JP5681955B2 - 短いrna分子 - Google Patents
短いrna分子 Download PDFInfo
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- JP5681955B2 JP5681955B2 JP2013532277A JP2013532277A JP5681955B2 JP 5681955 B2 JP5681955 B2 JP 5681955B2 JP 2013532277 A JP2013532277 A JP 2013532277A JP 2013532277 A JP2013532277 A JP 2013532277A JP 5681955 B2 JP5681955 B2 JP 5681955B2
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- cells
- rna
- short
- strand
- insulin
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Description
a)標的RNA転写物と標的遺伝子のプロモーター領域の長さが同一であり、その長さ全体にわたって重なる(つまり、相補的である)。
b)標的RNA転写物は標的遺伝子のプロモーター領域より短く、かつその長さ全体にわたって標的遺伝子のプロモーター領域と重なる(すなわち、その長さ全体にわたって標的遺伝子のプロモーター領域内の配列と相補的である。)。
c)標的RNA転写物は標的遺伝子のプロモーター領域より長く、標的遺伝子のプロモーター領域はそれによって完全に重複している、すなわち、標的遺伝子のプロモーター領域はその長さ全体にわたって、標的RNA転写物内の配列と相補的である)。
d)標的RNA転写物および標的遺伝子のプロモーター領域は、同じまたは異なる長さであり、重なる領域は、標的RNA転写物の長さおよび標的遺伝子のプロモーター領域の長さのいずれよりも短い。
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4. Halvorsen T & Levine F (2001) Diabetes mellitus-cell transplantation and gene therapy approaches Curr Mol Med 1, 273-286.
5. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, & Goff JP (1999) Bone marrow as a potential source of hepatic oval cells Science 284, 1168-1170.
6. Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, & Krause DS (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation Hepatology 31, 235-240.
7. Oh SH, Miyazaki M, Kouchi H, Inoue Y, Sakaguchi M, Tsuji T, Shima N, Higashio K, & Namba M (2000) Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro Biochem Biophys Res Commun 279, 500-504.
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9. Gordon MY (1994) Plastic-adherent cells in human bone marrow generate long-term hematopoiesis in vitro Leukemia 8, 865-870.
10. Hellerstrom C (1984) The life story of the pancreatic B cell Diabetologia 26, 393-400.
11. Rosenberg L & Vinik AI (1992) Trophic stimulation of the ductular-islet cell axis: a new approach to the treatment of diabetes Adv Exp Med Biol 321, 95-104; discussion 105-109.
12. Swenne I (1992) Pancreatic beta-cell growth and diabetes mellitus Diabetologia 35, 193-201.
13. Zaret KS (2008) Genetic programming of liver and pancreas progenitor s: lessons for stem-cell differentiation Nat Rev Genet 9, 329-340.
14. Jonsson J, Carlsson L, Edlund T, & Edlund H (1994) Insulin-promoter- factor 1 is required for pancreas development in mice Nature 371, 606-609.
15. Guz Y, Montminy MR, Stein R, Leonard J, Gamer LW, Wright CV, & Teitelman G (1995) Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny Development 121, 11-18.
16. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, & Wright CV (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum Development 122, 983-995.
17. Ahlgren U, Jonsson J, & Edlund H (1996) The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice Development 122, 1409-1416.
18. Ahlgren U, Pfaff SL, Jessell TM, Edlund T, & Edlund H (1997) Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells Nature 385, 257-260.
19. Gradwohl G, Dierich A, LeMeur M, & Guillemot F (2000) neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas Proc Natl Acad Sci U S A 97, 1607-1611.
20. Murtaugh LC (2007) Pancreas and beta-cell development: from the actual to the possible Development 134, 427-438.
21. Smith SB, Qu HQ, Taleb N, Kishimoto NY, Scheel DW, Lu Y, Patch AM, Grabs R, Wang J, Lynn FC, et al. Rfx6 directs islet formation and insulin production in mice and humans Nature 463, 775-780.
22. Gordon MY, Levicar N, Pai M, Bachellier P, Dimarakis I, Al-Allaf F, 'Hamdi H, Thalji T, Welsh JP, Marley SB, et al. (2006) Characterization and clinical application of human CD34+ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor Stem Cells 24, 1822-1830.
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PDX1の発現を活性化するための短いRNAの設計
上述の方法を用いてsaRNAの設計を実施した。さらに具体的な詳細を以下に示す。
ニューロゲニン3の発現を活性化させるための短いRNAの設計
設計は、実施例1のように実施したが、以下の相違があった。
Rfx6の発現を活性化するための短いRNAの設計
設計は、実施例1のように実施したが、以下の相違があった。
MafAの発現を活性化するための短いRNAの設計
設計は、実施例1のように実施したが、以下の相違があった。
特殊化の誘導―インスリン産生細胞の作製
材料および方法
以下のようにして多能性細胞(omnicytes)を得た:顆粒球コロニー刺激因子(G−CSF)に動員された末梢血液細胞を、Stem Cell Laboratory, Hammersmith Hospitalによって処理された白血球除去輸血から、臨床的必要量を超えて得た。すべてのケースについて、インフォームドコンセントおよび地元の研究倫理委員会の承認を得た。CD34+細胞をハンクス緩衝食塩水(HBSS;Gibco, Paisley, U.K)で、1:4に希釈してから、Lymphoprep(Axis−Shield, Kimbolton, Cambridgeshire,UK)密度勾配にわたる1800rpmでの遠心分離に30分間かけて単核細胞(MNC)を分離した(Heraeus、Hanau、Germany)。MNC部分を回収し、まずHBSS中で、次に、0.5%のヒト血清アルブミンおよび5mMのEDTA、pH7.2)を補充したMACS(磁気細胞選別)緩衝液(リン酸緩衝生理食塩水)を用いて洗浄した。CD34+陽性細胞選択キット(LargeMacs; Miltenyl Biotec, Bergisch Glasbach Germany)を用いて,MNCからCD34+細胞を単離した。単離したCD34+細胞を、24ウェル培養皿(Corning, USA)上.1ウェルにつき500μlのα−最小必須培地(α−MEM)を入れて、2.5x105細胞の密度で平板培養し、37℃および5%CO2で30分間インキュベートした。このインキュベーションの後、プレートをPBSで4回洗浄することにより、この非接着性の細胞集団を除去した。
RNA分析用に、9日目に細胞を採取し、遠心分離により粒状化した。RNAqueous−Microキット(Ambion)をメーカーの指示に従って用いて、全RNAを回収した。RNAを、Nanodrop 1000マイクロサンプル定量装置を用いて定量した。QiagenのOne Step RT−PCRキットをメーカー推奨に従って用いて、各サンプルから1μgの全RNAを逆転写した。PDX1、Rfx6、MafA、およびインスリンの発現を、半定量的に測定した。ローディング対照としてアクチンを用いた。
4%パラホルムアルデヒドで細胞を20分間、1cmのガラス製カバースリップ上に固定し、続いて0.2%トリトンX100で20分間透過化処理をした。カバースリップを3回洗浄し、続いて10%ウサギ血清で45分間ブロックした。ウサギで育てた抗ヒトインスリン(1:200)(シグマ)を10%血清中の細胞に1時間添加した。PBSで細胞を3回洗浄し、続いてさらに15分間10%血清を添加した後、Alexa−488抱合抗ウサギ二次抗体(1:600)(Cell Signaling Technology)とともに1時間インキュベートした。PBS中で5回洗浄した後、カバースリップを4’6’−ジアミジノ−2−フェニルインドール(DAPI)(Vector labs)を含有するVectashieldとともにガラススライド上に載せた。スライドをLeica DM4000上で60倍の倍率で視覚化した。平均で5つの画像を撮影し、二次抱合抗体単独での染色と比較して、自己蛍光が存在しないことを確認した。
9日目に、10mMのニコチンアミド、50ngのIGF−II、10ngのHGF、25ngのエキセンジン4、および10ngのアクチビンAを補充した前条件付けのα−MEM培地に細胞を移した(付属書類E)。37℃および5%のCO2で16時間、細胞をインキュベートした後、グルコース勾配を添加した。2.8mMのグルコースを3時間添加し、続いて20mMのグルコースをさらに3時間添加した。培地を単離し、メーカー指示に従って全ヒトプロインスリンELISA(Millipore)用に処理した。キットが供給する陽性対照は、7.9pMのインスリンを含有する。
omnicyteは、膵臓β細胞の特殊化に必要な因子を発現する。プラスチック付着性の骨髄由来幹細胞の初期の性質は、Gordon MY et al.,(1987)およびGordon MY(1994)(8,9)によってすでに証明されている。われわれは、単離されたばかりのomnicyteからNeuroD、PDX1、およびRfx6をはじめとする膵臓β細胞への特殊化に必要な転写因子の存在を確認した。これらの因子の発現は、当然、培養3日のうちに下方制御された(図5A)。
成熟β細胞の特殊化に必要な遺伝子一式を補完するために、ニューロゲニン3(図2)、Rfx6(図3)、MafA(図4)を標的とするsaRNAを細胞にトランスフェクトした。mRNA分析は、細胞培養9日目までにはプレプロインスリンおよびプロインスリンの発現が有意に上方制御されたことを明らかにしている(図6A)。
saRNAオリゴヌクレオチドのトランスフェクションに続くmRNA分析によってPDX1、Rfx6、ニューロゲニン3(Ngn3)、およびMafAの標的上方制御が確認されたため、われわれはこれらのオリゴヌクレオチドの組み合わせをomnicytesに添加して、この細胞が一層成熟したインスリン分泌性β細胞へと発達する兆候が示されるかどうかを評価した。3日目および6日目にトランスフェクトされたRfx6、Ngn3、およびMafA単独の組み合わせ、またはPDX1+Rfx6、PDX1+Ngn3、もしくはPDX1+MafAに続いて、9日目のグルコース反応テストのために材料および方法に記載したように細胞を準備し、続いて全プロインスリン分泌のためのELISAを実施した。示された数値は、ELISAキット(Millipore)が提供する7.9pMインスリンの陽性対照に対する割合であり、24ウェルプレートの単一ウェル中で培養された細胞を表す(図6B)。
この10年間にわたって幹細胞の理解と操作に関して得られた進歩は、研究者が再生医療にこのような細胞を用いることを可能にした。胚細胞は催腫瘍性のリスクを抱えていることから、その臨床応用への使用に対する倫理的な制約は、成体前駆細胞の使用に多くの焦点が当たることを意味した。この分野の研究は、臨床的使用に移すことのできない遺伝子組み換え成分に依存することから、かつては制限されてきた。しかしながら、omnicytesは、治療で補充用幹細胞として使用するために必要な安全基準をすべて満たすものである(22)。自己細胞から単離することが可能であり、無血清環境で力強い拡大能力があり、かつ肝細胞、膵細胞、心血管細胞、および神経細胞をはじめとする様々な系統への拘束に必要な転写因子をすでに発現していることから優れた発生的柔軟性を示す(22)。本明細書に示すデータは、新規の小型活性化RNA分子の使用により、omnicytesをインスリン分泌細胞中に誘導し得ることを明らかにしている。このような分子は疑いなく、ウイルス性のバックボーンを持つベクターに依存する標準技術を用いるよりも安全なアプローチを可能にする。インスリン産生の誘導に必要な下流の遺伝子を制御するカスケード効果を生じさせるには、標的転写因子を2つ3つだけ活性化すれば十分だということは、すでに明らかにした。
(プロ)インスリンの発現を活性化させるための短いRNAの設計
実施例1のように設計を行ったが、以下の相違があった。
MafAを上方制御するためのsaRNAを用いたインスリン産生の誘導
a)CD34+細胞をインスリン分泌表現型へと誘導するための最適化プロトコル
誘導プロトコルは、0日目に72時間、付着性のCD34+細胞集団(Omnicytes)を10ng/mlのアクチビンA(Sigma、UK)および2.5nMのグルコース(Sigma、USA)とともに、0.5%ペニシリン/ストレプトマイシン抗生物質中にStem Cell Factor(Invitrogen、USA)、250ng/mlのインターロイキン3およびインターロイキン6を含有する無血清CellGro培地(CellGenix、UK)に補充することで開始された。細胞を80%の密集度に拡大させてから、11nMのグルコース、5ngのエキセンジン4(Sigma、USA)、50ngのノギン(Sigma、USA)、5ngのbFGF(Sigma、USA)、および5ngのEGF(Sigma、USA)を72時間おきに添加し、それとともに、α−MEM培地を伴うNanofectamine(PAA、UK)(抗生物質なし)をメーカー指示に従って用いて、MafAを上方制御するために設計された150ngの二本鎖saRNAのトランスフェクションを実施した。このプロセスを、2/3日目、5/6日目、および8/9日目に3回繰り返した(適切な細胞密度の確立に依存)。最大のトランスフェクション効率を達成するため、24時間ごとに細胞をピペットでそっと取ることにより、細胞のクラスター形成を防いだ。
未分化CD34+細胞(対照)からの全タンパク質抽出物10μgおよびトランスフェクトしたCD34+細胞(上記プロトコルに従って調製)を、SDS−PAGE(Invitrogenからの4〜12%TrisGlycine変性アクリルアミドゲル)上で分離した。ニトロセルロース膜上に移した後、ブロットを抗アクチン(Sigma)(1:8000)および抗インスリン(Abcam)、続いてHRP抱合二次抗体(1:10000)(Dako)とともにインキュベートした。インスリンは、小さいαサブユニットとそれより大きいβサブユニットを含み、分子重量が約6kDa(キロダルトン)の小型ペプチドである。結果を図10に示す。
トランスフェクトしたCD34+細胞(上記プロトコルに従って調製)との比較による分化した(トランスフェクトしていない)CD34+細胞(対照)からの転写物の相対量(RQ)および全ヒト正常膵臓RNAを評価した。結果を図11に示す。
未分化CD34+細胞およびトランスフェクト細胞からの培地を、低グルコース(2.8nM)パルス、およびそれに続く高グルコース(16nM)パルスに細胞を暴露した後に、回収した。次に、この培地を、メーカーの指示に従って、ヒトインスリンおよびヒトC−ペプチド(Millipore)に対して特異的な免疫吸収ELISAプレート上に移した。図13に示すデータは、トランスフェクト細胞がインスリンを分泌することによってグルコース勾配に反応することができたことを示している。
本発明の短いRNAを用いたMafAの上方制御は、肝細胞に関しても達成された。肝上皮細胞を、Pr−1およびPr−2と表わされるsaRNAでトランスフェクトした(表4参照)。結果は、それぞれのsaRNAが単独でMafAの発現を上方制御することができ、かつ両方のsaRNAの組み合わせの方が、各saRNAの単独よりも大幅な上方制御を達成したことを示している。図14を参照されたい。
表
表中、Locは位置を、Eはエキソンを表す。
Claims (15)
- ヒト細胞中でのMafAの発現を上方制御することができる短いRNAであって、19〜25ヌクレオチド長の、(a)配列AUCUGUACUGGAUGAGCGG(配列番号1)を含むか、または(b)配列UUUCCCGCAGGAGAUUGAC(配列番号2)を含む第1の鎖を含み、かつ19〜25ヌクレオチド長の、前記第1の鎖とともに二本鎖を形成する第2の鎖を含む、短いRNA。
- 前記RNAのそれぞれの鎖が、その3’末端に対をなさない1〜3のヌクレオチドを持ち、オーバーハングを形成している、請求項1に記載の短いRNA。
- 前記3’オーバーハングが1または複数のウラシルヌクレオチドを含む、請求項2に記載の短いRNA。
- 前記3’オーバーハングが1または複数のウラシルヌクレオチドから成る、請求項2に記載の短いRNA。
- インスリンの産生および分泌をインビトロのヒト細胞によるグルコース反応性の方式で誘導するための、請求項1〜4のいずれか1項に定義された短いRNAの使用。
- 請求項1の(a)の部分に定義された短いRNAと、請求項1の(b)の部分に定義された短いRNAとが組み合わせて用いられる、請求項5に記載の使用。
- インスリンの産生および分泌をヒト細胞によるグルコース反応性の方式で誘導するインビトロの方法であって、前記細胞を、請求項1〜4のいずれか1項に定義された短いRNAに接触させることを含む方法。
- 請求項1の(a)の部分で定義された短いRNAと、請求項1の(b)の部分で定義された短いRNAとが組み合わせて用いられる、請求項7に記載の方法。
- 前記ヒト細胞が幹細胞、肝臓細胞、または膵臓細胞である、請求項7〜8のいずれか1項に記載の方法。
- 請求項1〜4のいずれか1項に定義された短いRNAを1または複数含む、エクスビボまたはインビトロのヒト細胞。
- 糖尿病または病的肥満の治療での使用のための、請求項10に記載の細胞。
- 糖尿病、脂肪肝、または病的肥満の治療での使用のための、請求項1〜4のいずれか1項に記載の短いRNA。
- 請求項1〜4のいずれか1項に定義された短いRNA、および/または請求項10に定義された細胞、ならびに薬学的に許容し得る希釈剤、担体、または賦形剤を含む、医薬組成物。
- 糖尿病、脂肪肝、病的肥満の治療での使用のための、請求項13に記載の医薬組成物。
- (i)ヒト細胞中のMafAの発現を上方制御することができ、19〜25ヌクレオチド長の鎖であって、配列AUCUGUACUGGAUGAGCGG(配列番号1)を含む第1の鎖を含み、かつ19〜25ヌクレオチド長の、前記第1の鎖とともに二本鎖を形成する第2の鎖を含む、短いRNA、および
(ii)ヒト細胞中のMafAの発現を上方制御することができ、19〜25ヌクレオチド長の鎖であって、配列UUUCCCGCAGGAGAUUGAC(配列番号2)を含む第1の鎖を含み、かつ19〜25ヌクレオチド長の、前記第1の鎖とともに二本鎖を形
成する第2の鎖を含む、短いRNA
を含む、キット。
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US10774331B2 (en) | 2020-09-15 |
US20130323211A1 (en) | 2013-12-05 |
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CA2817191A1 (en) | 2012-04-12 |
CN103314108A (zh) | 2013-09-18 |
US20180112222A1 (en) | 2018-04-26 |
US20200362352A1 (en) | 2020-11-19 |
WO2012046085A3 (en) | 2012-07-19 |
US20220282256A1 (en) | 2022-09-08 |
JP2014501492A (ja) | 2014-01-23 |
EP2625273A2 (en) | 2013-08-14 |
US20150176008A1 (en) | 2015-06-25 |
US11365414B2 (en) | 2022-06-21 |
US20130289258A1 (en) | 2013-10-31 |
EP2625272B1 (en) | 2015-09-16 |
KR20130132795A (ko) | 2013-12-05 |
EP2625273B1 (en) | 2015-01-07 |
EP2625272A2 (en) | 2013-08-14 |
US8835400B2 (en) | 2014-09-16 |
WO2012046084A3 (en) | 2012-09-27 |
US8916534B2 (en) | 2014-12-23 |
WO2012046085A2 (en) | 2012-04-12 |
CN103314108B (zh) | 2015-08-05 |
AU2011311344B2 (en) | 2016-09-08 |
AU2011311344A1 (en) | 2013-05-30 |
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