming particularly high cleavage activity.

[0050] Example 2: Study of High-Expression Vector Constructs. High-expression plasmid vectors for ZFN were studied. Generally, methods for expressing two genes include expressing two genes using a single promoter (bicistronic expression) and expressing each gene using two different promoters. In this example, as an example of the former, a plasmid vector (hereinafter referred to as "bicistronic") was constructed in which ZF-S-ND1DDD and ZF-AS-ND1RRR were linked with an autolytic peptide (Furin-P2A) under the control of a CMV promoter. As an example of the latter, a plasmid vector (hereinafter referred to as "dual promoter") in which the CMV promoter and human EF1a (Elongation Factor alpha-1) promoter were arranged in the same direction (dual promoter) and a plasmid vector in which they were arranged in opposite directions (bidirectional promoter) were constructed. The structure of each plasmid vector is shown in Figure 2. The dual promoter and bidirectional promoter express ZF-S-ND1DDD under the control of the EF1a promoter and ZF-AS-ND1RRR under the control of the CMV promoter. The ZF target binding sequences (ZF-S, ZF-AS) used were pair 22 (5461S, 5562AS) in Table 1. The dual promoter contains an insulator sequence to prevent interference between the expression of the two genes.

[0051] Using each of the bicistronic, dual promoter, and bidirectional promoter plasmid vectors, SSA analysis was performed in the same manner as in Example 1. As a positive control, the ZF-ND1 expression plasmid (positive control) of pair 22 in Example 1 was used.

[0052] The results are shown in Figure 3. Almost no EGFP expression was observed with the dual promoter. Furthermore, the EGFP signal was attenuated with the bicistronic promoter compared to the positive control. On the other hand, the bidirectional promoter produced EGFP expression equivalent to that of the positive control.

[0053] The frequencies of InDel (insertion and deletion) in the target RHO sequence were compared by Cel-I analysis using the bicistronic and bidirectional promoter plasmid vectors. The ZF-ND1 expression plasmid (positive control) pair 22 from Example 1 was used as a positive control. Genomic DNA from HEK293 cells transfected with each plasmid construct was used for Cel-I analysis according to the recommended protocol for the GeneArt Genomic Cleavage Detection kit (ThermoFisher Scientific). The target RHO sequence amplified by PCR was treated with Cel-I nuclease and electrophoresed. The results are shown in Figure 4. The PCR amplification products were also directly sequenced, and the resulting electrophenograms were quantified using the Tide (Tracking of Indels by DEcomposition) analysis site (http: / / shinyapps.datacurators.nl / tide / ). The results are shown in Figure 5. Figure 5 shows a histogram of InDels. The X-axis indicates the same sequence as WT (no InDels), the positive direction indicates the number of inserted bases, and the negative direction indicates the number of deleted bases. The Y-axis indicates the frequency (percentage). Total eff indicates the sum of InDel frequencies (%).

[0054] A band at 345 bp was observed in the genomic DNA of untransfected cells (see Figure 4, lane 2). In the positive control, two bands (220 bp and 130 bp) were observed due to cleavage by Cel-I nuclease due to the presence of indels (see Figure 4, lanes 3 and 5). The frequency of indels was 25.7% (see Figure 5). In the bicistronic transfection, the amount of cleaved DNA was reduced compared to the positive control (see Figure 4, lane 4), and the frequency of indels was also low at 4.6% (see Figure 5). On the other hand, the bidirectional promoter transfection showed the same amount of cleaved DNA (see Figure 4, lane 6) and the same frequency of indels as the positive control (see Figure 5).

[0055] The results of this example demonstrated that the bidirectional promoter enables two types of genes to be integrated into one construct, making it useful for industrial applications such as AAV gene therapy preparations.

[0056] SEQ ID NO: 1: Recognition sequence of the zinc finger array of ZFN 4350S SEQ ID NO: 2: Recognition sequence of the zinc finger array of ZFN 4350AS SEQ ID NO: 3: Recognition sequence of the zinc finger array of ZFN 4653S SEQ ID NO: 4: Recognition sequence of the zinc finger array of ZFN 4653AS SEQ ID NO: 5: Recognition sequence of the zinc finger array of ZFN 4956S SEQ ID NO: 6: Recognition sequence of the zinc finger array of ZFN 4956AS SEQ ID NO: 7: Recognition sequence of the zinc finger array of ZFN 5158S SEQ ID NO: 8: Recognition sequence of the zinc finger array of ZFN 5158AS SEQ ID NO: 9: Recognition sequence of the zinc finger array of ZFN 5259S SEQ ID NO: 10: Recognition sequence of the zinc finger array of ZFN 5259AS SEQ ID NO: 11: Recognition sequence of the zinc finger array of ZFN 5461S SEQ ID NO: 12: Zinc finger array recognition sequence of ZFN 5461AS SEQ ID NO: 13: Zinc finger array recognition sequence of ZFN 5562S SEQ ID NO: 14: Zinc finger array recognition sequence of ZFN 5562AS SEQ ID NO: 15: Zinc finger array recognition sequence of ZFN 5764S SEQ ID NO: 16: Zinc finger array recognition sequence of ZFN 5764AS SEQ ID NO: 17: Zinc finger array recognition sequence of ZFN 5865S SEQ ID NO: 18: Zinc finger array recognition sequence of ZFN 5865AS SEQ ID NO: 19: Zinc finger array recognition sequence of ZFN 6067S SEQ ID NO: 20: Zinc finger array recognition sequence of ZFN 6067AS SEQ ID NO: 21: Zinc finger array recognition sequence of ZFN 6168S SEQ ID NO: 22: ZFN SEQ ID NO: 23: Recognition sequence of the zinc finger array of ZFN 7178S SEQ ID NO: 24: Recognition sequence of the zinc finger array of ZFN 7178AS SEQ ID NO: 25: Recognition sequence of the zinc finger array of ZFN 7481S SEQ ID NO: 26: Recognition sequence of the zinc finger array of ZFN 7481AS SEQ ID NO: 27: Recognition sequence of the zinc finger array of ZFN 7784S SEQ ID NO: 28: Recognition sequence of the zinc finger array of ZFN 7784AS SEQ ID NO: 29: Recognition sequence of the zinc finger array of ZFN 8087SSEQ ID NO: 30: Zinc finger array recognition sequence of ZFN 8087AS SEQ ID NO: 31: Zinc finger array recognition sequence of ZFN 8289S SEQ ID NO: 32: Zinc finger array recognition sequence of ZFN 8289AS SEQ ID NO: 33: Zinc finger array recognition sequence of ZFN 8592S SEQ ID NO: 34: Zinc finger array recognition sequence of ZFN 8592AS SEQ ID NO: 35: Zinc finger array recognition sequence of ZFN 8895S SEQ ID NO: 36: Zinc finger array recognition sequence of ZFN 8895AS SEQ ID NO: 37: Zinc finger array recognition sequence of ZFN 4955S SEQ ID NO: 38: Zinc finger array recognition sequence of ZFN 5157S SEQ ID NO: 39: Zinc finger array recognition sequence of ZFN 6874S SEQ ID NO: 40: ZFN SEQ ID NO: 41: Recognition sequence of the zinc finger array of ZFN 7177S SEQ ID NO: 42: Recognition sequence of the zinc finger array of ZFN 7480S SEQ ID NO: 43: Recognition sequence of the zinc finger array of ZFN 7783S SEQ ID NO: 44: Recognition sequence of the zinc finger array of ZFN 8086S SEQ ID NO: 45: Recognition sequence of the zinc finger array of ZFN 8288S SEQ ID NO: 46: Recognition sequence of the zinc finger array of ZFN 8591S SEQ ID NO: 47: Recognition sequence of the zinc finger array of ZFN 8892S SEQ ID NO: 48: Recognition sequence of the zinc finger array of ZFN 9197S SEQ ID NO: 49: Recognition sequence of the zinc finger array of ZFN 9197AS SEQ ID NO: 50: Recognition sequence of the zinc finger array of ZFN 9410S SEQ ID NO: 51: Recognition sequence of zinc finger array of ZFN 9410AS SEQ ID NO: 52: Amino acid sequence of full-length protein (derived from Bacillus SGD-V-76) containing ND1 SEQ ID NO: 53: Nucleotide sequence of full-length protein (derived from Bacillus SGD-V-76) containing ND1 SEQ ID NO: 54: Amino acid sequence of full-length protein (derived from Clostridium botulinum) containing ND2 SEQ ID NO: 55: Nucleotide sequence of amino acid sequence of full-length protein (derived from Clostridium botulinum) containing ND2 SEQ ID NO: 56: Amino acid sequence of peptide linker SEQ ID NO: 57: Amino acid sequence of ND1DDD SEQ ID NO: 58: Amino acid sequence of ND1RRRSEQ ID NO: 59: Amino acid sequence of the zinc finger array of ZFN 4350S SEQ ID NO: 60: Amino acid sequence of the zinc finger array of ZFN 4350AS SEQ ID NO: 61: Amino acid sequence of the zinc finger array of ZFN 4653S SEQ ID NO: 62: Amino acid sequence of the zinc finger array of ZFN 4653AS SEQ ID NO: 63: Amino acid sequence of the zinc finger array of ZFN 4956S SEQ ID NO: 64: Amino acid sequence of the zinc finger array of ZFN 4956AS SEQ ID NO: 65: Amino acid sequence of the zinc finger array of ZFN 5158S SEQ ID NO: 66: Amino acid sequence of the zinc finger array of ZFN 5158AS SEQ ID NO: 67: Amino acid sequence of the zinc finger array of ZFN 5259S SEQ ID NO: 68: Amino acid sequence of the zinc finger array of ZFN 5259AS SEQ ID NO: 69: ZFN SEQ ID NO:70: Amino acid sequence of the zinc finger array of ZFN 5461AS SEQ ID NO:71: Amino acid sequence of the zinc finger array of ZFN 5562S SEQ ID NO:72: Amino acid sequence of the zinc finger array of ZFN 5562AS SEQ ID NO:73: Amino acid sequence of the zinc finger array of ZFN 5764S SEQ ID NO:74: Amino acid sequence of the zinc finger array of ZFN 5764AS SEQ ID NO:75: Amino acid sequence of the zinc finger array of ZFN 5865S SEQ ID NO:76: Amino acid sequence of the zinc finger array of ZFN 5865AS SEQ ID NO:77: Amino acid sequence of the zinc finger array of ZFN 6067S SEQ ID NO:78: Amino acid sequence of the zinc finger array of ZFN 6067AS SEQ ID NO:79: Amino acid sequence of the zinc finger array of ZFN 6168S SEQ ID NO: 80: Amino acid sequence of the zinc finger array of ZFN 6168AS SEQ ID NO: 81: Amino acid sequence of the zinc finger array of ZFN 7178S SEQ ID NO: 82: Amino acid sequence of the zinc finger array of ZFN 7178AS SEQ ID NO: 83: Amino acid sequence of the zinc finger array of ZFN 7481S SEQ ID NO: 84: Amino acid sequence of the zinc finger array of ZFN 7481AS SEQ ID NO: 85: Amino acid sequence of the zinc finger array of ZFN 7784S SEQ ID NO: 86: ZFNSEQ ID NO: 87: Amino acid sequence of the zinc finger array of ZFN 8087S SEQ ID NO: 88: Amino acid sequence of the zinc finger array of ZFN 8087AS SEQ ID NO: 89: Amino acid sequence of the zinc finger array of ZFN 8289S SEQ ID NO: 90: Amino acid sequence of the zinc finger array of ZFN 8289AS SEQ ID NO: 91: Amino acid sequence of the zinc finger array of ZFN 8592S SEQ ID NO: 92: Amino acid sequence of the zinc finger array of ZFN 8592AS SEQ ID NO: 93: Amino acid sequence of the zinc finger array of ZFN 8895S SEQ ID NO: 94: Amino acid sequence of the zinc finger array of ZFN 8895AS SEQ ID NO: 95: Amino acid sequence of the zinc finger array of ZFN 4955S SEQ ID NO: 96: Amino acid sequence of the zinc finger array of ZFN 5157S SEQ ID NO: 97: Amino acid sequence of the zinc finger array of ZFN 6874S SEQ ID NO: 98: Amino acid sequence of the zinc finger array of ZFN 6874AS SEQ ID NO: 99: Amino acid sequence of the zinc finger array of ZFN 7177S SEQ ID NO: 100: Amino acid sequence of the zinc finger array of ZFN 7480S SEQ ID NO: 101: Amino acid sequence of the zinc finger array of ZFN 7783S SEQ ID NO: 102: Amino acid sequence of the zinc finger array of ZFN 8086S SEQ ID NO: 103: Amino acid sequence of the zinc finger array of ZFN 8288S SEQ ID NO: 104: Amino acid sequence of the zinc finger array of ZFN 8591S SEQ ID NO: 105: Amino acid sequence of the zinc finger array of ZFN 8892S SEQ ID NO: 106: Amino acid sequence of the zinc finger array of ZFN 9197S SEQ ID NO: 107: ZFN SEQ ID NO: 108: Amino acid sequence of the zinc finger array of ZFN 9410S SEQ ID NO: 109: Amino acid sequence of the zinc finger array of ZFN 9410AS

Claims

1. A combination of a pair of zinc finger nucleases (ZFNs) for genome editing of the rhodopsin (RHO) locus, the combination being selected from the following group: (1) A combination of a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO: 1 and a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO:

2. (2) A combination of a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO:5 and a ZFN having a zinc finger array that recognizes the nucleic acid sequence of SEQ ID NO:

6. (3) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 7 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:

8. (4) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 9 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:

10. (5) A combination of a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO: 11 and a ZFN having a zinc finger array recognizing the nucleic acid sequence of SEQ ID NO:

12. (6) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 13 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

14. (7) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 15 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

16. (8) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 31 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

32. (9) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 35 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

36. (10) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 37 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

6. (11) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 38 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

8. (12) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 7 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

10. (13) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 11 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

14. (14) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 15 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

18. (15) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 45 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

32. (16) A combination of a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO: 47 and a ZFN having a zinc finger array recognizing a nucleic acid sequence of SEQ ID NO:

36.

2. The combination of claim 1, wherein the nucleic acid cleavage domain of the ZFN is ND1.

3. A vector comprising a nucleic acid sequence encoding a pair of ZFNs according to claim 1.

4. The vector described in claim 3, which is a bidirectional expression vector, comprising a nucleic acid sequence encoding a sense strand ZFN and a promoter sequence that controls the transcription of the nucleic acid sequence, and a nucleic acid sequence encoding an antisense strand ZFN and a promoter sequence that controls the transcription of the nucleic acid sequence, wherein the two promoters are arranged consecutively without a spacer sequence between them.

5. A therapeutic agent for a disease caused by the RHO gene, comprising the vector according to claim 3 or 4.

6. A method for genome editing of the RHO locus, comprising the step of introducing the vector described in claim 3 or 4 into photoreceptor cells.

7. A method for producing photoreceptor cells in which the RHO gene has been genome-edited, comprising the step of introducing the vector described in claim 3 or 4 into photoreceptor cells.