JP2020535834A - 遺伝子修飾されたベータ細胞による糖尿病の治療 - Google Patents
遺伝子修飾されたベータ細胞による糖尿病の治療 Download PDFInfo
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Abstract
Description
本出願は、2017年10月3日に出願の米国仮出願第62/567,604号;2017年10月4日に出願の第62/568,117号;2018年3月2日に出願の第62/637,913号;2018年4月25日に出願の第62/662,651号;2018年7月6日に出願の第62/694,634号;2018年7月11日に出願の第62/696,603号;2018年8月10日に出願の第62/717,587号;2018年8月20日に出願の第62/719,975号;および2018年9月21日に出願の第62/734,910号の優先権を主張し;それぞれの全体を参照により本明細書に組み込む。
配列表
本願は、ASCII形式で電子的に提出された配列表を含有し、ここにその全体を参照により組み込む。2018年9月25日に作成された前記ASCIIコピーは、054610-501001WO_SL.txtと命名され、サイズは8,668バイトである。
技術分野
本発明は、遺伝子修飾されたヒトベータ細胞およびそのような細胞を使用する方法を対象とする。遺伝子修飾された(トランスジェニック)ヒトベータ細胞は、逃避走性量(fugetactic amount)の逃避走性剤(fugetactic agent)を発現し、それによりヒト単核免疫細胞に対する保護をもたらす。一実施形態において、逃避走性剤は、例えば、CXCL12またはCXCL13である。一実施形態において、トランスジェニックベータ細胞は、ベクターを含み、ベクターは、逃避走性剤、好ましくはヒト逃避走性剤をコードする核酸配列を含む。一実施形態において、トランスジェニックベータ細胞は、老化するように更に修飾される。本発明の方法は、糖尿病患者、特に1型糖尿病患者に見られる環境を含めた高血糖環境においてインスリンを発現させるためのこれら細胞の使用を含む。
(a)ヒト対象由来のヒトプロジェニター細胞またはヒト多能性幹細胞の集団を入手するステップと;
(b)対象のプロジェニター細胞または多能性幹細胞をベータ細胞に分化させるステップと;
(c)ベータ細胞に逃避走性剤をコードする核酸分子を導入するステップとによって入手される。
本発明は、トランスジェニックであり、ヒト逃避走性剤(例えば、CXCL12、CXCL13)をコードする導入遺伝子を含み、または遺伝子修飾されて逃避走性量で内在性(ヒト)逃避走性剤(例えば、CXCL12、CXCL13)を発現もしくは過剰発現する自家/同種ヒトベータ細胞を提供する。好ましい実施形態において、本明細書に記載されるトランスジェニックベータ細胞は、老化するように更に修飾される。その方法の別の態様において、ベータ細胞は、トランスジェニックヒトベータ細胞の免疫破壊を阻害し、高血糖環境に応答してインスリンを産生するように修飾または処理されて、逃避走性剤(例えば、CXCL12、CXCL13)の有効量を発現する。
MKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP(配列番号7)を含むアミノ酸配列を有する。一実施形態において、CXCL13ポリペプチドは、Q53X90に対して少なくとも約85%、90%、92%、95%、96%、97%、98%、99%、または100%のアミノ酸配列同一性を有し、ケモカインまたは逃避走性活性を有する。一実施形態において、CXCL13ポリペプチドは、配列番号7に対して少なくとも約85%、90%、92%、95%、96%、97%、98%、99%、または100%のアミノ酸配列同一性を有し、ケモカインまたは逃避走性活性を有する。
FLAG=DYKDDDDKタンパク質タグ(配列番号10)
g/L=リットル当たりのグラム
HRP=ホースラディッシュペルオキシダーゼ
LDH=乳酸脱水素酵素
iBLOT=半乾燥タンパク質転写装置(Invitrogen)
MES=2−(N−モルホリノ)エタンスルホン酸
mL=ミリリットル
N/A=適用せず
nM=ナノモル濃度
PBMC=末梢血単核細胞
PBS=リン酸緩衝食塩水
TMB=3,3’,5,5’,−テトラメチルベンジジン
μL=マイクロリットル
μg=マイクログラム
×g=×重力
CXCL12−aおよび−bアイソフォームの発現レベルを評価するために使用したモデル細胞
HEK293細胞を、各アイソフォームに対して市販されているプラスミド(GenScriptから利用可能なプラスミド)を使用してCXCL12の異なる2つのアイソフォーム(アルファおよびベータ)でトランスフェクトした。トランスフェクトした細胞を、250μg/mL G418(ThermoFisherから市販されている)で選択し、各アイソフォームについて安定なプールを作製した。細胞を、適切な培地に3日間馴化させた。CXCL12アルファおよびCXCL12ベータを発現するトランスフェクトしたHEK293細胞からの馴化培地を、アッセイ希釈緩衝液で1:1に希釈した。2つの別々のプールを、各アイソフォームについて確立し、次いで溶液中の各アイソフォームの濃度を、標準化した濃度曲線を使用して吸収によって入手した。この実験を2回繰り返し、結果は以下の通りである:
CXCL12アルファ CXCL12ベータ
1. 310nM 1410nM
2. 274nM 1330nM
CXCL12の他のアイソフォームの発現レベルを評価するために使用したモデル細胞
HEK293細胞を、各アイソフォームに対して市販されているプラスミド(GenScriptから利用可能なプラスミド)を使用してCXCL12の異なる5つのアイソフォーム(アルファおよびベータ)でトランスフェクトした。トランスフェクトした細胞を、250μg/mL G418(ThermoFisherから市販されている)で選択し、各アイソフォームについて安定なプールを作製した。細胞を、適切な培地に3日間馴化させた。馴化培地を、MES緩衝液を用いて4〜8% NuPageゲル(ThermoFisherから市販されている)で分離し、ニトロセルロース(iBLOT)に移した。
トランスジェニックベータ細胞の調製
ヒト誘導多能性幹細胞から得られた膵臓ベータ細胞を、TAKARA Bio USA社(Mountain View、CA)から購入し、提供された指示に従って培養した。
トランスジェニックベータ細胞は、PBMCを忌避する
実施例3のトランスジェニックベータ細胞を、比30:1(PBMC対ベータ細胞)でヒト末梢血単核細胞(PBMC、Innovative Research、Novi、MI)と接触させた。簡潔には、PBMCをベータ完全培養培地に再懸濁し、計数し、調整して、(発現されるCXCL12の希釈を最小限にするために)PBMC 100μLの添加によりPBMC:ベータ細胞比30:1を可能にした。最終容量は、1.1mLであった。PBMCを含まないベータ細胞のバックグラウンド対照およびベータ細胞を含まないPBMCも作製した。各サンプルから培地150μLを直ちに取り出し、1200×gで10分間遠心分離した。上清を取り出し、4℃で貯蔵した(時間0)。細胞をインキュベーターに戻し、24および48時間後両方で時間0のサンプルと類似の方法でサンプル採取した。
トランスジェニックベータ細胞の別の調製
ベータ細胞を、1型糖尿病の対象から単離し、CXCL12をコードするレトロウイルス発現ベクターもしくはCXCL12をコードしない対照レトロウイルスベクターでin vitroでトランスフェクトするまたは感染させる。CXCL12をコードするレトロウイルスベクターを保有しているトランスジェニックベータ細胞を、Poznansky et al., Journal of Clinical Investigation, 109, 1101 (2002)内の前述のボイデンチャンバーアッセイを使用して逃避走性量のCXCL12の発現についてアッセイする。少なくとも100nM CXCL12を発現しているトランスジェニックベータ細胞は、本アッセイにおいて免疫細胞を忌避することになると予想される。
トランスジェニックサイトカイン発現に対するトランスジェニックベータ細胞の強制的老化の効果
ベータ細胞を、実施例3に記載の通り調製した。SDF1a/CXCL12aおよびSDF1b/CXCL12bの発現レベルをマイトマイシンC(Santa Cruz Biotechnologyから利用可能)処理前にELISAによってアッセイして、ベースライン発現(「前」)を決定した。培地を、老化を誘導することが公知の薬剤であるマイトマイシンC 10μg/mLを含有する新鮮な培地と置きかえた。細胞を、インキュベーターに2時間戻した。マイトマイシンC含有培地を、穏やかなピペット操作によって除去した。細胞を、PBSで2回洗浄した。2回目の洗浄後に、細胞に新鮮な完全培地を与えた。SDF1a/CXCL12aまたはSDF1b/CXCL12b発現を、ELISAアッセイによって決定した。
PBMC負荷に対するトランスジェニックベータ細胞の強制的老化の効果
ベータ細胞を、実施例3に記載の通り調製した。細胞を、実施例4に記載の通りマイトマイシンCまたは対照で処理した。細胞を、実施例2に記載の通りPBMCと接触させた。
インスリン産生に対するトランスジェニックベータ細胞の強制的老化の効果
ベータ細胞を、実施例3に記載の通り調製した。細胞を実施例4に記載の通りマイトマイシンCまたは対照で処理した。
トランスジェニックベータ細胞のin vivo評価
ヒト化免疫系を有するヒト化マウス、例えば、N. Walsh, "Humanized mouse models of clinical disease," Annu Rev Pathol 2017, 12, 187-215; E. Yoshihara et alを参照、に逃避走性量のCXCL12を発現しているトランスジェニックヒトベータ細胞または対照トランスジェニックヒトベータ細胞のいずれかを投与し、マウスにおけるインスリンの産生およびトランスジェニックベータ細胞の生存を、初回投与後の様々な時点でアッセイする。逃避走性量のCXCL12を発現しているトランスジェニックヒトベータ細胞は、対照トランスジェニックヒトベータ細胞より長期間生存することになると考えられる。逃避走性量のCXCL12を発現しているトランスジェニックベータ細胞を受けたマウスは、対照トランスジェニックヒトベータ細胞を受けたマウスより多量のヒトインスリンを有することにもなり、より高いレベルのヒトインスリンは、対照トランスジェニックヒトベータ細胞を投与したマウスにおけるレベルと比較して、より長い期間持続することになることも考えられる。
Claims (23)
- ヒトベータ細胞をヒト免疫細胞に対して抵抗性にするのに十分な量でヒト逃避走性剤(fugetactic agent)を発現または過剰発現するヒトベータ細胞であって、インスリンを発現することができ、細胞分裂できない、ヒトベータ細胞。
- ヒトCXCL12をコードする核酸配列をそれ自体に含むベクターを含むヒトベータ細胞であって、ヒト免疫細胞に対して抵抗性である、ヒトベータ細胞。
- 1型糖尿病の対象から入手した自家ベータ細胞である、請求項1または2に記載のヒトベータ細胞。
- 同種ベータ細胞である、請求項1または2に記載のヒトベータ細胞。
- 前記ヒト免疫細胞が、NK細胞、細胞障害性T細胞および/またはB細胞を含む、請求項1から4のいずれか一項に記載のヒトベータ細胞。
- 逃避走性量(fugetactic amount)でヒトCXCL12を発現する、請求項1から5のいずれか一項に記載のヒトベータ細胞。
- 前記CXCL12が、CXCL12アルファおよびCXCL12ベータからなる群から選択される、請求項6に記載のヒトベータ細胞。
- 内在性CXCL12コード領域の上流にトランスジェニック調節領域を含むヒトベータ細胞であって、ヒト免疫細胞に抵抗性である、ヒトベータ細胞。
- 自家ベータ細胞である、請求項8に記載のヒトベータ細胞。
- 同種ベータ細胞である、請求項8に記載のヒトベータ細胞。
- 1型糖尿病の対象から入手または由来する自家ベータ細胞である、請求項9に記載のヒトベータ細胞。
- 1型糖尿病でない対象から入手または由来する同種ベータ細胞である、請求項10に記載のヒトベータ細胞。
- 前記トランスジェニック調節領域が、外来性構成的または誘導可能なプロモーターである、請求項8から12のいずれか一項に記載のヒトベータ細胞。
- 逃避走性量でCXCL12を発現する、請求項8から10のいずれか一項に記載のヒトベータ細胞。
- 前記CXCL12が、CXCL12アルファおよびCXCL12ベータからなる群から選択される、請求項14に記載のヒトベータ細胞。
- CXCL12ベータを発現する、請求項15に記載のヒトベータ細胞。
- 細胞分裂できない、請求項2から16のいずれか一項に記載のヒトベータ細胞。
- 前記ヒトCXCL12が、CXCL12アルファ、CXCL12ベータ、CXCL12デルタおよびCXCL12ガンマからなる群から選択される、請求項1から17のいずれか一項に記載のヒトベータ細胞。
- 前記ヒト免疫細胞が、NK細胞、細胞障害性T細胞およびB細胞を含む、請求項8から18のいずれか一項に記載のヒトベータ細胞。
- 高血糖環境に応答してインスリンを産生させる方法であって、請求項1から19のいずれか一項に記載のヒトトランスジェニックベータ細胞の集団を前記環境と接触させるステップを含む、方法。
- 前記高血糖環境が、ヒト免疫細胞を含む、請求項20に記載の方法。
- 前記ヒト免疫細胞が、NK細胞、T細胞およびB細胞を含む、請求項21に記載の方法。
- 糖尿病の治療に使用する組成物であって、請求項1から19のいずれか一項に記載のヒトトランスジェニックベータ細胞を含む、組成物。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170196913A1 (en) * | 2016-01-07 | 2017-07-13 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Enhancement of glucose-stimulated insulin secretion by cells through induction of cellular senescence |
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AU4230900A (en) | 1999-04-08 | 2000-10-23 | General Hospital Corporation, The | Purposeful movement of human migratory cells away from an agent source |
US6903073B2 (en) * | 1999-12-10 | 2005-06-07 | The General Hospital Corporation | Methods to stimulate insulin production by pancreatic beta-cells |
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ES2587395T3 (es) | 2008-06-04 | 2016-10-24 | Cellular Dynamics International, Inc. | Procedimientos para la producción de células IPS usando un enfoque no vírico |
US9683215B2 (en) | 2008-08-22 | 2017-06-20 | President And Fellows Of Harvard College | Methods of reprogramming cells |
EP3450545B1 (en) | 2008-10-24 | 2023-08-23 | Wisconsin Alumni Research Foundation | Pluripotent stem cells obtained by non-viral reprogramming |
US20120053119A1 (en) * | 2009-02-11 | 2012-03-01 | Habener Joel F | Therapeutic method for increasing pancreatic beta cell mass |
US9109245B2 (en) | 2009-04-22 | 2015-08-18 | Viacyte, Inc. | Cell compositions derived from dedifferentiated reprogrammed cells |
WO2012025925A1 (en) * | 2010-08-24 | 2012-03-01 | Rappaport Family Institute For Research In The Medical Sciences | Methods of improving transplantation using sdf-1alpha |
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US9775816B2 (en) | 2013-11-07 | 2017-10-03 | The General Hospital Corporation | Eluting matrix and uses thereof |
US9371516B2 (en) * | 2014-09-19 | 2016-06-21 | Regenerative Medical Solutions, Inc. | Compositions and methods for differentiating stem cells into cell populations comprising beta-like cells |
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-
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170196913A1 (en) * | 2016-01-07 | 2017-07-13 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Enhancement of glucose-stimulated insulin secretion by cells through induction of cellular senescence |
Non-Patent Citations (2)
Title |
---|
PLOS ONE, vol. Vol.8, Issue 3, e59679, JPN6022037531, 2013, ISSN: 0005063896 * |
TRANSPLANTATION, vol. Vol.83, No.2, pp.174-183, JPN6022037530, 2007, ISSN: 0005063895 * |
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