WO2023140409A1 - Composition pour le traitement de la dysplasie de la hanche canine à l'aide d'un éditeur primaire - Google Patents

Composition pour le traitement de la dysplasie de la hanche canine à l'aide d'un éditeur primaire Download PDF

Info

Publication number
WO2023140409A1
WO2023140409A1 PCT/KR2022/001180 KR2022001180W WO2023140409A1 WO 2023140409 A1 WO2023140409 A1 WO 2023140409A1 KR 2022001180 W KR2022001180 W KR 2022001180W WO 2023140409 A1 WO2023140409 A1 WO 2023140409A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant vector
hip dysplasia
gene
dogs
seq
Prior art date
Application number
PCT/KR2022/001180
Other languages
English (en)
Korean (ko)
Inventor
김민규
박강선
김동언
지국빈
이지혜
김은영
박연배
길태영
Original Assignee
주식회사 엠케이바이오텍
충남대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 엠케이바이오텍, 충남대학교산학협력단 filed Critical 주식회사 엠케이바이오텍
Publication of WO2023140409A1 publication Critical patent/WO2023140409A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to a method for treating canine hip dysplasia using prime editing technology, and uses a recombinant vector containing a guide RNA, a primer binding site, and a reverse transcriptase polymerase template gene to correct a gene involved in canine hip dysplasia efficiently and with high accuracy compared to the existing CRISPR/Cas9 system.
  • Hip dysplasia has very similar clinical manifestations and pathogenesis in both humans and dogs.
  • Hip dysplasia is a musculoskeletal disease caused by an incomplete connection between the femoral head and the acetabulum. It is accompanied by severe pain and is a common disease in medium-large dogs such as retrievers. It is known that individuals with hip dysplasia cause osteoarthritis, lameness, reduced mobility, and the like. The causes of hip dysplasia include both genetic and environmental influences. To reduce the prevalence of genetically induced hip dysplasia, breeding strategies can be used according to well-established selection plans, and these strategies have been used to produce healthy individuals in all species. However, research on treating the disease by directly regulating the gene that causes hip dysplasia in dogs has not yet been attempted.
  • Prime editing has been developed and validated in mice and plants. Unlike the method using CRISPR/Cas9-HDR (homology-directed repair), prime editing does not induce double-strand breaks and does not require oligo DNA.
  • Prime editing is designed to generate nickase in double strands using CRISPR/Cas9 (H840A) and edit DNA at the nickase-generating site using reverse transcriptase (Moloney murine leukemia virus: M-MLV).
  • M-MLV reverse transcriptase
  • Prime editing allows low off-target efficiency and precise transitions, insertions, and removals. Because of this low off-target efficiency, prime editing has great potential for correcting pathogenic alleles.
  • Prime editing was originally developed in human cells, but has recently been used to develop plant strains, and mice and fruit flies have been used as human disease models.
  • One aspect provides a recombinant vector comprising a guide RNA represented by SEQ ID NO: 1, a primer binding site represented by SEQ ID NO: 3, and a reverse transcription polymerase template represented by SEQ ID NO: 4.
  • Another aspect provides a composition for treating or preventing canine hip dysplasia comprising the recombinant vector.
  • Another aspect provides a method for treating or preventing hip dysplasia in dogs, comprising introducing the recombinant vector into a subject.
  • One aspect provides a recombinant vector comprising a guide RNA represented by SEQ ID NO: 1, a primer binding site represented by SEQ ID NO: 3, and a reverse transcription polymerase template represented by SEQ ID NO: 4.
  • the recombinant vector is for treating or preventing canine hip dysplasia, and may be for targeting a gene involved in canine hip dysplasia.
  • the recombinant vector may be defined as a gene cassette or may be a genome editing vector.
  • the recombinant vector of the present invention includes not only gRNA, but also a scaffold RT template and a primer binding site, so that each component is operably linked so as to form a set. Therefore, targeting with gRNA can be the same as the previous Cas9, but overall it can perform more stable and accurate gene editing function compared to conventional CRISPR/Cas9.
  • the term "vector” refers to a genetic construct comprising a nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a gene of interest in a suitable host, and the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence regulating the termination of transcription and translation.
  • the term "recombinant vector" when the coding gene of the target polypeptide to be expressed is operably linked, can be used as an expression vector of the target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell, and the recombinant vector can be expressed in the host cell.
  • the host cell may preferably be a eukaryotic cell, and depending on the type of host cell, expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways depending on the purpose.
  • the vector may be an expression vector or a genome editing vector, and may be, for example, a non-viral vector or a viral vector.
  • the non-viral vector is preferably plasmid DNA
  • the viral vector may be a lentivirus, retrovirus, adenovirus, herpes virus or avipox virus vector, but is not limited thereto.
  • the expression vector preferably further includes a gene encoding a selectable marker such as a fluorescent protein to facilitate selection of transformed cells.
  • markers conferring selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents or expression of surface proteins, for example, fluorescent proteins, puromycin, neomycin, hygromycin, histidinol dehydrogenase (hisD) and guanine phosphoribosyltransferase (Gpt).
  • the fluorescent protein may be, for example, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or far-red fluorescent protein, but is not limited thereto.
  • the recombinant vector may further include a PAM gene represented by the polynucleotide sequence of SEQ ID NO: 2.
  • the PAM gene may be specifically located between the guide RNA and the primer binding site.
  • the guide RNA, PAM gene, primer binding site, and reverse transcription polymerase template are operably linked.
  • guide RNA, PAM gene, primer binding site, and reverse transcription polymerase template may be defined as prime editor RNA, and the recombinant vector may further include components of CRISPR/Cas9 and RNA polymerase to serve as genetic scissors.
  • Cas9 protein and RNA polymerase can be commonly known.
  • the virus may be a lentivirus, a retrovirus, an adenovirus, a herpes virus, or an avipox virus, but is not limited thereto.
  • Another aspect provides a composition for treating hip dysplasia comprising the recombinant vector.
  • Another aspect provides a composition for breeding management of dogs comprising the recombinant vector.
  • the breeding management of the dog may mean obtaining a second-generation individual whose hip dysplasia is corrected from an individual who is likely to have hip dysplasia or has hip dysplasia.
  • prevention means reversing, alleviating, or inhibiting, delaying, or preventing the symptoms of hip dysplasia, and the “treatment” may refer to the act of treating in the sense of "treatment method”.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not stimulate organisms and does not inhibit the biological activity and properties of the administered compound.
  • acceptable pharmaceutical carriers are sterile and biocompatible, and saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary.
  • diluents such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • Another aspect provides a method of treating canine hip dysplasia comprising introducing the recombinant vector into the subject.
  • Another aspect provides a dog breeding management method comprising introducing the recombinant vector into an individual.
  • the breeding management method may be for correcting a hip dysplasia inducing gene in a dog determined to have hip dysplasia.
  • the subject may be a dog (Canis lupus familiaris).
  • the dogs may be more specifically Labrador retrievers.
  • the expression vector containing the recombinant vector may be introduced into cells together with delivery reagents including G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions or liposomes, or by conjugating biocompatible polymers such as polyethylene glycol to increase intracellular uptake, using a virus
  • delivery reagents including G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions or liposomes, or by conjugating biocompatible polymers such as polyethylene glycol to increase intracellular uptake, using a virus
  • delivery reagents including G-fectin, Mirus TrasIT-TKO lipophilic reagent, lip
  • the step of injecting the recombinant vector into the organism may be more specifically, a method of introducing the recombinant vector into somatic cells to obtain knocked-in somatic cells, and then obtaining cloned embryos of somatic cells; a method of directly injecting a recombinant vector into a fertilized egg; A method of making fertilized eggs through nuclear transfer in which a recombinant vector is injected into somatic cells of the subject and the nucleus of the somatic cells into which the recombinant vector is injected is injected into an egg, but is not limited thereto.
  • the genetic construct of the present invention canine hip dysplasia can be effectively and successfully treated.
  • the genetic construct of the present invention is applied with prime editing technology, and has the advantage of being simple and highly efficient compared to CRISPR/Cas9-HDR without the need for cell division or introduction of oligo DNA.
  • Figure 1 shows the genetic construct of the virus for the treatment of hip dysplasia.
  • peg RNA contains CRISPR/Cas9 guide RNA, RNA polymerase RT template, and RNA binding site.
  • Figure 2 shows the genetic structure of peg RNA in detail.
  • [AGG] is the PAM sequence
  • the left side of the PAM sequence is the gRNA
  • the right side is the RT template and primer binding site
  • the red base is the mutation site.
  • Figure 3 shows the results of analyzing the nucleotide sequence of cells corrected using the genetic construct of the present invention.
  • Figure 4 shows the results of analyzing the nucleotide sequence of cells corrected using the genetic construct of the present invention.
  • Figure 5 confirms the image and GFP expression of an individual delivered after correcting using the genetic construct of the present invention.
  • FIG. 7 is a sequencing result showing that SNPs causing hip dysplasia were corrected in the genomic DNA of an individual delivered after correction using the genetic construct of the present invention.
  • FIG. 8 is an X-ray image of a hip joint of a cell donor dog and a cloned dog. Specifically, FIG. 8A is the hip joint of an 18-month-old cell donor dog, FIG. 8B is the hip joint of a 38-month-old cell donor dog, FIG. 8C is the hip joint of cloned dog #1 at 11 months, and FIG. 8D is an X-ray image of the hip joint of cloned dog #2 at 11 months.
  • the present invention relates to a recombinant vector comprising a guide RNA represented by SEQ ID NO: 1, a primer binding site represented by SEQ ID NO: 3, and a reverse transcription polymerase template represented by SEQ ID NO: 4.
  • the guide RNA for prime editing and the vector construct containing it were designed as follows.
  • SNPs single nucleotide polymorphisms
  • the SNP selected as a target has the feature of point mutation of T to C at the BISF2S23030416 locus.
  • the pegRNA used for Lyme editing was designed to start with a 13 nt primer binding site and a 14 nt RT template (Bioneer, Inc) (Fig. 2).
  • a lentivirus-derived vector was used (addgene #135955), and it was designed to include CRISPR/Cas9, RNA polymerase, and pegRNA, and include a GFP gene to confirm the insertion with fluorescence (FIG. 1).
  • a virus containing the gene cassette as described above was prepared by a company (Lugen SCI, Inc).
  • Fibroblasts were obtained from the ear of an 18-month-old dog suffering from hip dysplasia to confirm hip dysplasia correction in a cell line. Then, the cells were cultured in a culture medium containing DMEM-Glutamax, 15% FBS and 1% penicillin/streptomycin. After infecting the cultured fibroblasts by treating the virus at 100 MOI in a 12-well plate, the presence or absence of infection was confirmed by GFP and sequencing analysis. As a result, it was confirmed that the prime editing was working as the cell lines treated with the virus showed a heterogeneous type with C and T, whereas the cells without treatment had the C sequence (Figs. 3 and 4).
  • somatic cell nuclear transfer SCNT
  • embryo transfer methods were used.
  • a Labrador retriever was selected as a dog breed, matured oocytes of an individual with hip dysplasia having first polarity were obtained, metaphase chromosomes were removed, and the somatic cells of Example 2 were transferred to the perivitelline space of the extracted oocytes. Then, each donor cell-cytoplasmic bond was fused with two direct current pulses (24-26V for 15 ⁇ sec) in an electro-cell fusion device. The fused SCNT embryos were cultured in 10 ⁇ M calcium ion channel (Sigma) to induce chemical activation, washed and then incubated in synthetic oviduct fluid medium (mSOF) containing 1.9 mM 6-dimethylaminopurine (6-DMAP).
  • mSOF synthetic oviduct fluid medium
  • a cell donor dog (dog name: Miwoo) with hip dysplasia disease was diagnosed with hip dysplasia at 18 months, and an individual that maintained the findings of hip dysplasia even after 38 months was used.
  • X-rays were taken at 11 months of age for two cloned dogs produced through gene editing technology and somatic cell nuclear transfer technology in the same manner as in Test Example 1 above.
  • FIG. 8 The results are shown in FIG. 8 .
  • Figure 8A shows the hip joint of an 18-month-old cell donor dog
  • Figure 8B shows the hip joint of a 38-month-old cell donor dog.
  • Fig. 8C shows the hip joint of cloned dog #1 at 11 months old
  • Fig. 8D shows the hip joint of cloned dog #2 at 11 months old.
  • the present invention relates to a method for treating canine hip dysplasia using prime editing technology, and uses a recombinant vector containing a guide RNA, a primer binding site, and a reverse transcriptase polymerase template gene to correct a gene involved in canine hip dysplasia efficiently and with high accuracy compared to the existing CRISPR/Cas9 system.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Immunology (AREA)
  • Rheumatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de traitement de la dysplasie de la hanche canine utilisant une technique d'édition primaire. En utilisant le vecteur recombiné de la présente invention, les gènes associés à la dysplasie de la hanche canine peuvent être comparés à un système CRISPR/Cas9 existant pour effectuer des corrections très précises et efficaces, et la présente invention peut donc être utilisée pour traiter la dysplasie de la hanche canine ou pour gérer l'élevage des canidés.
PCT/KR2022/001180 2022-01-20 2022-01-24 Composition pour le traitement de la dysplasie de la hanche canine à l'aide d'un éditeur primaire WO2023140409A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2022-0008306 2022-01-20
KR20220008306 2022-01-20

Publications (1)

Publication Number Publication Date
WO2023140409A1 true WO2023140409A1 (fr) 2023-07-27

Family

ID=87348439

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/001180 WO2023140409A1 (fr) 2022-01-20 2022-01-24 Composition pour le traitement de la dysplasie de la hanche canine à l'aide d'un éditeur primaire

Country Status (1)

Country Link
WO (1) WO2023140409A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120043793A (ko) * 2010-10-27 2012-05-07 대한민국(농촌진흥청장) 개 고관절 이형성증 조기 진단용 ｓｎｐ 및 이의 용도
KR102124770B1 (ko) * 2018-12-12 2020-06-19 대한민국 개의 고관절탈구 조기 예측 또는 진단용 조성물

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120043793A (ko) * 2010-10-27 2012-05-07 대한민국(농촌진흥청장) 개 고관절 이형성증 조기 진단용 ｓｎｐ 및 이의 용도
KR102124770B1 (ko) * 2018-12-12 2020-06-19 대한민국 개의 고관절탈구 조기 예측 또는 진단용 조성물

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHOI BONG-HWAN, SEULGI KWON , JAE-GYU YOO , SEUNG HWAN LEE: "Genome-wide Association Study using the Canine SNP20 Beadchip for Canine Hip Dysplasia in Labrador Retriever", JOURNAL OF ANIMAL BREEDING AND GENOMICS, vol. 3, no. 1, 1 March 2019 (2019-03-01), pages 7 - 15, XP093079326, ISSN: 1226-5543, DOI: 10.12972/jabng.20190002 *
KIM, LEE-KYUNG: "Development of single nucleotide polymorphism (SNP) markers for the diagnosis of hip dysplasia in Labrador retriever", THESIS, 1 August 2013 (2013-08-01), KR, pages 1 - 33, XP009547755 *
MIKKOLA LEA, HOLOPAINEN SAILA, PESSA-MORIKAWA TIINA, LAPPALAINEN ANU K., HYTÖNEN MARJO K., LOHI HANNES, IIVANAINEN ANTTI: "Genetic dissection of canine hip dysplasia phenotypes and osteoarthritis reveals three novel loci", BMC GENOMICS, vol. 20, no. 1, 1 December 2019 (2019-12-01), pages 1 - 13, XP093079329, DOI: 10.1186/s12864-019-6422-6 *
YAN JUN; CIRINCIONE ANN; ADAMSON BRITT: "Prime Editing: Precision Genome Editing by Reverse Transcription", MOLECULAR CELL, vol. 77, no. 2, 16 January 2020 (2020-01-16), AMSTERDAM, NL, pages 210 - 212, XP086007503, ISSN: 1097-2765, DOI: 10.1016/j.molcel.2019.12.016 *

Similar Documents

Publication Publication Date Title
CN106535630B (zh) 多重基因编辑
WO2018177351A1 (fr) Procédé de préparation d'un animal à invalidation génique non chimérique basé sur la technologie crispr/cas9
WO2018110805A1 (fr) Modèle animal de diabète avec inactivation de gène ins ou de complications diabétiques et procédé de fabrication correspondant
EP0506945B1 (fr) Procede de transfert de genes au moyen de retrotransposons
US6027722A (en) Vectors for gene transfer
US8921332B2 (en) Chromosomal modification involving the induction of double-stranded DNA cleavage and homologous recombination at the cleavage site
CN111278972B (zh) 用于细胞遗传修饰的非整合型dna载体
US20180051298A1 (en) Efficient non-meiotic allele introgression
US7960525B2 (en) Gene repair involving in vivo excision of targeting DNA
AU699706B2 (en) Improved vectors for gene therapy
CN106459951A (zh) 驯养的大型动物的受精卵中的靶向基因组编辑
WO2018030630A1 (fr) Vecteur non viral en minicercle portant un gène sox et procédé pour sa construction
WO2023140409A1 (fr) Composition pour le traitement de la dysplasie de la hanche canine à l'aide d'un éditeur primaire
Allen et al. Using phiC31 integrase to make transgenic Xenopus laevis embryos
KR102472344B1 (ko) 프라임 편집을 이용한 개의 고관절 이형성증 치료용 조성물
EP3954765A1 (fr) Chromosome recombiné artificiel et son utilisation
US6936465B1 (en) Plasmid vector comprising a retroviral integrase gene and an integrase recognition region
KR101105248B1 (ko) 형질전환된 복제개의 생산방법
CN113444722B (zh) 单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用
WO2023085505A1 (fr) Vecteur induisant la maladie de parkinson et procédé de construction d'un modèle canin l'utilisant
WO2022114815A1 (fr) Composition pour l'édition d'amorces comprenant un vecteur de virus adéno-associé de trans-épissage
HUT77343A (hu) Transzgenikus szervezetek és alkalmazásuk
US20030110524A1 (en) Transgenic organisms and their uses
CN115838719B (zh) 特异性促进腺嘌呤碱基编辑器活性的化合物、化学调控方法及其应用
US11530424B1 (en) Efficient CRISPR/HDR-mediated knock-in system and method of use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22922271

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE