JP7431812B2 - 自己免疫疾患における新規な遺伝子分類とその使用 - Google Patents
自己免疫疾患における新規な遺伝子分類とその使用 Download PDFInfo
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Description
本出願は、2018年5月9日に出願された米国仮特許出願第62/669,297号の利益を主張し、その出願全体は引用によって本明細書に組み込まれる。
いくつかの実施形態では、乾癬に関連付けられる遺伝子分類からの遺伝子の発現レベルを検出する方法が本明細書で開示される。いくつかの例では、上記方法は、インターロイキン17A(IL-17A)、インターロイキン17F(IL-17F)、インターロイキン8(IL-8)、C-X-Cモチーフケモカインリガンド5(CXCL5)、S100カルシウム結合タンパク質A9(S100A9)、デフェンシンβ4A(DEFB4A)、またはこれらの組み合わせの発現レベルを検出する工程を含む。いくつかの例では、上記方法は、(a)被験体から得られた皮膚サンプルから核酸を単離する工程であって、皮膚サンプルが(例えば、角質層からの細胞を含む)、工程と;(b)プローブのセットに、単離された核酸を接触させることによって、IL-17A、IL-17F、IL-8、CXCL5、S100A9、DEFB4A、またはこれらの組み合わせの発現レベルを検出する工程であって、プローブのセットがIL-17A、IL-17F、IL-8、CXCL5、S100A9、DEFB4A、またはこれらの組み合わせを認識し、および、IL-17A、IL-17F、IL-8、CXCL5、S100A9、DEFB4A、またはこれらの組み合わせとプローブのセット間の結合を検出する、工程を含む。
いくつかの実施形態では、アトピー性皮膚炎に関連付けられる遺伝子分類からの遺伝子の発現レベルを検出する方法が本明細書で開示される。いくつかの例では、方法は、インターロイキン13(IL-13)、インターロイキン31(IL-31)、胸腺間質性リンパ球新生因子(TSLP)、またはその組み合わせの発現レベルを検出する工程を含む。いくつかの例では、上記方法は、(a)被験体から得られた皮膚サンプルから核酸を単離する工程であって、皮膚サンプルが(例えば、角質層からの細胞を含む)、工程と;(b)プローブのセットに、単離された核酸を接触させることによって、IL-13、IL-31、TSLP、またはこれらの組み合わせの発現レベルを検出する工程であって、プローブのセットが、IL-13、IL-31、TSLP、またはこれらの組み合わせを認識し、および、IL-13、IL-31、TSLP、またはこれらの組み合わせとプローブのセットとの間の結合を検出する、工程を含む。
いくつかの実施形態では、エリテマトーデスに関連付けられる遺伝子分類からの遺伝子の発現レベルを検出する方法が本明細書で開示される。いくつかの例では、方法は、インターフェロンアルファ1(IFNA1)、インターフェロンアルファ2(IFNA2)、インターフェロンアルファ4(IFNA4)、インターフェロンアルファおよびベータ受容体サブユニット1(IFNR1)、インターフェロンアルファおよびベータ受容体サブユニット2(IFNR2)、C-Cモチーフケモカインリガンド5(CCL5)、またはこれらの組み合わせの発現レベルを検出する工程を含む。いくつかの例では、方法は:(a)被験体から得られた皮膚サンプルから核酸を単離する工程であって、皮膚サンプルが(例えば、角質層からの細胞を含む)、工程と;(b)プローブのセットに、単離された核酸を接触させることによって、IFNA1、IFNA2、IFNA4、IFNR1、IFNR2、CCL5、またはこれらの組み合わせの発現レベルを検出する工程であって、プローブのセットが、IFNA1、IFNA2、IFNA4、IFNR1、IFNR2、CCL5、またはこれらの組み合わせを認識し、および、IFNA1、IFNA2、IFNA4、IFNR1、IFNR2、CCL5、またはこれらの組み合わせとプローブのセットとの間の結合を検出する、工程を含む。
いくつかの実施形態では、1つ以上の遺伝子は一組のプローブにより検出される。いくつかの実施形態では、前記一組のプローブは、少なくともまたは約1、2、3、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、または30より多くのプローブを含む。いくつかの実施形態では、前記一組のプローブは約6のプローブを含む。いくつかの実施形態では、前記一組のプローブは約7のプローブを含む。いくつかの実施形態では、前記一組のプローブは約8のプローブを含む。いくつかの実施形態では、前記一組のプローブは約9のプローブを含む。いくつかの実施形態では、前記一組のプローブは約10のプローブを含む。いくつかの実施形態では、前記一組のプローブは約13のプローブを含む。いくつかの実施形態では、前記一組のプローブは約15のプローブを含む。いくつかの実施形態では、前記一組のプローブは約20のプローブを含む。
いくつかの実施形態では、本明細書に記載されるサンプル採取キットの粘着パッチは、粘着マトリクスを含む第1の採取領域と、該第1の採取領域の周囲から延在する第2の領域とを備えている。粘着マトリクスは第1の採取領域の皮膚対向面に位置する。第2の領域はタブとして機能し、粘着パッチの適用と取り外しに適している。このタブの大きさは、皮膚表面に粘着パッチを適用する間に、適用者が第1の採取領域のマトリクス材料に接触しないほど十分なものである。いくつかの実施形態では、粘着パッチは第2の領域タブを備えていない。いくつかの例では、粘着パッチは、使用前に接着マトリックスの汚染を減らすために手袋で取り扱われる。
本明細書に提供される方法とデバイスは、ある実施形態では、有効または十分な量の皮膚サンプルなどの組織が粘着パッチの粘着マトリクスに付着するような方法で、皮膚に粘着パッチまたはその他同様のパッチを適用する工程を含む。例えば、皮膚サンプルの有効または十分な量は、マトリクスまたは粘着パッチなどの材料に移動可能に付着する量である。付着された皮膚サンプルは、ある実施形態では核酸を含む細胞物質を含んでいる。いくつかの例では、核酸はRNAまたはDNAである。有効な量の皮膚サンプルは、診断アッセイの実行に十分な量の細胞材料を含んでいる。いくつかの例では、診断アッセイは、使用済み粘着パッチに付着された皮膚サンプルから単離される細胞材料を使用して実行される。いくつかの例では、診断アッセイは、使用済み粘着パッチに付着された細胞材料に実行される。いくつかの実施形態では、有効量の皮膚サンプルは、遺伝子発現解析の実行に十分な量のRNAを含む。十分な量のRNAは、ピコグラム、ナノグラム、マイクログラムの各量を含むが、これらに限定されるものではない。
別段の定めのない限り、本明細書で使用される技術用語と科学用語はすべて、主張される発明特定事項が属する当該技術分野の当業者により一般に理解されるのと同じ意味を持つ。詳細な記載は例示的かつ説明的なものに過ぎず、主張される発明特定事項を制限するものではないことを理解されたい。本出願では、単数形の使用は、特に別記しない限り複数を含む。本明細書で使用されるように、単数形「a」、「an」、および「the」は、文脈が明確に定めていない限り、複数の指示対象を含む。本出願では、「または」の使用は、特に明記しない限り「および/または」を意味する。さらに、用語「含むこと(including)」の使用は、「含む(include)」、「含む(includes)」、および「含まれる(included)」といった他の形態と同じく、制限はない。
TNFα、IL-17A、およびIL-23の遮断により中等度から重度の乾癬を処置することに進歩が見られている。罹患した経路を図6に表す。さらに、疾患のモニタリング、再発の予測、および処置の選択が依然として困難とされている。以下、乾癬における遺伝子発現を評価し、処置反応を予測する非侵襲的方法について記述する。
本明細書に記載されるような粘着パッチベースのデバイスを使用して、非侵襲的処置により被験体(対照として健康な人、および乾癬が未処置の患者)から表皮細胞を採取した(乾癬患者から、病変と非病変両方の皮膚も採取した)。全RNAをこれらサンプルから抽出し、TaqMan RT-qPCRでのサイトカイン遺伝子発現解析に使用した。主にTh17経路において乾癬に関与する13のサイトカインのパネルを調べて、qPCRから閾値サイクル数(Ct)を通じてそれらの発現レベルを算出した。
本明細書に記載される粘着パッチを用いたデバイスを使用してサンプルを採取した。それらの受容体を含むサイトカイン標的に対して選択的な抗体を、24の乾癬病変の皮膚サンプルと15の非病変の皮膚サンプルにおける発現レベルについて評価した。IL-17RC、IL-26、IL-22、IL-17A、IL-17F、IL-17C、TNFα、IL-6、IL-23A、DEFB4A、S100A9、CXCL5、およびIL-8への結合についてサンプルを検査した。図5はスクリーニングの結果を示すヒートマップである。乾癬サンプルでは、DEFB4A、S100A9、CXCL5、およびIL-8に対する低レベルの抗体結合を検出した。非乾癬サンプルでは、IL-17RC、IL-26、IL-22、IL-17A、IL-17F、IL-17C、TNFα、IL-6、およびIL-23Aに対する結合の増加を検出した。
本明細書に記載される粘着パッチ収集方法を使用して乾癬皮膚から2つのRNAサンプルを採取した。表1は、IL-23/TH17軸からの乾癬サイトカインの発現レベル上昇を示す。
サンプルを採取し、本明細書に記載される方法に従ってアッセイした。正常な皮膚と比較した乾癬病変皮膚の遺伝子発現レベル、および正常な皮膚と比較された非病変皮膚の遺伝子発現レベルの倍率変化を算出した。図25は、遺伝子発現の増加が病変皮膚および非病変領域の両方に検出されたサイトカインを示す。図26は、未関与非病変皮膚では遺伝子発現は減少したが、乾癬病変皮膚では遺伝子発現が増加したサイトカインを示す。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してサンプルを採取し、アッセイした。qRT-PCRアッセイのモジュール構造により、乾癬、アトピー性皮膚炎、または狼瘡を含む多数の炎症性皮膚疾病にそれを使用することが可能になる。アトピー性皮膚炎では、このアッセイを、拡大されたTH2経路に関与した18の標的に焦点を当てた(図7を参照)。標的にはIL-4、IL-13、IL-17、IL-22、IL-31、TSLP、CXCL9、CXCL10、CXCL11、S100A7、S100A8、S100A9、CCL17、CCL18、CCL19、CCL26、CCL27、およびNOS2が含まれていた。
AD被験体由来のサンプルを採取し、本明細書に記載される方法に従ってアッセイした。病変領域由来の40のサンプル、非病変領域由来の17のサンプル、および正常な皮膚由来の20のサンプルを、IL-31RA、CCL17、IL-23A、IL-4R、IL22、IL-13、およびIL-13RA1の発現レベルについてアッセイした。図27A~27Iを参照。
16週間にわたり隔週でデュピルマブ300mgを皮下投与することでAD被験体を処置した。実施例5に記載されるようにサンプルを採取した。
12週間にわたり毎週125mgのレブリキズマブ、IL-13遮断モノクローナル抗体、およびコルチコステロイドを投与することでAD被験体を処置した。実施例5に記載されるようにサンプルを採取した。
血液中のDPP-4レベルについてAD被験体を検査した。DPP-4レベル上昇について処置群とプラセボ群をさらに選択した。処置群には12周間にわたりトラロキヌマブ、IL-13遮断モノクローナル抗体、および局所ステロイドを投与した。実施例5に記載されるようにサンプルを採取した。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してADサンプルを採取し、アッセイした。病変領域および非病変領域のほか、正常な皮膚もCCL17発現について検査した(図12Aおよび図12B)。ΔCt解析は、病変皮膚サンプルの変化がより重度であったが、この変化のパターンはAD患者の病変サンプルと非病変サンプル両方において同様であることを示しており、このことから非病変サンプルは疾患診断の方法として使用されてもよいことが示唆される。ΔCt=正常化された遺伝子発現変化(=CTtarget gene-CtACTB)。より大きなΔCt値は、標的遺伝子の発現が少ないことを意味する。より小さなΔCt値は、標的遺伝子の発現が多いことを意味する。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してADサンプルを採取し、アッセイした。病変領域および非病変領域のほか、正常な皮膚もIL-13、IL-22、およびIL-23Aの各発現レベルについて検査した(図13A、13B、14A、14B、15A、および15B)。ΔCt解析は、3つすべてのサイトカインが健康な皮膚サンプルにおいて非常に低い遺伝子発現レベルを呈したが(三角形)、異なるADサンプルでは異なる遺伝子発現パターンを示す(菱形)ことを示す。発現レベルは、低いCtにより示されるように著しく増加したか、または高いCtにより示されるように不変のままであった。差次的遺伝子発現は疾患の「応答者」または「非応答者」に関連する場合があり、そのため、IL-13、IL-22、IL-23Aの各発現は非応答者から応答者をスクリーニングする可能性があることが考慮される。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してADサンプルを採取し、アッセイした。病変領域および非病変領域のほか、正常な皮膚もIL-31およびIL-31Rの各発現レベルについて検査した(図16A、16B、17A、17B、18A、および18B)。サイトカインIL-4と同様に、IL-31はAD疾患の調節に重要な別のサイトカインである。IL-31におけるある程度の増加が病変サンプル中に検出される。IL-31受容体(IL-31R)は病変サンプル中の遺伝子発現の減少を呈する。
IL-13およびIL-4は、図19に表される経路に従いADにおいて機能すると提唱されている。本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してADサンプルを採取し、アッセイした。病変領域および非病変領域のほか、正常な皮膚もIL-13、IL-13RA1、IL4Rの各発現レベルについて検査した。結果を図20A、20B、21A、21B、22A、および22Bに示す。IL-13発現の増加を、その受容体IL-13RA1の遺伝子発現の減少と共に検出した。サンプル中でIL-4遺伝子発現は検出されなかったが、IL-4Rは、正常な皮膚と比較してADサンプル中で不変のままであった発現レベルを呈した。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してADサンプルを採取し、NOS2発現レベルについてアッセイした。図23の結果は正常な皮膚サンプルと比較して低い平均Ctを持つADサンプルを示しており、このことからADサンプル中の発現レベルの増加が示される。
本明細書に記載される粘着パッチを用いた皮膚生検プラットフォームを使用してサンプルを採取し、アッセイした。qRT-PCRアッセイのモジュール構造により、乾癬、アトピー性皮膚炎、または狼瘡を含む多数の炎症性皮膚疾病にそれを使用することが可能になる。狼瘡では、アッセイを、好中球媒介性の発赤に関与した21の標的に焦点を当てた(図24を参照)。標的にはIFNA1、IFNA2、IFNA4、IFNAR1、IFNAR2、IFNB1、IFNE、IFNW1、ADAR、CCL5、IFIT’s、IFI’s、IRF’s、OAS1、IRAK1、TNFAIP3、ATG5、TYK2、STAT4、OPN、およびKRTが含まれる。
角質層からの細胞を含む皮膚サンプルから核酸を単離することによって、および、
IL-17A、IL-17F、IL-8、CXCL5、S100A9、ならびにDEFB4Aのうち少なくとも2つを認識し、および、IL-17A、IL-17F、IL-8、CXCL5、S100A9、ならびにDEFB4Aのうち少なくとも2つとプローブのセットとの間の結合を検出する上記プローブのセットに、単離された核酸を接触させることにより、上記皮膚サンプル上で発現分析を行うか、行ったことによって、
上記被験体が改変された遺伝子発現レベルを有しているか否かを決定する工程を含み;
上記被験体が、IL-17A、IL-17F、IL-8、CXCL5、S100A9、ならびにDEFB4Aのうち少なくとも2つの改変された遺伝子発現レベルを有する場合、TNFα、IL-17A、あるいはIL-23の阻害剤を被験体に投与し、または上記阻害剤を用いた処置のレベルを増大させ、ならびに、
上記被験体が、IL-17A、IL-17F、IL-8、CXCL5、S100A9、ならびにDEFB4Aのうち少なくとも2つの改変された遺伝子発現レベルを有していない場合、上記阻害剤を投与しないか、または上記阻害剤を用いた処置を中止する、方法。
角質層からの細胞を含む皮膚サンプルからの単離核酸を得るか、得たことによって、ならびに、
IL-13、IL-31、および、TSLPのうち少なくとも2つを認識し、および、IL-13、IL-31、およびTSLPのうち少なくとも2つとプローブのセットとの間の結合を検出する上記プローブのセットを、上記単離された核酸と接触させることにより、上記皮膚サンプル上で発現分析を行うか、行ったことによって、
上記被験体が改変された遺伝子発現レベルを有しているか否かを決定する工程を含み;
上記被験体が、IL-13、IL-31、またはTSLPのうち少なくとも2つの改変された遺伝子発現レベルを有する場合、IL-13あるいはIL-13Rの阻害剤を上記被験体に投与し、および、
上記被験体が、IL-13、IL-31、またはTSLPのうち少なくとも2つの改変された遺伝子発現レベルを有しない場合、IL-13あるいはIL-13Rの阻害剤を上記被験体に投与しない、方法。
Claims (13)
- 乾癬を抱えている疑いのある被験体のIL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの少なくとも2つの遺伝子発現レベルを検出する方法であって、前記方法は、
a)前記被験体から得られた皮膚サンプルから核酸を単離する工程であって、皮膚サンプルが角質層からの細胞を含む、工程と、
b)プローブのセットに、単離された核酸を接触させることによって、IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの少なくとも2つの発現レベルを検出する工程であって、前記プローブのセットがIL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの少なくとも2つを認識し、ならびに、IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aと、前記プローブのセットとの間の結合を検出する、工程と、
c)前記細胞を接着パッチへ充分接着させるように前記被験体の皮膚領域に前記接着パッチを適用することと、接着された細胞を前記接着パッチに充分保持するように前記皮膚領域から前記接着パッチを取り除く、工程と、
d)前記発現レベルは、対照サンプルからの同等の遺伝子の遺伝子発現レベルと比較して、アップレギュレートされた遺伝子発現レベルである、工程、
を含む、方法。 - 前記方法は、IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの少なくとも3つ、少なくとも4つ、または少なくとも5つの発現レベルを検出する工程を含む、請求項1に記載の方法。
- 前記方法が、
IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの発現レベルを検出する工程、
IL-17A、IL-17F、IL-8、CXCL5、およびS100A9の発現レベルを検出する工程、
IL-17A、IL-17F、IL-8、およびCXCL5の発現レベルを検出する工程、
IL-17A、IL-17F、およびIL-8の発現レベルを検出する工程、または、
IL-17AとIL-17Fの発現レベルを検出する工程、
を含む、請求項1に記載の方法。 - IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの遺伝子発現レベルがアップレギュレートされる、請求項1に記載の方法。
- 前記プローブのセットは2つ以上6つ以下の遺伝子を認識する、請求項1に記載の方法。
- 検出する工程は、プローブの追加のセットに、単離された核酸を接触させることを含み、前記プローブの追加のセットが、IL-17C、S100A7、IL-17RA、IL-17RC、IL-23A、IL-22、IL-26、IL-24、IL-6、CXCL1、TNFα、LCN2、CCL20、TNFRSF1A、またはこれらの組み合わせを認識する、請求項1に記載の方法。
- 前記プローブの追加のセットは1以上14以下の遺伝子を認識する、請求項6に記載の方法。
- 乾癬を抱えている疑いのある被験体の第1の遺伝子分類と第2の遺伝子分類から遺伝子発現レベルを検出する方法であって、前記方法は、
a)前記被験体から得られた皮膚サンプルから核酸を単離する工程であって、皮膚サンプルが角質層からの細胞を含む、工程と、
b)プローブのセットに、単離された核酸を接触させることによって、第1の遺伝子分類、IL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aから1つ以上の遺伝子の発現レベルを検出する工程であって、前記プローブのセットが、第1の遺伝子分類からの1以上の遺伝子を認識し、および、第1の遺伝子分類からの1以上の遺伝子と前記プローブのセットとの間の結合を検出する、工程と、
c)プローブの追加のセットに、単離された核酸を接触させることによって、第2の遺伝子分類、IL-17C、S100A7、IL-17RA、IL-17RC、IL-23A、IL-22、IL-26、IL-24、IL-6、CXCL1、IFN-ガンマ、IL-31、IL-33、TNFα、LCN2、CCL20、およびTNFRSF1Aから1つ以上の遺伝子の発現レベルを検出する工程であって、前記プローブの追加のセットが第2の遺伝子分類から1つ以上の遺伝子を検出し、ならびに、第2の遺伝子分類からの1つ以上の遺伝子と前記プローブの追加のセットとの間の結合を検出する、工程と、
d)細胞を接着パッチへ充分接着させるように前記被験体の皮膚領域に前記接着パッチを適用することと、接着された細胞を前記接着パッチに充分保持するように前記皮膚領域から前記接着パッチを取り除く、工程と、
e)前記発現レベルは、対照サンプルからの同等の遺伝子の遺伝子発現レベルと比較して、アップレギュレートされた遺伝子発現レベルである、工程を、
含む、方法。 - 前記方法は、
第1の遺伝子分類からIL-17AとIL-17Fの発現レベルを検出する工程、
前記第1の遺伝子分類からIL-8、CXCL5、S100A9、およびDEFB4Aの発現レベルを検出する工程、
前記第1の遺伝子分類からIL-17A、IL-8、およびDEFB4Aの発現レベルを検出する工程、
前記第1の遺伝子分類からIL-17F、CXCL5、およびS100A9の発現レベルを検出する工程、または、
前記第1の遺伝子分類からIL-17A、IL-17F、IL-8、CXCL5、S100A9、およびDEFB4Aの発現レベルを検出する工程、
を含む、請求項8に記載の方法。 - IL-17A、IL-17F、IL-8、CXCL5、S100A9、またはDEFB4Aの遺伝子発現レベルはアップレギュレートされる、請求項8に記載の方法。
- 前記方法は、第2の分類からの1つ以上の遺伝子の発現レベルがアップレギュレートされていることを判定する工程をさらに含む、請求項8に記載の方法。
- 角質層からの細胞が角化細胞を含む、請求項1-11のいずれか1つに記載の方法。
- 皮膚サンプルから単離された核酸の量は、約100ピコグラムから約100マイクログラム、約200ピコグラムから約10マイクログラム、または約500ピコグラムから約1マイクログラムである、請求項1-12のいずれか1つに記載の方法。
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US20210222247A1 (en) | 2021-07-22 |
WO2019217478A1 (en) | 2019-11-14 |
AU2019266226A1 (en) | 2020-12-03 |
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