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1 대한내분비학회지 : 제 20 권제 4 호 2005 지상강좌 아데노바이러스를이용한내분비질환의재생요법 시카고노스웨스턴의과대학내분비내과 이은직 Endocrine Regeneration Therapy using Adenoviral vector Eun Jig Lee Endocrinology, Metabolism & Molecular Medicine Northwestern University, Feinberg School of Medicine 303 East Superior Street 서론재생요법 (regeneration therapy) 은내분비호르몬결핍질환의궁극적인치료로미래에널리이용될전망인데, 크게시험관내 (in vitro), 생체외 (ex vivo), 및생체내 (in vivo) 재생요법등 3가지범주로나뉜다 [1]. 시험관내재생요법은이미확립된세포주나태아줄기세포 (embryonic stem cell) 및조직특이성성체줄기세포에서분화된세포를생체에이식하는방법이며, 대부분면역억제제를병용하여야한다. 생체외재생요법은환자자신의줄기세포를획득하여체외에서분화를유도한뒤다시환자에게이식하는방법으로면역억제제를쓰지않아도되는장점이있다. 생체내재생요법은손상된조직을환자몸안에서재생을유도하거나줄기세포로부터새롭게분화를유도하는방법으로약물요법및유전자치료법을이용할수있다. 재생요법이이용될수있는내분비호르몬결핍질환은뇌하수체호르몬결핍, 갑상선호르몬결핍, 원발성부신피질호르몬결핍, 및인슐린의절대결핍에의한제1형당뇨병등이며, 위에서열거한방법의단독및병용한연구가많이진행되고있으며, 본문에서는유전자치료법을응용한생체내재생요법에대해소개하고자한다. Current status and preclinical models 최근까지유전자치료는전달된유전자에서발현된단백질이주로치료작용을하는개념이었다. 즉만성질환및유전성결핍질환에서해당세포에결핍된유전자를전달하면, 그유전자에서생성된단백질이세포의기능을정상화시키게된다. 그러나발현된단백질의양이조절되지않기때문에과잉및과소치료를가져올수있다. 예를들어인슐린결핍성제1형당뇨병의치료로인슐린유전자를전달하여혈 당을조절한실험동물모델 [2] 에서혈당이때에따라서는충분한조절이되지않거나, 또는인슐린이과량생성되어저혈당을보인경우가있다. 이를극복하기위해인슐린의생성을조절할수있는 promoter를사용하기도하였으나 [3], 시시각각변하는실제혈당의조절은인슐린생성의조절보다는췌장소도의베타세포에서생성된인슐린분비의조절에의하므로이또한혈당조절을완전하게하지못한다는단점이있다. 이에반해베타세포의분화에필요한유전자를유전자전달벡터를통해생체의줄기세포에전달한다면분화된완전한세포를얻을가능성이있어, 이에대한연구가활발히이뤄지고있다. 최근 Ferber 등 [4] 은췌장의발생과소도세포기능에중요한역할을하는 pancreas-duodenum homeobox-1 (PDX-1) 유전자를아데노바이러스를이용하여당뇨병이유발된생쥐의간에전달하여혈당강하를관찰한바있는데, 생쥐의간에서추출한 mrna에서췌장베타세포에서볼수있는인슐린유전자의활성화및파라핀조직에서면역염색상인슐린을검출한바있다. 그후로 Kojima 등 [5] 은면역반응이적은삼세대아데노바이러스를이용하여 PDX-1을전달한결과췌장내분비세포분화뿐만아니라외분비세포의분화도촉진됨을발견하였고, 이결과생성된트립신 (trypsin) 이심한간손상을유발하고분화된인슐린분비세포를파괴함을관찰하였다. 한편소도세포의발달에중요한 NeuroD (BETA2) 유전자및소도성장촉진유전자인 betacellulin을당뇨병이유발된생쥐에동시에전달하여 4개월간혈당이조절됨을보고했는데, 간조직에서인슐린분비세포및소도의분화를관찰했다. 인슐린분비세포는문맥삼각 (portal triads) 의주변에서검출되었고, 소도는간의캡슐 (capsule) 밑에서관찰되었는데, 분화된소도에는인슐린뿐만아니라다른소도세포호르몬인글루카곤 (glucagons) 및소마토스타틴 (somatostain) 등이검출되었다

- 대한내분비학회지 : 제 20 권제 4 호 2005 - 이렇게전달된전사인자유전자는생쥐전체간세포의 50~70% 에서발현되나인슐린분비세포는간조직의 1.0% 미만에서발견되는데, 이는어떤특정세포즉분화능력이있는줄기세포에서기원되었음을암시한다. 문맥삼각주위에는간줄기세포가존재하는것으로알려져있다 [6,7].

2 - 대한내분비학회지 : 제 20 권제 4 호 이렇게전달된전사인자유전자는생쥐전체간세포의 50~70% 에서발현되나인슐린분비세포는간조직의 1.0% 미만에서발견되는데, 이는어떤특정세포즉분화능력이있는줄기세포에서기원되었음을암시한다. 문맥삼각주위에는간줄기세포가존재하는것으로알려져있다 [6,7]. 저자등 [8] 은같은방법을사용하였을때다른호르몬생성도촉진하는지, 또이런분화과정을규명하기위하여뇌하수체전사인자 (pituitary transcription factor-1, Pit-1) 유전자를아데노바이러스를이용하여생쥐의간에전달한결과프로락틴분비세포 (lactotrope) 의분화를관찰한바있는데, 초기에문맥삼각주위에서발견된프로락틴분비세포는간줄기세포의표지자들을발현하고있었다 (Fig. 1). 이는 Pit-1 유전자가전달된간줄기세포에서부터분화가시작됨을의미한다. 프로락틴분비세포는시간이지남에따라증식을하고, 점차로중심정맥으로이동함을관찰했는데, 이는정상간세포의생성과성장과정 [9,10] 을밟는것으로생각된다. Kojima 등 (5) 이 4개월후발견한분화된소도는간의캡슐하부인데정확한기원을추적하기위해서는더연구가필요하다. 한편 Pit-1유전자는뇌하수체성장호르몬, 갑상선자극호르몬분비세포의분화에도필수적인데, Pit-1이전달된암컷생쥐의간에서프로락틴분비세포로분화됨을관찰하였는데, 이는성장호르몬분비세포나갑상선자극호르몬분비세포로분화를위해서는다른유전자의발현 [11,12] 도필요함을의미한다. 최근베타세포특이성전사인자유전자들의상호보완적인작용이인슐린유전자활성화에상승적으로작용한다는보고가있었으며 [13,14], 따라서 2~3가지전사인자유전자를동시에전달한다면보다효과적인내분비호르몬분비세포의분화를유도할가능성이있다. 실제 PDX-1, neurogenin 3 (NGN3), 및 NeuroD 등을아데노바이러스를이용하여당뇨병생쥐에단독및병용투여한결과, 병용투여한경우혈당조절및인슐린유전자활성화에효과적임을관 Fig. 1. Colocalization of prolactin (PRL) with stem cell markers in livers of mice treated with Ad-Pit-1. Paraffin sections of livers of mice at day 5 after treatment were analyzed by double immunofluorescence staining for PRL with c-kit, thy-1, or cytokeratin 14. Cells with red fluorescent cytoplasm express PRL (yellow arrow in left panel) and cells with green fluorescent cytoplasm (red arrow in center panel) express c-kit (upper), thy-1 (middle) or cytokeratin 14 (lower). PV: Portal vein. Upper and middle panels are 630 x magnification and lower panel is 1000 x magnification. Lactotrope cells express hepatic stem cells marker at the initial stage of differentiation. Reprinted from (8) with permission

3 - 이은직 : 아데노바이러스를이용한내분비질환의재생요법 - 찰하였다 [15]. 하지만병용투여하는경우과량의바이러스투여로치명적이므로, 여러전사인자유전자를 1개의벡터에서발현시키는방법개발등실제당뇨병의치료에사용되기까지는많은연구가필요하다. Strategy overcoming disadvantage of adenoviral vector 아데노바이러스에의해전달된유전자는숙주세포의게놈 (genome) 에통합되지않기때문에장기간유전자의발현이필요한만성및유전병질환의치료에는적합하지않다. 최근에개발된제3세대아데노바이러스벡터는약 1년동안유전자의발현이관찰되었으나 [16], 숙주의유전자에통합되지않는다면, 빠르게분화및증식하는세포에서는궁극적으로는전달된유전자가소멸된다. 이러한단점을극복하기위해숙주의게놈에통합 (integration) 능이있는다른바이러스의통합단위 (unit) 를아데노바이러스의게놈에삽입한 hybrid 벡터가개발되었다. 즉치료유전자발현카세트를레트로바이러스 (retrovirus) 의 long terminal repeat (LTR), 혹은아데노바이러스의존바이러스 (adeno-associated virus, AAV) 의 inverted terminal repeat (ITR) 로둘러싸고, 이들을다시아데노바이러스의게놈에삽입하게되면 hybrid 벡터 (Fig. 2) 가완성된다 [17,18]. AAV는레트로바이러스와는달리질병을일으키지않고인체세포의제 19번염색체에선택적으로통합되므로, 만성질환의유전자치료벡터로도입되었다 [19~22]. 이런 AAV의장점에도불구하고, 실제임상에적용할때많은양의바이러스를증식시키고정제하는과정이쉽지않고, 증식을위해사용했던아데노바이러스를제거해야하는문제도있다. AAV와아데노바이러스의각각장점을통합한아데노바이러스- 아데노바이러스의존바이러스 (Ad/AAV) hybrid 벡터는숙주세포의게놈에유전자를통합시킬수있고, 많은양의바이러스를용이하게증식및정제할수있어향후유전자전달벡터로많이사용될것으로기대된다. 특히 AAV의 ITR과 Rep78을같이병용할경우인체세포의제 19번염색체에선택적으로삽입되며 (Fig. 2)[17,18,23], 제3세대아데노바이러스를기본으로한 hybrid 벡터나 mini-hybrid 벡터는면역성을제거하였으므로숙주의면역반응에의해유전자를발현하는세포가제거될가능성을줄일수있다 [24]. Fig. 2. Adenoviral vector and Ad/AAV hybrid vectors carrying gene of interest. Infection of cells with Ad/AAV hybrid vector enables precise excision of the AAV inverted terminal repeat (ITR)-flanked gene from adenoviral genome, subsequently integration into the host genome occurs. In the presence of Rep78 expression unit, transgene is predictably integrated into the AAVS1 locus on human chromosome

- 대한내분비학회지 : 제 20 권제 4 호 2005-3 weeks (fixed) 1 month (living) Ad-PDX1- IRES-GFP x 50 Hyb-PDX1- IRES-GFP GFP DAPI GFP X 200 X 400 Fig. 3.

4 - 대한내분비학회지 : 제 20 권제 4 호 weeks (fixed) 1 month (living) Ad-PDX1- IRES-GFP x 50 Hyb-PDX1- IRES-GFP GFP DAPI GFP X 200 X 400 Fig. 3. GFP expression in HepG2 cells infected with adenovirus or Ad/AAV hybrid vectors carrying pancreas-duodenum homeobox 1 (PDX1)-internal ribosomal entry site (IRES)-green fluorescent protein (GFP). GFP was more abundantly expressed in HepG2 cells infected with Ad/AAV hybrid vector. (not published data) 최근저자는 PDX-1유전자와 green fluorescent protein (GFP) 유전자를 encephalomyocarditis 바이러스의 internal ribosomal entry site (IRES) 로연결한 bicistronic cassette를 Ad/AAV hybrid 벡터에삽입한뒤이를 HepG2세포에감염한결과, 장기간 GFP의발현을관찰한바있는데 (Fig. 3), 이를이용해서줄기세포분화를유도한다면분화된인슐린분비세포의기원과운명을추적할수있는가능성이있다. 또한 IRES[25] 를이용하면여러필수전사인자유전자를한개의벡터 (multi-cistronic vector) 에서발현시킬수있는장점이있다. 결론및전망본문에서소개한세포재생요법은유전자치료방법을응용한세포치료법으로아직초기의시도단계에머물고있다. 향후줄기세포의분화에필요한전사인자유전자와, 분화및성장촉진유전자등에대한지속적인연구가필요하며, 생체내각장기의줄기세포만선택적으로표적하여결핍된호르몬분비세포로분화를촉진하는방향으로연구가진행될것으로기대된다. 참고문헌 1. Yamaoka T: Regeneration therapy of pancreatic beta cells: towards a cure for diabetes? Biochem Biophys Res Commun 296: , Matsumoto T, Yamaguchi M, Kuzume M, Matsumiya A, Kumada K: Insulin gene transfer with adenovirus vector via the spleen safely and effectively improves posthepatectomized conditions in diabetic rats. J Surg Res 110: , Lee HC, Kim SJ, Kim KS, Shin HC, Yoon JW: Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue. Nature 408: , Ferber S, Halkin A, Cohen H, Ber I, Einav Y, Goldberg I, Barshack I, Seijffers R, Kopolovic J, Kaiser N, Karasik A: Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med 6: , Kojima H, Fujimiya M, Matsumura K, Younan P, Imaeda H, Maeda M, Chan L: NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat Med 9: , Zhang Y, Bai XF, Huang CX: Hepatic stem cells: existence and origin. World J Gastroenterol 9: , Falkowski O, An HJ, Ianus IA, Chiriboga L, Yee H, West AB, Theise ND: Regeneration of hepatocyte `buds' in cirrhosis from intrabiliary stem cells. J Hepatol 39: , Lee EJ, Russell T, Hurley L, Jameson JL: Pit-1 induces transient differentiation of adult hepatic stem

5 - 이은직 : 아데노바이러스를이용한내분비질환의재생요법 - cells into prolactin-producing cells in vivo. Mol Endocrinol 19: , Zajicek G, Ariel I, Arber N: The streaming liver. III. Littoral cells accompany the streaming hepatocyte. Liver 8: , Arber N, Zajicek G, Ariel I: The streaming liver. II. Hepatocyte life history. Liver 8:80-87, Drolet DW, Scully KM, Simmons DM, Wegner M, Chu KT, Swanson LW, Rosenfeld MG: TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 5: , Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA, Mayo KE: GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat Genet 4: , Glick E, Leshkowitz D, Walker MD: Transcription factor BETA2 acts cooperatively with E2A and PDX1 to activate the insulin gene promoter. J Biol Chem 275: , Aramata S, Han SI, Yasuda K, Kataoka K: Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription factors MafA, Beta2, and Pdx1. Biochim Biophys Acta 1730:41-46, Kaneto H, Nakatani Y, Miyatsuka T, Matsuoka TA, Matsuhisa M, Hori M, Yamasaki Y: PDX-1/VP16 fusion protein, together with NeuroD or Ngn3, markedly induces insulin gene transcription and ameliorates glucose tolerance. Diabetes 54: , Schiedner G, Morral N, Parks RJ, Wu Y, Koopmans SC, Langston C, Graham FL, Beaudet AL, Kochanek S: Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expr ession and decreased toxicity. Nat Genet 18: , Surosky RT, Urabe M, Godwin SG, McQuiston SA, Kurtzman GJ, Ozawa K, Natsoulis G: Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol 71: , Sun BD, Chen YT, Bird A, Amalfitano A, Koeberl DD: Long-term correction of glycogen storage disease type II with a hybrid Ad-AAV vector. Mol Ther 7: , Samulski RJ, Sally M, Muzyczka N: Adeno-associate viral vectors. In The development of human gene therapy Friedmann T, Ed. Cold Spring harbor, Cold Spring Harbor Laboratory Press, 1999, p Buning H, Nicklin SA, Perabo L, Hallek M, Baker AH: AAV-based gene transfer. Curr Opin Mol Ther 5: , Carter PJ, Samulski RJ: Adeno-associated viral vectors as gene delivery vehicles. Int J Mol Med 6:17-27, Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA: Targeted integration of adeno-associated virus (AAV) into human chromosome 19. Embo J 10: , Recchia A, Parks RJ, Lamartina S, Toniatti C, Pieroni L, Palombo F, Ciliberto G, Graham FL, Cortese R, La Monica N, Colloca S: Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proc Natl Acad Sci U S A 96: , Lieber A, Steinwaerder DS, Carlson CA, Kay MA: Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes. J Virol 73: , Douin V, Bornes S, Creancier L, Rochaix P, Favre G, Prats AC, Couderc B: Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors. BMC Biotechnol 4:16,

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract