JP6812478B2 - ヌクレアーゼ媒介性遺伝子破壊を増強するための方法および組成物 - Google Patents
ヌクレアーゼ媒介性遺伝子破壊を増強するための方法および組成物 Download PDFInfo
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Description
この出願は、2013年2月25日に出願された米国仮出願第61/769,038号(この開示は、その全体が参考として本明細書に援用される)の利益を主張する。
本発明の実施形態において、例えば以下の項目が提供される。
(項目1)
細胞における標的化されたゲノム破壊のための方法であって、前記方法は:
少なくとも1種のヌクレアーゼを前記細胞に投与するステップであって、前記ヌクレアーゼが、前記細胞における内因性ゲノム配列を開裂する、ステップと、
典型的または代替的非相同末端結合(NHEJ)の少なくとも1種の阻害剤を前記細胞に投与するステップと
を含む、方法。
(項目2)
前記阻害剤が、DNA依存性プロテインキナーゼ触媒サブユニット(DNA−PKcs)、ポリ−(ADP−リボース)ポリメラーゼ1/2(PARP1/2)およびこれらの組合せからなる群より選択されるタンパク質を阻害する、項目1に記載の方法。
(項目3)
前記タンパク質が、PARP1、Ku70/80、DNA−PKcs、XRCC4/XLF、リガーゼIV、リガーゼIII、XRCC1、アルテミス、ポリヌクレオチドキナーゼ(PNK)およびこれらの組合せからなる群より選択される、項目2に記載の方法。(項目4)
前記標的化されたゲノム破壊が、欠失を含む、項目1〜3のいずれかに記載の方法。
(項目5)
前記標的化されたゲノム破壊が、挿入を含む、項目1〜3のいずれかに記載の方法。
(項目6)
前記方法が、外因性配列を前記細胞に投与するステップをさらに含み、前記外因性配列は、前記ヌクレアーゼによる開裂後に、相同組換え修復(HDR)機構により前記ゲノムに組み込まれる、項目5に記載の方法。
(項目7)
前記外因性配列が、タンパク質をコードする配列、調節配列、構造RNAをコードする配列およびこれらの組合せからなる群より選択される、項目6に記載の方法。
(項目8)
HDRまたはマイクロホモロジー媒介末端結合(MMEH)を活性化する小分子またはポリペプチドを投与するステップをさらに含む、項目1〜7のいずれかに記載の方法。
(項目9)
前記活性化因子が、Mre11、Rad50、NSB1、Rad52およびこれらの組合せからなる群より選択されるタンパク質を活性化する、項目8に記載の方法。
(項目10)
前記ヌクレアーゼが、ジンクフィンガータンパク質、CRISPR/Cas系およびこれらの組合せを含むヌクレアーゼからなる群より選択される、項目1〜9のいずれかに記載の方法。
(項目11)
前記ヌクレアーゼが、発現ベクターを使用して投与されるか、またはmRNAとして投与される、項目10に記載の方法。
(項目12)
項目1〜11のいずれかに記載の方法によって作製された細胞。
(項目13)
真核細胞である、項目12に記載の細胞。
(項目14)
前記真核細胞が、哺乳動物細胞または植物細胞である、項目13に記載の細胞。
(項目15)
項目12に記載の細胞を産生するためのキットであって、前記キットは、前記細胞における標的部位に結合する少なくとも1種のヌクレアーゼと、典型的または代替的NHEJに関与するタンパク質の1種または複数の阻害剤とを含み、そして任意選択で、HDRもしくはMMEJ修復経路の活性化因子および/または外因性配列をさらに含む、キット。
Res.;34巻(21号):6170〜82頁を参照)。
本明細書に開示されている方法の実施ならびに組成物の調製および使用は、他に断りがなければ、分子生物学、生化学、クロマチン構造および解析、計算化学、細胞培養、組換えDNAならびに関連分野における従来技法を当業者の技能範囲内で用いる。これらの技法は、文献中で十分に説明されている。例えば、Sambrookら、MOLECULAR CLONING: A LABORATORY MANUAL、第2版、Cold Spring Harbor Laboratory Press、1989年および第3版、2001年;Ausubelら、CURRENT PROTOCOLS IN MOLECULAR BIOLOGY、John Wiley & Sons、New York、1987年および定期更新;METHODS IN ENZYMOLOGYシリーズ、Academic Press、San Diego;Wolffe、CHROMATIN STRUCTURE AND FUNCTION、第3版、Academic
Press、San Diego、1998年;METHODS IN ENZYMOLOGY、304巻、「Chromatin」(P.M. WassarmanおよびA. P. Wolffe編)、Academic Press、San Diego、1999年;ならびにMETHODS IN MOLECULAR BIOLOGY、119巻、「Chromatin Protocols」(P.B. Becker編)Humana Press、Totowa、1999年を参照されたい。
用語「核酸」、「ポリヌクレオチド」および「オリゴヌクレオチド」は、互換的に使用されており、直鎖状または環状コンフォメーションの、一本または二本鎖型のいずれかのデオキシリボヌクレオチドまたはリボヌクレオチドポリマーを指す。本開示の目的のために、これらの用語は、ポリマーの長さに関して限定的に解釈するべきではない。この用語は、天然ヌクレオチドの公知のアナログと共に、塩基、糖および/またはリン酸部分で修飾されたヌクレオチド(例えば、ホスホロチオエート骨格)を包含することができる。一般に、特定のヌクレオチドのアナログは、同じ塩基対形成特異性を有する、即ち、Aのアナログは、Tと塩基対形成する。
1種または複数のヌクレアーゼによる細胞ゲノムの開裂後に遺伝子破壊(例えば、欠失、付加および/または標的化組込み)を増加させるための組成物および方法が、本明細書に記載されている。記載されている組成物および方法は、ゲノム修飾が必要とされる種々の細胞型における遺伝子破壊の増加において有効である。本明細書に記載されている方法において、天然修復機構が阻害され、ヌクレアーゼに媒介される遺伝子破壊が増強されるように、ヌクレアーゼの前、後またはこれと同時発生的にDNA修復阻害剤が投与される。加えて、本明細書に記載されている方法において、ヌクレアーゼおよび/または阻害剤の複数回投与(いずれかの順序の)を使用することができる。ヌクレアーゼにより遺伝子破壊を増加させるための迅速かつ効率的な方法を使用して、ノックアウト細胞株の作製を容易にし、細胞および/またはトランスジェニック生物の作出における標的遺伝子へのドナー分子の挿入を増加させ、種々の細胞型におけるヌクレアーゼの治療適用を増加させることができる。
本明細書に記載されている組成物および方法は、ヌクレアーゼ媒介性遺伝子修飾を増加させる。したがって、本明細書において、ヌクレアーゼ、例えば、DNAに結合する結合ドメインおよび開裂(ヌクレアーゼ)ドメインを含む融合タンパク質が提供される。そのようなものとして、遺伝子修飾は、ヌクレアーゼ、例えば、操作されたヌクレアーゼを使用して達成することができる。操作されたヌクレアーゼの技術は、天然起源のDNA結合タンパク質の操作に基づく。
ジンクフィンガーDNA結合ドメイン、TALE DNA結合ドメインまたはメガヌクレアーゼ由来のDNA結合ドメインが挙げられるがこれらに限定されない、いずれかのDNA結合ドメインが、本明細書に開示されている組成物および方法において使用されるヌクレアーゼにおいて使用され得る。
Biochem.70巻:313〜340頁;Isalanら(2001年)Nature Biotechnol.19巻:656〜660頁;Segalら(2001年)Curr. Opin. Biotechnol.12巻:632〜637頁;Chooら(2000年)Curr. Opin. Struct. Biol.10巻:411〜416頁;米国特許第6,453,242号、第6,534,261号、第6,599,692号、第6,503,717号、第6,689,558号、第7,030,215号、第6,794,136号、第7,067,317号、第7,262,054号、第7,070,934号、第7,361,635号、第7,253,273号および米国特許出願公開第2005/0064474号、第2007/0218528号、第2005/0267061号を参照されたい。
本明細書に記載されているいずれかのDNA結合ドメインと共に、いずれかのヌクレアーゼを使用することができる。ヌクレアーゼは、異種DNA結合および開裂ドメイン(例えば、ジンクフィンガーヌクレアーゼ;TALENおよびメガヌクレアーゼDNA結合ドメインと異種開裂ドメイン)を含むことができる、あるいは、天然起源のヌクレアーゼのDNA結合ドメインは、選択された標的部位に結合するよう変更することができる(例えば、同族結合部位とは異なる部位に結合するよう操作されたメガヌクレアーゼ)。例えば、DNA結合特異性が調整されたホーミングエンドヌクレアーゼの操作について記載されている、Chamesら(2005年)Nucleic Acids Res 33巻(20号):e178頁;Arnouldら(2006年)J. Mol. Biol.355巻:443〜458頁およびGrizotら(2009年)Nucleic Acids Res 7月7日電子出版を参照されたい。加えて、ZFPの操作も記載されている。例えば、米国特許第6,534,261号、第6,607,882号、第6,824,978号、第6,979,539号、第6,933,113号、第7,163,824号および第7,013,219号を参照されたい。
Biotechnol.19巻:656〜660頁;Segalら(2001年)Curr. Opin. Biotechnol.12巻:632〜637頁;Chooら(2000年)Curr. Opin. Struct. Biol.10巻:411〜416頁を参照されたい。操作されたジンクフィンガー結合ドメインは、天然起源のジンクフィンガータンパク質と比較して、新規結合特異性を有することができる。操作方法として、合理的設計および様々な種類の選択が挙げられるがこれらに限定されない。合理的設計は、例えば、三つ組(または四つ組)ヌクレオチド配列および個々のジンクフィンガーアミノ酸配列を含むデータベースの使用を含み、データベースにおいて、各三つ組または四つ組ヌクレオチド配列は、特定の三つ組または四つ組配列に結合するジンクフィンガーの1種または複数のアミノ酸配列に関連付けられている。例えば、参照によりその全体が本明細書に組み込まれる、米国特許第6,453,242号および第6,534,261号を参照されたい。
上に詳細に記載されている通り、ヌクレアーゼ(ZFN、TALENおよび/またはCRISPR/CasのRNA)におけるDNAドメインは、遺伝子座におけるいずれかの選択した配列に結合するよう操作することができる。操作されたDNA結合ドメインは、天然起源のDNA結合ドメインと比較して、新規結合特異性を有することができる。操作方法として、合理的設計および様々な種類の選択が挙げられるがこれらに限定されない。合理的設計は、例えば、三つ組(または四つ組)ヌクレオチド配列および個々の(例えば、ジンクフィンガー)アミノ酸配列を含むデータベースの使用を含み、該データベースにおいて、各三つ組または四つ組ヌクレオチド配列は、特定の三つ組または四つ組配列に結合するDNA結合ドメインの1種または複数のアミノ酸配列に関連付けられている。例えば、参照によりその全体が本明細書に組み込まれる、米国特許第6,453,242号および第6,534,261号を参照されたい。TALエフェクタードメインの合理的設計を行うこともできる。例えば、米国特許第8,586,526号を参照されたい。
本発明の実施において、DNA修復経路のいずれかの阻害剤を使用することができる。典型的には、阻害剤は、誤りのない典型的NHEJおよび/またはPARP1媒介性代替的NHEJ修復、または例えば、リン酸化、ユビキチン化およびSUMO化による翻訳後修飾によるそれらの上流調節に関与する酵素に向けられる。阻害剤は、小分子であってもよく、その例として、DNA修復に関与する1種または複数のタンパク質を阻害する市販の小分子を含む小分子が挙げられるがこれに限定されない。
上に記す通り、例えば、変異体遺伝子の補正のためまたは野生型遺伝子の発現増加のために、外因性配列(「ドナー配列」または「ドナー」とも呼ばれる)の挿入を行うこともできる。ドナー配列は、典型的に、該ドナー配列が配置されるゲノム配列と同一ではないことが容易に明らかとなる。ドナー配列は、相同性の2領域に隣接する非相同配列を含有して、目的の位置における効率的HDRを可能にすることができる。その上、ドナー配列は、細胞クロマチンにおける目的の領域と相同ではない配列を含有するベクター分子を含むことができる。ドナー分子は、細胞クロマチンに対し相同性のいくつかの不連続領域を含有することができる。例えば、目的の領域に通常存在しない配列の標的化挿入のため、前記配列は、ドナー核酸分子に存在し、目的の領域における配列に対し相同性の領域に隣接することができる。
本明細書で記載されているように使用されるタンパク質(例えば、ZFP、TALEN、CRISPR/Cas)および/またはこれをコードするポリヌクレオチド、いずれかのドナーポリヌクレオチドならびにDNA修復阻害剤(例えば、小分子)は、いずれか適した手段により標的細胞に送達することができる。
6巻(10号):1149〜1154頁(1988年);Vigne、Restorative Neurology and Neuroscience 8巻:35〜36頁(1995年);KremerおよびPerricaudet、British Medical Bulletin 51巻(1号):31〜44頁(1995年);Haddadaら、Current Topics in Microbiology and
Immunology内、DoerflerおよびBoehm(編)(1995);ならびにYuら、Gene Therapy 1巻:13〜26頁(1994年)を参照されたい。
Ther.2巻:291〜297頁(1995年);Behrら、Bioconjugate Chem.5巻:382〜389頁(1994年);Remyら、Bioconjugate Chem.5巻:647〜654頁(1994年);Gaoら、Gene
Therapy 2巻:710〜722頁(1995年);Ahmadら、Cancer Res.52巻:4817〜4820頁(1992年);米国特許第4,186,183号、第4,217,344号、第4,235,871号、第4,261,975号、第4,485,054号、第4,501,728号、第4,774,085号、第4,837,028号および第4,946,787号を参照)。
上に記す通り、DNA構築物は、種々の従来技法により所望の植物宿主(例えば、そのゲノム)に導入することができる。このような技法の総説に関して、例えば、WeissbachおよびWeissbach、Methods for Plant Molecular Biology(1988年、Academic Press、N.Y.)第VIII節、421〜463頁;ならびにGriersonおよびCorey、Plant Molecular Biology(1988年、第2版)、Blackie、London、7〜9章を参照されたい。
4巻:1495〜1505頁)が挙げられるがこれらに限定されない。植物細胞形質転換のための追加的な方法は、マイクロインジェクション、シリコンカーバイド媒介性DNA取込み(Kaepplerら(1990年)Plant Cell Reporter
9巻:415〜418頁)および微粒子銃(Kleinら(1988年)Proc. Nat. Acad. Sci. USA 85巻:4305〜4309頁;およびGordon−Kammら(1990年)Plant Cell 2巻:603〜618頁を参照)を含む。
Plant Cell Culture内、124〜176頁、Macmillian
Publishing Company、New York、1983年;およびBinding、Regeneration of Plants, Plant Protoplasts、21〜73頁、CRC Press、Boca Raton、1985年に記載されている。再生は、植物カルス、外植片、器官、花粉、胚またはこれらの部分から得ることもできる。このような再生技法は、Kleeら(1987年)Ann. Rev. of Plant Phys.38巻:467〜486頁に全般的に記載されている。
開示されている組成物および方法は、臨床背景で実行可能な臨床適用のヌクレアーゼに基づく治療法および農業(植物)適用を含む、いずれかの細胞型においてヌクレアーゼ媒介性ゲノム修飾を増加させることが望まれる、いずれかの適用のために使用することができる。例えば、本明細書に記載されている方法は、次のシナリオにおけるZFN、TALENおよび/またはCRISPR/Cas系の治療効果を改善する:CD34+細胞におけるex vivoおよびin vivo遺伝子破壊(CCR5)(例えば、米国特許第7,951,925号を参照);CD34+細胞における異常ヘモグロビン症のex vivoおよびin vivo遺伝子補正(例えば、米国特許出願第61/694,693号を参照);および/またはリソソーム蓄積症および血友病の治療法のためのアルブミン遺伝子座のex vivoおよびin vivo遺伝子付加(例えば、米国特許出願公開第20140017212号および第20130177983号を参照)。
上述の方法のいずれかを行うためのキットも提供される。キットは、典型的に、1種もしくは複数のヌクレアーゼをコードするポリヌクレオチド、1種もしくは複数のDNA修復阻害剤、および/または本明細書に記載されているドナーポリヌクレオチド、ならびにヌクレアーゼおよび/またはドナーポリヌクレオチドが導入される細胞にDNA修復阻害剤を投与するための説明書を含有する。キットは、細胞、細胞の形質転換のためのバッファー、細胞のための培養培地、および/またはアッセイを行うためのバッファーを含有することもできる。典型的には、キットは、キットの他の構成成分に添付または他の仕方で付随した説明書、包装または宣伝チラシ等、いずれかの材料を含むラベルも含有する。
図2Aに示す通り、Lipofectamine(商標)RNAiMAXにより、アルブミン特異的ZFN対(米国特許出願公開第20130177983号および第20130177968号を参照)をコードするmRNAをHepa 1−6細胞にトランスフェクトし、小分子阻害剤で2回処理した。特に、mRNA送達の3時間後に、それぞれ5μMおよび3〜5μMの濃度で、阻害剤オラパリブ(PARP1阻害剤)およびNU7441(DNA−PKcs阻害剤)を添加した。対照として、10μMの濃度のATM阻害剤KU55933を添加した(ATMは、DSB修復における直接的な役割を持たないが、DNA−PKcsに関係する)。15時間後に、新鮮な阻害剤を培地に添加して、細胞培養培地における阻害剤の減衰を相殺した。
A.ZFN
Amaxa(登録商標)エレクトロポレーションシステムにより、CCR5特異的ZFN対(米国特許第7,951,925号を参照)をコードするプラスミドDNAと、標的領域に対し相同性を有し特有の制限部位(AvrII)を有する一本鎖オリゴヌクレオチド(ssオリゴ)(120bp)をK562細胞にトランスフェクトした。次に、実施例1に記載されている(図2Aに概要を示す)小分子阻害剤で細胞を2回処理した。特に、ZFNコードDNA送達の5時間後に、それぞれ5μMおよび3μMの濃度の阻害剤オラパリブ(PARP1/2阻害剤)およびNU7441(DNA−PKcs阻害剤)を添加した。対照として、10μMの濃度のATM阻害剤KU55933を添加した。15時間後に、新鮮な阻害剤を培地に添加して、細胞培養培地における阻害剤の減衰を相殺した。さらに54時間後に、細胞を収集し、Surveyor(商標)/CelIアッセイ、RFLP解析およびDNA配列決定による3日目の解析のためにゲノムDNAを調製した。
TALEN媒介性標的化組込みも、DNA修復阻害剤の使用によって増強され得ることを実証するために、本発明者らは、同様にヒトCCR5遺伝子座を標的とするTALEN対101028:101036を用いて同様の実験を行った。例えば、米国特許第8,586,526号を参照されたい。AMAXAにより、hCCR5 TALENまたはZFNのいずれかをコードするプラスミドDNAをK562細胞にトランスフェクトし、続いて細胞をDMSOまたはDNA−PKcs阻害剤NU7441で処理した。3日目および10日目に細胞を収集し、ディープシーケンシングによりPCR後にゲノムPCRを解析した。
次に、本発明者らは、阻害剤を使用して、一本鎖ドナーの標的化組込みに対するその効果を測定した。簡潔に説明すると、Amaxa(登録商標)機器において、NucleofectorキットVを用いてHEK293およびMCF7細胞にトランスフェクトし、Nuleofector(商標)キットLを用いてMCF10A細胞にトランスフェクトした。トランスフェクション構成成分およびその投薬量を次に示す:100μLのnucleofection溶液に懸濁した80万個の細胞;ヒト細胞におけるAAVS1遺伝子座にDSBを誘導するために操作された、それぞれ3μgの2種のin vitro転写されたZFN mRNA(例えば、米国特許第8,110,379号を参照);0.3nmolの100nt ssDNAオリゴドナー。オリゴドナーは、ZFN開裂部位の20bp上流の位置にHindIII制限部位を保有し、RFLPによる標的組込み解析のためのマーカーとしてHindIII部位を使用した。
Amaxaエレクトロポレーションシステムにより、CCR5特異的ZFN対(米国特許第7,951,925号を参照)をコードするプラスミドDNAと、標的領域に対し相同性を有し特有の制限部位(AvrII)を有する一本鎖オリゴヌクレオチド(ssオリゴ)(120bp)をK562細胞にトランスフェクトした。以前の実験とは対照的に、ZFN対は、野生型FokI ZFN(8196−WT)および変異体Fok1 ZFN(8267−D450N)(米国特許第7,914,796号、第8034,598号および第8,623,618号を参照)を含み、本実験において使用されているZFN対は、標的部位におけるDNAの一方の鎖を開裂し、したがって、「ニッカーゼ」として機能する。米国特許出願公開第20100047805号を参照されたい。この種類のDNA損傷の低い有害性質のため、ssDNA切断に媒介される組換え事象は、DSBよりも好ましくなり得る。しかし、ssDNA切断により媒介される遺伝子修飾の頻度は非常に低い。ssDNA切断(「ニック」)により媒介される遺伝子修飾の頻度を増加させる試みにおいて、DNA−PKcsおよびPARP阻害剤を使用した。
Claims (11)
- 細胞におけるマイクロホモロジー媒介末端結合(MMEJ)を介した標的化されたゲノム破壊のためのインビトロの方法であって、前記方法は:
少なくとも1種のヌクレアーゼを前記細胞に投与するステップであって、前記ヌクレアーゼが、前記細胞における内因性ゲノム配列を開裂し、前記ヌクレアーゼが、TALEエフェクタードメインヌクレアーゼ(TALEN)および/またはCRISPR/Cas系である、ステップと、
少なくとも1つのDNA依存性プロテインキナーゼ触媒サブユニット(DNA−PKcs)タンパク質の小分子阻害剤、および少なくとも1つのポリ−(ADP−リボース)ポリメラーゼ1/2(PARP1/2)タンパク質の小分子阻害剤を含む培地中で前記細胞を増殖させるステップと
を含み、
前記PARP1/2タンパク質の小分子阻害剤が、ニコチンアミド;イソキノリノン;ジヒドロイソキノリノン;ベンズイミダゾール;インドール;フタラジン−1(2H)−オン;キナゾリノン;イソインドリノン;フェナントリジン;フェナントリジノン;ベンゾピロン;不飽和ヒドロキサム酸誘導体;ピリダジン;カフェイン、テオフィリン、およびチミジンからなる群から選択され、前記DNA−PKcsタンパク質の小分子阻害剤が、NU7026およびNU7441からなる群から選択され、
前記細胞における前記内因性ゲノム配列は、前記少なくとも1種のヌクレアーゼによる開裂の後にMMEJを介して破壊される、
方法。 - 前記細胞が、Rad52 mRNAを含む、請求項1に記載の方法。
- 前記標的化されたゲノム破壊が、欠失を含む、請求項1または2に記載の方法。
- 前記標的化されたゲノム破壊が、挿入を含む、請求項1または2に記載の方法。
- 前記方法が、外因性配列を前記細胞に投与するステップをさらに含み、前記外因性配列は、前記ヌクレアーゼによる開裂後に、相同組換え修復(HDR)機構により前記ゲノムに組み込まれる、請求項4に記載の方法。
- 前記ヌクレアーゼが、発現ベクターを使用して投与されるか、またはmRNAとして投与される、請求項1に記載の方法。
- 前記細胞が真核細胞である、請求項1〜6のいずれか一項に記載の方法。
- 前記真核細胞が、哺乳動物細胞または植物細胞である、請求項7に記載の方法。
- 請求項1〜8のいずれか一項に記載の方法において使用するためのキットであって、前記キットは、前記細胞における標的部位に結合する少なくとも1種のヌクレアーゼと、DNA依存性プロテインキナーゼ触媒サブユニット(DNA−PKcs)タンパク質の1種または複数の小分子阻害剤および/またはポリ−(ADP−リボース)ポリメラーゼ1/2(PARP1/2)タンパク質の1種または複数の小分子阻害剤とを含み、
前記PARP1/2タンパク質の小分子阻害剤が、ニコチンアミド;イソキノリノン;ジヒドロイソキノリノン;ベンズイミダゾール;インドール;フタラジン−1(2H)−オン;キナゾリノン;イソインドリノン;フェナントリジン;フェナントリジノン;ベンゾピロン;不飽和ヒドロキサム酸誘導体;ピリダジン;カフェイン、テオフィリン、およびチミジンからなる群から選択され、前記DNA−PKcsタンパク質の小分子阻害剤が、NU7026およびNU7441からなる群から選択され、
そして任意選択で、MMEJ修復経路の活性化因子および/または外因性配列をさらに含む、キット。 - 処置の方法において使用するための組成物を製造する方法であって、該組成物は、標的化されたゲノム破壊を含む細胞を含み、
該製造する方法は、請求項1〜8のいずれか1項に記載の方法によって該細胞において標的化されたゲノム破壊を導入するステップを含む、
製造する方法。 - 前記細胞が、前記細胞において標的化されたゲノム破壊を導入するステップの前に個々の患者から外植されているか、または前記細胞が幹細胞である、請求項10に記載の方法。
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