KR102108107B1 - Producing method of dominant intermediate Charcot-Marie-Tooth disease type C animal model and uses thereof - Google Patents
Producing method of dominant intermediate Charcot-Marie-Tooth disease type C animal model and uses thereof Download PDFInfo
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- KR102108107B1 KR102108107B1 KR1020170078755A KR20170078755A KR102108107B1 KR 102108107 B1 KR102108107 B1 KR 102108107B1 KR 1020170078755 A KR1020170078755 A KR 1020170078755A KR 20170078755 A KR20170078755 A KR 20170078755A KR 102108107 B1 KR102108107 B1 KR 102108107B1
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Abstract
본 발명은 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase, YARS)를 포함하는 벡터를 이용한 DI-CMTC 형 동물 모델 제조 방법에 관한 것으로, 본 발명에서 제공하는 DI-CMTC 동물 모델 제조 방법은 샤르코-마리에-투스 질환과 관련한 말초신경병증에 대한 분자해부학적 원인을 구명하는 학술적 효과를 가질 수 있으며, 샤르코-마리에-투스 질환을 치료하는 치료기술 분야의 발전에 기여할 수 있을 것이다.The present invention relates to a method for preparing a DI-CMTC type animal model using a vector containing a tyrosyl-tRNA synthetase (YARS), and the method for producing a DI-CMTC animal model provided by the present invention is Sharco- It may have an academic effect to investigate the molecular anatomical causes for peripheral neuropathy associated with Marie-Tooth disease, and may contribute to the development of the field of treatment technology for treating Sharko-Marie-Tooth disease.
Description
본 발명은 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase, YARS)를 포함하는 벡터를 이용한 DI-CMTC 동물 모델 제조 방법에 관한 것이다.The present invention relates to a method for preparing a DI-CMTC animal model using a vector containing a tyrosyl-tRNA synthetase (YARS).
유전성 말초 신경병은 크게 유전운동감각신경병(hereditary motor and sensory neuropathy; HMSN), 유전운동신경병(herediatary motor neuropathy; HMN) 및 유전감각신경병(herediatary sensory neuropathy; HSN)의 3가지로 구분된다. 이 중 유전운동감각신경병이 대부분을 차지하며, 이는 샤르코-마리에-투스 질환(Charcot-Marie-Tooth disease; CMT)으로 더 잘 알려져 있다. 샤르코-마리에-투스 질환은 신경전도검사상 운동신경과 감각신경에 이상이 있는 모든 유전질환을 총칭하며, 희귀질환 중 가장 높은 발병 빈도(2,500명 중 1명)를 갖는다. 과거에는 하지 원위부의 근육위축으로 인하여 샴페인 병을 거꾸로 세운 듯한 다리 모양을 하는 질환으로 비교적 단순하게 인식되어 왔으나, 현재는 단일 질환 보다는 하나의 질환군(syndrome)으로 인식되고 있다. 최근에는 CMT의 새로운 발병 기전들이 많이 밝혀져서 병태 생리학적 연구뿐만 아니라 복잡한 임상형과 유전형의 분류에 큰 도움을 주었다 (Kennerson, M. L., et al., The American Journal of Human Genetics 69.4 (2001): 883-888.).Hereditary peripheral neuropathy is largely divided into three categories: hereditary motor and sensory neuropathy (HMSN), herediatary motor neuropathy (HMN), and herediatary sensory neuropathy (HSN). Of these, genetic motor sensation neuropathy accounts for the majority, which is better known as Charcot-Marie-Tooth disease (CMT). Sharko-Marie-Tooth disease is a collective term for all genetic diseases that have abnormalities in motor nerves and sensory nerves on nerve conduction examination, and has the highest incidence of rare diseases (1 in 2,500 patients). In the past, due to muscle atrophy in the distal lower extremity, it has been recognized as relatively simple as a disease in the shape of a leg with a champagne bottle upside down, but it is now recognized as a syndrome rather than a single disease. In recent years, many new mechanisms of onset of CMT have been uncovered, helping to categorize complex clinical and genotypes as well as pathophysiological studies (Kennerson, ML, et al., The American Journal of Human Genetics 69.4 (2001): 883- 888.).
샤르코-마리에-투스 질환 중 하나인 우성 중간형 샤르코-마리에-투스 신경병증 C형(dominant intermediate CMT neuropathy type C, DI-CMTC)은 CMT1형과 2형의 중간형으로 신경병증의 느린 진행 속도, 중간 수준의 신경 전도 속도를 나타내며 탈수초성 특징과 축삭형 신경병증의 특징을 모두 가진다.The dominant intermediate CMT neuropathy type C (DI-CMTC), one of the Charco-Marie-Tooth diseases, is a medium-to-lower progression of neuropathy with type CMT1 and type II. It shows the speed and the level of nerve conduction at a moderate level, and has both demyelinating and axonal neuropathy.
현재 DI-CMTC의 동물 모델은 유전자 변형 생쥐를 사용하고 있으나, 이는 수정란에 유전자를 도입하여 생쥐를 수득하는 방식으로 시간과 비용이 많이 들 뿐만 아니라 도입된 유전자가 다양한 유전적/환경적 요인에 의해 표현형이 나타나지 않는 경우도 발생하기 때문에 DI-CMTC 동물 모델을 간편하게 구축하기 위한 기술이 필요한 실정이다.Currently, the animal model of DI-CMTC uses genetically modified mice, but this is a method of obtaining mice by introducing the gene into the fertilized egg, which is not only time and costly, but also the introduced gene is caused by various genetic / environmental factors. Since the phenotype does not appear, there is a need for a technique to easily construct a DI-CMTC animal model.
이에 본 발명자들은 샤르코-마리에-투스 질환에 대한 맞춤형 의료기술을 개발하기 위해 노력한 결과, hYARS 단백질의 196번째 글루탐산(Glutamic acid)이 라이신(Lysine)으로 치환된 변이체 hYARS(E196K) 단백질을 발현하는 벡터가 주입된 생쥐의 좌골신경 축삭의 퇴행을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors tried to develop a customized medical technology for Sharko-Marie-Tus disease, and as a result, the 196 th glutamic acid of the hYARS protein expresses a variant hYARS (E196K) protein substituted with Lysine. The present invention was completed by confirming the degeneration of the sciatic nerve axon of the mouse injected with the vector.
본 발명의 하나의 목적은 1) 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase, YARS)의 아미노산 서열에서 N-말단으로부터 196번째 아미노산인 글루탐산(Glutamic acid)이 라이신(Lysine)으로 변이된 YARS 변이체를 발현하는 유전자를 포함하는 벡터를 제조하는 단계; 및 2) 상기 벡터를 동물에 주입하는 단계를 포함하는 우성 중간형 샤르코-마리에-투스 신경병증 C형(dominant intermediate CMT neuropathy type C, DI-CMTC) 동물 모델의 제조방법을 제공하는 것이다.One object of the present invention is 1) a tyrosyl-tRNA synthetase (tyrosyl-tRNA synthetase, YARS) amino acid sequence from the N-terminus 196 amino acid glutamic acid (Glutamic acid) lysine (Lysine) mutant YARS variant Preparing a vector comprising a gene expressing; And 2) injecting the vector into an animal to provide a method for producing a dominant intermediate CMT neuropathy type C (DI-CMTC) animal model.
본 발명의 다른 목적은 E196K 변이 YARS 유전자 또는 이를 함유하는 벡터를 포함하는 DI-CMTC 동물 모델 제조용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for preparing a DI-CMTC animal model comprising the E196K mutant YARS gene or a vector containing the same.
본 발명의 또 다른 목적은 상기 제조방법으로 제조된 DI-CMTC 동물 모델을 제공하는 것이다.Another object of the present invention is to provide a DI-CMTC animal model prepared by the above manufacturing method.
본 발명의 또 다른 목적은 1) DI-CMTC 동물 모델에 후보물질을 투여하는 단계; 2) 상기 1)단계의 동물 모델의 축삭과 후보물질을 투여하지 않은 대조군 동물 모델의 축삭을 비교하여 축삭 퇴행이 개선된 경우, 후보물질을 DI-CMTC 예방 치료제로 선별하는 단계를 포함하는 DI-CMTC 예방 또는 치료제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is 1) administering a candidate substance to the DI-CMTC animal model; 2) When the axon regression is improved by comparing the axon of the animal model of step 1) with the axon of the control animal model without administration of the candidate substance, DI-comprising the step of selecting the candidate substance as a preventive treatment for DI-CMTC It is to provide a method for screening for CMTC prevention or treatment.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 1) 티로실-tRNA 합성효소(tyrosyl-tRNA synthetase, YARS)의 아미노산 서열에서 N-말단으로부터 196번째 아미노산인 글루탐산(Glutamic acid)이 라이신(Lysine)으로 변이된 YARS 변이체를 발현하는 유전자를 포함하는 벡터를 제조하는 단계; 및 2) 상기 벡터를 동물에 주입하는 단계를 포함하는 우성 중간형 샤르코-마리에-투스 신경병증 C형(dominant intermediate CMT neuropathy type C, DI-CMTC) 동물 모델의 제조방법을 제공한다.In one aspect for achieving the above object, the present invention is 1) tyrosyl-tRNA synthetase (tyrosyl-tRNA synthetase, YARS) in the amino acid sequence of the 196th amino acid from the N- terminal glutamic acid (Glutamic acid) lysine (Lysine) to prepare a vector containing a gene expressing the mutated YARS variant; And 2) injecting the vector into an animal. A method of manufacturing a dominant intermediate CMT neuropathy type C (DI-CMTC) animal model is provided.
이하, 본 발명에서의 DI-CMTC형 동물 모델 제조방법을 구체적으로 설명한다.Hereinafter, a method of manufacturing a DI-CMTC type animal model in the present invention will be described in detail.
본 발명에서 사용되는 용어, “티로실-tRNA 합성효소(tyrosyl-tRNA synthetase, YARS)”는 tRNA의 아미노아실화를 촉매하는 효소로서 단백질 생합성에 필수적인 역할을 하는 효소이다. The term used in the present invention, “tyrosyl-tRNA synthetase (YARS)” is an enzyme that catalyzes aminoacylation of tRNA and plays an essential role in protein biosynthesis.
상기 YARS 단백질을 코딩하는 유전자의 구체적인 염기서열 및 단백질 정보는 NCBI에 공지되어 있다.The specific nucleotide sequence and protein information of the gene encoding the YARS protein is known from NCBI.
본 발명에서 YARS는 당해 활성을 가지는 한 특별히 제한되지 않으나, N-말단으로부터 196번째 아미노산이 글루탐산인 아미노산 서열을 가질 수 있다. 또한, 상기 YARS는 N-말단으로부터 196번째 아미노산이 글루탐산인 아미노산 서열을 가지는 한, 아미노산 서열에 한정되지 않으나, 서열번호 1의 아미노산 서열 및 이와 80%이상, 구체적으로는 90% 이상, 더욱 구체적으로는 95% 이상, 보다 더욱 구체적으로는 97% 이상의 상동성을 갖는 아미노산 서열을 가질 수 있다. 이러한 상동성을 갖는 서열로서 실질적으로 서열번호 1로 기재된 아미노산 서열을 가지는 단백질과 동일하거나 상응하는 생물학적 활성을 갖는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환, 또는 부가된 아미노산 서열을 갖는 경우도 본 발명의 범위에 포함됨은 자명하다. In the present invention, YARS is not particularly limited as long as it has the activity, but may have an amino acid sequence in which the 196th amino acid from the N-terminus is glutamic acid. In addition, the YARS is not limited to the amino acid sequence, as long as the 196th amino acid from the N-terminus has an amino acid sequence of glutamic acid, but the amino acid sequence of SEQ ID NO: 1 and 80% or more thereof, specifically 90% or more, more specifically May have an amino acid sequence having at least 95% homology and more specifically at least 97% homology. If the sequence having the homology is an amino acid sequence having substantially the same or corresponding biological activity as the protein having the amino acid sequence set forth in SEQ ID NO: 1, some sequences may have deletion, modification, substitution, or added amino acid sequences. It is obvious that it is included in the scope of the present invention.
본원에서 “아미노산 서열을 포함하는 단백질”은 “아미노산 서열을 가지는 단백질”, 또는 “아미노산 서열로 구성되는 단백질”이라는 표현과 혼용되어 사용될 수 있다.As used herein, “a protein comprising an amino acid sequence” may be used interchangeably with the expression “a protein having an amino acid sequence” or “a protein consisting of an amino acid sequence”.
본 발명에서 YARS를 코딩하는 폴리뉴클레오티드 서열이라면 본 발명의 범주에 포함될 수 있다. 예를 들어, 상기 서열번호 1의 아미노산 서열을 코딩하는 폴리뉴클레오티드 서열 및 이와 80%이상, 구체적으로는 90%이상, 더욱 구체적으로는 95%이상, 보다 더욱 구체적으로는 97%이상의 상동성을 갖는 폴리뉴클레오티드 서열일 수 있다. 또한, 상기 YARS를 코딩하는 폴리뉴클레오티드 서열은 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 YARS를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 YARS의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있다.Any polynucleotide sequence encoding YARS in the present invention may be included in the scope of the present invention. For example, the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 and 80% or more thereof, specifically 90% or more, more specifically 95% or more, and more specifically 97% or more homology It may be a polynucleotide sequence. In addition, the polynucleotide sequence encoding the YARS does not change the amino acid sequence of the YARS expressed from the coding region due to the degeneracy of the codon or considering the preferred codon in the organism to express the YARS. Various modifications can be made to the coding region within the range.
또한, 공지의 유전자 서열로부터 조제될 수 있는 프로브, 예를 들면, 상기 폴리뉴클레오티드 서열의 전체 또는 일부에 대한 상보 서열과 엄격한 조건 하에 하이드리드화하여, 상기 3-디하이드로퀴네이트 디하이드라타제, 2-디하이드로-3-데옥시포스포헵토네이트 알돌라제, 포스포에놀피루베이트 신테타제, 트랜스케톨라제 및 3-디하이드로퀴네이트 신타제 효소 단백질의 활성을 가지는 단백질을 암호화하는 서열이라면 제한없이 포함될 수 있다.In addition, probes that can be prepared from known gene sequences, such as the complementary sequence for all or part of the polynucleotide sequence, and hybridized under stringent conditions, the 3-dihydroquinate dehydratase, If it is a sequence encoding a protein having activity of 2-dihydro-3-deoxyphosphoheptonate aldolase, phosphoenolpyruvate synthetase, transketolase and 3-dihydroquinate synthase enzyme protein It can be included without limitation.
본원에 사용된 용어 "상동성"은 주어진 아미노산 서열 또는 뉴클레오티드 서열과 일치하는 정도를 의미하며 백분율로 표시될 수 있다. 본 명세서에서, 주어진 아미노산 서열 또는 뉴클레오티드 서열과 동일하거나 유사한 활성을 가지는 그의 상동성 서열이 "% 상동성"으로 표시된다. 예를 들면, 점수(score), 동일성(identity) 및 유사도(similarity) 등의 매개 변수(parameter)들을 계산하는 표준 소프트웨어, 구체적으로 BLAST 2.0을 이용하거나, 정의된 엄격한 조건(stringent condition)하에서 썼던 혼성화 실험에 의해 서열을 비교함으로써 확인할 수 있으며, 정의되는 적절한 혼성화 조건은 해당 기술 범위 내이고, 당업자에게 잘 알려진 방법(예컨대, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York)으로 결정될 수 있다. 상기에서 용어 “엄격한 조건”이란 폴리뉴클레오티드 간의 특이적 혼성화를 가능하게 하는 조건을 의미한다. 예를 들어, 이러한 조건은 문헌 (예컨대, J. Sambrook et al., 상동)에 구체적으로 기재되어 있다.As used herein, the term “homology” refers to the degree to which a given amino acid sequence or nucleotide sequence is consistent and may be expressed as a percentage. In the present specification, a homology sequence thereof having the same or similar activity to a given amino acid sequence or nucleotide sequence is indicated as "% homology". For example, standard software that calculates parameters such as score, identity and similarity, specifically hybridization using BLAST 2.0 or under defined stringent conditions Appropriate hybridization conditions, which can be confirmed by comparing sequences by experiment, are defined and are well within the scope of the art, and are well known to those skilled in the art (e.g. J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring) Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term "strict conditions" in the above refers to conditions that enable specific hybridization between polynucleotides. For example, these conditions are specifically described in J. Sambrook et al., Supra.
본 발명의 YARS는 N-말단으로부터 196번째 아미노산이 글루탐산인 아미노산 서열을 가지는 한, 유래에 한정되지 않으나, 구체적으로 인간, 원숭이, 돼지, 소, 말, 양, 개, 토끼 또는 생쥐 등의 유래일 수 있으며, 더욱 구체적으로 인간 유래일 수 있다.The YARS of the present invention is not limited to origin, as long as the 196th amino acid from the N-terminus has an amino acid sequence of glutamic acid, but specifically, the date of origin of human, monkey, pig, cow, horse, sheep, dog, rabbit or mouse It may be, and more specifically, may be of human origin.
본 발명의 YARS 변이체는 YARS를 구성하는 아미노산 서열에서 N-말단으로부터 196번째 아미노산이 글루탐산에서 라이신으로 변이된 YARS를 의미한다.The YARS variant of the present invention refers to YARS in which the amino acid sequence constituting YARS is changed from glutamic acid to lysine by the 196th amino acid from the N-terminus.
본 발명에서 사용되는 용어, “벡터(vector)”는 DNA 재조합 실험에 있어서 목적하는 DNA 단편을 숙주균 등에 도입시켜 주고 증식할 수 있는 DNA로서 클로닝 운반체(cloning vehicle)라고도 한다. 벡터는 다른 DNA 단편이 결합하여 결합된 단편의 복제를 가져올 수 있는 복제단위(replicon)일 수 있다. "복제단위"란 생체 내에서 DNA 복제의 자가 유닛으로서 기능하는, 즉, 스스로의 조절에 의해 복제가능한, 임의의 유전적 단위(예를 들면, 플라스미드, 파지, 코스미드, 염색체, 바이러스)를 말한다. 또한 목적하는 단편의 발현 조절을 위한 프로모터를 벡터에 함께 포함시킬 수 있다. 본 발명의 벡터는 YARS 변이체를 코딩하는 유전자를 가지고 있어, 상기 벡터가 목적 동물에 주입되었을 경우 YARS 변이체가 발현되어, DI-CMTC 동물 모델이 제조될 수 있다.The term “vector” used in the present invention is a DNA capable of introducing and propagating a desired DNA fragment in a host DNA bacterium in a DNA recombination experiment, and is also referred to as a cloning vehicle. The vector may be a replication unit (replicon) capable of binding to other DNA fragments to obtain replication of the combined fragments. “Replication unit” refers to any genetic unit (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autologous unit of DNA replication in vivo, ie, is capable of replication by self-regulation. . In addition, a promoter for regulating the expression of a desired fragment may be included in a vector. Since the vector of the present invention has a gene encoding a YARS variant, when the vector is injected into a target animal, a YARS variant is expressed, and a DI-CMTC animal model can be prepared.
상기 벡터에 포함되는 프로모터는 신경 특이적으로 발현하기를 원하는 목적 유전자의 발현을 크게 증가시킬 수 있어 본 발명의 YARS 유전자를 신경 특이적으로 발현시킬 수 있다.The promoter included in the vector can significantly increase the expression of a desired gene desired to be expressed specifically for the nerve, and thus the YARS gene of the present invention can be expressed specifically for the nerve.
구체적으로, 본 발명의 벡터에 포함되는 프로모터는 신경 특이적 발현의 조절요소이며 콜린 특이적 인핸서와 뉴런 제한적 사일렌서 요소를 포함하는 콜린 아세틸트랜스퍼레이즈(Choline acetyltransferase, ChAT) 프로모터일 수 있다. Specifically, the promoter included in the vector of the present invention may be a choline acetyltransferase (ChAT) promoter, which is a modulator of neuro-specific expression and includes choline specific enhancer and neuron limited silencer elements.
상기 벡터는 예를 들어, 파지 벡터 또는 코스미드 벡터로서 pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, 및 Charon21A 등을 사용할 수 있으며, 플라스미드 벡터로서 pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등을 사용할 수 있다. 구체적으로는 pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC 벡터 등을 사용할 수 있으나, 이에 제한되지 않는다.The vector may be, for example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., as a phage vector or a cosmid vector, and a pBR system or a pUC system as a plasmid vector. , pBluescriptII, pGEM, pTZ, pCL and pET. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used, but are not limited thereto.
본 발명의 벡터는 아데노바이러스, AAV바이러스 및 렌티바이러스로 이루어진 군에서 선택되는 어느 하나의 유전인자일 수 있으나, 이에 제한되지 않는다. The vector of the present invention may be any genetic factor selected from the group consisting of adenovirus, AAV virus and lentivirus, but is not limited thereto.
상기 동물 모델 제조방법 2)단계는 상기 벡터를 동물의 신경에 주입하는 것일 수 있다. 상기 신경은 유전운동감각신경병과 관련된 신경세포라면 모두 제한 없이 포함될 수 있으나, 구체적으로 슈반 세포(schwann cell), 말초 신경 축삭돌기(peripheral nerve axons), 감각/운동 신경세포체(sensory/motor nuron cell body), 척수 등/배쪽 뿔(spinal dorsal/ventral horn), 신경근접합부(neuromuscular junction), 좌골신경(sciatic nerve)일 수 있으며 더욱 구체적으로 좌골신경일 수 있다.The method of manufacturing the animal model 2) may be injecting the vector into the animal's nerve. The nerve may be included without limitation as long as all neurons associated with hereditary motor sensory neuropathy are specifically, Schwann cells, peripheral nerve axons, and sensory / motor nuron cell bodies. ), Spinal dorsal / ventral horn, neuromuscular junction, sciatic nerve, and more specifically sciatic nerve.
본 발명의 일 실시예에서는 전사영역 E1 및 E3 가 이중 결실된 초나선 아데노바이러스 골격 벡터에 신경 특이적 발현을 위한 ChAT 프로모터를 삽입하고 Flag가 표지된 각각의 인간 YARS유전자(WT, E196K, G41R)를 삽입하여 상동 재조합체를 제작하였다. 또한, 상기 벡터 및 벡터에 도입된 유전자의 발현을 확인하기 위해 웨스턴 블롯을 이용하여 그 발현을 확인한 결과, 각 hYARS 유전자(WT, E196K, G41R)가 도입된 벡터 모두 표지된 Flag 단백질이 검출되어 벡터가 정상적으로 기능하는 것을 확인하였다. In an embodiment of the present invention, the human YARS gene (WT, E196K, G41R), each of which is labeled with a ChAT promoter for neurospecific expression, is inserted into a super-helical adenovirus skeleton vector in which the transcription regions E1 and E3 are double deleted. Was inserted to prepare a homologous recombinant. In addition, as a result of confirming the expression using the Western blot to confirm the expression of the vector and the gene introduced into the vector, all the vectors introduced with each hYARS gene (WT, E196K, G41R) are labeled Flag proteins are detected and vector Was confirmed to function normally.
본 발명에서 사용되는 용어, “우성 중간형 샤르코-마리에-투스 신경병증 C형(dominant intermediate CMT neuropathy type C, DI-CMTC)”은 샤르코-마리에-투스 질환 중 하나인 CMT1형과 2형의 중간형으로 신경병증의 느린 진행 속도, 중간 수준의 신경 전도 속도를 나타내며 탈수초성 특징과 축삭형 신경병증의 특징을 모두 가진다. The term used in the present invention, “dominant intermediate CMT neuropathy type C (DI-CMTC)” is a type of CMT1 and type 2, one of the Sharco-Marie-Tooth diseases. It is an intermediate type of neuropathy that exhibits a slow progression rate and a moderate level of nerve conduction, and has both demyelinating and axonal neuropathy characteristics.
본 발명에서 사용되는 용어, “동물 모델”은 사람에서와 같이 특유한 질환의 병증을 나타내는 것이 가능한 질환 모델 동물을 통해 질환을 나타낸 질환 모델을 의미하며, 대표적인 예로 인슐린의존성 당뇨병의 모델로서 BB쥐나 자연발증 당뇨생쥐(비비만 당뇨병생쥐, NOD mouse)가 있다.As used in the present invention, the term “animal model” refers to a disease model capable of representing a pathology of a specific disease as in humans, and refers to a disease model representing a disease through animals. As a representative example, insulin-dependent diabetes mellitus is a BB mouse or spontaneous onset. There are diabetic mice (obese obese diabetic mice, NOD mice).
본 발명의 DI-CMTC 동물 모델은 도입된 본 발명의 벡터로 인해 YARS 변이체가 발현된 동물로서, 축삭의 퇴행 및 말초신경의 기능 저해를 특징으로 하는 동물일 수 있다.The DI-CMTC animal model of the present invention is an animal expressing a YARS variant due to the introduced vector of the present invention, and may be an animal characterized by axonal degeneration and peripheral nerve function inhibition.
본 발명의 일 실시예에서는 E196K 변이 YARS의 신경세포 특이적 발현을 확인하기 위해 면역형광염색법을 통해 신경조직(척수, 배근신경절, 좌골신경)에서 벡터 및 각각의 hYARS 유전자의 발현을 GFP와 Flag 표지로 측정한 결과, E196K군이 척수 및 배근신경절에서는 정상 발현이 되나, 좌골신경에서 발현이 되지 않는 것을 확인함으로서 E196K 변이 hYARS 유전자를 갖는 아데노바이러스 벡터를 주입한 생쥐에서 축삭 퇴행이 일어난 것을 확인하였다. 다만, 동일한 조건에서 실험한 G41R군의 경우, Flag 발현을 측정할 수 없어 축삭의 퇴행에 대해서는 확인할 수 없었다.In an embodiment of the present invention, to express neuron-specific expression of E196K mutant YARS, the expression of vectors and respective hYARS genes in nerve tissues (spinal cord, dorsal ganglion, sciatic nerve) through immunofluorescence staining is labeled with GFP and Flag. As a result, it was confirmed that axon regression occurred in mice injected with an adenovirus vector carrying the E196K mutation hYARS gene by confirming that the E196K group has normal expression in the spinal cord and dorsal ganglion, but not in the sciatic nerve. However, in the case of the G41R group tested under the same conditions, Flag expression could not be measured, so it was not possible to confirm the degeneration of the axon.
본 발명의 DI-CMTC 동물 모델은 신경병증의 일환으로 축삭이 퇴행될 수 있으며 척수에 비해 말초신경의 기능이 저해될 수 있다. 따라서, 상기 동물 모델은 좌골신경에서 변이체의 발현이 척수, 배근신경절의 발현보다 낮을 수 있다.In the DI-CMTC animal model of the present invention, axons may be degenerated as part of neuropathy and peripheral nerve function may be inhibited compared to the spinal cord. Therefore, in the animal model, the expression of the variant in the sciatic nerve may be lower than that of the spinal cord and dorsal ganglion.
본 발명에서 DI-CMTC 동물 모델은 YARS 변이에 의한 DI-CMTC 동물 모델일 수 있다. YARS 변이에 의한 동물 모델 내의 유전자 프로파일은 DI-CMTC 원인 유전자 네트워크 및 발병(pathogenesis) 연구에 사용될 수 있다.In the present invention, the DI-CMTC animal model may be a DI-CMTC animal model due to YARS mutation. Gene profiles in animal models due to YARS mutations can be used in studies of DI-CMTC causal gene networks and pathogenesis.
상기 동물 모델은 DI-CMTC를 유도하여, DI-CMTC의 치료 후보 물질 또는 치료제의 효과를 판정할 수 있는, 인간을 제외한 동물을 포함할 수 있다.The animal model may include animals other than humans capable of inducing DI-CMTC to determine the effect of a candidate therapeutic agent or therapeutic agent for DI-CMTC.
상기 동물은 이에 제한되지는 않으나, 예컨대 원숭이, 개, 고양이, 토끼, 모르모트, 생쥐, 소, 양, 돼지, 염소 등을 포함한다. 본 발명의 일 실시예에서는 생쥐를 사용하였으나, 이에 제한되지 않는다.The animals include, but are not limited to, monkeys, dogs, cats, rabbits, mormots, mice, cows, sheep, pigs, goats, and the like. In one embodiment of the present invention, mice were used, but are not limited thereto.
본 발명의 일 실시예에서는 상기 벡터를 유리모세관 피펫을 이용하여 생쥐의 좌골신경에 미세주입하는 방법으로 DI-CMTC 질환을 유발하여 동물 모델을 제조하였다.In one embodiment of the present invention, an animal model was prepared by inducing DI-CMTC disease by microinjecting the vector into the sciatic nerve of a mouse using a glass capillary pipette.
본 발명에서 “주입”은 본 발명의 벡터를 동물에 도입시킬 수 있는 한 제한되지 않으며, 당업계에 통상적으로 사용되는 방법을 이용할 수 있다.In the present invention, "injection" is not limited as long as the vectors of the present invention can be introduced into animals, and methods commonly used in the art can be used.
다른 양태로서, 본 발명은 E196K 변이 YARS 유전자 또는 이를 함유하는 벡터를 포함하는 DI-CMTC 동물 모델 제조용 조성물을 제공한다.In another aspect, the present invention provides a composition for preparing a DI-CMTC animal model comprising an E196K variant YARS gene or a vector containing the same.
상기 “YARS”, “DI-CMTC”, “동물 모델”은 전술한 바와 같다.The “YARS”, “DI-CMTC”, and “animal model” are as described above.
또 다른 양태로서, 본 발명은 상기 제조방법으로 제조된 DI-CMTC 동물 모델을 제공한다. As another aspect, the present invention provides a DI-CMTC animal model prepared by the above manufacturing method.
상기 “DI-CMTC”, “동물 모델”은 전술한 바와 같다.The “DI-CMTC” and “animal model” are as described above.
또 다른 양태로서, 본 발명은 1) DI-CMTC 동물 모델에 후보물질을 투여하는 단계; 2) 상기 1)단계의 동물 모델의 축삭과 후보물질을 투여하지 않은 대조군 동물 모델의 축삭을 비교하여 축삭 퇴행이 개선된 경우, 후보물질을 DI-CMTC 예방 치료제로 선별하는 단계를 포함하는 DI-CMTC 예방 또는 치료제의 스크리닝 방법을 제공한다.In another aspect, the present invention provides a method of administering a candidate substance to a DI-CMTC animal model; 2) When the axon regression is improved by comparing the axon of the animal model of step 1) with the axon of the control animal model without administration of the candidate substance, DI-comprising the step of selecting the candidate substance as a preventive treatment for DI-CMTC Provides a screening method for CMTC prevention or treatment.
상기 “DI-CMTC”, “동물 모델”은 전술한 바와 같다.The “DI-CMTC” and “animal model” are as described above.
본 발명에서 사용되는 용어, “후보물질(test agent)”은 임의의 물질(substance), 분자(molecule), 원소(element), 화합물(compound), 실재물(entity) 또는 이들의 조합을 포함한다. 예를 들어, 이들로 한정되지는 않으나, 단백질, 폴리펩티드, 소 유기분자(small organic molecule), 다당류(polysaccharide), 폴리뉴클레오티드 등을 포함한다. 또한, 천연 산물(natural product), 합성 화합물 또는 2개 이상의 물질의 조합일 수도 있다. As used herein, the term “test agent” includes any substance, molecule, element, compound, entity, or combinations thereof. For example, but not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound, or a combination of two or more substances.
본 발명의 후보물질은 이에 제한되지는 않으나, 예컨대 말초신경의 수초 이상 또는 축색의 손상을 완화시키거나, 예방, 지연시킬 수 있는 모든 물질을 포함한다.Candidate materials of the present invention include, but are not limited to, all materials capable of alleviating, preventing, or delaying damage to axons or axons of the peripheral nerve, for example.
상기 후보물질을 투여하는 방법으로는 경구 투여, 정맥 투여, 스와빙(swabbing), 피하 투여, 피내 투여, 국소 투여, 비내 투여, 폐내 투여, 직장 투여 또는 복강 투여 등이 이용될 수 있으나 이에 한정되지 않으며, 실험 동물의 증상 또는 후보물질의 성질 등에 맞추어 적당히 선택할 수 있다. 또한, 후보물질의 투여 시기나 투여 회수 등의 투여 조건은 후보물질의 성질, 시험 및 평가 목적 등에 따라 적절하게 최적이 되도록 설정하면 되며 특별히 제한되지 않는다. 예컨대, 투여 시기에 대해서는 모델 동물 제작 직후부터 30분 마다 수회 투여한 후, 그 다음 1시간 마다 수회 투여하고, 그때마다 증상의 변화를 관찰, 평가하는 방법이 많이 이용된다. 투여 회수에 대해서는 후보물질의 성질에 따라 다르고, 1회 이상 수회가 될 수도 있다. 본 발명의 모델 동물을 이용한 후보물질의 스크리닝 방법은 원하는 목적을 달성시키는 방법이면 어떠한 방법을 이용할 수도 있다Oral administration, intravenous administration, swabing, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration or intraperitoneal administration may be used as a method of administering the candidate substance, but are not limited thereto. It can be appropriately selected according to the symptoms of the experimental animal or the nature of the candidate substance. In addition, the administration conditions such as the administration time and the number of administrations of the candidate substances may be set to be appropriately optimal according to the properties of the candidate substances, test and evaluation purposes, and are not particularly limited. For example, the administration timing is often administered several times every 30 minutes immediately after the model animal is produced, and then administered several times every hour, and a method of observing and evaluating the change in symptoms is often used. The number of administrations depends on the nature of the candidate substance, and may be one or more times. The method for screening a candidate substance using the model animal of the present invention may be any method as long as it achieves a desired purpose.
본 발명에서 사용된 용어, “대조군”은 상기 후보 물질이 처리되지 않은 동물 모델을 의미한다.As used in the present invention, the term “control” refers to an animal model in which the candidate substance is not treated.
본 발명에서 사용되는 용어, “축삭”은 신경세포의 축색소구에서 나오는 돌기상의가늘고 긴 신경섬유이며, 원형질의 축삭형질을 중심으로, 쉬반 세포(Schwann's cell)로 이루어지는 나선상의 신경초가 그 주위를 둘러싸는 구조를 이루고 있다. 신경세포에서 일어난 활동전위를 그 말초에 전달한다.As used in the present invention, the term "axon" is a long elongated nerve fiber on the projections coming out of the axons of the neurons, and around the axon plasm of the protoplasm, Schwann's cell (Schwann's cell) is composed of a spiral nerve sheath surrounding it. Has a structure. It transmits the action potential generated in the nerve cell to its periphery.
상기 축삭 퇴행은 손과 발의 근육 위축으로 인한 근력 저하 및 형태 변화를 표현형으로 나타낼 수 있는 것으로, 후보물질을 동물 모델에 투여시 손과 발의 근력 저하 또는 형태 변화가 개선된 경우 상기 후보물질을 DI-CMTC 예방 또는 치료제로 선별할 수 있다.The axonal degeneration may represent a decrease in muscle strength and morphology due to muscle atrophy in the hands and feet as a phenotype. When administration of a candidate substance to an animal model results in a decrease in muscle strength or shape change in the hands and feet, the candidate substance is DI- It can be selected as a CMTC prevention or treatment.
이러한 스크리닝 방법에 의하여 얻어진 물질은 이후의 DI-CMTC 예방 또는 치료제 개발 과정에서 선도 물질(leading compound)로 작용하게 되며, 선도 물질을 변형시키고 최적화함으로써, 새로운 신경병증의 예방 또는 치료제를 개발할 수 있다.The material obtained by this screening method acts as a leading compound in the course of the subsequent DI-CMTC prevention or treatment development, and by modifying and optimizing the leading substance, a new neuropathy prevention or treatment can be developed.
따라서, 본 발명에 따른 스크리닝 방법은 인간을 포함하는 동물의 DI-CMTC와 관련된 생리학적, 분자생물학적, 생화학적 연구의 수행에 유용하게 사용될 수 있다.Therefore, the screening method according to the present invention can be usefully used in the performance of physiological, molecular biological, and biochemical studies related to DI-CMTC in animals including humans.
본 발명에서 제공하는 DI-CMTC 동물 모델 제조 방법은 샤르코-마리에-투스 질환과 관련한 말초신경병증에 대한 분자해부학적 원인을 구명하는 학술적 효과를 가질 수 있으며, 샤르코-마리에-투스 질환을 치료하는 치료기술 분야의 발전에 기여할 수 있을 것이다.The method for preparing an animal model of DI-CMTC provided by the present invention may have an academic effect to investigate molecular anatomical causes for peripheral neuropathy associated with Sharco-Marie-Tus disease, and treat Charco-Marie-Tus disease It can contribute to the development of the field of treatment technology.
도 1은 제조된 각각의 아데노바이러스 프로모터 및 발현 유닛(YARS WT, E196K, G41R)의 도식화를 나타낸다.
도 2는 웨스턴 블롯을 통해 분석된 아데노바이러스 감염 생쥐의 좌골 신경 유래 단백질 용해물을 나타낸다 (Con, 바이러스 없음; WT, hYARS 야생형; (-), ChAT 프로모터가 없는 빈 바이러스; E, E196K 변이 hYARS; (+) ChAT 프로모터를 가진 빈 바이러스; G, G41R 변이 hYARS.)
도 3은 척수 내 hYARS 단백질의 분포 패턴을 나타낸다; (A) GFP 발현(녹색) 및 Flag의 면역형광 표지(적색)를 이용한 척수 절편 내 야생형 hYARS의 신경 특이적 발현; 황색은 적색 및 녹색 신호가 겹쳐진 것을 의미한다. GFP/Flag 이중 양성 신호는 척수 내 Flag 표지된 hYARS 융합 단백질의 발현을 의미한다 (Empty, 삽입되지 않은 빈 바이러스; WT, 야생형; E196K, E196K변이 hYARS; G41R, G41R 변이 hYARS; 척도: 500μm), (B) 척수 내 E196K와 G41R 변이 hYARS 발현 결과를 확대한 사진 (척도: 100μm.)
도 4는 좌골신경 내 hYARS 단백질의 신경 특이적 발현을 나타낸다; (A) GFP 발현(녹색) 및 Flag의 면역형광 표지(적색)를 이용한 좌골신경 횡단면 내 hYARS 단백질의 신경 특이적 발현; 황색은 Flag 표지된 hYARS 융합 단백질에 의해 적색 및 녹색 신호가 겹쳐진 것을 의미한다 (GFP/Flag 이중 염색, 가는 화살표), 두꺼운 화살표는 Flag 발현이 없는 GFP 발현 신경 섬유를 표시한다 (척도: 40μm), (B) 300 × 300 μm2 확대 면적에서의 GFP/Flag 이중 양성 신경 섬유의 정량 (P***<0.0001, 야생형(n=5)과 비교한 유의성), (C) 좌골신경의 횡단면 내 감염된 야생형 유전자, E196K, G41R 변이에서 항-Flag 항체를 포함하는 면역형광 단계; 황색은 Flag 표지된 hYARS 융합 단백질에 의해 적색 및 녹색 신호가 겹쳐진 것을 의미한다 (척도: 50μm), (D) 야생형 및 변이 내 Flag/GFP 이중 양성 액손의 수; 300 × 300 μm2의 면적에서 측정을 진행하였다 (P***<0.0001; n=5).
도 5는 배근신경절(DRG) 신경세포체 내 Flag 표지된 hYARS 융합 단백질의 발현을 나타낸다; (A) 배근신경절 신경세포체 내 Flag 표지된 hYARS 융합 단백질의 발현. DRG 내 Flag(적색) 및 GFP(녹색) 발현의 면역형광 분석을 나타냄 (척도: 40μm), (B) 전체 아데노바이러스에서 Flag/GPF 이중 양성 DRG 뉴런의 백분율; 300 × 300 μm2 확대 면적에서의 정량.1 shows the schematic of each adenovirus promoter and expression unit prepared (YARS WT, E196K, G41R).
Figure 2 shows sciatic nerve derived protein lysates of adenovirus infected mice analyzed via Western blot (Con, no virus; WT, hYARS wild type; (-), empty virus without ChAT promoter; E, E196K mutation hYARS; (+) Empty virus with ChAT promoter; G, G41R mutation hYARS.)
3 shows the distribution pattern of hYARS protein in the spinal cord; (A) Neurospecific expression of wild-type hYARS in spinal cord sections using GFP expression (green) and Flag's immunofluorescence labeling (red); Yellow means overlapping red and green signals. GFP / Flag double positive signal means expression of Flag labeled hYARS fusion protein in the spinal cord (Empty, empty virus not inserted; WT, wild type; E196K, E196K mutation hYARS; G41R, G41R mutation hYARS; scale: 500μm), (B) E196K and G41R mutant hYARS expression results in the spinal cord enlarged photo (scale: 100μm.)
4 shows neuro-specific expression of hYARS protein in the sciatic nerve; (A) Neurospecific expression of hYARS protein in the sciatic nerve cross section using GFP expression (green) and Flag's immunofluorescent label (red); Yellow indicates overlapping red and green signals by Flag labeled hYARS fusion protein (GFP / Flag double staining, thin arrow), thick arrows indicate GFP expressing nerve fibers without Flag expression (scale: 40 μm), (B) Quantification of GFP / Flag double positive nerve fibers at 300 × 300 μm 2 enlarged area (P *** <0.0001, significance compared to wild type (n = 5)), (C) Infection in the sciatic nerve cross section An immunofluorescence step comprising an anti-Flag antibody in the wild-type gene, E196K, G41R variant; Yellow means overlapping red and green signals by Flag labeled hYARS fusion protein (scale: 50 μm), (D) number of Flag / GFP double positive axons in wild type and mutation; Measurement was performed in an area of 300 × 300 μm 2 (P *** <0.0001; n = 5).
5 shows the expression of Flag-labeled hYARS fusion protein in the dorsal ganglion (DRG) neuronal cell body; (A) Expression of the flag-labeled hYARS fusion protein in the dorsal ganglion neuron cell body. Immunofluorescence analysis of Flag (red) and GFP (green) expression in DRG (scale: 40 μm), (B) percentage of Flag / GPF double positive DRG neurons in total adenovirus; Quantification at 300 × 300 μm 2 enlarged area.
이하 본 발명을 하기 예에 의해 상세히 설명한다. 다만, 하기 예는 본 발명을 예시하기 위한 것일 뿐, 하기 예에 의해 본 발명의 범위가 제한되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.
실시예Example 1. 실험동물의 준비 1. Preparation of laboratory animals
모든 실험에 사용된 6주령의 C57BL/6 생쥐는 습도 및 온도가 조절되며 물과 음식을 자유롭게 섭취할 수 있는 환경에서 사육되었다. 모든 동물 실험 절차 및 희생은 대한의학회의 가이드라인 및 경희대학교 동물 연구 위원회의 승인 (KHUASP[SE]-16-043)에 의해 진행되었다. The 6-week-old C57BL / 6 mice used in all experiments were kept in an environment where humidity and temperature were controlled and water and food could be freely consumed. All animal testing procedures and sacrifices were conducted with the guidelines of the Korean Medical Association and the approval of Kyung Hee University Animal Research Committee (KHUASP [SE] -16-043).
실시예Example 2. 신경 특이적 재조합 2. Neurospecific recombination 아데노바이러스Adenovirus 제작 making
YARS의 E196K 및 G41R 변이체를 가진 아데노바이러스를 제작하기 위해, 본 발명은 신경 특이적 발현의 조절요소이며 콜린 특이적 인핸서와 뉴런 제한적 사일렌서 요소(neuron-restrictive silencer element, NRSE)를 포함하는 생쥐 콜린 아세틸트랜스퍼레이즈(Choline acetyltransferase, ChAT) 프로모터(Gene bank 번호 NC_000080)를 사용하였다. 추가로, pAD Track-ChAT-CMV/GFP 벡터에 들어가는 인간 티로실-tRNA 합성효소(human tyrosyl-tRNA synthetase, hYARS) 클론이 사용되었다. 재조합을 위해 전사 영역 중 E1 및 E3이 이중 결실된 초나선 아데노바이러스 골격 벡터(pADEasy-1, Stratagene; Santa Clara, CA, USA)를 이용하여 야생형 hYARS(서열번호 1)가 삽입된 pADTrack-ChAT-hYARSWT-CMG/GFP, E196K 변이체를 포함하는 hYARS가 삽입된 pADTrack-ChAT-hYARSE196K-CMG/GFP, 및 G41R 변이체를 포함하는 hYARS가 삽입된 pADTrack-ChAT-hYARSG41R-CMG/GFP의 안정된 상동 재조합체가 생성되었다. 실험에 사용된 역가는 각각 Ad/ChAT 6.3 × 109 (pfu/mL), AdhYARSWT/ChAT 4.8 × 109 (pfu/mL), AdhYARSE196K/ChAT 4.3 × 109 (pfu/mL), AdhYARSG41R/ChAT 7.1 × 109 (pfu/mL)이다.To construct an adenovirus with E196K and G41R variants of YARS, the present invention is a mouse choline acetyl that is a modulator of neuron specific expression and contains a choline specific enhancer and a neuron-restrictive silencer element (NRSE). A transferase (Choline acetyltransferase, ChAT) promoter (Gene bank number NC_000080) was used. Additionally, human tyrosyl-tRNA synthetase (hYARS) clones that enter pAD Track-ChAT-CMV / GFP vectors were used. For recombination, pADTrack-ChAT- with wild type hYARS (SEQ ID NO: 1) inserted using a super-helix adenovirus skeleton vector (pADEasy-1, Stratagene; Santa Clara, CA, USA) with double deletion of E1 and E3 among transcription regions hYARS -CMG WT / GFP, with a hYARS containing the E196K mutant insert pADTrack-ChAT-hYARS E196K -CMG / GFP, and variants of the G41R hYARS is inserted pADTrack-ChAT-hYARS G41R -CMG / GFP containing stable homologous Recombinant was produced. The titers used in the experiment were Ad / ChAT 6.3 × 109 (pfu / mL), AdhYARS WT / ChAT 4.8 × 109 (pfu / mL), AdhYARS E196K / ChAT 4.3 × 109 (pfu / mL), AdhYARS G41R / ChAT 7.1, respectively. X 109 (pfu / mL).
실시예Example 3. 3. DIDI -- CMTCCMTC 동물 모델 제조 Animal model manufacturers
모든 생쥐들은 졸레틸(Vibrac; Carrors Seedees, France) 30mg/kg 및 롬푼(Bayer; Leverkusen, Germany) 10mg/kg을 이용하여 마취시켰다. 대둔근 표면(musculus gluteus superficialis)의 절개를 통해 좌골신경(sciatic nerve)을 노출시켰으며, 입체경을 이용하여 해밀턴 주사기와 유리 마이크로피펫을 통해 좌골신경에 재조합 아데노바이러스를 주입하였다. 0.1%의 fast green(트레이서)을 포함하는 AdhYARSWT/ChAT (1μl), AdhYARSE196K/ChAT (1μl), AdhYARSG41R/ChAT (1μl)의 희석된 바이러스 용액이 각 동물의 좌골신경에 주입되었으며, 절개된 피부는 Silkam 4.0 sutures를 이용하여 봉합하였다.All mice were anesthetized with 30 mg / kg of zoletyl (Vibrac; Carrors Seedees, France) and 10 mg / kg of rompun (Bayer; Leverkusen, Germany). The sciatic nerve was exposed through the incision of the gluteus gluteus superficialis, and a recombinant adenovirus was injected into the sciatic nerve through a Hamilton syringe and a glass micropipette using a stereoscope. AdhyaRS WT / ChAT (1μl), AdhYARS E196K / ChAT (1μl), AdhYARS G41R / ChAT (1μl) diluted virus solution containing 0.1% fast green (tracer) was injected into the sciatic nerve of each animal The skin was sutured using Silkam 4.0 sutures.
실시예Example 4. 동물 조직 적출 4. Animal tissue extraction
바이러스 투여 7일 후, 생쥐에서 좌골신경, L4/L5 배근신경절(DRG), 척수를 적출하였다. 조직 시료는 면역조직화학(immunohistochemistry) 과정을 위해 0.1M 인산 완충액(PB) 내에 준비된 4% 파라포름알데히드(PFA)를 이용하여 4℃에서 밤새 고정되었다. 그 후, 0.1M PB 내의 30% 자당(sucrose)으로 옮겨져 3일간 침지하였으며, 최종적으로 OCT 화합물(Leica Biosystems Richmond, Inc. USA)에 끼워넣었다. 시료는 드라이 아이스를 사용하여 얼렸으며 동결절편(16μm)으로 잘려 젤라틴 코팅된 유리슬라이드에 배치하였다. 인산완충식염수를 사용하여 좌골신경초(sciatic nerve sheaths)를 제거하였다. 척수의 경우, L4-L6 부분을 잘라내었다 (등쪽 뿔(dorsal horn) 및 배쪽 뿔(ventral horn)).Seven days after virus administration, the sciatic nerve, L4 / L5 dorsal ganglion (DRG) and spinal cord were removed from the mice. Tissue samples were fixed overnight at 4 ° C. using 4% paraformaldehyde (PFA) prepared in 0.1 M phosphate buffer (PB) for immunohistochemistry. Then, it was transferred to 30% sucrose in 0.1 M PB and immersed for 3 days, and finally embedded in an OCT compound (Leica Biosystems Richmond, Inc. USA). Samples were frozen using dry ice and cut into frozen sections (16 μm) and placed on a gelatin coated glass slide. Sciatic nerve sheaths were removed using phosphate buffered saline. For the spinal cord, L 4 -L 6 sections were cut (dorsal horn and ventral horn).
실험예Experimental Example 1. One. hYARShYARS 유전자 발현 확인을 위한 To confirm gene expression 웨스턴Western 블롯Blot 분석 analysis
단백질 용해물을 분리하기 위해 생쥐의 베타-액틴(1:5000, Sigma; St. Louis, MO, USA)과 rabbit anti-Flag(1:1000, Cell Signaling Technology) 일차 항체를 이용하여 10% 도데실 황산 나트륨 폴리아크릴아마이드 겔 전기영동 (SDS-PAGE) 절차가 진행되었다. 일차 항체 염색을 진행한 후, Tween 20을 포함하는 트리스 완충 식염수(tris-buffered saline with Tween 20, TBST)에서 3회 세척한 다음, 일차 항체와 어울리는 horseradish peroxidase(HRP)-결합 이차 항체 염색을 진행하였다. 그 후, 화학 발광 증폭 (enhanced chemiluminescence, ECL) 절차(Amersham; Buckinghamshire, England)를 적용하여, SDS-PAGE를 위한 막을 완성하였다.To isolate the protein lysate, 10% dodecyl was used using mouse beta-actin (1: 5000, Sigma; St. Louis, MO, USA) and rabbit anti-Flag (1: 1000, Cell Signaling Technology) primary antibody. Sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) procedure was performed. After primary antibody staining, washed three times in tris-buffered saline with Tween 20 (TBST), followed by horseradish peroxidase (HRP) -binding secondary antibody staining with primary antibody Did. Then, the chemiluminescence amplification (enhanced chemiluminescence, ECL) procedure (Amersham; Buckinghamshire, England) was applied to complete the membrane for SDS-PAGE.
그 결과, 도 2에 나타난 바와 같이, 아데노바이러스 벡터에 삽입된 야생형과 E196K 및 G41R 변이 hYARS 유전자에 표지된 Flag가 모두 발현한 것을 확인하여 벡터가 정상적으로 hYARS 유전자를 발현하는 것을 확인할 수 있었다.As a result, as shown in FIG. 2, it was confirmed that both the wild type inserted in the adenovirus vector and the flags labeled in the E196K and G41R mutant hYARS genes were expressed, and that the vector normally expressed the hYARS gene.
실험예Experimental Example 2. 2. 면역형광염색법을Immunofluorescence staining method 이용한 발현 확인 Expression confirmation
2-1. 척수 내 2-1. In the spinal cord hYARShYARS 단백질의 발현 분석 Protein expression analysis
면역형광염색을 위해, 젤라틴 코팅된 유리슬라이드에 배치된 조직 절편을 4% PFA에 10분간 고정시킨 후 PBS에 3회 세척하였다. 세척한 조직은 0.3% Triton-X 100을 포함하는 PBS에서 투과시켰다. 그 후, 4℃에서 밤새 10% 소 혈청 알부민 (BSA)으로 차단하였다. 다음으로, 슬라이드를 일차 항체로 사용된 rabbit anti-flag (1:1000, Cell Signaling Technology; Danvers, MA, USA)에서 한 시간 동안 상온에 배양한 후, PBS에 3회 세척하였다. 그 후, 시료는 Alexa-Fluor 594 donkey anti-rabbit secondary antibody (1:1000, Invitrogen, Life Technologies; Madison, WI, USA)에 상온에서 두 시간 동안 침지한 후 PBS로 3회 세척하고 고정 겔을 이용하여 고정되었다. 슬라이드의 면역형광을 찾기 위해 레이저 현미경(LSM 700, Carl Zeiss; Oberkochen, Germany)이 사용되었다.For immunofluorescence staining, tissue sections placed on gelatin coated glass slides were fixed in 4% PFA for 10 minutes and then washed 3 times in PBS. The washed tissue was permeated in PBS containing 0.3% Triton-X 100. It was then blocked with 10% bovine serum albumin (BSA) overnight at 4 ° C. Next, the slides were incubated at room temperature for 1 hour in rabbit anti-flag (1: 1000, Cell Signaling Technology; Danvers, MA, USA) used as the primary antibody, and then washed 3 times in PBS. Then, the sample was immersed in Alexa-Fluor 594 donkey anti-rabbit secondary antibody (1: 1000, Invitrogen, Life Technologies; Madison, WI, USA) for 2 hours at room temperature, washed 3 times with PBS and fixed gel was used. Was fixed. A laser microscope (LSM 700, Carl Zeiss; Oberkochen, Germany) was used to find the immunofluorescence of the slide.
그 결과, 도 3A에 나타난 바와 같이, 모든 실험군에서 GFP 표지를 통해 척수 내 발현이 되어 아데노바이러스 벡터가 정상적으로 기능한 것을 확인할 수 있다. 그러나 Flag 표지의 경우, 아데노바이러스 벡터 내 hYARS 유전자의 발현 유무를 확인할 수 있는 표지로서 hYARS유전자가 없는 음성대조군(Empty)를 제외한 야생형, E196K군, G41R군이 유전자를 가지고 있으나 G41R군은 발현이 일어나지 않은 것을 확인할 수 있다. 도 3B에서도 마찬가지로 E196K에서는 Flag 표지의 발현을 확인할 수 있으나, G41R에서는 발현하지 않는 것을 확인할 수 있다.As a result, as shown in FIG. 3A, it can be confirmed that the adenovirus vector functioned normally by being expressed in the spinal cord through the GFP label in all experimental groups. However, in the case of the flag marker, the wild type, E196K group, and G41R group, except for the negative control group (Empty), which does not have the hYARS gene, have genes, but the G41R group does not express. You can confirm that it is not. In FIG. 3B, the expression of the flag label can be confirmed in E196K, but it can be confirmed that it is not expressed in G41R.
2-2. 좌골신경 내 2-2. Sciatic nerve hYARShYARS 단백질의 발현 분석 Protein expression analysis
실험예 2-1과 동일한 방법으로 분석을 진행하였다. Analysis was conducted in the same manner as in Experimental Example 2-1.
그 결과, 도 4A 및 4C에 나타난 바와 같이, 모든 실험군에서 GFP는 좌골신경 내 발현이 나타나 아데노바이러스 벡터가 정상적으로 기능한 것을 확인하였다. 그러나 Flag 표지의 경우, G41R은 발현이 일어나지 않았으며, E196K에서도 발현이 거의 일어나지 않은 것을 확인할 수 있다. 이를 정량화하여 나타낸 도 4B 및 4D를 통해 발현의 정도를 자세히 확인할 수 있다. As a result, as shown in FIGS. 4A and 4C, in all experimental groups, GFP showed expression in the sciatic nerve, confirming that the adenovirus vector functioned normally. However, in the case of the Flag label, it was confirmed that the expression of G41R did not occur, and the expression of E196K hardly occurred. The degree of expression can be confirmed in detail through FIGS. 4B and 4D, which are quantified.
2-3. 2-3. 배근신경절Ganglion (dorsal root ganglia) 내 (dorsal root ganglia) my hYARShYARS 단백질의 발현 분석 Protein expression analysis
실험예 2-1과 동일한 방법으로 분석을 진행하였다. Analysis was conducted in the same manner as in Experimental Example 2-1.
그 결과, 도 5A에 나타난 바와 같이, 모든 실험군에서 GFP는 배근신경절 내 발현이 나타나 아데노바이러스 벡터가 정상적으로 기능한 것을 확인하였다. 그러나 Flag 표지의 경우, 야생형과 E196K군은 발현을 나타내고 있으나 G41R군은 발현이 일어나지 않은 것을 확인하였다.As a result, as shown in Fig. 5A, in all experimental groups, GFP showed expression in the dorsal ganglion and confirmed that the adenovirus vector functioned normally. However, in the case of the flag marker, the wild type and the E196K group showed expression, but the G41R group confirmed that the expression did not occur.
상기 실험예 2-1 내지 2-3의 결과를 종합해보면, 야생형의 경우, 척수, 좌골신경, 배근신경절 모두에서 GFP/Flag 이중 양성을 나타내어 아데노바이러스 벡터와 hYARS유전자가 정상적으로 기능함을 보여주었다. 그러나 G41R군의 경우, GFP는 모든 신경 부위에서 확인이 가능하지만 Flag는 확인되지 않아, hYARS의 G41R 아미노산 변이에 의해 발현이 저해됨을 확인하였다. E196K군의 경우, 척수 및 배근신경절에서는 GFP/Flag 이중 양성을 나타내었으나 좌골신경에서는 Flag의 발현이 매우 낮은 것을 확인하였다. 이는 주 신경에서는 정상 발현을 하지만 주변 신경에서는 발현하지 않는 것이므로, 뉴런 부분에서 문제가 생긴 것으로 판단가능하며, 특히 배근신경절 내에서는 발현한 유전자가 신경 섬유에서 발현하지 않은 것은 축삭의 기능에 문제가 생긴 것인바, 이는 축삭 퇴행을 의미한다. Summarizing the results of Experimental Examples 2-1 to 2-3, in the wild type, the spinal cord, sciatic nerve, and dorsal ganglion showed GFP / Flag double positive, indicating that the adenovirus vector and the hYARS gene function normally. However, in the case of the G41R group, GFP can be confirmed in all nerve regions, but the Flag is not confirmed, confirming that the expression is inhibited by the amino acid mutation of G41R in hYARS. In the E196K group, GFP / Flag double positive was observed in the spinal cord and dorsal ganglion, but the expression of Flag was very low in the sciatic nerve. Since this is normal expression in the main nerve but not in the peripheral nerve, it can be judged that there is a problem in the neuron section. Especially, the expression of the gene expressed in the nerve fibers in the nerve fibers does not cause expression in the nerve fibers. This means degeneration of axons.
이를 통해, G41R 변이 hYARS 유전자와 달리, E196K 변이 hYARS 유전자를 동물의 신경에서 발현시키는 경우, DI-CMTC 동물 모델을 제조할 수 있음을 알 수 있었다.Through this, it was found that, unlike the G41R mutant hYARS gene, when the E196K mutant hYARS gene is expressed in the animal's nerve, a DI-CMTC animal model can be prepared.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.
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Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365 Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375 380 Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385 390 395 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln 405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 495 Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr 500 505 510 Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 515 520 525 <110> University-Industry Cooperation Group of Kyung Hee University <120> Producing method of dominant intermediate Charcot-Marie-Tooth disease type C animal model and uses thereof <130> KPA170084-KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 528 <212> PRT <213> Homo sapiens <400> 1 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg 1 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys 20 25 30 Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro 35 40 45 His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys 50 55 60 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu 65 70 75 80 Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 85 90 95 Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu 100 105 110 Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu 115 120 125 Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp 130 135 140 Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu 145 150 155 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Glu Tyr 165 170 175 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe 180 185 190 Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val 195 200 205 His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser 210 215 220 Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 235 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu 245 250 255 Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys 260 265 270 Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr 275 280 285 Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val 290 295 300 His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 315 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu 325 330 335 Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys 340 345 350 Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Arg Leu 355 360 365 Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala 370 375 380 Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385 390 395 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln 405 410 415 Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg 420 425 430 Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile 435 440 445 Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly 450 455 460 Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu 465 470 475 480 Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 495 Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr 500 505 510 Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser 515 520 525
Claims (7)
2) 상기 벡터를 인간을 제외한 동물의 좌골신경에 주입하는 단계를 포함하는 우성 중간형 샤르코-마리에-투스 신경병증 C형(dominant intermediate CMT neuropathy type C, DI-CMTC) 동물 모델의 제조방법으로,
상기 동물 모델은 생쥐(mouse)인 것인, DI-CMTC 동물모델 제조방법.
1) In the amino acid sequence of a tyrosyl-tRNA synthetase (YARS), the 196th amino acid glutamic acid (Glutamic acid) from the N-terminus contains a gene expressing a YARS variant that has been transformed into Lysine. Preparing a vector; And
2) A method of manufacturing a dominant intermediate CMT neuropathy type C, DI-CMTC animal model comprising injecting the vector into the sciatic nerve of an animal other than a human. ,
The animal model is a mouse (mouse), DI-CMTC animal model manufacturing method.
상기 벡터는 아데노바이러스, AAV바이러스 및 렌티바이러스로 이루어진 군에서 선택되는 어느 하나의 유전인자인 것인, 제조방법.
According to claim 1,
The vector is any one of the genetic factors selected from the group consisting of adenovirus, AAV virus and lentivirus, production method.
상기 동물 모델은 생쥐인 것인, DI-CMTC 동물모델.
A DI-CMTC animal model excluding humans manufactured by the method of claim 1 or 2,
The animal model is a mouse, DI-CMTC animal model.
상기 동물 모델은 좌골신경에서 변이체의 발현이 척수 또는 배근신경절의 발현보다 낮은 것인, 동물 모델.
The method of claim 5,
In the animal model, the expression of the variant in the sciatic nerve is lower than that of the spinal cord or dorsal ganglion.
1) administering a candidate substance to the DI-CMTC animal model except for the human of claim 5; 2) When axon regression is improved by comparing axons of the animal model of step 1) with axons of a control animal model without administration of a candidate substance, DI-comprising the step of selecting the candidate substance as a DI-CMTC preventive treatment Methods of screening for CMTC prevention or treatment.
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Non-Patent Citations (4)
| Title |
|---|
| Bioessays. vol.38(9), pp.818-29(Epub 2016 Jun 28) |
| Bioessays. vol.38(9), pp.818-829(2016.06.28). |
| Nature Genetics, vol.38(2), pp..197-202(Epub 2006 Jan 22) |
| PNAS vol.106 (28) pp.11782-11787(July 14, 2009)* |
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