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KR-20260062936-A - EYS Gene Editing Prime Editor and Its Uses

KR20260062936AKR 20260062936 AKR20260062936 AKR 20260062936AKR-20260062936-A

Abstract

One of the most important technical problems to be solved by this specification is to select a prime editor that exhibits the effect of correcting the c.4957dupA mutation in the EYS gene of actual human retinal cells from among numerous prime editors that can be theoretically designed, specify its composition, and provide a prime editor capable of demonstrating an actual therapeutic effect. To solve the above technical problem, this specification provides a prime editor and its components that are screened through a high-throughput screening method and verified in actual human retinal cells, and are capable of actually correcting the c.4957dupA mutation in the EYS gene. The prime editor disclosed in this specification acts on human retinal cells containing the c.4957dupA mutation in the EYS gene to correct the EYS gene to a normal state. Therefore, the prime editor can be used as a therapeutic agent for retinal epithelial pigmentation caused by the c.4957dupA mutation in the EYS gene.

Inventors

  • 김희권
  • 이준원
  • 변석호
  • 정한
  • 김지성

Assignees

  • 성균관대학교산학협력단
  • 연세대학교 산학협력단

Dates

Publication Date
20260507
Application Date
20240826
Priority Date
20230829

Claims (6)

  1. A pegRNA for a prime editor to correct the c.4957dupA variant of the EYS gene, having the following structure: 5' - [Guide Domain] - [Scaffold] - [Prime Editing Domain] - 3' Here, the scaffold can interact with the prime editor to form a composite, and The above guide domain and the above prime editing domain consist of a combination selected from the following: A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11534 to 11552 and SEQ ID NOs 12104 to 12122, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 3343 to 3482; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11553 to 11571 and SEQ ID NOs 12123 to 12141, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 3483 to 3573; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11572 to 11590 and SEQ ID NOs 12142 to 12160, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 3574 to 3710; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11591 to 11609 and SEQ ID NOs 12161 to 12179, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 3711 to 3829; A guide domain targeting a target sequence composed of a nucleic acid sequence selected from SEQ ID NOs 11610 to 11628 and SEQ ID NOs 12180 to 12198, and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NOs 3830 to 3888; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11629 to 11647 and SEQ ID NOs 12199 to 12217, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 3889 to 4022; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11648 to 11666 and SEQ ID NOs 12218 to 12236, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4023 to 4169; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11667 to 11685 and SEQ ID NOs 12237 to 12255, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4170 to 4309; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11686 to 11704 and SEQ ID NOs 12256 to 12274, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4310 to 4450; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11705 to 11723 and SEQ ID NOs 12275 to 12293, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4451 to 4578; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11724 to 11742 and SEQ ID NOs 12294 to 12312, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4579 to 4608; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11743 to 11761 and SEQ ID NOs 12313 to 12331, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4609 to 4671; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11762 to 11780 and SEQ ID NOs 12332 to 12350, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4672 to 4736; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11781 to 11799 and SEQ ID NOs 12351 to 12369, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4737 to 4878; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11800 to 11818 and SEQ ID NOs 12370 to 12388, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 4879 to 5024; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11819 to 11837 and SEQ ID NOs 12389 to 12407, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5025 to 5101; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11838 to 11856 and SEQ ID NOs 12408 to 12426, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5102 to 5214; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11857 to 11875 and SEQ ID NOs 12427 to 12445, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5215 to 5311; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11876 to 11894 and SEQ ID NOs 12446 to 12464, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5312 to 5441; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11895 to 11913 and SEQ ID NOs 12465 to 12483, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5442 to 5550; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11914 to 11932 and SEQ ID NOs 12484 to 12502, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5551 to 5681; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11933 to 11951 and SEQ ID NOs 12503 to 12521, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5682 to 5804; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11952 to 11970 and SEQ ID NOs 12522 to 12540, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5805 to 5831; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11971 to 11989 and SEQ ID NOs 12541 to 12559, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5832 to 5923; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 11990 to 12008 and SEQ ID NOs 12560 to 12578, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5924 to 5966; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 12009 to 12027 and SEQ ID NOs 12579 to 12597, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 5967 to 5993; A guide domain targeting a target sequence composed of a nucleic acid sequence selected from SEQ ID NOs 12028 to 12046 and SEQ ID NOs 12598 to 12616, and a prime editing domain composed of a nucleic acid sequence selected from SEQ ID NOs 5994 to 6040; A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 12047 to 12065 and SEQ ID NOs 12617 to 12635, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 6041 to 6054; and A guide domain targeting a target sequence composed of a selected nucleic acid sequence among SEQ ID NOs 12066 to 12103, and a prime editing domain composed of a selected nucleic acid sequence among SEQ ID NOs 6055 to 6057.
  2. In claim 1, the scaffold is a pegRNA composed of a selected nucleic acid sequence among SEQ ID NOs 11531 to 11533.
  3. A prime editor composition for correcting the c.4957dupA variant of the EYS gene, comprising the following: A prime editor protein, or a nucleic acid encoding the prime editor protein; and The pegRNA of claim 1, or the nucleic acid encoding the pegRNA, Here, the prime editor protein comprises a Cas9 protein derived from Streptococcus pyogenes or a variant thereof, and a reverse transcriptase, and The scaffold of the above pegRNA interacts with the above prime editor protein to form a complex.
  4. In paragraph 3, the prime editor composition comprises the prime editor protein and the pegRNA, wherein the prime editor protein and the pegRNA bind to form a complex.
  5. In paragraph 3, the prime editor composition comprises a nucleic acid encoding the prime editor and a nucleic acid encoding the pegRNA.
  6. A prime editor composition according to any one of claims 3 to 5, wherein the prime editor protein is selected from the following: NRCH-hyPE2max; NRCH-PE2max; NRCH-PE2; and PE2max.

Description

EYS Gene Editing Prime Editor and Its Uses **EYS Gene Editing Prime Editor and Its Uses** The invention disclosed in this specification relates to a technology for treating genetic diseases using a Prime Editor. Retinitis pigmentosa Retinitis pigmentosa Retinitis pigmentosa is a hereditary ophthalmic disease that is a serious condition ranging from night blindness and visual field constriction to bilateral blindness. It is a relatively common ophthalmic disease with a high prevalence, affecting approximately 1 in 3,000 people worldwide. However, there are currently very few effective treatments for retinitis pigmentosa. c.4957dupA mutation of the EYS gene With the recent advancement of Next Generation Sequencing (NGS) technology, more than 50 diverse gene mutations causing retinal pigment epithelium have been discovered. Among these, mutations in the Eyes Shut Homolog (EYS) gene are known to cause retinal pigment epithelium as a single variant. Mutations in the EYS gene are found particularly frequently in Asian patients with retinal pigment epithelium. Among the single gene mutations that cause retinochromocytic epithelium, there is the c.4957dupA mutation, which is caused by a duplication of adenine bases at position 4957 of the EYS gene. Research results showed that a large number of Asian patients with retinochromocytic epithelium, particularly those in Korea and Japan, developed the disease due to the c.4957dupA mutation in the EYS gene. Therefore, it is expected that many patients will be able to treat retinal pigment epitheliosis by correcting the c.4957dupA mutation in the EYS gene. Limitations of existing treatment strategies For diseases caused by gene mutations, the conventional strategy has mainly involved delivering the wild-type gene of the mutated gene into cells. The methods for delivering the wild-type gene include 1) using adeno-associated viruses, 2) using lentiviruses, and 3) directly delivering mRNA to cells. However, there are problems such as: 1) adeno-associated viruses can only deliver genes smaller than 4.8kb, but since the EYS gene exceeds 10kb, it must be divided into multiple viruses and delivered separately, resulting in a drastic decrease in delivery efficiency; 2) lentiviruses are free in terms of gene size that can be delivered, but there is a risk of random insertion into the cell genome; and 3) mRNA is unstable, so the gene expression time is limited, which has the disadvantage of requiring continuous administration of the treatment. With the use of CRISPR/Cas gene scissors, which have recently gained prominence, it is possible to employ 1) a therapeutic strategy that aims for a knockout or knockdown effect by "damaging" the target gene through the repeated cutting of the double-stranded DNA of the target gene to induce indels, and 2) a therapeutic strategy that uses donor DNA to insert information from the donor DNA into the gene via the Homology-Directed Repair (HDR) mechanism to achieve a correction effect. However, there are limitations: 1) gene knockout/knockdown strategies are not suitable for treating retinal pigment epitheliosis with EYS gene mutations, and 2) the method of correcting genes using donor DNA has too low an efficiency to achieve a significant therapeutic effect. Prime Editor Prime Editor refers to a protein-nucleic acid complex used in a gene editing technology called prime editing. The prime editor comprises a prime editor protein containing a reverse transcriptase and a CRISPR/Cas protein with mutated double-strand cleavage activity, and a prime editing guide RNA (pegRNA). Prime editing is a technology that corrects a gene as intended by 1) binding the prime editor protein to a target gene by the guidance of the pegRNA, 2) reverse transcribing a correction sequence contained in the 3' end of the pegRNA into cDNA, and 3) inserting the reverse transcribed cDNA instead of the mutated sequence of the target gene. Figure 1 is a schematic diagram showing the structures of pegRNA and extended pegRNA. Figure 2 is a schematic diagram illustrating the relationship between the guide domain, primer binding site (PBS), reverse transcriptase template (RTT), and target nucleic acid of pegRNA. Specifically, it shows the state in which the prime editor recognizes the PAM of the non-target strand of the target nucleic acid and the guide domain binds complementarily to the target sequence of the target strand of the target nucleic acid. Figure 3 is a schematic diagram illustrating the relationship between the guide domain, primer binding site (PBS), reverse transcriptase template (RTT), and target nucleic acid of pegRNA. Specifically, it shows the positional relationship of the PBS and RTT sequences after nicking the target nucleic acid by the prime editor. The best modes for carrying out the invention are disclosed below by way of example. These include some embodiments of the invention disclosed herein, but not all embodiments. The embodiments described in this par