보안과제 ( ), 일반과제 ( ) 과제번호 108080-03-3-SB010 유전자적중및핵이식에의한레쉬 - 니한증후군질환모델복제돼지의생산 레쉬 - 니한증후군질환모델 HPRT1 유전자적중복제돼지의생산 비외과적수정란이식에의한복제돼지생산기술개발
농림수산식품부장관귀하 이보고서를 유전자적중및핵이식에의한레쉬 - 니한증후군질환모델복제돼지의생 산 ( 세부과제 레쉬 - 니한증후군질환모델 HPRT1 유전자적중복제돼지의생산, 협동과제 비외과적수정란이식에의한복제돼지생산기술개발 ) 의보고서로제출합니다. 2011 년 7 월일 주관연구기관명 : 단국대학교 주관연구책임자 : 심호섭 연 구 원 : 강지현 연 구 원 : 김영준 연 구 원 : 김은영 협동연구기관명 : 노아바이오텍 협동연구책임자 : 손중호
Ⅰ. 제목 유전자적중및핵이식에의한레쉬 - 니한증후군질환모델복제돼지의생산 Ⅱ. 연구개발의목적및필요성인간유전질환의발병기전을이해하고신약및새로운치료법을개발하기위해서특정유전질환의모델동물이널리활용되고있다. 현재까지마우스의배아줄기세포를이용한유전자적중방법에의해특정유전자의기능을상실한여러종류의유전자적중마우스가개발되었다. 그러나마우스는해부와생리가인간과크게달라서다수의질환에있어서인간과동일한유전질환의특성을보이지않는경우가많은실정이다. 따라서본연구에서는체세포의유전자적중및핵이식기술을이용하여보다인간과해부생리학적으로유사한미니돼지에서인간유전질환모델을개발하고자한다. Ⅲ. 연구개발내용및범위본연구에서는미니돼지섬유아세포의유전자적중에의하여레쉬-니한증후군의원인유전자로알려진 HPRT1 유전자및모세혈관확장성운동실조증의원인유전자인 ATM 유전자를결손시킨후핵이식에의하여특정유전질환의모델로이용될수있는유전자적중복제미니돼지의생산을시도하였다. Ⅳ. 연구개발결과미니돼지섬유아세포에유전자적중벡터를도입하여유전자적중을시도한결과 HPRT1 유전자는유전자적중효율이약 1% 였으며 ATM 유전자의경우 2.2% 였다. 선별된유전자적중세포를이용하여핵이식을실시하고각각 2두및 5두의대리모에이식한결과 HPRT1 유전자적중복제수정란에서는임신이확인되지않았으나 ATM 유전자적중의경우 3두가임신하였고그중 2두가각각 1두및 4두의산자를분만하였다. 생산된총 5두의복제미니돼지를 PCR에의하여분석한결과모두 ATM 유전자적중이이루어진것으로확인되었다. Ⅴ. 연구성과및성과활용계획본연구를통하여개발된 ATM 유전자적중복제미니돼지는장차모세혈관확장성운동실조증의질환모델로활용될수있을것이며, ATM 유전자의돌연변이가다양한신경계, 면역계, 번식기계의질환을유발하고암의유병률을높이는것으로알려져있어서이러한질환의기전을이해하고신약및치료법을개발하는대기여할수있을것이다. 또한본연구에서개발된기술을활용하여전임상단계에서사용될수있는다양한질환모델돼지의공급이가능할것이다. 인간과생리적으로유사한질환모델돼지를생산할수있는기초를제공함으로서개발중인신약을현재의질환모델 mouse to human 방식보다효과적인 pig to human 시스템을이용하여검증할수있을것이다. 1
SUMMARY ( 영문요약문 ) 2
CONTENTS Chapter 1. Introduction 7 Section 1. Objectives 7 Section 2. Rationale 7 1. Transgenic animals as models for human genetic disease 7 2. Gene-targeted clone animals using nuclear transfer 7 3. Gene-targeted clone animals as models for human genetic disease 8 4. Importance of technical development in model animals for human genetic disease 10 Chapter 2. Status of technology 11 Section 1. International status 11 Section 2. Domestic status 11 Chapter 3. Experimental results and discussion 12 Section 1. Experimental methods 12 Section 2. Experimental results 14 1. Experimental animals 14 2. Culture of fetal fibroblasts 14 3. Preparation of gene-targeting vectors 14 4. HPRT1 gene targeting in fibroblasts 15 5. Nuclear transfer using HPRT1 gene-targeted fibroblasts 17 6. Analysis of HPRT1 gene targeting in nuclear transfer embryos 18 7. ATM gene targeting in fibroblasts 19 8. Nuclear transfer using ATM gene-targeted fibroblasts 19 9. Analysis of HPRT1 gene targeting in nuclear transfer embryos 19 10. Transfer of nuclear transfer embryos into surrogate gilts 20 11. Development of non-surgical embryo transfer techniques in pigs 21 Chapter 4. Achievements and contributions 23 Section 1. Research objectives and points of evaluation 23 Section 2. Achievements and contributions 23 Chapter 5. Research outcomes and applications 24 Section 1. Research outcomes 24 1. Publications 24 3
2. Patents 24 Section 2. Applications of research outcomes 25 1. Ongoing applications 25 2. Future plan 26 Chapter 6. Scientific and technical information obtained from international collaboration 27 Chapter 7. References 28 4
제 1 장연구개발과제의개요 7 제 1 절연구개발의목적 7 제 2 절연구개발의필요성 7 1. 인간유전질환모델형질전환동물 7 2. 핵이식기술을이용한유전자적중복제동물생산 7 3. 인간유전질환모델유전자적중복제돼지 8 4. 인간유전질환모델동물생산기술의중요성 10 제 2 장국내외기술개발현황 11 제 1 절국외현황 11 제 2 절국내현황 11 제 3 장연구개발수행내용및결과 12 제 1 절연구방법 12 제 2 절연구결과 14 1. 공시동물 14 2. 섬유아세포주의수립 14 3. 유전자적중벡터의작성 14 4. 체세포의 HPRT1 유전자적중 15 5. HPRT1 유전자적중체세포의핵이식 17 6. 복제수정란의 HPRT1 유전자적중검정 18 7. 체세포의 ATM 유전자적중 19 8. ATM 유전자적중체세포의핵이식 19 9. 복제수정란의 ATM 유전자적중검정 19 10. 복제수정란의이식 20 11. 비외과적수정란이식방법의개발 21 제 4 장목표달성도및관련분야에의기여도 23 제 1 절연구목표및평가의착안점 23 제 2 절목표달성도및관련분야에의기여도 23 제 5 장연구개발성과및연구성과활용계획 24 제 1 절연구개발성과 24 1. 논문 24 5
2. 특허 24 제 2 절성과활용계획 25 1. 성과활용실적 25 2. 성과활용계획 26 제 6 장연구개발과정에서수집한해외과학기술정보 27 제 7 장참고문헌 28 6
제 1 장연구개발과제의개요 7
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Disease Mutated gene Symptom Lesch- Nyhan syndrome (LNS) Ataxia telangiectasia (AT) Lowe syndrome Adrenoleukodystrophy Fabry's disease hypoxanthineguanine phosphoribosyl transferase (HPRT); X-linked Ataxia-telangiectasi a mutated (ATM) Antibody injection; Desferrioxamine Oculocerebrorenal syndrome of Lowe 1 (OCRL1); X-linked ATP-binding cassette, subfamily D, member 1 (ABCD1); X-linked α-galactosidase A (α-gal A); X-linked - Build-up of uric acid - Arthritis - Poor muscle control - Renal failure - Mental retardation - Progressive immunological and neurological dysfunction - Cataracts - Hypotonia - Mental impairment - Renal tubular dysfunction - Degenerative disorder of myelin - Adrenal insufficiency - Diability - Globotriaosylcermide accumulation - Skin lesions - Kidney complication Frequency/ Expected lifespan 1/380,000 Lethal <20 yrs 1/40,000-100,000 Lethal <early 20s 1/500,000 Lethal if severe 1/20,000 Lethal 1/40,000 Lethal Treatment Allopurinol to control excessive amounts of uric acid. Alkali supplement; Vit D; Kidney transplantation No cure; Lorenzo's oil; Low VLCFA diet; BM transplantation Enzyme replacement therapy Mouse model KO mice wo disease symptoms KO mice w partial disease symptoms KO mice wo disease symptoms KO mice wo disease symptoms KO mice wo disease symptoms Galactosemia Galactose-1- phosphate uridyl transferase (GALT); galactokinase (GALK1); UDP galactose epimerase (GALE) - Galactose metabolism failure - Neurological impairment - Ovarian failure 1/40,000-60,000 Not lethal Restricted diet wo galactose and lactose KO mice wo disease symptoms 9
Glycogen storage disease type II (Pompe's disease) Acid maltase - Defect in lysosomal metabolism - Muscle weakness 1/40,000-300,000 Lethal if severe Enzyme replacement therapy w Myozyme; Gene therapy KO mice wo disease symptoms Metachromatic leukodystrophy (MLD) Arylsulfatase A - Myelin degeneration - Muscle rigidity - Paralysis 1/40,000 Lethal No cure; Enzyme replacement therapy; BM transplantation; Cell therapy KO mice wo disease symptoms Tay-Sachs disease (TSD) Cystic fibrosis (CF) Atherosclerosis Kallmann's syndrome Gaucher's disease Huntington's disease Hexosaminidase A (HEXA) Cystic fibrosis transmembrane conductance regulator (CFTR) Low-density lipoprotein receptor (LDLR) KAL1 (10%); X-linked Lysosomal gluco-cerebrosidase (a,k.a β-glucosidase) Trinucleotide repeat expansion in the Huntingtin (Htt) gene - Ganglioside accumulate in the nerve cells - Mental and physical disabilities - Difficulty in breathing - Insufficient enzyme production in the pancreas - Artery inflammation by deposition of lipoproteins - Heart attack - Hypogonadism - Lack of GnRH - Anosmia (inability to smell) - Delayed puberty - Accumulation of glucocerebro- side - Spleen and liver malfunction - Skeletal disorders - Severe neurologic complications - Degeneration of neuronal cells - Abnormal body movements - Lack of coordination 1/20,000 Lethal Infantile <5 yrs Juvenile <15 yrs 1/3,900 children in the US Lethal <30 yrs 65% and 47% in the US affected Not lethal 1/10,000 1/50,000 Not lethal 1/40,000 Lethal if acute (Type 2) 1/12,000 No cure; Enzyme replacement therapy; Substrate reduction therapy Antibiotics; Lung transplantation; Gene therapy Excercise; Diet; Niacin Hormone replacement therapy (hcg/t for ; E2/P4 for Enzyme replacement therapy w Cerezyme Antidepressants; Omega III fatty acid; dopamine receptor blockers KO mice wo disease symptoms KO mice wo disease symptoms KO mice but small arteries No mouse homolog KO mice available KO mice available 10
제 2 장국내외기술개발현황 현재인간유전질환모델형질전환돼지의예는국내에서는전무하다. 그러나이와같은연구는선진국에서조차도매우초기단계에있기때문에적절한투자가이루어진다면단기간에세계시장에서기술적우위를점할수있을것으로예상된다. 특히국내에서도다수의연구팀에서형질전환돼지의생산에성공한바있으므로 ( 표 2) 적절한인프라가제공된다면국내기술만으로도인간유전질환모델돼지의개발을국제적으로선도할수있으리라보여진다. 특히최근에인간유전질환모델돼지의생산에필수적인유전자적중기술을활용한복제돼지의생산에도성공한바있어국내의연구력도크게성장하였다. α 11
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제 4 장목표달성도및관련분야에의기여도 23
제 5 장연구개발성과및성과활용계획 24
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제 6 장연구개발과정에서수집한해외과학기술정보 27
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