JP2022051772A - Dna結合ドメインと切断ドメインとを連結するための組成物 - Google Patents
Dna結合ドメインと切断ドメインとを連結するための組成物 Download PDFInfo
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Abstract
Description
本出願は、2016年2月2日に出願された米国仮出願第62/290,065号の利益を請求し、その開示は、その全体が参照として本明細書により援用される。
該当なし。
本開示は、ゲノム編集およびタンパク質工学の分野におけるものである。
人工ヌクレアーゼ、例えば、操作されたジンクフィンガーヌクレアーゼ(ZFN)、転写活性化因子様エフェクターヌクレアーゼ(TALEN)、操作されたcrRNA/tracrRNA(「一本鎖ガイドRNA」)を用いるCRISPR/Casシステムおよび/またはArgonauteシステムに基づくヌクレアーゼ(例えば、T.thermophilusに由来し、「TtAgo」として知られるもの(Swartsら(2014)Nature 507(7491):258-261)は、切断ドメインに作動可能に連結されたDNA結合ドメイン(ヌクレオチドまたはポリペプチド)を含み、ゲノム配列の標的化変更に使用されている。例えば、ジンクフィンガーヌクレアーゼは、外因性配列を挿入するため、1つまたはそれを超える内因性遺伝子を不活性化するため、遺伝子発現パターンが変更された生物(例えば、作物)および細胞株を作製するためなどに使用されている。例えば、9,255,250;9,045,763;9,005,973;8,956,828;8,945,868;8,703,489;8,586,526;6,534,261;6,599,692;6,503,717;6,689,558;7,067,317;7,262,054;7,888,121;7,972,854;7,914,796;7,951,925;8,110,379;8,409,861;米国特許公開20030232410;同20050208489;同20050026157;同20050064474;同20060063231;同20080159996;同201000218264;同20120017290;同20110265198;同20130137104;同20130122591;同20130177983および同20130177960および同20150056705を参照のこと。例えば、ゲノム配列を切断するためにヌクレアーゼ(例えば、ジンクフィンガーヌクレアーゼ、TALEN)の対が、使用され得る。その対の各メンバーは、通常、ヌクレアーゼの1つまたはそれを超える切断ドメイン(またはハーフドメイン)に連結された操作された(天然に存在しない)DNA結合タンパク質を含む。DNA結合タンパク質が、それらの標的部位に結合するとき、それらのDNA結合タンパク質に連結された切断ドメインは、二量体化およびその後のゲノムの切断が生じることができるように、通常、ジンクフィンガーヌクレアーゼまたはTALENの対の間に置かれる。
ヌクレアーゼ対の切断活性は、ジンクフィンガーと切断ドメインとをつないでいるリンカー(「ZC」リンカー)の長さ、アミノ酸組成および標的部位(結合部位)間の距離(ギャップ)に関係することが示された。例えば、米国特許第9,394,531号;同第8,772,453号;同第7,888,121号および同第8,409,861号;Smithら(2000)Nucleic Acids Res.28:3361-3369;Bibikovaら(2001)Mol.Cell.Biol.21:289-297;米国公開第20150064789号を参照のこと。ヌクレアーゼ融合タンパク質の対を使用すると、その融合タンパク質に対する結合部位が(各結合部位の近傍端から計測して)5または6ヌクレオチド離れて配置されているとき、現在利用可能なZCリンカーおよび切断ハーフドメインで最適な切断が得られた。例えば、米国特許第7,888,121号を参照のこと。米国特許公開20090305419および同20150064789には、種々のリンカー配列(種々のギャップ間隔に関して)を使用し、および/またはFokI切断ドメインのN末端残基を修飾することによるDNA結合ドメインと切断ドメインとの連結が記載されている。
しかしながら、人工ヌクレアーゼが、代替的な構成で、かつ結合部位が6、7、8塩基対またはそれを超える塩基対だけ離れている内因性ゲノム配列を切断し得る、標的化された改変を可能にする方法および組成物がなおも必要とされている。
TF、TALE TFまたはCRISPR/Cas TF)である。
特定の実施形態では、例えば以下が提供される:
(項目1)
DNA結合ドメイン、野生型または操作された切断ドメイン、および該DNA結合ドメインと該切断ドメインとの間のアミノ酸リンカーを含む融合分子であって、該リンカーが、配列番号6~281のいずれかに示されているとおりの配列を含む、融合分子。
(項目2)
前記DNA結合ドメインが、ジンクフィンガータンパク質、TAL-エフェクタードメイン、または一本鎖ガイドRNA(sgRNA)を含む、項目1に記載の融合分子。
(項目3)
前記リンカーが、前記DNA結合ドメインのN末端と前記切断ドメインのC末端との間に伸びている、項目1または項目2に記載の融合分子。
(項目4)
項目1から3のいずれかに記載の第1の融合分子と、第2のDNA結合ドメインおよび第2の野生型または操作された切断ドメインを含む第2の融合分子とを含む、二量体。
(項目5)
前記第2の融合分子が、前記DNA結合ドメインと前記切断ドメインとの間のリンカーをさらに含み、該リンカーが、配列番号6~281のいずれかに示されているとおりの配列を含む、項目4に記載の二量体。
(項目6)
前記DNA結合ドメインが、二本鎖標的DNAにおいてDNAの逆鎖または同じ鎖に結合する、項目4または項目5に記載の二量体。
(項目7)
標的二本鎖DNAにおいて二本鎖または一本鎖の切断部を作る、項目4から6のいずれかに記載の二量体。
(項目8)
前記第1および第2の融合分子の前記DNA結合ドメインが、6~11塩基対だけ離れた標的部位に結合する、項目4から7のいずれかに記載の二量体。
(項目9)
項目1から3のいずれかに記載の融合分子または項目4から7のいずれかに記載の二量体をコードするポリヌクレオチド。
(項目10)
項目1から3のいずれかに記載の1つもしくはそれを超える融合分子、または項目4から7のいずれかに記載の二量体を含む、単離された細胞。
(項目11)
細胞内の細胞クロマチンを改変する方法であって、該方法が、
該細胞クロマチンが改変されるように項目4から7のいずれかに記載の二量体で該細胞クロマチンを切断する工程を含み、ここで、該二量体が、1つまたはそれを超えるポリヌクレオチドを使用して該細胞に導入される、方法。
(項目12)
前記改変が、挿入および/または欠失を前記細胞クロマチンに導入する工程を含む、項目11に記載の方法。
(項目13)
前記改変が、ドナー配列のインテグレーションを含む、項目11または12に記載の方法。
(項目14)
項目1から3のいずれかに記載の融合分子を含むキット。
(項目15)
ヒトまたは動物の被験体を処置する方法において使用するための項目1から3のいずれかに記載の融合分子または項目4から7のいずれかに記載の二量体であって、ここで、該方法が、該融合分子または二量体を該被験体の細胞に導入する工程を含み、該融合分子または二量体が、該細胞の内因性遺伝子座が改変されるように該内因性遺伝子座を切断する、融合分子または二量体。
DNA結合ドメインと切断ドメインとを連結することにより人工ヌクレアーゼを形成するための組成物、および例えば、標的化切断に続く非相同末端結合によって;標的化切断に続く、外因性ポリヌクレオチド(細胞のヌクレオチド配列と相同な1つまたはそれを超える領域を含む)とゲノム配列との間の相同組換えによって;1つまたはそれを超える内因性遺伝子の標的化された不活性化によって、細胞のヌクレオチド配列を標的化変更するためにこれらのヌクレアーゼを使用する方法が、本明細書中に記載される。
本方法の実施、ならびに本明細書中に開示される組成物の調製および使用は、別段示されない限り、分子生物学、生化学、クロマチンの構造および解析、計算機化学、細胞培養、組換えDNAならびに当該分野の技術範囲内である関連分野における従来の手法を用いる。これらの手法は、文献において十分に説明されている。例えば、Sambrookら、MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989およびThird edition,2001;Ausubelら、CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons,New York,1987および定期的な改訂版;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATIN
STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS IN ENZYMOLOGY,Vol.304,“Chromatin” (P.M.Wassarman and A.P.Wolffe,eds.),Academic Press,San Diego,1999;ならびにMETHODS IN MOLECULAR
BIOLOGY,Vol.119,“Chromatin Protocols”(P.B.Becker,ed.)Humana Press,Totowa,1999を参照のこと。
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、交換可能に使用され、直鎖状または環状の立体配座であり、一本鎖または二本鎖の形態である、デオキシリボヌクレオチドポリマーまたはリボヌクレオチドポリマーのことを指す。本開示の目的では、これらの用語は、ポリマーの長さに関して限定すると解釈されるべきでない。これらの用語は、天然のヌクレオチドの公知のアナログ、ならびに塩基、糖および/またはリン酸の部分(例えば、ホスホロチオエート骨格)において改変されたヌクレオチドを包含し得る。一般に、特定のヌクレオチドのアナログは、同じ塩基対形成の特異性を有し;すなわち、Aのアナログは、Tと塩基対形成する。
Laboratory Manual,Second Edition,(1989)Cold Spring Harbor,N.Y.を参照のこと)を用いて評価され得る。そのようなアッセイは、様々な程度の選択性を用いて、例えば、低ストリンジェンシーから高ストリンジェンシーまで様々な条件を用いて、行うことができる。低ストリンジェンシーの条件を使用する場合、非特異的結合事象の非存在下では、その第2のプローブは標的にハイブリダイズしないような部分的な程度の配列同一性さえも欠く第2のプローブ(例えば、標的分子と約30%未満の配列同一性しか有しないプローブ)を使用して、非特異的結合が存在しないことが評価され得る。
は変動する)が、ヌクレオソームコア間に伸びている。通常、ヒストンH1の分子がリンカーDNAと会合している。本開示の目的では、用語「クロマチン」は、原核生物と真核生物の両方のすべてのタイプの細胞核タンパク質を包含すると意味される。細胞クロマチンには、染色体クロマチンとエピソームクロマチンの両方が含まれる。
(i)哺乳動物における疾患もしくは状態の発生を、特に、かかる哺乳動物がその状態の素因を有しているが、未だそれを有するとは診断されていないときに防止すること、
(ii)疾患もしくは状態を阻害すること、すなわち、その発生を停止すること、
(iii)疾患もしくは状態を緩和すること、すなわち、疾患もしくは状態の後退を起こさせること、または
(iv)疾患もしくは状態から生じる症状を緩和すること、すなわち、根底にある疾患もしくは状態に対処することなく疼痛を緩和すること。本明細書中で使用されるとき、用語「疾患」および「状態」は、交換可能に使用される場合もあれば、特定の病気または状態が公知の原因物質を有しないかもしれず(そのため病因が未だ解明されておらず)、したがって未だ疾患としては認識されていないが、単に望ましくない状態または症候群として認識されており、臨床医によって多少具体的な一群の症状が同定されているという点で、異なっている場合もある。
DNA結合ドメイン(例えば、ジンクフィンガータンパク質、TALE、sgRNAなど)とヌクレアーゼ(例えば、切断ドメインまたは切断ハーフドメイン)とを融合する(連結する)アミノ酸配列が、本明細書中に記載される。
本明細書中に記載されるリンカー配列を有益に使用することにより、DNA結合ドメイン、例えば、ジンクフィンガータンパク質、TALE、ホーミングエンドヌクレアーゼ、CRISPR/CasガイドRNAおよび/またはTtagoガイドRNAが、ヌクレアーゼ切断ドメインまたはハーフドメインに連結され、それにより、特異的に標的化された天然に存在しないヌクレアーゼが形成される。DNA結合ドメインは、任意のゲノム配列における12またはそれを超えるヌクレオチドの任意の標的配列を含むがこれに限定されない、遺伝子内の任意の標的配列に結合し得る。
任意のDNA結合ドメインが、本明細書中に開示される方法において使用され得る。ある特定の実施形態において、DNA結合ドメインは、ジンクフィンガータンパク質を含む。好ましくは、そのジンクフィンガータンパク質は、最適な標的部位に結合するように操作されているという点において、天然に存在しない。例えば、Beerliら(2002)Nature Biotechnol.20:135-141;Paboら(2001)Ann.Rev.Biochem.70:313-340;Isalanら(2001)Nature Biotechnol.19:656-660;Segalら(2001)Curr.Opin.Biotechnol.12:632-637;Chooら(2000)Curr.Opin.Struct.Biol.10:411-416を参照のこと。操作されたジンクフィンガー結合ドメインは、天然に存在するジンクフィンガータンパク質と比べて、新しい結合特異性を有し得る。操作する方法としては、合理的なデザインおよび様々なタイプの選択が挙げられるが、これらに限定されない。合理的なデザインとしては、例えば、トリプレット(またはクワドルプレット)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を使用することが挙げられ、ここで、トリプレットヌクレオチド配列またはクワドルプレットヌクレオチド配列の各々は、特定のトリプレット配列またはクワドルプレット配列に結合するジンクフィンガーの1つまたはそれを超えるアミノ酸配列に会合される。例えば、共同所有の米国特許第6,453,242号および同第6,534,261号(全体として参照により本明細書に援用される)を参照のこと。
7:49-66;米国特許公開番号20070117128を参照のこと。ホーミングエンドヌクレアーゼおよびメガヌクレアーゼのDNA結合ドメインは、そのヌクレアーゼに照らして全体として変化し得る(すなわち、そのヌクレアーゼは、同種の切断ドメインを含む)か、または異種の切断ドメインに融合され得る。
and Envir Micro 73(13):4379-4384を参照のこと)。これらの遺伝子は、ヌクレオチド配列において互いと98.9%同一であるが、hpx17の反復ドメインにおける1,575bpの欠失が異なる。しかしながら、両方の遺伝子産物が、XanthomonasのAvrBs3ファミリータンパク質と40%未満の配列同一性を有する。例えば、米国特許第8,586,526号(全体として参照により本明細書に援用される)を参照のこと。
本明細書中に記載されるヌクレアーゼ(例えば、ZFN、TALEN、CRISPR/Casヌクレアーゼ)は、あるヌクレアーゼ(切断ドメイン、切断ハーフドメイン)も含む。ヌクレアーゼ(複数可)は、標的DNAにおける二本鎖断裂(DSB)または一本鎖断裂(ニック)を誘導し得る。いくつかの実施形態において、2つのニックを導入することによってDSBをもたらすために、2つのニッカーゼが使用される。場合によっては、ニッカーゼはZFNであるが、一方、その他においては、ニッカーゼはTALENまたはCRISPR/Casニッカーゼである。
上で詳細に説明されたように、本明細書中に記載されるようなリンカーを含む融合分子のDNA結合ドメインは、任意の最適な配列に結合するように操作され得る。操作されたDNA結合ドメインは、天然に存在するDNA結合ドメインと比べて、新しい結合特異性を有し得る。
ある特定の実施形態において、本開示は、細胞(例えば幹細胞)のゲノムのヌクレアーゼ媒介性改変に関する。上で述べたように、外因性配列(「ドナー配列」または「ドナー」または「トランスジーン」とも呼ばれる)の挿入は、例えば、指定の領域の欠失のためおよび/もしくは変異遺伝子の修正のため、または野生型遺伝子の発現の増大のためである。そのドナー配列は、通常、それが配置されるゲノム配列と同一でないことは容易に明らかである。ドナー配列は、目的の位置における効率的なHDRを可能にするために相同な2つの領域に隣接した非相同配列を含み得るか、または非相同性特異的修復機構(non-homology directed repair mechanisms)によってインテグレートされ得る。さらに、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ない配列を含むベクター分子を構成し得る。ドナー分子は、細胞クロマチンと相同ないくつかの不連続な領域を含み得る。さらに、目的の領域に通常存在しない配列を標的化挿入するために、前記配列は、ドナー核酸分子に存在し得、目的の領域における配列に相同な領域に隣接し得る。
ヌクレアーゼ、これらのヌクレアーゼをコードするポリヌクレオチド、ドナーポリヌクレオチド、ならびに本明細書中に記載されるタンパク質および/またはポリヌクレオチドを含む組成物は、任意の好適な手段によって送達され得る。ある特定の実施形態において、ヌクレアーゼおよび/またはドナーは、インビボにおいて送達される。他の実施形態において、ヌクレアーゼおよび/またはドナーは、患者へのエキソビボ送達において有用な改変された細胞(例えば、幹細胞)を提供するために、単離された細胞(例えば、自己または異種の幹細胞)に送達される。
rh10を含む任意のAAV血清型、ならびに偽型AAV、例えば、AAV2/8、AAV2/5およびAAV2/6を使用することができる。
1702-3(1998),Kearnsら、Gene Ther.9:748-55(1996))。AAV1、AAV3、AAV4、AAV5、AAV6、AAV8、AAV9およびAAVrh10を含む他のAAV血清型ならびにそれらのすべてのバリアントもまた、本発明に従って使用され得る。
5-10(1996);Stermanら、Hum.Gene Ther.9:7 1083-1089(1998);Welshら、Hum.Gene Ther.2:205-18(1995);Alvarezら、Hum.Gene Ther.5:597-613(1997);Topfら、Gene Ther.5:507-513(1998);Stermanら、Hum.Gene Ther.7:1083-1089(1998)が挙げられる。
ed.,1989を参照のこと)。
本明細書中に記載される任意のリンカーを含むキットおよび/または上記方法のいずれかを実施するためのキットもまた提供される。それらのキットは、代表的には、本明細書中に記載されるようなリンカー配列(または本明細書中に記載されるようなリンカーをコードするポリヌクレオチド)を含む。そのキットは、リンカーだけを供給してもよいし、最適なDNA結合ドメインおよび/またはヌクレアーゼが容易に挿入され得るベクターを提供してもよい。それらのキットは、細胞、細胞を形質転換するための緩衝液、細胞用の培養液および/またはアッセイを行うための緩衝液も含み得る。代表的には、それらのキットは、そのキットの他の構成要素に付着されたまたはその他の方法で伴う任意の材料(例えば、指示書、包装または広告リーフレット)を含むラベルも含む。
開示されるリンカーは、切断ドメインを有する操作されたDNA結合ドメインと連結して、DNAを切断するためのヌクレアーゼを形成するために都合よく使用される。本明細書中に記載されるようなリンカーは、切断のために使用されるヌクレアーゼ対の標的部位が、様々な間隔であるとき、例えば、標的部位が5または6塩基対ではない塩基対だけ離れている(例えば、7、8、9塩基対またはそれを超える塩基対だけ離れている)とき、DNAの切断を可能にする。切断は、ゲノム配列(例えば、細胞クロマチンにおける目的の領域)を相同であるが同一でない配列で置き換えるため(すなわち、標的化された組換え);ゲノム内の1つまたはそれを超える部位においてDNAを切断し、次いで、その切断部位を非相同末端結合(NHEJ)によってつなげることによってゲノム配列を欠失させるため;相同組換えを促進する細胞性因子についてスクリーニングするため;および/または野生型配列を変異体配列で置き換えるため、もしくは一方の対立遺伝子を異なる対立遺伝子に変換するための、細胞クロマチンにおける目的の領域(例えば、ゲノム内の、例えば、変異体または野生型の遺伝子内の、所望のまたは所定の部位)での切断であり得る。そのような方法は、例えば、米国特許第7,888,121号(全体として参照により本明細書に援用される)に詳細に記載されている。
リンカーの選択
20150064789に記載されるように細菌選択系からリンカーを選択した。完全にランダム化された4~22アミノ酸のリンカーライブラリーを、ZFN二量体のZFNのうちの一方のN末端FokIドメインとC末端ZFP結合ドメインとの間にクローニングした(図2)。2つのDNA結合ドメイン(ZFN)結合部位間に6、7、8、9、10、または11塩基対の間隔をあけた標的に選択を行った(これらの結合部位はDNAの同じ鎖上にあった)。
インビボ活性
次いで、リンカーをインビボでの活性についてスクリーニングした。米国特許第7,951,925号(CCR5)および同第8,110,379号(AAVS1)に開示される、ジンクフィンガーDNA結合ドメインに切断ドメイン(野生型または操作されたFokI)を融合する選択されたリンカーを使用して、CCR5およびAAVS1標的部位(しかし同じDNA鎖上にある)を標的とするジンクフィンガーヌクレアーゼ構築物を調製した。
可搬性研究
前出の2つのスクリーニングから、6および7塩基対間隔それぞれにつき上位8つのリンカー(図11に示す)をさらなる試験のために選択した。この可搬性研究の実験デザインは図12に示されている。次の3つの構成のそれぞれについて、CTLA4遺伝子に対する10個のZFN対をデザインした:1)テール・トゥ・テール、2)6塩基対間隔のヘッド・トゥ・テール、および3)7塩基対間隔のテール・トゥ・テール。ヘッド・トゥ・テール構成については、10個一組のZFN対を各間隔につき上位8つのリンカーのそれぞれを用いて試験した。7塩基対間隔では、一定のZFNは、標準的リンカー(L0)または拡張されたリンカー(L7c5)のいずれかを含んだ。8塩基対間隔のために選択されたさらなる2つのリンカーも含めた。トランスフェクションはK562細胞内で行った。
ヘッド・トゥ・ヘッド構成
次いで、6および7塩基対間隔のそれぞれについて、実施例3において同定された2つの最良のリンカーを、ヘッド・トゥ・ヘッド構成で試験するために選択した(図1Cを参照のこと)。5、6、7、8、または9塩基対間隔のそれぞれにつき、11個の対をデザインした。6、7、および8塩基対間隔は、4つすべてのリンカーを使用したが、5塩基対間隔は、最良の6塩基対リンカー2つのみを使用し、9塩基対間隔は、最良の7塩基対リンカー2つのみを使用した。
間隔が拡大されたテール・トゥ・テール構成
5~6塩基対間隔を使用する従来のZFNと比べて、7、8、または9塩基対間隔のいずれかを用いるテール・トゥ・テール構成のZFNについても、選択を行った。この選択におけるZFNの設定は図19Aに示されている。両方のZFNが同じ選択されたリンカーを含む場合(図19A上部)、この構成を対称テール・トゥ・テールと呼ぶ。この構成は、リンカーライブラリーおよびホモ二量体標的部位を含む単一のZFNを使用して選択した。一方のZFNが選択されたリンカーを含み、他方のZFNがL0リンカー(すなわち、LRGSQLVKS、配列番号283)を含む場合、この構成を非対称テール・トゥ・テールと呼ぶ。この構成は、L0リンカーを含む一定のZFNと、ヘテロ二量体標的部位上にリンカーライブラリーを含むものとを使用して選択した。選択は、実施例1および米国特許公開番号20150064789に記載のように行った。
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