JP6855579B2 - T細胞を得る方法及び使用 - Google Patents
T細胞を得る方法及び使用 Download PDFInfo
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Description
本発明の目的は、転写因子Hoxb5を用いてBリンパ系細胞のTリンパ系細胞への分化転換を誘導する方法、その関連製品及び使用を提供することにある。
上記目的を達成するために、第1の態様において、本発明は、Hoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物の、(i)Bリンパ系細胞を機能性T細胞に分化転換するための製剤、(ii)免疫応答を増強するため、好ましくはT細胞関連免疫応答を増強する医薬、及び/又は(iii)免疫不全症を予防又は治療するため、好ましくはT細胞免疫不全症を予防又は治療するための医薬の調製における使用を提供する。
(1)Hoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物を前記Bリンパ系細胞に導入し、Hoxb5が過剰発現したBリンパ系細胞を得ることと、
(2)分化転換を誘導するために、ステップ(1)で得られたBリンパ系細胞を被験者の体内に移植してT細胞前駆細胞を得てから、分化によって機能性T細胞を得ることと、を含むBリンパ系細胞を機能性T細胞に分化転換するための方法を提供する。
まず、Hoxb5を用いてB細胞を体内で分化転換して再生したT細胞を産生する全実験フローチャートを設計した(図1A)。次に、LasergeneソフトウェアによってHoxb5遺伝子の切断部位の情報を分析し、上流、下流の組換え酵素切断部位としてXhoI/SnaBIを選択し、Hoxb5遺伝子をレトロウイルスベクター(pMYs−IRES−EGFP)に組換え構築した。DH5αコンピテントが連結産物へ形質転換した後、アンピシリンプレートを用いて陽性組換えクローンをスクリーニングした。さらに菌液PCR及びスクリーニングした組換えプラスミを参照しながらDNAシークエンシングし、組み換えが成功したpMY−Hoxb5−IRES−EGFPレトロウイルスプラスミドを確定し、内毒素除去による大規模抽出を将来の使用のために行った。続いて、リン酸カルシウムトランスフェクションを用いてHoxb5組換えベクターをレトロウイルスパッケージング細胞株(Plat−E細胞)に移した。24時間後、トランスフェクション率を蛍光顕微鏡でチェックし、トランスフェクション率≧85%を確保した(図1B)。48時間後、Hoxb5を含有するレトロウイルス上清を将来の使用のために集めると同時に、4〜6週齢のマウスを殺し、マウスの骨髄を取り出して単一細胞懸濁液に調製した。次に、骨髄単一細胞懸濁液中のB220+細胞を磁気ビーズ富化法により富化した。Pro−/Pre−B抗体組合せ(CD19/B220/CD93/IgM)で染色した後、超高速フローソーター(Moflo Astrios)を用いてPro−/Pre−B細胞(CD19+B220+CD93+IgM−)を選別した。500gで低温遠心分離した後に選別されたPro−/Pre−B細胞を集めた。続いて、富化されたPro−/Pre−B細胞をBリンパ系細胞完全培地に入れ、12〜14時間予備刺激し、レトロウイルスを1:1の体積でopti−MEM基礎培地に添加すると同時に、8μg/ml polybreneを添加し、将来の使用のために均一に混合した。350gで5分間遠心分離によって予備刺激したPro−/Pre−B細胞を集めた。続いて、Pro−/Pre−B細胞を、再構成したレトロウイルス溶液を用いて再懸濁して、低接着性6穴培養プレートに入れて、35℃の恒温水平遠心分離機において805gで90分間遠心分離して感染させた。遠心分離終了後、6穴培養プレートを細胞インキュベーター(37℃、5%CO2)に戻させ、2時間静置培養した。続いて、Pro−/Pre−B細胞を350gでの遠心分離によって集めた。予熱した完全培地を用いてPro−/Pre−B細胞を再懸濁して、1ml当たり2〜4百万個の細胞の密度を保持した。24時間後、遠心感染を1回繰り返した。2回目の感染の24時間後、少量の細胞を採取し、Pro−/Pre−B細胞感染率をトリパンブルーで計数し、フローサイトメトリーによって分析した結果において、対照群とHoxb5群のいずれか一方の感染率が50%を超えることを示した(図1C)。続いて、懸濁細胞を350gでの遠心分離によって集め、レシピエントマウス1匹当たり6〜8百万個の生存細胞の用量で移植し、レシピエントマウスは4時間前に致死量未満(6.5Gy)の照射処理を行う。同時に、照射後のマウスの腸感染を予防するためにネオマイシン硫酸塩1.14g/Lをマウスの給水に添加した。
レトロウイルス組込み部位の不確実性及び発現レベルの不均一性などの潜在的な問題を排除するために、本発明者らは、Hoxb5ノックイン動物モデル(LSL−Hoxb5)を更に構築した(図4A)。Hoxb5ノックインマウスとBリンパ系細胞特異的発現CreモデルマウスCD19−Creをヘテロ接合することにより、LSL−Hoxb5 CD19−Cre(以下、Hoxb5マウスと略記する)を得る。Hoxb5マウスの胸腺を分析することで、その中に少量のCD3+EGFP+の再生したT細胞を有することを発見した(図4B)。同様に、再生したT細胞を選別してB細胞Ig重鎖VDJ及び軽鎖(κ、λ)再配列を行って同定した(図4C)。Tベクターと組み合わせてシークエンシングした結果は、再生したT細胞がB細胞特徴の再配列を有し、それらがB細胞の分化転換に由来することを示した。
Claims (9)
- Hoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物の、Pro−/Pre−B細胞を機能性T細胞に分化転換するための製剤の調製における使用。
- 形質転換したBリンパ系細胞であって、前記Bリンパ系細胞において、Hoxb5を過剰発現させるようにHoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物を導入し、且つ前記Bリンパ系細胞は、T細胞に分化転換する潜在能力を有する、ことを特徴とする形質転換したBリンパ系細胞。
- 前駆B細胞又はプレB細胞である、ことを特徴とする請求項2に記載の形質転換したBリンパ系細胞。
- 請求項2又は3に記載の形質転換したBリンパ系細胞の、T細胞を再生するための医薬の調製における使用であって、前記Bリンパ系細胞はPro−/Pre−B細胞である使用。
- 活性成分としての請求項2又は3に記載の形質転換したBリンパ系細胞、及び薬学的に許容可能であるベクター、賦形剤又は希釈剤を含む、医薬組成物であって、前記Bリンパ系細胞はPro−/Pre−B細胞である医薬組成物。
- Bリンパ系細胞を機能性T細胞に分化転換するための方法であって、
(1)Hoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物を前記Bリンパ系細胞に導入し、Hoxb5が過剰発現したBリンパ系細胞を得ることと、
(2)分化転換を誘導するために、ステップ(1)で得られたBリンパ系細胞を被験マウスの体内に移植してT細胞前駆細胞を得てから、分化によって機能性T細胞を得ることとを含む、方法。 - 前記Bリンパ系細胞は、前駆B細胞又はプレB細胞である、ことを特徴とする請求項6に記載の方法。
- ステップ(1)において、前記Hoxb5、Hoxb5をコードする核酸分子、又は前記核酸分子を含む構築物は、トレーサー、好ましくは蛍光タンパク質トレーサー、より好ましくはEGFP蛍光タンパク質トレーサーを担持する、ことを特徴とする請求項6に記載の方法。
- ステップ(1)において、トランスフェクション又はウイルス感染、好ましくはレトロウイルス感染により、Hoxb5をコードする核酸分子又は前記核酸分子を含む構築物をBリンパ系細胞に導入する、ことを特徴とする請求項6〜8のいずれか一項に記載の方法。
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