JP5883265B2 - ヒト肝細胞を担持するキメラ非ヒト動物 - Google Patents
ヒト肝細胞を担持するキメラ非ヒト動物 Download PDFInfo
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- JP5883265B2 JP5883265B2 JP2011226233A JP2011226233A JP5883265B2 JP 5883265 B2 JP5883265 B2 JP 5883265B2 JP 2011226233 A JP2011226233 A JP 2011226233A JP 2011226233 A JP2011226233 A JP 2011226233A JP 5883265 B2 JP5883265 B2 JP 5883265B2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Description
[1] (i)免疫不全であること、(ii)肝障害を受けていること、および(iii)内在性のCyp3a遺伝子の機能が欠失していること、を特徴とする非ヒト動物に、ヒト肝細胞を移植することを含む、該非ヒト動物の薬物代謝系が欠失または抑制され、かつ該ヒト肝細胞による薬物代謝系を備えるキメラ非ヒト動物の作出方法。
[2] 前記非ヒト動物がマウスである、[1]の方法。
[3] 前記非ヒト動物が、遺伝的免疫不全症の非ヒト動物またはその子孫、遺伝的に肝障害を有する非ヒト動物またはその子孫、ならびに内在性のCyp3a遺伝子の機能が遺伝的に欠失している非ヒト動物またはその子孫を用いて三元交配する工程を含む作出方法により得られる、[1]または[2]の方法。
[4] 前記非ヒト動物が、遺伝的免疫不全症であり、かつ遺伝的に肝障害を有する非ヒト動物またはその子孫と内在性のCyp3a遺伝子の機能が遺伝的に欠失している非ヒト動物またはその子孫を用いて交配する工程を含む作出方法により得られる、[1]または[2]の方法。
[5] 前記非ヒト動物が、uPA(+/+)/SCID(+/+)マウスとcyp3a(KO/KO)マウスを用いて交配する工程、ならびに各遺伝的要素をホモに有する動物をスクリーニングする工程を含む作出方法により得られる、[1]〜[4]のいずれかの方法。
[6] 前記非ヒト動物が、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウスである、[1]〜[5]のいずれかの方法。
[7] [1]〜[6]のいずれかの方法によって得られる、キメラ非ヒト動物。
[8] 内在性のCyp3a遺伝子の機能が欠失しており、かつヒト肝細胞を体内に担持することを特徴とするキメラ非ヒト動物。
[9] 非ヒト動物の薬物代謝系が欠失または抑制されており、かつヒト肝細胞による薬物代謝系を備える、[8]のキメラ非ヒト動物。
[10] 前記非ヒト動物がマウスである、[8]または[9]のキメラ非ヒト動物。
[11] [7]〜[10]のいずれかのキメラ非ヒト動物に、被験物質を投与する工程、および該被験物質がヒト肝細胞に与える影響を評価する工程を含む、該被験物質の毒性試験方法。
[12] [7]〜[10]のいずれかのキメラ非ヒト動物に、被験物質を投与する工程、および該被験物質に対するヒト肝細胞の代謝能を評価する工程を含む、ヒト肝細胞の該被験物質に対する代謝能試験方法。
ヒト肝細胞は、上記非ヒト動物の脾臓または門脈に注入することによって、脾臓または門脈を経由して非ヒト動物の肝臓へ移植することができる。移植に用いるヒト肝細胞の数は、約1〜200万個、好ましくは約10万〜100万個とすることができる。移植したヒト肝細胞のうち約5〜15%、好ましくは約10%が非ヒト動物の類洞から肝細胞索へ侵入し、生着・増殖する。
本発明者が作製したcyp3a(KO/KO)マウス(WO2009/063722)の凍結精子を融解し、本発明者が作製したuPA(+/+)/SCID(+/+)マウス(WO2008/001614)の未受精卵と人工授精後、仮腹に戻した。生まれた子マウスのうち、遺伝子型がcyp3a(KO/wt)/uPA(+/wt)/SCID(+/wt)マウス[F1]を選択し、自然交配にてuPA(+/+)/SCID(+/+)マウスに2度目のバッククロスを行い、cyp3a(KO/wt)/uPA(+/+)/SCID(+/+)マウス[N2]を得た。
ヒト肝細胞としては、BD Gentest社より購入した肝細胞(Lot No.BD85、男児、2才)を使用した。この凍結肝細胞を、従来公知の手法(Chise Tatenoら、Am J Pathol 165:901-912, 2004)に従って融解して用いた。
上記実施例2で作製したcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]およびPXBマウスならびに非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]の肝臓における、ヒトまたはマウスの各種CYPおよびβ-actin(補正遺伝子)をコードするmRNAの発現量を、RT-PCR法を用いて測定した。
肝臓におけるこれらCYPをコードするmRNAの発現量について、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]とPXBマウスを比較した場合、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]における発現量は、PXBマウスにおける発現量のおよそ70%〜117%であり、ほぼ同程度の遺伝子発現量が認められた。
cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]および非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]においては、マウスにおいて代表的なCyp3a分子種であるCyp3a11をコードするmRNA発現は検出されなかった(図3(f))。cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]とPXBマウスを比べた場合、Cyp1a2、2b10および2c37をコードするmRNA発現量は、両者においてほぼ同程度であり(図3(a)、(b)、(d))、Cyp2c29および2c55をコードするmRNA発現量はcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]の方が、PXBマウスよりも高値を示した(図3(c)、(e))。また、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]と非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]を比べた場合、非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]の方が、Cyp1a2、2b10、2c29、2c37および2c55をコードするmRNAについていずれも高い発現量を示した(図3(a)−(e))。
上記実施例2で作製したcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]およびPXBマウスならびに非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]の小腸における、マウスの各種CYPをコードするmRNAの発現量を、RT-PCR法を用いて測定した。
cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]および非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]においては、マウスにおいて代表的なCyp3a分子種であるCyp3a11をコードするmRNA発現は検出されなかった(図4(c))。また、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]と非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]を比べた場合、非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]の方が、Cyp2b10および2c55をコードするmRNAがいずれも高い発現量を示した(図4(a)、(b))。
上記実施例2で作製したcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]およびPXBマウスの外側左葉の凍結切片を作製し、ヒト特異的サイトケラチン8/18(hCK8/18)抗体(PROGEN)およびマウスCyp3a抗体(SANTA CRUZ)を用いて、肝臓の免疫染色を行った。
肝臓においては、PXBマウスでは、hCK8/18陰性のマウス肝細胞に一致してマウスCyp3aが陽性であった(図5(a))。一方、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]では、hCK8/18陰性のマウス肝細胞においてマウスCyp3aは陰性であった(図5(b))。
上記実施例2で作製したcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]およびPXBマウスの小腸の凍結切片を作製し、マウスCyp3a抗体を用いて、小腸の免疫染色を行った。
小腸においては、PXBマウスでは、小腸上皮においてマウスCyp3aが陽性であった(図6(a))。一方、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]では、マウスCyp3aは陰性であった(図6(b))。
上記実施例2にて調製した各マウスの肝臓および小腸に由来するミクロソームを用いてMDZ代謝活性を測定した。肝ミクロソームを用いた実験では、終濃度50 umol/LのMDZを終濃度0.1 mg/mL肝ミクロソーム中にて37℃下で5分間インキュベーションさせた。小腸ミクロソームを用いた実験では、終濃度50 umol/LのMDZを終濃度0.5 mg/mL小腸ミクロソーム中にて37℃で10分間インキュベーションさせた。MDZの1’位および4位水酸化代謝物はLC-MS/MSを用いて測定した。
cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]とPXBマウスを比べた場合、肝ミクロソーム中のMDZ代謝活性は両者において同程度であった(図7(a))。一方、小腸ミクロソーム中のMDZ代謝活性については、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウス[N2F1]は、PXBマウスよりも低く、非移植のcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N2F1]とほぼ同程度であった(図7(b))。cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)キメラマウスではPXBマウスに比べ、小腸におけるマウスCyp3aによる代謝が抑制されていることが示された。
上記実施例2と同様にヒト肝細胞を調製し、実施例1にて得られたcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N4F1](生後2〜4週齢)8匹に、上記実施例2と同様に5.0×105個のヒト肝細胞を移植した。
上記実施例8で得られた、ヒト肝細胞が移植されたcyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウス[N4F1](移植後8週(11週齢))4匹(雄2匹、雌2匹)に用量10 mg free/kg b.w. になるよう、0.5%メチルセルロースに懸濁した8 mg free/mL塩酸ネファゾドンを5 mL/kg b.w.の容量で2回強制経口投与した。
Claims (12)
- (i)免疫不全であること、(ii)肝障害を受けていること、および(iii)内在性のCyp3a遺伝子の機能が欠失していること、を特徴とする非ヒト動物に、ヒト肝細胞を移植することを含む、該非ヒト動物の薬物代謝系が欠失または抑制され、かつ該ヒト肝細胞による薬物代謝系を備えるキメラ非ヒト動物の作出方法。
- 前記非ヒト動物がマウスである、請求項1に記載の方法。
- 前記非ヒト動物が、遺伝的免疫不全症の非ヒト動物またはその子孫、遺伝的に肝障害を有する非ヒト動物またはその子孫、ならびに内在性のCyp3a遺伝子の機能が遺伝的に欠失している非ヒト動物またはその子孫を用いて三元交配する工程を含む作出方法により得られる、請求項1または2に記載の方法。
- 前記非ヒト動物が、遺伝的免疫不全症であり、かつ遺伝的に肝障害を有する非ヒト動物またはその子孫と内在性のCyp3a遺伝子の機能が遺伝的に欠失している非ヒト動物またはその子孫を用いて交配する工程を含む作出方法により得られる、請求項1または2に記載の方法。
- 前記非ヒト動物が、uPA(+/+)/SCID(+/+)マウスとcyp3a(KO/KO)マウスを用いて交配する工程、ならびに各遺伝的要素をホモに有する動物をスクリーニングする工程を含む作出方法により得られる、請求項1〜4のいずれか1項に記載の方法。
- 前記非ヒト動物が、cyp3a(KO/KO)/uPA(+/+)/SCID(+/+)マウスである、請求項1〜5のいずれか1項に記載の方法。
- 請求項1〜6のいずれか1項に記載の方法によって得られる、キメラ非ヒト動物。
- 内在性のCyp3a遺伝子の機能が欠失しており、かつヒト肝細胞を体内に担持することを特徴とするキメラ非ヒト動物。
- 非ヒト動物の薬物代謝系が欠失または抑制されており、かつヒト肝細胞による薬物代謝系を備える、請求項8に記載のキメラ非ヒト動物。
- 前記非ヒト動物がマウスである、請求項8または9に記載のキメラ非ヒト動物。
- 請求項7〜10のいずれか1項に記載のキメラ非ヒト動物に、被験物質を投与する工程、および該被験物質がヒト肝細胞に与える影響を評価する工程を含む、該被験物質の毒性試験方法。
- 請求項7〜10のいずれか1項に記載のキメラ非ヒト動物に、被験物質を投与する工程、および該被験物質に対するヒト肝細胞の代謝能を評価する工程を含む、ヒト肝細胞の該被験物質に対する代謝能試験方法。
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EP12840420.9A EP2767160B1 (en) | 2011-10-13 | 2012-10-12 | Chimeric non-human animal carrying human hepatocyte |
PCT/JP2012/077019 WO2013054949A1 (ja) | 2011-10-13 | 2012-10-12 | ヒト肝細胞を担持するキメラ非ヒト動物 |
US14/351,481 US9420769B2 (en) | 2011-10-13 | 2012-10-12 | Chimeric non-human animal carrying human hepatocyte |
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