Method for the therapeutic administration of messenger ribonucleic acid drugs
Patent Information
- Application Number
- JP2021129834
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2016-06-14
- Filing Date
- 2021-08-06
- Publication Date
- 2026-09-14
- Estimated Expiration
- 2036-10-05
AI Technical Summary
【0074】 本開示の多様な実施形態についての詳細を、下記の記載に明示する。本開示の他の特色、目的、および利点は、記載および図面、ならびに特許請求の範囲から明らかであろう。 本発明は、例えば、以下の項目を提供する。 (項目1) 被験体における抗薬物抗体応答を低減または阻害する方法であって、前記被験体へと、目的のポリペプチドをコードする修飾メッセンジャーRNA(mmRNA)を投与するステップを含み、前記被験体における、前記目的のポリペプチドに対する抗薬物抗体応答を低減または阻害するように、前記mmRNAが、(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を含み、前記mmRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目2) 前記mmRNAを、脂質ナノ粒子内に封入して、静脈内投与する、項目1に記載の方法。 (項目3) 前記mmRNAを、毎週1回の注入により投与する、項目2に記載の方法。 (項目4) 前記mmRNAが、5’UTR、前記目的のポリペプチドをコードする、コドン最適化オープンリーディングフレーム、(i)前記少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)前記少なくとも1つのmiR-126結合部位;または(iii)前記少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を含む3’UTR、ならびに連結されるヌクレオシドの3’テーリング領域を含む、先行する項目のいずれかに記載の方法。 (項目5) 前記mmRNAが、コード領域に対して異種性である、5’UTRおよび3’UTRを含む、先行する項目のいずれかに記載の方法。 (項目6) 前記mmRNAを、全修飾する、先行する項目のいずれかに記載の方法。 (項目7) 前記mmRNAが、シュードウリジン(ψ)、シュードウリジン(ψ)および5-メチルシチジン(m5C)、1-メチルシュードウリジン(m1ψ)、1-メチルシュードウリジン(m1ψ)および5-メチルシチジン(m5C)、2-チオウリジン(s2U)、2-チオウリジンおよび5-メチルシチジン(m5C)、5-メトキシウリジン(mo5U)、5-メトキシウリジン(mo5U)および5-メチルシチジン(m5C)、2’-O-メチルウリジン、2’-O-メチルウリジンおよび5-メチルシチジン(m5C)、N6-メチルアデノシン(m6A)、またはN6-メチルアデノシン(m6A)および5-メチルシチジン(m5C)を含む、先行する項目のいずれかに記載の方法。 (項目8) 前記mmRNAが、シュードウリジン(ψ)、N1-メチルシュードウリジン(m1ψ)、2-チオウリジン、4’-チオウリジン、5-メチルシトシン、2-チオ-1-メチル-1-デアザシュードウリジン、2-チオ-1-メチルシュードウリジン、2-チオ-5-アザウリジン、2-チオジヒドロシュードウリジン、2-チオジヒドロウリジン、2-チオシュードウリジン、4-メトキシ-2-チオシュードウリジン、4-メトキシシュードウリジン、4-チオ-1-メチルシュードウリジン、4-チオシュードウリジン、5-アザウリジン、ジヒドロシュードウリジン、5-メトキシウリジン、もしくは2’-O-メチルウリジン、またはこれらの組合せを含む、先行する項目のいずれかに記載の方法。 (項目9) 前記mmRNAが、1-メチルシュードウリジン(m1ψ)、5-メトキシウリジン(mo5U)、5-メチルシチジン(m5C)、シュードウリジン(ψ)、α-チオグアノシン、もしくはα-チオアデノシン、またはこれらの組合せを含む、先行する項目のいずれかに記載の方法。 (項目10) 前記目的のポリペプチドが、治療用タンパク質、サイトカイン、増殖因子、抗体、または融合タンパク質である、先行する項目のいずれかに記載の方法。 (項目11) 脂質ナノ粒子が、リポソームである、先行する項目のいずれかに記載の方法。 (項目12) 脂質ナノ粒子が、カチオン性脂質および/またはイオン化脂質を含む、先行する項目のいずれかに記載の方法。 (項目13) 前記カチオン性脂質および/またはイオン化脂質が、DLin-KC2-DMAまたはDLin-MC3-DMAである、項目12に記載の方法。 (項目14) 前記mmRNAが、配列番号3に示される配列を含む、少なくとも1つのmiR-142-3pマイクロRNA結合部位を含む、項目1から13のいずれか一項に記載の方法。 (項目15) 前記mmRNAが、少なくとも2つのマイクロRNA結合部位を含み、前記マイクロRNA結合部位のうちの少なくとも1つが、miR-142-3pマイクロRNA結合部位である、項目1から13のいずれか一項に記載の方法。 (項目16) 前記mmRNAが、miR-142-3p結合部位と、miR-142-5p、miR-146-3p、miR-146-5p、miR-155、miR-126、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択される、miRに対する第2のマイクロRNA結合部位とを含む、項目15に記載の方法。 (項目17) 前記mmRNAが、配列番号26に示される配列を含む、少なくとも1つのmiR-126マイクロRNA結合部位を含む、項目1から13のいずれか一項に記載の方法。 (項目18) 前記mmRNAが、少なくとも2つのマイクロRNA結合部位を含み、前記マイクロRNA結合部位のうちの少なくとも1つが、miR-126マイクロRNA結合部位である、項目1から13のいずれか一項に記載の方法。 (項目19) 前記mmRNAが、miR-126結合部位と、miR-142-3p、miR-142-5p、miR-146-3p、miR-146-5p、miR-155、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択される、miRに対する第2のマイクロRNA結合部位とを含む、項目18に記載の方法。 (項目20) mmRNA構築物が、miR-126結合部位およびmiR-142-3p結合部位を含む、項目1から13のいずれか一項に記載の方法。 (項目21) mmRNA構築物が、3つのmiR-142-3p結合部位を含む、項目1から13のいずれか一項に記載の方法。 (項目22) mmRNA構築物が、3つのmiR-126結合部位を含む、項目1から13のいずれか一項に記載の方法。 (項目23) 前記目的のポリペプチドをコードする、前記コドン最適化オープンリーディングフレームが、終止コドンを含み、前記(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を、前記終止コドンの後の、3’UTRの30~50ヌクレオチドの中に配置する、項目1から13のいずれか一項に記載の方法。 (項目24) 前記目的のポリペプチドをコードする、前記コドン最適化オープンリーディングフレームが、終止コドンを含み、前記(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を、前記終止コドンの後の、3’UTRの少なくとも50ヌクレオチドの中に配置する、項目1から13のいずれか一項に記載の方法。 (項目25) 前記(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を、前記mmRNA構築物の5’UTR内に配置する、項目1から13のいずれか一項に記載の方法。 (項目26) 目的のポリペプチドの、被験体への反復投与後における、抗薬物抗体応答を低減または阻害する方法であって、前記被験体へと、LNP内に封入された、目的のポリペプチドをコードする修飾mRNA(mmRNA)の第1の用量を静脈内投与するステップであり、前記mmRNAが、(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を含み、前記mmRNAが、1または複数の修飾ヌクレオ塩基を含むステップと; 前記被験体における、前記目的のポリペプチドに対する抗薬物抗体応答を低減または阻害するように、前記被験体へと、LNP内に封入された前記mmRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目27) 目的のポリペプチドの、被験体への反復投与後における、抗薬物抗体応答を低減または阻害する方法であって、 (i)前記被験体へと、LNP内に封入された、目的のポリペプチドをコードする修飾mRNA(mmRNA)の第1の用量を静脈内投与するステップであって、前記mmRNAが、(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を含み、前記mmRNAが、1または複数の修飾ヌクレオ塩基を含むステップと; (ii)前記被験体に由来する試料中の抗薬物抗体のレベルを検出するステップと; (iii)前記試料中の抗薬物抗体の前記レベルが低下したら、前記被験体における、前記目的のポリペプチドに対する抗薬物抗体応答を低減または阻害するように、前記被験体へと、LNP内に封入された前記mmRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目28) 被験体における薬物関連毒性を低減または阻害する方法であって、前記被験体へと、目的のポリペプチドをコードする修飾メッセンジャーRNA(mmRNA)を投与するステップを含み、前記被験体における、前記目的のポリペプチドに対する薬物関連毒性を低減または阻害するように、前記mmRNAが、(i)少なくとも1つのmiR-142-3pマイクロRNA結合部位;(ii)少なくとも1つのmiR-126結合部位;または(iii)少なくとも1つのmiR-142-3pマイクロRNA結合部位および少なくとも1つのmiR-126結合部位を含み、前記mmRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目29) 目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を投与された被験体における、望ましくない免疫細胞の活性化を低減または阻害する方法であって、前記被験体へと、前記目的のポリペプチドをコードする化学修飾mRNAを投与するステップを含み、前記被験体における、望ましくない免疫細胞の活性化を低減または阻害するように、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目30) 望ましくない免疫細胞の活性化の低減または阻害を、前記少なくとも1つのマイクロRNA結合部位を欠く化学修飾mRNAの対照投与と比較して決定する、項目29に記載の方法。 (項目31) 前記望ましくない免疫細胞の活性化の低減または阻害が、リンパ球の活性化の低減または阻害である、項目29に記載の方法。 (項目32) 前記リンパ球の活性化の低減または阻害が、B細胞の活性化の低減または阻害である、項目31に記載の方法。 (項目33) B細胞の活性化の低減または阻害を、CD19+CD86+CD69+B細胞の頻度により決定する、項目32に記載の方法。 (項目34) 前記望ましくない免疫細胞の活性化の低減または阻害が、サイトカイン産生の低減または阻害を引き起こす、項目29から33のいずれか一項に記載の方法。 (項目35) 免疫細胞の活性化を、前記化学修飾mRNAによりコードされる、前記目的のポリペプチドの発現の、対応する減少を伴わずに減少させる、項目29から34のいずれか一項に記載の方法。 (項目36) 目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を投与された被験体における、望ましくないサイトカインの産生を低減または阻害する方法であって、前記被験体へと、前記目的のポリペプチドをコードする化学修飾mRNAを投与するステップを含み、前記被験体における、望ましくないサイトカインの産生を低減または阻害するように、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目37) 望ましくないサイトカインの産生の低減または阻害を、免疫細胞内で発現するマイクロRNAに対する、前記少なくとも1つのマイクロRNA結合部位を欠く化学修飾mRNAの対照投与と比較して決定する、項目36に記載の方法。 (項目38) 前記サイトカインの産生の低減または阻害が、インターロイキン6(IL-6)、腫瘍壊死因子α(TNF-α)、またはインターフェロンγ(IFN-γ)の産生の低減または阻害である、項目36に記載の方法。 (項目39) 前記サイトカインの産生の低減または阻害が、インターロイキン6(IL-6)の産生の低減または阻害である、項目36に記載の方法。 (項目40) サイトカインの産生を、前記化学修飾mRNAによりコードされる、前記目的のポリペプチドの発現の、対応する減少を伴わずに減少させる、項目36から39のいずれか一項に記載の方法。 (項目41) 前記化学修飾mRNAを、脂質ナノ粒子内に封入して、静脈内投与する、項目29から40のいずれか一項に記載の方法。 (項目42) 前記化学修飾mRNAを、毎週1回の注入により投与する、項目41に記載の方法。 (項目43) 前記化学修飾mRNAが、5’UTR、前記目的のポリペプチドをコードする、コドン最適化オープンリーディングフレーム、前記少なくとも1つのマイクロRNA結合部位を含む3’UTR、および連結されるヌクレオシドの3’テーリング領域を含む、項目29から42のいずれか一項に記載の方法。 (項目44) 前記化学修飾mRNAが、前記オープンリーディングフレームに対して異種性である、5’UTRおよび3’UTRを含む、項目43に記載の方法。 (項目45) 前記mRNAを、全修飾する、項目29から44のいずれか一項に記載の方法。 (項目46) 前記mRNAが、シュードウリジン(ψ)、シュードウリジン(ψ)および5-メチルシチジン(m5C)、1-メチルシュードウリジン(m1ψ)、1-メチルシュードウリジン(m1ψ)および5-メチルシチジン(m5C)、2-チオウリジン(s2U)、2-チオウリジンおよび5-メチルシチジン(m5C)、5-メトキシウリジン(mo5U)、5-メトキシウリジン(mo5U)および5-メチルシチジン(m5C)、2’-O-メチルウリジン、2’-O-メチルウリジンおよび5-メチルシチジン(m5C)、N6-メチルアデノシン(m6A)、またはN6-メチルアデノシン(m6A)および5-メチルシチジン(m5C)を含む、項目29から45のいずれかに記載の方法。 (項目47) 前記mRNAが、シュードウリジン(ψ)、N1-メチルシュードウリジン(m1ψ)、2-チオウリジン、4’-チオウリジン、5-メチルシトシン、2-チオ-1-メチル-1-デアザシュードウリジン、2-チオ-1-メチルシュードウリジン、2-チオ-5-アザウリジン、2-チオジヒドロシュードウリジン、2-チオジヒドロウリジン、2-チオシュードウリジン、4-メトキシ-2-チオシュードウリジン、4-メトキシシュードウリジン、4-チオ-1-メチルシュードウリジン、4-チオシュードウリジン、5-アザウリジン、ジヒドロシュードウリジン、5-メトキシウリジン、もしくは2’-O-メチルウリジン、またはこれらの組合せを含む、項目29から46のいずれか一項に記載の方法。 (項目48) 前記mRNAが、1-メチルシュードウリジン(m1ψ)、5-メトキシウリジン(mo5U)、5-メチルシチジン(m5C)、シュードウリジン(ψ)、α-チオグアノシン、もしくはα-チオアデノシン、またはこれらの組合せを含む、項目29から47のいずれか一項に記載の方法。 (項目49) 前記マイクロRNA結合部位が、骨髄系細胞内で発現するマイクロRNAに結合する、項目29から48のいずれか一項に記載の方法。 (項目50) 前記マイクロRNA結合部位が、形質細胞様樹状細胞内で発現するマイクロRNAに結合する、項目29から48のいずれか一項に記載の方法。 (項目51) 前記マイクロRNA結合部位が、マクロファージ内で発現するマイクロRNAに結合する、項目29から48のいずれか一項に記載の方法。 (項目52) 前記マイクロRNA結合部位が、miR-126マイクロRNA結合部位である、項目29から48のいずれか一項に記載の方法。 (項目53) 前記miR-126マイクロRNA結合部位が、配列番号26に示される配列を含む、項目52に記載の方法。 (項目54) 前記マイクロRNA結合部位が、miR-142マイクロRNA結合部位である、項目29から48のいずれか一項に記載の方法。 (項目55) 前記miR-142マイクロRNA結合部位が、配列番号3に示される配列を含む、項目54に記載の方法。 (項目56) 前記マイクロRNA結合部位が、miR-155マイクロRNA結合部位である、項目29から48のいずれか一項に記載の方法。 (項目57) 前記miR-155マイクロRNA結合部位が、配列番号35に示される配列を含む、項目56に記載の方法。 (項目58) 前記目的のポリペプチドが、治療用タンパク質、サイトカイン、増殖因子、抗体、または融合タンパク質である、項目29から57のいずれか一項に記載の方法。 (項目59) 前記化学修飾mRNAが、少なくとも2つのマイクロRNA結合部位を含む、項目29から58のいずれか一項に記載の方法。 (項目60) 前記マイクロRNA結合部位のうちの少なくとも1つが、miR-126マイクロRNA結合部位である、項目59に記載の方法。 (項目61) 前記化学修飾mRNAが、miR-126結合部位と、miR-142-3p、miR-142-5p、miR-146-3p、miR-146-5p、miR-155、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択される、miRに対する第2のマイクロRNA結合部位とを含む、項目60に記載の方法。 (項目62) 前記化学修飾mRNAが、miR-126結合部位およびmiR-142結合部位を含む、項目60に記載の方法。 (項目63) 前記脂質ナノ粒子が、リポソームである、項目41に記載の方法。 (項目64) 前記脂質ナノ粒子が、カチオン性脂質および/またはイオン化脂質を含む、項目41に記載の方法。 (項目65) 前記カチオン性脂質および/またはイオン化脂質が、DLin-KC2-DMAまたはDLin-MC3-DMAである、項目64に記載の方法。 (項目66) 目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を投与された被験体における、望ましくない免疫細胞の活性化を低減または阻害する方法であって、前記被験体へと、脂質ナノ粒子(LNP)内に封入された化学修飾mRNAの第1の用量を、静脈内投与するステップであり、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含むステップと; 前記被験体における、望ましくない免疫細胞の活性化を低減または阻害するように、前記被験体へと、LNP内に封入された前記化学修飾mRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目67) 目的のポリペプチドをコードするメッセンジャーRNA(mRNA)の、被験体への反復投与後の前記被験体における、望ましくない免疫細胞の活性化を低減または阻害する方法であって、 (i)前記被験体へと、脂質ナノ粒子(LNP)内に封入された化学修飾mRNAの第1の用量を、静脈内投与するステップであり、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、 前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含むステップと; (ii)前記被験体に由来する試料中の免疫細胞の活性化のレベルを検出するステップと; (iii)前記試料中の免疫細胞の活性化の前記レベルが低下したら、前記被験体における、望ましくない免疫細胞の活性化を低減または阻害するように、前記被験体へと、LNP内に封入された前記化学修飾mRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目68) 前記望ましくない免疫細胞の活性化の低減または阻害が、B細胞の活性化の低減または阻害である、項目66または67に記載の方法。 (項目69) 前記望ましくない免疫細胞の活性化の低減または阻害が、サイトカインの産生の低減または阻害を引き起こす、項目66または67に記載の方法。 (項目70) 脂質ナノ粒子(LNP)内に封入された、目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を反復投与された被験体における、血中クリアランスの加速化を低減または阻害する方法であって、前記被験体へと、脂質ナノ粒子(LNP)内に封入された、前記目的のポリペプチドをコードする化学修飾mRNAを投与するステップを含み、反復投与後の前記被験体における血中クリアランスの加速化を低減または阻害するように、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目71) 脂質ナノ粒子(LNP)内に封入された、目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を投与された被験体における、血中クリアランスの加速化を低減または阻害する方法であって、前記被験体へと、脂質ナノ粒子(LNP)内に封入された化学修飾mRNAの第1の用量を、静脈内投与するステップであり、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含む、ステップと; 前記被験体における血中クリアランスの加速化を低減または阻害するように、前記被験体へと、LNP内に封入された前記化学修飾mRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目72) 脂質ナノ粒子(LNP)内に封入された、前記目的のポリペプチドをコードするmRNAが、B細胞を活性化させず、かつ/または前記LNPに結合することが可能なIgM分子の産生を誘導しない、項目70から71のいずれか一項に記載の方法。 (項目73) 血中クリアランスの加速化の低減または阻害を、脂質ナノ粒子(LNP)内に封入された、前記少なくとも1つのマイクロRNA結合部位を欠く化学修飾mRNAの対照投与と比較して決定する、項目70から72のいずれか一項に記載の方法。 (項目74) 血中クリアランスの加速化を、前記化学修飾mRNAによりコードされる、前記目的のポリペプチドの発現の、対応する低下または阻害を伴わずに低減または阻害する、項目70から73のいずれか一項に記載の方法。 (項目75) 2回にわたる連続投与の間の間隔が、2週間未満である、項目70から74のいずれか一項に記載の方法。 (項目76) 2回にわたる連続投与の間の間隔が、1週間未満である、項目75に記載の方法。 (項目77) 脂質ナノ粒子(LNP)内に封入された、目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を反復投与された被験体における、ポリエチレングリコール(PEG)を認識するIgM分子の産生を低減または阻害する方法であって、前記被験体へと、脂質ナノ粒子(LNP)内に封入された、前記目的のポリペプチドをコードする化学修飾mRNAを投与するステップを含み、反復投与後の前記被験体における、PEGを認識するIgM分子の産生を低減または阻害するように、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含む、方法。 (項目78) 脂質ナノ粒子(LNP)内に封入された、目的のポリペプチドをコードするメッセンジャーRNA(mRNA)を投与された被験体における、ポリエチレングリコール(PEG)を認識するIgM分子の産生を低減または阻害する方法であって、前記被験体へと、脂質ナノ粒子(LNP)内に封入された化学修飾mRNAの第1の用量を、静脈内投与するステップであり、前記化学修飾mRNAが、免疫細胞内で発現するマイクロRNAに対する、少なくとも1つのマイクロRNA結合部位を含み、前記化学修飾mRNAが、1または複数の修飾ヌクレオ塩基を含むステップと; 前記被験体における、PEGを認識するIgM分子の産生を低減または阻害するように、前記被験体へと、LNP内に封入された前記化学修飾mRNAの第2の用量を、静脈内投与するステップと を含む方法。 (項目79) 前記化学修飾mRNAが、5’UTR、前記目的のポリペプチドをコードする、コドン最適化オープンリーディングフレーム、前記少なくとも1つのマイクロRNA結合部位を含む3’UTR、および連結されるヌクレオシドの3’テーリング領域を含む、項目70から78のいずれか一項に記載の方法。 (項目80) 前記化学修飾mRNAが、前記オープンリーディングフレームに対して異種性である、5’UTRおよび3’UTRを含む、項目79に記載の方法。 (項目81) 前記mRNAを、全修飾する、項目70から80のいずれか一項に記載の方法。 (項目82) 前記mRNAが、シュードウリジン(ψ)、シュードウリジン(ψ)および5-メチルシチジン(m5C)、1-メチルシュードウリジン(m1ψ)、1-メチルシュードウリジン(m1ψ)および5-メチルシチジン(m5C)、2-チオウリジン(s2U)、2-チオウリジンおよび5-メチルシチジン(m5C)、5-メトキシウリジン(mo5U)、5-メトキシウリジン(mo5U)および5-メチルシチジン(m5C)、2’-O-メチルウリジン、2’-O-メチルウリジンおよび5-メチルシチジン(m5C)、N6-メチルアデノシン(m6A)、またはN6-メチルアデノシン(m6A)および5-メチルシチジン(m5C)を含む、項目70から81のいずれか一項に記載の方法。 (項目83) 前記mRNAが、シュードウリジン(ψ)、N1-メチルシュードウリジン(m1ψ)、2-チオウリジン、4’-チオウリジン、5-メチルシトシン、2-チオ-1-メチル-1-デアザシュードウリジン、2-チオ-1-メチルシュードウリジン、2-チオ-5-アザウリジン、2-チオジヒドロシュードウリジン、2-チオジヒドロウリジン、2-チオシュードウリジン、4-メトキシ-2-チオシュードウリジン、4-メトキシシュードウリジン、4-チオ-1-メチルシュードウリジン、4-チオシュードウリジン、5-アザウリジン、ジヒドロシュードウリジン、5-メトキシウリジン、もしくは2’-O-メチルウリジン、またはこれらの組合せを含む、項目70から82のいずれか一項に記載の方法。 (項目84) 前記mRNAが、1-メチルシュードウリジン(m1ψ)、5-メトキシウリジン(mo5U)、5-メチルシチジン(m5C)、シュードウリジン(ψ)、α-チオグアノシン、もしくはα-チオアデノシン、またはこれらの組合せを含む、項目70から83のいずれか一項に記載の方法。 (項目85) 前記マイクロRNA結合部位が、骨髄系細胞内で発現するマイクロRNAに結合する、項目70から84のいずれか一項に記載の方法。 (項目86) 前記マイクロRNA結合部位が、miR-142マイクロRNA結合部位である、項目70から84のいずれか一項に記載の方法。 (項目87) 前記miR-142マイクロRNA結合部位が、配列番号3に示される配列を含む、項目86に記載の方法。 (項目88) 前記マイクロRNA結合部位が、形質細胞様樹状細胞内で発現するマイクロRNAに結合する、項目70から84のいずれか一項に記載の方法。 (項目89) 前記マイクロRNA結合部位が、miR-126マイクロRNA結合部位である、項目70から84のいずれか一項に記載の方法。 (項目90) 前記miR-126マイクロRNA結合部位が、配列番号26に示される配列を含む、項目89に記載の方法。 (項目91) 前記マイクロRNA結合部位が、マクロファージ内で発現するマイクロRNAに結合する、項目70から84のいずれか一項に記載の方法。 (項目92) 前記マイクロRNA結合部位が、miR-155マイクロRNA結合部位である、項目70から84のいずれか一項に記載の方法。 (項目93) 前記miR-155マイクロRNA結合部位が、配列番号35に示される配列を含む、項目92に記載の方法。 (項目94) 前記目的のポリペプチドが、治療用タンパク質、サイトカイン、増殖因子、抗体、または融合タンパク質である、項目70から93のいずれか一項に記載の方法。 (項目95) 前記化学修飾mRNAが、少なくとも2つのマイクロRNA結合部位を含む、項目70から94のいずれか一項に記載の方法。 (項目96) 前記マイクロRNA結合部位のうちの少なくとも1つが、miR-126マイクロRNA結合部位である、項目95に記載の方法。 (項目97) 前記化学修飾mRNAが、miR-126結合部位と、miR-142-3p、miR-142-5p、miR-146-3p、miR-146-5p、miR-155、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択される、miRに対する第2のマイクロRNA結合部位とを含む、項目96に記載の方法。 (項目98) 前記化学修飾mRNAが、miR-126結合部位およびmiR-142結合部位を含む、項目96に記載の方法。 (項目99) 前記脂質ナノ粒子が、リポソームである、項目70から98のいずれか一項に記載の方法。 (項目100) 前記脂質ナノ粒子が、カチオン性脂質および/またはイオン化脂質を含む、項目70から98のいずれか一項に記載の方法。 (項目101) 前記カチオン性脂質および/またはイオン化脂質が、DLin-KC2-DMAまたはDLin-MC3-DMAである、項目100に記載の方法。 (項目102) 目的のポリペプチドをコードする修飾メッセンジャーRNA(mmRNA)であって、前記mmRNAが、少なくとも2つの異なるマイクロRNA(miR)結合部位を含み、前記マイクロRNAが、造血系免疫細胞内で、またはTLR7および/もしくはTLR8を発現し、炎症促進性のサイトカインおよび/もしくはケモカインを分泌する細胞内で発現され、前記mmRNAが、1または複数の修飾ヌクレオ塩基を含む、mmRNA。 (項目103) 前記造血系免疫細胞が、T細胞、B細胞、またはNK細胞などのリンパ系細胞である、項目102に記載のmmRNA。 (項目104) 前記造血系免疫細胞が、単球、マクロファージ、好中球、好塩基球、好酸球、赤血球、樹状細胞、巨核球、または血小板などの骨髄系細胞である、項目102に記載のmmRNA。 (項目105) 前記造血系免疫細胞が、造血前駆細胞である、項目102に記載のmmRNA。 (項目106) 前記TLR7および/もしくはTLR8を発現し、炎症促進性のサイトカインおよび/もしくはケモカインを分泌する細胞が、内皮細胞である、項目102に記載のmmRNA。 (項目107) 前記マイクロRNAが、同じ目的の細胞型内または異なる目的の細胞型内で夥多である、項目102から106のいずれか一項に記載のmmRNA。 (項目108) 前記マイクロRNAが、複数の目的の細胞型内で夥多である、項目102から106のいずれか一項に記載のmmRNA。 (項目109) 前記mmRNAが、造血系免疫細胞内で夥多なマイクロRNAの少なくとも1つの第1のマイクロRNA結合部位および、内皮細胞内で夥多なマイクロRNAの少なくとも1つの第2のマイクロRNA結合部位を含む、項目102に記載のmmRNA。 (項目110) 前記mmRNAが、B細胞内で夥多なマイクロRNAの少なくとも1つの第1のマイクロRNA結合部位と、内皮細胞内で夥多なマイクロRNAの、少なくとも1つの第2のマイクロRNA結合部位とを含む、項目102に記載のmmRNA。 (項目111) 前記mmRNAが、形質細胞様樹状細胞内で夥多なマイクロRNAの、少なくとも1つの第1のマイクロRNA結合部位と、内皮細胞内で夥多なマイクロRNAの、少なくとも1つの第2のマイクロRNA結合部位とを含む、項目102に記載のmmRNA。 (項目112) 第1のマイクロRNA結合部位の複数のコピーと、第2のマイクロRNA結合部位の少なくとも1つのコピーとを含む、項目102から111のいずれか一項に記載のmmRNA。 (項目113) 前記第1のマイクロRNA結合部位の2つのコピーを含む、項目112に記載のmmRNA。 (項目114) 同じマイクロRNAの第1のマイクロRNA結合部位および第2のマイクロRNA結合部位を含む、項目102から111のいずれか一項に記載のmmRNA。 (項目115) 前記マイクロRNA結合部位が、同じマイクロRNAの3pアームおよび5pアームのものである、項目114に記載のmmRNA。 (項目116) 前記マイクロRNAが、miR-126、miR-142、miR-144、miR-146、miR-150、miR-155、miR-16、miR-21、miR-223、miR-24、miR-27、およびmiR-26aからなる群から選択される、項目102に記載のmmRNA。 (項目117) 前記マイクロRNAが、miR126-3p、miR-142-3p、miR-142-5p、およびmiR-155からなる群から選択される、項目102に記載のmmRNA。 (項目118) 少なくとも1つのマイクロRNA結合部位が、miR-126結合部位である、項目102に記載のmmRNA。 (項目119) 少なくとも1つのマイクロRNA結合部位が、miR-142結合部位である、項目102に記載のmmRNA。 (項目120) 1つのマイクロRNA結合部位が、miR-126結合部位であり、前記第2のマイクロRNA結合部位が、miR-142-3p、miR-142-5p、miR-146-3p、miR-146-5p、miR-155、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択されるマイクロRNAに対するものである、項目102に記載のmmRNA。 (項目121) 少なくとも1つのmiR-126-3p結合部位と、少なくとも1つのmiR-142-3p結合部位とを含む、項目102に記載のmmRNA。 (項目122) 少なくとも1つのmiR-142-3p結合部位と、少なくとも1つのmiR-142-5p結合部位とを含む、項目102に記載のmmRNA。 (項目123) 少なくとも3つの異なるマイクロRNA結合部位を含み、前記マイクロRNA結合部位のうちの少なくとも1つが、miR-126結合部位である、項目102に記載のmmRNA。 (項目124) 少なくとも3つの異なるマイクロRNA結合部位を含み、前記マイクロRNA結合部位のうちの少なくとも1つが、miR-142結合部位である、項目102に記載のmmRNA。 (項目125) 少なくとも1つのmiR-126-3p結合部位と、少なくとも1つのmiR-142-3pと、miR-146-3p、miR-146-5p、miR-155、miR-16、miR-21、miR-223、miR-24、およびmiR-27からなる群から選択されるマイクロRNAに対する第3のマイクロRNA結合部位とを含む、項目102に記載のmmRNA。 (項目126) 少なくとも1つのmiR-126-3p結合部位と、少なくとも1つのmiR-142-3p結合部位と、少なくとも1つのmiR-155結合部位とを含む、項目102に記載のmmRNA。 (項目127) 少なくとも1つのmiR-126-3p結合部位と、少なくとも1つのmiR-142-3p結合部位と、少なくとも1つのmiR-142-5p結合部位と、少なくとも1つのmiR-155結合部位とを含む、項目102に記載のmmRNA。 (項目128) 前記マイクロRNA結合部位を、前記mmRNAの前記5’UTR内、3’UTR内、または前記5’UTR内および3’UTR内の両方に配置した、先行する項目のいずれかに記載のmmRNA。 (項目129) 前記マイクロRNA結合部位を、前記mmRNAの前記3’UTR内に配置する、項目128に記載のmmRNA。 (項目130) 前記マイクロRNA結合部位を、前記mmRNAの前記5’UTR内に配置する、項目128に記載のmmRNA。 (項目131) 前記マイクロRNA結合部位を、前記mmRNAの前記5’UTR内および3’UTR内の両方に配置する、項目128に記載のmmRNA。 (項目132) 少なくとも1つのマイクロRNA結合部位を、前記mmRNAのコード領域の終止コドンと直に隣接する前記3’UTR内に配置する、項目128に記載のmmRNA。 (項目133) 少なくとも1つのマイクロRNA結合部位を、前記mmRNAのコード領域の終止コドンの70~80塩基下流の前記3’UTR内に配置する、項目128に記載のmmRNA。 (項目134) 少なくとも1つのマイクロRNA結合部位を、前記mmRNAのコード領域の開始コドンに直に先行する前記5’UTR内に配置する、項目128に記載のmmRNA。 (項目135) 少なくとも1つのマイクロRNA結合部位を、前記mmRNAのコード領域の開始コドンに15~20ヌクレオチド先行する前記5’UTR内に配置する、項目128に記載のmmRNA。 (項目136) 少なくとも1つのマイクロRNA結合部位を、前記mmRNAのコード領域の開始コドンに70~80ヌクレオチド先行する前記5’UTR内に配置する、項目128に記載のmmRNA。 (項目137) 互いと直に隣接するか、または5ヌクレオチド未満、5~10、10~15、もしくは15~20ヌクレオチドのスペーサーを伴って位置する、同じマイクロRNA結合部位の複数のコピーを含む、項目128に記載のmmRNA。 (項目138) 前記3’UTR内に配置された、同じマイクロRNA結合部位の複数のコピーを含み、前記第1のマイクロRNA結合部位が、終止コドンと直に隣接して位置し、前記第2のマイクロRNA結合部位および第3のマイクロRNA結合部位が、前記第1のマイクロRNA結合部位の30~40塩基下流に位置する、項目128に記載のmmRNA。 (項目139) 前記3’UTR内に配置された、第1のマイクロRNA結合部位の2つのコピーと、第2のマイクロRNA結合部位の1つのコピーとを含み、前記第1のマイクロRNA結合部位の前記第1のコピーが、終止コドンと直に隣接して位置し、前記第2のマイクロRNA結合部位が、前記第1のマイクロRNA結合部位の前記第1のコピーの30~40塩基下流に位置し、前記第1のマイクロRNA結合部位の前記第2のコピーが、前記第2のマイクロRNA結合部位の30~40塩基下流に位置する、項目128に記載のmmRNA。 (項目140) 前記mmRNAが全修飾された、先行する項目のいずれかに記載のmmRNA。 (項目141) シュードウリジン(ψ)、シュードウリジン(ψ)および5-メチルシチジン(m5C)、1-メチルシュードウリジン(m1ψ)、1-メチルシュードウリジン(m1ψ)および5-メチルシチジン(m5C)、2-チオウリジン(s2U)、2-チオウリジンおよび5-メチルシチジン(m5C)、5-メトキシウリジン(mo5U)、5-メトキシウリジン(mo5U)および5-メチルシチジン(m5C)、2’-O-メチルウリジン、2’-O-メチルウリジンおよび5-メチルシチジン(m5C)、N6-メチルアデノシン(m6A)、またはN6-メチルアデノシン(m6A)および5-メチルシチジン(m5C)を含む、先行する項目のいずれかに記載のmmRNA。 (項目142) シュードウリジン(ψ)、N1-メチルシュードウリジン(m1ψ)、2-チオウリジン、4’-チオウリジン、5-メチルシトシン、2-チオ-1-メチル-1-デアザシュードウリジン、2-チオ-1-メチルシュードウリジン、2-チオ-5-アザウリジン、2-チオジヒドロシュードウリジン、2-チオジヒドロウリジン、2-チオシュードウリジン、4-メトキシ-2-チオシュードウリジン、4-メトキシシュードウリジン、4-チオ-1-メチルシュードウリジン、4-チオシュードウリジン、5-アザウリジン、ジヒドロシュードウリジン、5-メトキシウリジン、もしくは2’-O-メチルウリジン、またはこれらの組合せを含む、先行する項目のいずれかに記載のmmRNA。 (項目143) 1-メチルシュードウリジン(m1ψ)、5-メトキシウリジン(mo5U)、5-メチルシチジン(m5C)、シュードウリジン(ψ)、α-チオグアノシン、もしくはα-チオアデノシン、またはこれらの組合せを含む、先行する項目のいずれかに記載のmmRNA。 (項目144) 前記目的のポリペプチドが、治療用タンパク質、サイトカイン、増殖因子、抗体、または融合タンパク質である、先行する項目のいずれかに記載のmmRNA。 (項目145) 先行する項目のいずれかに記載のmmRNAを含む、脂質ナノ粒子。 (項目146) リポソームを含む、項目145に記載の脂質ナノ粒子。 (項目147) カチオン性脂質および/またはイオン化脂質を含む、項目145に記載の脂質ナノ粒子。 (項目148) 前記カチオン性脂質および/またはイオン化脂質が、DLin-KC2-DMAまたはDLin-MC3-DMAである、項目147に記載の脂質ナノ粒子。 (項目149) 項目102から144のいずれか一項に記載のmmRNA、または項目145から148のいずれか一項に記載の脂質ナノ粒子と、薬学的に許容される担体、希釈剤、または賦形剤とを含む医薬組成物。 (項目150) 抗薬物抗体応答の低減もしくは阻害、または薬物関連毒性の阻害を必要とする被験体における、抗薬物抗体応答の低減もしくは阻害、または薬物関連毒性の阻害における使用のための、項目102から144のいずれか一項に記載のmmRNA、項目145から148のいずれか一項に記載の脂質ナノ粒子、または項目149に記載の医薬組成物。 (項目151) 望ましくない免疫細胞の活性化の低減もしくは阻害、または望ましくないサイトカインの産生の低減もしくは阻害を必要とする被験体における、望ましくない免疫細胞の活性化の低減もしくは阻害、または望ましくないサイトカインの産生の低減もしくは阻害における使用のための、項目102から144のいずれか一項に記載のmmRNA、項目145から148のいずれか一項に記載の脂質ナノ粒子、または項目149に記載の医薬組成物。 (項目152) 血中クリアランスの加速化の低減または阻害を必要とする被験体における、血中クリアランスの加速化の低減または阻害における使用のための、項目102から144のいずれか一項に記載のmmRNA、項目145から148のいずれか一項に記載の脂質ナノ粒子、または項目149に記載の医薬組成物。 (項目153) ポリエチレングリコール(PEG)を認識するIgM分子の産生の低減または阻害を必要とする被験体における、ポリエチレングリコール(PEG)を認識するIgM分子の産生の低減または阻害における使用のための、項目102から144のいずれか一項に記載のmmRNA、項目145から148のいずれか一項に記載の脂質ナノ粒子、または項目149に記載の医薬組成物。
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Abstract
Description
[Technical Field]
[0001] This application claims the benefits of U.S. Provisional Patent Application No. 62 / 237,462 filed on 5 October 2015, U.S. Provisional Patent Application No. 62 / 317,268 filed on 1 April 2016, U.S. Provisional Patent Application No. 62 / 317,271 filed on 1 April 2016, U.S. Provisional Patent Application No. 62 / 317,366 filed on 1 April 2016, U.S. Provisional Patent Application No. 62 / 338,385 filed on 18 May 2016, U.S. Provisional Patent Application No. 62 / 338,386 filed on 18 May 2016, U.S. Provisional Patent Application No. 62 / 338,388 filed on 18 May 2016, and U.S. Provisional Patent Application No. 62 / 350,149 filed on 14 June 2016. The entire contents of the above-mentioned referenced patent application are incorporated herein by reference. [Background technology]
[0002] Biological agents such as recombinant antibodies, cytokines, and growth factors have been shown to be effective in treating a wide variety of diseases, and the FDA has now approved a number of such agents for human use (see Kinch, MS (2015), Drug Discovery Today, Vol. 20: pp. 393–398 for a review). The vast majority of FDA-approved biological agents are protein-based. More recently, messenger RNA-based agents have been developed as a disruptive therapeutic modality. Several examples of effective mRNA-based vaccines have been reported, including both infectious disease vaccines and oncology vaccines (see Marc MA et al. (2015), Expert Opin Drug Deliv. Sep, Vol. 12: pp. 1–15 for reviews; and Sahin, U. et al. (2014), Nature Reviews Drug Discovery, Vol. 13: pp. 759–780 for reviews). More recently, the use of mRNA-based drugs has been pursued for therapeutic purposes, for example, by using mRNA constructs that encode the target therapeutic protein. [Prior art documents] [Non-patent literature]
[0003] [Non-Patent Document 1] Marc MA et al. (2015), Expert Opin Drug Deliv. Sep, Vol. 12: pp. 1-15. [Non-Patent Document 2] B. Sahin, U. et al. (2014), Nature Reviews Drug Discovery, Vol. 13: pp. 759-780. [Overview of the project] [Means for solving the problem]
[0004] Therefore, there is a need for new methods and techniques for the use of mRNA-based drugs in subjects, such as mRNA-based therapeutic agents, particularly methods that bring about advantageous characteristics regarding the safety and / or therapeutic efficacy of mRNA-based drugs in subjects. The present invention provides a method for the use of mRNA-based drugs administered to a subject, which brings advantageous features to the use of mRNA-based drugs in vivo. Surprisingly, it has now been found that administration of mRNA-based drugs encoding a target protein to non-human primates can lead to the development of an undesirable immune response in the animals, in which case antibodies against the mRNA-encoded protein may be detected in the animals. This is an unexpected result because the animals were administered an mRNA construct rather than a protein-based therapeutic agent, and it was not predicted that local production of the target protein in target tissue in vivo would result in a response to the encoded protein product. The response observed in non-human primates has also been studied in other involved animal model systems and is similar to the anti-drug antibody (ADA) response recognized in the art, which is still seen in both the field of recombinant protein-based therapeutics and the field of small molecule therapeutics. Those skilled in the art will recognize the recognizable distinction in terminology, as classical anti-drug antibody (ADA) responses are generally understood to be responses to systemic administration of recombinant protein therapies that can generate antibodies directed against them, for example. In the field of mRNA therapies, the antibody responses observed in the animal studies described herein are not responses to the mRNA-based drugs themselves. Conversely, the antibody responses are responses to the protein products encoded by the mRNA drugs. Those skilled in the art may refer to such phenomena as anti-protein antibodies (APAs), but for pharmacologically similar effects, this application utilizes the terminology recognized in the art for anti-drug antibodies (ADAs).
[0005] Notably, it has now been discovered that incorporating at least one microRNA (miR) binding site into an mRNA construct for a miR expressed in conventional immune cells, or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs and / or spleen cells and / or endothelial cells) results in dramatic inhibition of the ADA response in vivo. Accordingly, this disclosure presents a method for reducing or inhibiting the anti-drug antibody response, wherein the protein of interest is encoded by messenger RNA (mRNA) containing at least one microRNA (miR) binding site for a miR expressed in conventional immune cells, or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs, such as spleen cells, e.g., myeloid cells of the spleen, and / or endothelial cells). In exemplary embodiments, the Disclosure presents a method for reducing or inhibiting an anti-drug antibody response, wherein the protein of interest is encoded by messenger RNA (mRNA), e.g., modified messenger RNA (mmRNA), comprising one or more modified nucleobases, and the mRNA, e.g., mmRNA, further comprises at least one microRNA (miR) binding site to a miR expressed in conventional immune cells or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs, such as spleen cells, e.g., myeloid cells of the spleen, and / or endothelial cells). The Method of the Disclosure involves administering to a subject the mRNA, e.g., mmRNA, which encodes the polypeptide of interest and comprises at least one binding site to an immune cell-expressed miR, in order to reduce or inhibit the anti-drug antibody response to the polypeptide of interest in the subject. In exemplary embodiments, the miR binding site is for miRs that are highly or preferentially expressed within immune cells (e.g., within immune cells of peripheral lymphoid organs and / or spleen cells).In exemplary embodiments, the miR binding site is incorporated into mRNA encoding the protein of interest, for example, within the untranslated region (UTR) of the mRNA (e.g., within the 3'UTR, within the 5'UTR, or both within the 3'UTR and 5'UTR). Thus, the method of the present disclosure reduces or inhibits the anti-drug antibody response via post-transcriptional regulation by mRNA with possible components derived from translational repression by the incorporation of at least one miR binding site, without requiring alteration of the amino acid sequence of the protein of interest.
[0006] In one embodiment, at least one miR expressed in an immune cell is a miR-142-3p microRNA binding site. In another embodiment, at least one miR expressed in an immune cell is a miR-126 microRNA binding site, such as a miR-126-3p binding site. Accordingly, the present disclosure presents a method for reducing or inhibiting an anti-drug antibody response in a subject, comprising the step of administering to the subject a messenger RNA (mRNA), e.g., modified messenger RNA (mmRNA), encoding a polypeptide of interest, wherein the mRNA, e.g., mmRNA comprises at least one miR-142-3p microRNA binding site and / or at least one miR-126 microRNA binding site, and the mRNA, e.g., mmRNA comprises one or more modified nucleobases, to reduce or inhibit the anti-drug antibody response to the polypeptide of interest in the subject. In one embodiment, the miR-142-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 3. In one embodiment, the miR-126 binding site is the miR-126-3p binding site. In one embodiment, the miR-126-3p microRNA binding site includes the sequence shown in SEQ ID NO: 26.
[0007] In other embodiments, mRNA, e.g., mmRNA, includes at least one microRNA binding site to a miR selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27. In this specification, a numbered miR may refer to one of two mature microRNAs (e.g., 3p microRNA or 5p microRNA) derived from opposing arms of the same pre-miRNA. In this specification, all numbered miRs are intended to include both 3p and 5p arms per sequence.
[0008] In another embodiment, mRNA, e.g., mmRNA, includes at least two miR binding sites for microRNA expressed in an immune cell. In various embodiments, mRNA, e.g., mmRNA, includes one to four, one, two, three, or four miR binding sites for microRNA expressed in an immune cell. These miR binding sites may be for microRNA selected from the group consisting of miR-142 (including miR-142-3p and miR-142-5p), miR-146 (including miR-146-3p and miR-146-5p), miR-155, miR-126 (including miR-126-3p and miR-126-5p), miR-16, miR-21, miR-223, miR-24, miR-27, and combinations thereof. In another embodiment, mRNA, e.g., mmRNA, includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-142-3p. In various embodiments, mRNA, e.g., mmRNA, includes binding sites for miR-142-3p and miR-155, miR-142-3p, and miR-146, or miR-142-3p and miR-126 (e.g., miR-126-3p). In yet another embodiment, mRNA, e.g., mmRNA, includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-126 (e.g., miR-126-3p). In various embodiments, mRNA, for example, mmRNA, includes binding sites for miR-126 and miR-155, miR-126, and miR-146, or miR-126 and miR-142. In one embodiment, mRNA, for example, mmRNA, includes a miR-142-3p binding site and a miR-126 binding site.
[0009] In one embodiment, the mRNA, e.g., mmRNA, comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR containing at least one microRNA binding site for a miR expressed in an immune cell, and a 3' tailing region of a nucleoside to be ligated. In various embodiments, the 3'UTR contains one to four, at least one, two, three, or four microRNA binding sites for a miR expressed in an immune cell, preferably in immune cells (e.g., immune cells of peripheral lymphoid organs and / or spleen cells), which is abundant or preferentially expressed. In other embodiments, the 3'UTR contains at least one miR-142-3p microRNA binding site, or at least two miR binding sites for a miR expressed in an immune cell, in which case one of the miR binding sites is for miR-142-3p. In another embodiment, the 3'UTR includes at least one miR-126 microRNA binding site, or at least two miR binding sites for miRs expressed in immune cells, where one miR binding site is for miR-126. In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon, and at least one microRNA binding site (e.g., a miR-142-3p binding site and / or a miR-126 binding site) is located within 1 to 100 nucleotides of the 3'UTR after the stop codon. In another embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon, and at least one microRNA binding site (e.g., a miR-142-3p binding site and / or a miR-126 binding site) is located within 30 to 50 nucleotides of the 3'UTR after the stop codon. In another embodiment, a codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon, and at least one microRNA binding site (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site) is located within at least 50 nucleotides of the 3' UTR after the stop codon.
[0010] In another embodiment, an mRNA, for example an mmRNA, comprises 5'UTR and 3'UTR which are heterologous to the coding region.
[0011] In another embodiment, a chemically modified mRNA, for example an mmRNA, is fully modified. In another embodiment, a chemically modified mRNA, for example an mmRNA, comprises one or more modified nucleobases, as further described herein.
[0012] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methylcytidine (m 5 C). In some embodiments, the mRNA comprises 1-methylpseudouridine (m 1 ψ). In some embodiments, the mRNA comprises 1-methylpseudouridine (m 1 ψ) and 5-methylcytidine (m 5 C). In some embodiments, the mRNA comprises 2-thiouridine (s 2 U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methylcytidine (m 5 C). In some embodiments, the mRNA comprises 5-methoxyuridine (mo 5 U). In some embodiments, the mRNA comprises 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C). In some embodiments, the mRNA comprises 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methylcytidine (m 5 C). In some embodiments, the mRNA comprises N6-methyladenosine (m 6 A). In some embodiments, the mRNA comprises N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 C).
[0013] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudridine (m 1 ψ) is 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0014] In some embodiments, the modified nucleobase is 1-methylpseudolidine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) Pseudouridine(ψ), α-thioguanosine, or α-thioadenosine. In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0015] In one embodiment, mRNA, for example, mMRNA, is encapsulated within lipid nanoparticles and administered intravenously. In one embodiment, the lipid nanoparticles are liposomes. In one embodiment, the lipid nanoparticles include cationic lipids and / or ionized lipids. In one embodiment, the cationic lipids and / or ionized lipids are DLin-KC2-DMA or DLin-MC3-DMA.
[0016] In one embodiment, the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein. Further examples of the polypeptide of interest are described herein.
[0017] In one embodiment, mRNA, e.g., mmRNA, is administered by weekly infusion. In another embodiment, the infusion is intravenous. In yet another embodiment, mRNA, e.g., mmRNA, is administered by weekly infusion for at least four weeks. In yet another embodiment, mRNA, e.g., mmRNA, is administered intratumorally. Suitable dosage regimens are further described herein.
[0018] In another embodiment, the present disclosure presents a method for reducing or inhibiting an anti-drug antibody response to a polypeptide of interest after repeated administration to a subject, comprising the steps of: intravenously administering to the subject a first dose of mRNA, e.g., modified mRNA (mmRNA), encoding the polypeptide of interest encapsulated in an LNP, wherein the mRNA, e.g., mmRNA, comprises at least one miR-142-3p microRNA binding site and / or at least one miR-126 microRNA binding site, and the mRNA, e.g., mmRNA, comprises one or more modified nucleobases; and intravenously administering to the subject a second dose of mRNA, e.g., mmRNA, encapsulated in an LNP, to reduce or inhibit the anti-drug antibody response to the polypeptide of interest in the subject.
[0019] In another aspect, the present disclosure relates to a method for reducing or inhibiting an anti-drug antibody response after repeated administration of the target polypeptide to a subject, (i) A step of intravenously administering to a subject a first dose of mRNA, e.g., modified mRNA (mmRNA), which is encapsulated within an LNP and encodes the polypeptide of the target, wherein the mRNA, e.g., mmRNA contains at least one microRNA binding site (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site) for miRs expressed in immune cells, and the mRNA, e.g., mmRNA contains one or more modified nucleobases; (ii) The step of detecting the level of anti-drug antibodies in a sample derived from the subject; (iii) Once the level of anti-drug antibodies in the sample has decreased, the subject is intravenously administered a second dose of mRNA encapsulated in the LNP, for example, mMRNA, to reduce or inhibit the subject's anti-drug antibody response to the target polypeptide. I will present a method that includes this.
[0020] In another embodiment, the disclosure presents a method for reducing or inhibiting drug-related toxicity in a subject, comprising the step of administering to the subject a messenger RNA (mRNA), e.g., modified messenger RNA (mmRNA), encoding a polypeptide of interest, wherein the mRNA, e.g., mmRNA comprises at least one microRNA binding site (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site) to a miR expressed in an immune cell, and the mRNA, e.g., mmRNA comprises one or more modified nucleobases, to reduce or inhibit drug-related toxicity to the polypeptide of interest in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is a decrease in blood cell count (cytopenia) in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is autoimmunity in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is complement-mediated action in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is a decrease in hematopoiesis in the subject. In other embodiments, drug-related toxicity may be, for example, nephrotoxicity or hepatotoxicity.
[0021] In addition, it has now been discovered that incorporating at least one binding site, such as the miR-126 binding site and / or miR-142 binding site, into mRNA for microRNAs (miRs) expressed in conventional immune cells or in any cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs such as splenic cells, e.g., myeloid cells of the spleen, and / or endothelial cells), reduces or inhibits undesirable immune cell activation (e.g., B cell activation, cytokine secretion) in subjects to whom the mRNA is administered. Furthermore, it has been discovered that incorporating this at least one miR binding site into mRNA can reduce or inhibit accelerated blood clearance (ABC) of lipid-containing compounds or compositions to which the mRNA is administered. Furthermore, the incorporation of at least one miR binding site into mRNA may reduce or inhibit the growth and / or activation of plasmacytoid dendritic cells (pDCs), and / or reduce or inhibit IgM production by B cells in response to lipid-containing compounds or compositions to which mRNA is administered, such as IgM production by B cells in response to the phospholipid component (e.g., phosphatidylcholine) of lipid-containing compounds or compositions.
[0022] Accordingly, in one embodiment, the present disclosure presents a method for reducing or inhibiting unwanted immune cell activation in a subject administered RNA encoding a polypeptide of interest, e.g., mRNA, e.g., chemically modified messenger RNA (mmRNA), to a subject, the method comprising the step of administering to the subject RNA encoding a polypeptide of interest, e.g., mRNA, e.g., chemically modified messenger RNA (mmRNA), which includes at least one binding site to a microRNA (miR) expressed in conventional immune cells or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs, such as spleen cells, e.g., myeloid cells of the spleen, and / or endothelial cells), and which includes a binding site such as a miR-126 microRNA binding site and / or a miR-142 microRNA binding site, to reduce or inhibit unwanted immune cell activation in the subject. In another aspect, the present disclosure presents a method for reducing or inhibiting the production of undesirable cytokines in a subject administered with RNA encoding a polypeptide of interest, e.g., mRNA, e.g., chemically modified messenger RNA (mmRNA), comprising the step of administering to a subject, to reduce or inhibit the production of undesirable cytokines in the subject, RNA encoding a polypeptide of interest, e.g., mRNA, e.g., chemically modified messenger RNA (mmRNA), which includes at least one binding site to a microRNA (miR) expressed in conventional immune cells or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs, such as spleen cells, e.g., myeloid cells of the spleen, and / or endothelial cells), and which includes a binding site such as a miR-126 microRNA binding site and / or a miR-142 microRNA binding site. The RNA may be mRNA such as chemically modified mRNA (hereinafter referred to as mmRNA), which includes one or more modified nucleobases.mRNA can be fully modified (i.e., all nucleotides or nucleobases of a specific type within the mRNA are modified) or partially modified (i.e., some nucleotides or nucleobases of a specific type within the mRNA are modified, or the mRNA can be a chimeric mRNA containing a sequence of modified and unmodified nucleobases).
[0023] In one embodiment, the reduction or inhibition of undesirable immune cell activation and / or cytokine production is determined by comparing the administration of a control RNA, for example, mRNA lacking at least one binding site to microRNAs (miRs) expressed in immune cells, such as the miR-126 microRNA binding site and / or the miR-142 microRNA binding site, e.g., mMRNA. In one embodiment, immune cell activation is reduced by at least 10%. In another embodiment, immune cell activation is reduced by at least 25%. In yet another embodiment, immune cell activation is reduced by at least 50%. In yet another embodiment, immune cell activation is reduced without a corresponding reduction in the expression of the polypeptide of interest (e.g., therapeutic protein) encoded by mRNA.
[0024] In one embodiment, the activation of immune cells is the activation of lymphocytes. In one embodiment, the activation of lymphocytes is the activation of B cells. In one embodiment, the activation of B cells is CD19 + CD86 + CD69 +The activation of B cells is determined by the frequency of B cells. In another embodiment, B cell activation is determined, for example, by the secretion of cytokines from serum or whole spleen cells. In one embodiment, B cell activation is determined, for example, by the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) from serum or whole spleen cells. In one embodiment, B cell activation is determined, for example, by the secretion of IL-6 from serum or whole spleen cells. In one embodiment, the reduction or inhibition of cytokine production is determined by the reduction or inhibition of the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ). In another embodiment, the reduction or inhibition of cytokine production is determined by the reduction or inhibition of the production of interleukin-6 (IL-6).
[0025] In another embodiment, the Disclosure presents a method for reducing or inhibiting the acceleration of blood clearance in a subject who has been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject the chemically modified mRNA encoding the polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the acceleration of blood clearance in the subject after repeated administration.
[0026] In some embodiments, the present disclosure presents a method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of: intravenously administering to a subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of chemically modified mRNA encapsulated in LNPs to reduce or inhibit the acceleration of blood clearance in the subject.
[0027] In some embodiments, the Disclosure provides a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in subjects repeatedly administered with messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject after repeated administration.
[0028] In further embodiments, the disclosure presents a method for reducing or inhibiting B1a cell activation in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject the chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit B1a cell activation in the subject after repeated administration.
[0029] In some embodiments, the disclosure presents a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject after repeated administration.
[0030] In further embodiments, the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, so as to reduce or inhibit the acceleration of blood clearance in the subject during one or more subsequent doses. In some embodiments, the IgM molecules recognize polyethylene glycol (PEG).
[0031] In some embodiments, the reduction or inhibition of accelerated blood clearance is determined compared to a control dose of chemically modified mRNA lacking at least one microRNA binding site, encapsulated in lipid nanoparticles (LNPs). In further embodiments, the acceleration of blood clearance is reduced or inhibited without corresponding reduction or inhibition of the expression of the polypeptide of interest encoded by the chemically modified mRNA.
[0032] In some embodiments, the interval between two consecutive doses is less than two weeks. In other embodiments, the interval between two consecutive doses is less than one week.
[0033] In one embodiment, mRNA, e.g., mmRNA, comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR containing at least one binding site for microRNAs (miRs) expressed in immune cells, such as a miR-126 (e.g., miR-126-3p) microRNA binding site or a miR-142 (e.g., miR-142-3p) microRNA binding site, and a 3' tailing region of the nucleoside to be linked. In one embodiment, the codon-optimized open reading frame encoding the polypeptide of interest comprises a stop codon, and at least one binding site for microRNAs expressed in immune cells (e.g., a miR-142-3p binding site and / or a miR-126-3p binding site) is located within 1 to 100 nucleotides of the 3'UTR after the stop codon. In another embodiment, the codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon and at least one binding site for a microRNA (miR) expressed in an immune cell, such as a miR-126 microRNA binding site and / or a miR-142 microRNA binding site, which is located 30 to 50 nucleotides in the 3' UTR after the stop codon. In another embodiment, a codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon, and at least one binding site (e.g., a miR-142-3p binding site and / or a miR-126-3p binding site) for a microRNA expressed in an immune cell is located somewhere within the 3' UTR (e.g., 100 nucleotides after the stop codon).In another embodiment, mRNA, for example, mmRNA, includes a 5'UTR and a 3'UTR that are heterogeneous with respect to the open reading frame.
[0034] In various embodiments, mRNA, for example, mmRNA, comprises one to four, one, two, three, or four miR binding sites for microRNA expressed in an immune cell, where at least one of the miR binding sites is a miR-126 binding site. In one embodiment, mRNA, for example, mmRNA, comprises at least two microRNA binding sites for microRNA expressed in an immune cell, where at least one of the microRNA binding sites is a miR-126 binding site. In one embodiment, mRNA, for example, mmRNA, comprises a miR-126 binding site and a second microRNA binding site for a miR selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. In another embodiment, mRNA, e.g., mmRNA, includes a miR-126 (e.g., miR-126-3p) binding site and a miR-142 (e.g., miR-142-3p) binding site. In one embodiment, mRNA, e.g., mmRNA, includes at least three microRNA binding sites for microRNA expressed in an immune cell, in which case at least one of the microRNA binding sites is a miR-126 binding site. In one embodiment, mRNA, e.g., mmRNA, includes a miR-126 binding site, a miR-142 (e.g., miR-142-3p) binding site, and a third microRNA binding site for a miR selected from the group consisting of miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. In another embodiment, mRNA, e.g., mmRNA, includes a miR-126 binding site, a miR-142 (e.g., miR-142-3p) binding site, and a miR-155 binding site. In one embodiment, mRNA, e.g., mmRNA, includes at least four microRNA binding sites for microRNA expressed in immune cells.In another embodiment, mRNA, e.g., mmRNA, includes a miR-126 binding site, a miR-142-3p binding site, a miR-142-5p binding site, and a miR-155 binding site. In this specification, a numbered miR may refer to one of two mature microRNAs (e.g., 3p microRNA or 5p microRNA) derived from opposing arms of the same pre-miRNA. In this specification, all numbered miRs are intended to include both the 3p and 5p arms per sequence.
[0035] In one embodiment, the miR-126-3p binding site includes the sequence shown in SEQ ID NO: 26.
[0036] In one embodiment, the miR-142-3p binding site includes the sequence shown in Sequence ID No. 3.
[0037] In one embodiment, the miR-155 binding site includes the sequence shown in Sequence ID No. 35.
[0038] In some embodiments, the microRNA binding site binds to microRNA expressed in myeloid cells. In other embodiments, the microRNA binding site binds to microRNA expressed in plasmacytoid dendritic cells. In yet another embodiment, the microRNA binding site binds to microRNA expressed in macrophages.
[0039] In another embodiment, mRNA, for example, mmRNA, is fully modified for a specific chemical modification. The types of suitable chemical modifications are further described herein. In other embodiments, mRNA, for example, mmRNA, contains one or more modified nucleotides or nucleobases as further described herein.
[0040] In some embodiments, the mRNA contains pseudouridine (ψ). In some embodiments, the mRNA contains pseudouridine (ψ) and 5-methylcytidine (m 5 C) is included. In some embodiments, mRNA is 1-methylpseudridine (m1 It contains ψ). In some embodiments, mRNA is 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C) is included. In some embodiments, mRNA is 2-thiouridine (s 2 It contains U). In some embodiments, mRNA contains 2-thiouridine and 5-methylcytidine (m 5 C) is included. In some embodiments, mRNA is 5-methoxyuridine (mo 5 It contains U). In some embodiments, mRNA contains 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 Includes C). In some embodiments, mRNA contains 2'-O-methyluridine. In some embodiments, mRNA contains 2'-O-methyluridine and 5-methylcytidine (m 5 C) is included. In some embodiments, mRNA is N6-methyladenosine (m 6 A) includes. In some embodiments, mRNA contains N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 Includes C).
[0041] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudridine (m 1 ψ) is 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0042] In some embodiments, the modified nucleobase is 1-methylpseudolidine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) Pseudouridine(ψ), α-thioguanosine, or α-thioadenosine. In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0043] In one embodiment, mRNA, for example, mMRNA, is encapsulated within lipid nanoparticles and administered intravenously. In one embodiment, the lipid nanoparticles are liposomes. In one embodiment, the lipid nanoparticles include cationic lipids and / or ionized lipids. In one embodiment, the cationic lipids and / or ionized lipids are DLin-KC2-DMA or DLin-MC3-DMA.
[0044] In one embodiment, mRNA, e.g., mMRNA, encodes the polypeptide of interest. In various embodiments, the polypeptide of interest is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein. Further examples of the polypeptide of interest are described herein.
[0045] In one embodiment, mRNA, e.g., mmRNA, is administered by weekly infusion. In another embodiment, the infusion is intravenous. In yet another embodiment, mRNA, e.g., mmRNA, is administered by weekly infusion for at least four weeks. In yet another embodiment, mRNA, e.g., mmRNA, is administered intratumorally. Suitable dosage regimens are further described herein.
[0046] In another embodiment, the present disclosure presents a method for reducing or inhibiting unwanted immune cell activation (e.g., B cell activation) and / or production of unwanted cytokines in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest, comprising the steps of: intravenously administering to a subject a first dose of mRNA encoding a polypeptide of interest, e.g., modified mRNA (mmRNA), encapsulated in an LNP, wherein the mRNA, e.g., mmRNA comprises at least one binding site to a microRNA (miR) expressed in an immune cell, such as a miR-126 microRNA binding site and / or at least one miR-142 microRNA binding site, and the mRNA, e.g., mmRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of mRNA, e.g., mmRNA encapsulated in an LNP, to reduce or inhibit unwanted immune cell activation and / or production of unwanted cytokines in the subject.
[0047] In another aspect, the present disclosure relates to a method for reducing or inhibiting unwanted immune cell activation (e.g., B cell activation) and / or unwanted cytokine production after repeated administration of messenger RNA (mRNA) encoding a polypeptide of interest to a subject, (i) The step of intravenously administering to a subject a first dose of mRNA encoding the target polypeptide encapsulated within an LNP, for example, modified mRNA (mmRNA), wherein the mRNA, for example, mmRNA, includes at least one binding site to microRNA (miR) expressed in immune cells, such as a miR-126 microRNA binding site and / or at least one miR-142 microRNA binding site. The mRNA, for example, mMRNA, is a step in which one or more modified nucleobases are present; (ii) A step of detecting the level of activation of immune cells in a sample derived from the subject; (iii) Once the level of immune cell activation in the sample has decreased, the subject is intravenously administered a second dose of mRNA encapsulated in the LNP, for example, mMRNA, to reduce or inhibit the activation of undesirable immune cells and / or the production of undesirable cytokines in the subject. I will present a method that includes this.
[0048] In yet another embodiment, the disclosure presents a modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising at least two distinct microRNA (miR) binding sites, wherein the microRNA presents an mmRNA comprising one or more modified nucleobases, comprising miR binding sites expressed in hematopoietic immune cells or in cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines. In some embodiments, hematopoietic immune cells are lymphoid cells such as T cells, B cells, or NK cells. In some embodiments, hematopoietic immune cells are myeloid cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, or platelets. In some embodiments, hematopoietic immune cells are hematopoietic progenitor cells. In some embodiments, the cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines are endothelial cells.
[0049] In some aspects of this disclosure, the mMRNA comprises at least two different microRNA binding sites, and the microRNA is abundant within the same target cell type or within different target cell types. In some aspects, the microRNA is abundant within multiple target cell types.
[0050] In some embodiments, the disclosure presents an mmRNA comprising at least one first microRNA binding site of a hematopoietic immune cell and at least one second microRNA binding site of a microRNA of a large number of endothelial cells, and containing one or more modified nucleobases.
[0051] In some embodiments, the disclosure presents an mmRNA comprising at least one first microRNA binding site of a microRNA abundant in B cells and at least one second microRNA binding site of a microRNA abundant in endothelial cells, and containing one or more modified nucleobases.
[0052] In some embodiments, the disclosure presents an mMRNA containing one or more modified nucleobases, comprising at least one first microRNA binding site of a vast number of microRNAs in plasmacytoid dendritic cells and at least one second microRNA binding site of a vast number of microRNAs in endothelial cells.
[0053] In some aspects of this disclosure, the mMRNA comprises multiple copies (two, three, or four) of a first microRNA binding site and at least one copy of a second microRNA binding site. In some aspects, the mMRNA comprises two copies of the first microRNA binding site and one copy of the second microRNA binding site.
[0054] In some embodiments, the disclosure presents an mmRNA that includes a first microRNA binding site and a second microRNA binding site for the same microRNA, such as a microRNA binding site for the 3p arm and 5p arm of the same microRNA.
[0055] In some aspects of this disclosure, a modified messenger RNA (mmRNA) encoding a polypeptide of interest comprises at least two distinct microRNA (miR) binding sites, wherein the microRNA is selected from the group consisting of miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, and miR-26a, and presents an mmRNA containing one or more modified nucleobases. In some aspects, the microRNA is selected from the group consisting of miR126-3p, miR-142-3p, miR-142-5p, and miR-155. In some aspects, at least one microRNA binding site is a miR-126 binding site, such as that expressed in SEQ ID NO: 26. In some embodiments, at least one microRNA binding site is a miR-142 binding site, such as the one explicitly shown in Sequence ID No. 3.
[0056] In yet another embodiment, the disclosure presents a modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising at least two distinct microRNA (miR) binding sites, one of which is a miR-126 binding site, and the second microRNA binding site is for a microRNA selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27, and comprising one or more modified nucleobases.
[0057] In other embodiments, the disclosure presents modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising a miR-126-3p binding site and a miR-142-3p binding site, and containing one or more modified nucleobases. In some embodiments, the mmRNA comprises a single miR-126-3p binding site, as expressed in SEQ ID NO: 26, and a single miR-142-3p binding site, as expressed in SEQ ID NO: 3, within the 5'UTR or 3'UTR. In some embodiments, the mmRNA comprises at least one miR-142-3p binding site and at least one miR-142-5p binding site, such as those expressed in SEQ ID NO: 3 and SEQ ID NO: 51, respectively.
[0058] In yet another embodiment, the disclosure provides a modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising at least three distinct microRNA binding sites, at least one of which is a miR-126 binding site, and the mmRNA presents an mmRNA containing one or more modified nucleobases.
[0059] In certain embodiments, the disclosure provides a modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising at least three distinct microRNA binding sites, at least one of which is a miR-142 binding site, and the mmRNA presents an mmRNA containing one or more modified nucleobases.
[0060] In further embodiments, the disclosure presents a modified messenger RNA (mmRNA) encoding a polypeptide of interest, comprising at least one miR-126-3p binding site, at least one miR-142-3p, and a third microRNA binding site selected from the group consisting of miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27, and comprising one or more modified nucleobases. In some embodiments, the mmRNA comprises at least one miR-126-3p binding site, at least one miR-142-3p binding site, and at least one miR-155 binding site (e.g., a 155-5p binding site as explicitly shown in the sequence listing). In some embodiments, the mMRNA includes at least one miR-126-3p binding site, at least one miR-142-3p binding site, at least one miR-142-5p binding site, and at least one miR-155 binding site.
[0061] In any of the preceding and related embodiments, the disclosure presents an mmRNA comprising a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR, and a 3' tailing region of a ligated nucleoside, wherein the microRNA binding site is located within the 5'UTR, within the 3'UTR, or both within the 5'UTR and 3'UTR. In some embodiments, the microRNA binding site is located within the 3'UTR of the mmRNA. In some embodiments, the microRNA binding site is located within the 5'UTR of the mmRNA. In some embodiments, the microRNA binding site is located within both the 5'UTR and 3'UTR of the mmRNA. In some embodiments, at least one microRNA binding site is located within the 3'UTR directly adjacent to the stop codon of the coding region of the mmRNA. In some embodiments, at least one microRNA binding site is located within the 3'UTR 70–80 bases downstream of the stop codon of the coding region of the mmRNA. In some embodiments, at least one microRNA binding site is located in the 5' UTR immediately preceding the start codon of the coding region of the mmRNA. In some embodiments, at least one microRNA binding site is located in the 5' UTR 15 to 20 nucleotides preceding the start codon of the coding region of the mmRNA. In some embodiments, at least one microRNA binding site is located in the 5' UTR 70 to 80 nucleotides preceding the start codon of the coding region of the mmRNA.
[0062] In some embodiments, the disclosure presents an mmRNA containing multiple copies of the same microRNA binding site or different microRNA binding sites located within the 5'UTR, within the 3'UTR, or both, either directly adjacent to one another or separated by spacers of less than 5 nucleotides, 5-10, 10-15, or 15-20 nucleotides. In some embodiments, the mmRNA contains multiple copies of the same microRNA binding site located within the 3'UTR, in which case the first microRNA binding site is directly adjacent to the stop codon, and the second and third microRNA binding sites are located 30-40 bases downstream of the first microRNA binding site. In some embodiments, the mMRNA includes two copies of a first microRNA binding site and one copy of a second microRNA binding site, located within the 3'UTR, where the first copy of the first microRNA binding site is directly adjacent to the stop codon, the second microRNA binding site is located 30-40 bases downstream of the first copy of the first microRNA binding site, and the second copy of the first microRNA binding site is located 30-40 bases downstream of the second microRNA binding site.
[0063] In any of the embodiments described above or related embodiments, the disclosure presents modified mRNA in which the mRNA is fully modified.
[0064] In any of the embodiments described above or related embodiments, the Disclosure relates to pseudouridine(ψ), pseudouridine(ψ) and 5-methylcytidine(m5C), 1-methylpseudridine(m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine(s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 Present mmRNA containing C).
[0065] In any of the embodiments described above or related embodiments, the Disclosure relates to pseudouridine (ψ), N1-methylpseudridine (m 1 Present an mmRNA containing ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydropseuduridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine, or a combination thereof.
[0066] In any of the embodiments described above or related embodiments, the Disclosure relates to 1-methylpseuduridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) Present an mmRNA containing pseudouridine (ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof.
[0067] In any of the embodiments described above or related embodiments, the disclosure presents an mMRNA encoding a target polypeptide, wherein the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein.
[0068] In any of the embodiments described herein or related embodiments, the Disclosure presents lipid nanoparticles comprising modified mRNA as described herein. In some embodiments, the lipid nanoparticles comprise liposomes. In some embodiments, the lipid nanoparticles comprise cationic lipids and / or ionized lipids. In some embodiments, the cationic lipids and / or ionized lipids are DLin-KC2-DMA or DLin-MC3-DMA.
[0069] In any of the embodiments described herein or related embodiments, the present disclosure presents a pharmaceutical composition comprising a mMRNA or lipid nanoparticle and a pharmaceutically acceptable carrier, diluent, or excipient.
[0070] In any of the embodiments described herein or related embodiments, the Disclosure presents a mRNA, lipid nanoparticle, or pharmaceutical composition described herein for use in reducing or inhibiting an anti-drug antibody response or inhibiting drug-related toxicity in a subject requiring such use.
[0071] In any of the embodiments described herein or related embodiments, the Disclosure presents a mRNA, lipid nanoparticle, or pharmaceutical composition described herein for use in reducing or inhibiting unwanted immune cell activation or the production of unwanted cytokines in subjects requiring such use.
[0072] In any of the embodiments described herein or related embodiments, the Disclosure presents a mRNA, lipid nanoparticle, or pharmaceutical composition described herein for use in reducing or inhibiting the acceleration of blood clearance in subjects requiring such use.
[0073] In any of the embodiments described herein or related embodiments, the Disclosure presents a mRNA, lipid nanoparticle, or pharmaceutical composition described herein for use in reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in subjects requiring such reduction or inhibition.
[0074] Details of various embodiments of this disclosure are provided below. Other features, purposes, and advantages of this disclosure will be apparent from the description and drawings, as well as the claims. The present invention provides, for example, the following items: (Item 1) A method for reducing or inhibiting an anti-drug antibody response in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site, and the mmRNA comprises one or more modified nucleobases, so as to reduce or inhibit the anti-drug antibody response in the subject to the polypeptide of interest. (Item 2) The method according to item 1, wherein the aforementioned mMRNA is encapsulated within lipid nanoparticles and administered intravenously. (Item 3) The method according to item 2, wherein the aforementioned mMRNA is administered by injection once a week. (Item 4) The method according to any of the preceding items, wherein the mmRNA comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of the objective, (i) the at least one miR-142-3p microRNA binding site; (ii) the at least one miR-126 binding site; or (iii) a 3'UTR containing the at least one miR-142-3p microRNA binding site and the at least one miR-126 binding site, and a 3' tailing region of a ligated nucleoside. (Item 5) The method according to any of the preceding items, wherein the mmRNA includes a 5'UTR and a 3'UTR that are heterogeneous with respect to the coding region. (Item 6) A method for completely modifying the aforementioned mMRNA, or any of the preceding items. (Item 7) The aforementioned mmRNA is pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m5C), 1-methylpseudridine (m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine(s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 The method described in any of the preceding items, including C). (Item 8) The aforementioned mmRNA is pseudouridine (ψ), N1-methylpseudridine (m 1 The method described in any of the preceding items, comprising ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine, or a combination thereof. (Item 9) The aforementioned mmRNA is 1-methylpseudridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m5 C) The method described in any of the preceding items, comprising pseudouridine(ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof. (Item 10) The method according to any of the preceding items, wherein the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein. (Item 11) The method according to any of the preceding items, wherein the lipid nanoparticles are liposomes. (Item 12) The method according to any of the preceding items, wherein the lipid nanoparticles include cationic lipids and / or ionized lipids. (Item 13) The method according to item 12, wherein the cationic lipid and / or ionized lipid is DLin-KC2-DMA or DLin-MC3-DMA. (Item 14) The method according to any one of items 1 to 13, wherein the mmRNA includes at least one miR-142-3p microRNA binding site which includes the sequence shown in Sequence ID No. 3. (Item 15) The method according to any one of items 1 to 13, wherein the mmRNA comprises at least two microRNA binding sites, and at least one of the microRNA binding sites is a miR-142-3p microRNA binding site. (Item 16) The method according to item 15, wherein the mmRNA includes a miR-142-3p binding site and a second microRNA binding site for miR selected from the group consisting of miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 17) The method according to any one of items 1 to 13, wherein the mmRNA includes at least one miR-126 microRNA binding site which includes the sequence shown in SEQ ID NO: 26. (Item 18) The method according to any one of items 1 to 13, wherein the mmRNA comprises at least two microRNA binding sites, and at least one of the microRNA binding sites is a miR-126 microRNA binding site. (Item 19) The method according to item 18, wherein the mmRNA includes a miR-126 binding site and a second microRNA binding site for miR selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 20) The method according to any one of items 1 to 13, wherein the mmRNA construct includes a miR-126 binding site and a miR-142-3p binding site. (Item 21) The method described in any one of items 1 to 13, wherein the mmRNA construct contains three miR-142-3p binding sites. (Item 22) The method according to any one of items 1 to 13, wherein the mmRNA construct contains three miR-126 binding sites. (Item 23) The method according to any one of items 1 to 13, wherein the codon-optimized open reading frame encoding the target polypeptide comprises a stop codon, wherein (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site are located within 30 to 50 nucleotides of the 3'UTR after the stop codon. (Item 24) The method according to any one of items 1 to 13, wherein the codon-optimized open reading frame encoding the target polypeptide comprises a stop codon, wherein (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site are located within at least 50 nucleotides of the 3'UTR after the stop codon. (Item 25) The method according to any one of items 1 to 13, wherein (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site are located within the 5'UTR of the mmRNA construct. (Item 26) A method for reducing or inhibiting an anti-drug antibody response after repeated administration of a target polypeptide to a subject, comprising the steps of intravenously administering to the subject a first dose of a modified mRNA (mmRNA) encoding the target polypeptide, encapsulated within an LNP, wherein the mmRNA comprises (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site, and the mmRNA comprises one or more modified nucleobases; The step of intravenously administering a second dose of the mMRNA encapsulated within the LNP to the subject in order to reduce or inhibit the anti-drug antibody response to the target polypeptide in the subject. A method that includes this. (Item 27) A method for reducing or inhibiting the anti-drug antibody response after repeated administration of a target polypeptide to a subject, (i) Intravenously administering to the subject a first dose of a modified mRNA (mmRNA) encoding a target polypeptide, encapsulated within an LNP, wherein the mmRNA comprises (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site, and the mmRNA comprises one or more modified nucleobases; (ii) The step of detecting the level of anti-drug antibodies in a sample derived from the subject; (iii) Once the level of anti-drug antibodies in the sample has decreased, the subject is intravenously administered a second dose of the mMRNA encapsulated in the LNP to reduce or inhibit the subject's anti-drug antibody response to the target polypeptide. A method that includes this. (Item 28) A method for reducing or inhibiting drug-related toxicity in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises (i) at least one miR-142-3p microRNA binding site; (ii) at least one miR-126 binding site; or (iii) at least one miR-142-3p microRNA binding site and at least one miR-126 binding site, and the mmRNA comprises one or more modified nucleobases, to reduce or inhibit drug-related toxicity to the polypeptide of interest in the subject. (Item 29) A method for reducing or inhibiting unwanted immune cell activation in a subject administered with messenger RNA (mRNA) encoding a target polypeptide, comprising the step of administering to the subject a chemically modified mRNA encoding the target polypeptide, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit unwanted immune cell activation in the subject. (Item 30) The method according to item 29, for determining the reduction or inhibition of undesirable immune cell activation compared to a control dose of chemically modified mRNA lacking at least one microRNA binding site. (Item 31) The method according to item 29, wherein the reduction or inhibition of the undesirable activation of immune cells is the reduction or inhibition of lymphocyte activation. (Item 32) The method according to item 31, wherein the reduction or inhibition of lymphocyte activation is a reduction or inhibition of B cell activation. (Item 33) Reduced or inhibited B cell activation, CD19 + CD86 + CD69 + The method described in item 32, determined by the frequency of B cells. (Item 34) The method according to any one of items 29 to 33, wherein the reduction or inhibition of the activation of the undesirable immune cells results in a reduction or inhibition of cytokine production. (Item 35) The method according to any one of items 29 to 34, wherein the activation of immune cells is reduced without a corresponding decrease in the expression of the target polypeptide encoded by the chemically modified mRNA. (Item 36) A method for reducing or inhibiting the production of undesirable cytokines in a subject administered with messenger RNA (mRNA) encoding a target polypeptide, comprising the step of administering to the subject a chemically modified mRNA encoding the target polypeptide, wherein the chemically modified mRNA comprises at least one microRNA binding site for a microRNA expressed in an immune cell, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of undesirable cytokines in the subject. (Item 37) The method according to item 36, for determining the reduction or inhibition of undesirable cytokine production compared to a control dose of chemically modified mRNA lacking at least one microRNA binding site to microRNA expressed in immune cells. (Item 38) The method according to item 36, wherein the reduction or inhibition of cytokine production is the reduction or inhibition of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ). (Item 39) The method according to item 36, wherein the reduction or inhibition of cytokine production is a reduction or inhibition of interleukin-6 (IL-6) production. (Item 40) The method according to any one of items 36 to 39, wherein cytokine production is reduced without a corresponding reduction in the expression of the target polypeptide encoded by the chemically modified mRNA. (Item 41) The method according to any one of items 29 to 40, wherein the chemically modified mRNA is encapsulated in lipid nanoparticles and administered intravenously. (Item 42) The method according to item 41, wherein the chemically modified mRNA is administered by injection once a week. (Item 43) The method according to any one of items 29 to 42, wherein the chemically modified mRNA comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of the choice, a 3'UTR containing the at least one microRNA binding site, and a 3' tailing region of a ligated nucleoside. (Item 44) The method according to item 43, wherein the chemically modified mRNA includes a 5'UTR and a 3'UTR that are heterogeneous with respect to the open reading frame. (Item 45) The method according to any one of items 29 to 44, for completely modifying the mRNA. (Item 46) The mRNA is pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m5C), 1-methylpseudridine (m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine(s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 The method described in any of items 29 to 45, including C). (Item 47) The aforementioned mRNA is pseudouridine (ψ), N1-methylpseudridine (m 1The method described in any one of items 29 to 46, comprising ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine, or a combination thereof. (Item 48) The aforementioned mRNA is 1-methylpseudridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) The method described in any one of items 29 to 47, comprising pseudouridine(ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof. (Item 49) The method according to any one of items 29 to 48, wherein the microRNA binding site binds to microRNA expressed in myeloid cells. (Item 50) The method according to any one of items 29 to 48, wherein the microRNA binding site binds to a microRNA expressed in a plasmacytoid dendritic cell. (Item 51) The method according to any one of items 29 to 48, wherein the microRNA binding site binds to microRNA expressed within a macrophage. (Item 52) The method according to any one of items 29 to 48, wherein the microRNA binding site is the miR-126 microRNA binding site. (Item 53) The method according to item 52, wherein the miR-126 microRNA binding site includes the sequence shown in SEQ ID NO: 26. (Item 54) The method according to any one of items 29 to 48, wherein the microRNA binding site is the miR-142 microRNA binding site. (Item 55) The method according to item 54, wherein the miR-142 microRNA binding site includes the sequence shown in Sequence ID No. 3. (Item 56) The method according to any one of items 29 to 48, wherein the microRNA binding site is a miR-155 microRNA binding site. (Item 57) The method according to item 56, wherein the miR-155 microRNA binding site includes the sequence shown in SEQ ID NO: 35. (Item 58) The method according to any one of items 29 to 57, wherein the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein. (Item 59) The method according to any one of items 29 to 58, wherein the chemically modified mRNA comprises at least two microRNA binding sites. (Item 60) The method according to item 59, wherein at least one of the microRNA binding sites is a miR-126 microRNA binding site. (Item 61) The method according to item 60, wherein the chemically modified mRNA includes a miR-126 binding site and a second microRNA binding site for miR selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 62) The method according to item 60, wherein the chemically modified mRNA includes a miR-126 binding site and a miR-142 binding site. (Item 63) The method according to item 41, wherein the lipid nanoparticles are liposomes. (Item 64) The method according to item 41, wherein the lipid nanoparticles include cationic lipids and / or ionized lipids. (Item 65) The method according to item 64, wherein the cationic lipid and / or ionized lipid is DLin-KC2-DMA or DLin-MC3-DMA. (Item 66) A method for reducing or inhibiting undesirable immune cell activation in a subject administered with messenger RNA (mRNA) encoding a target polypeptide, comprising the steps of: intravenously administering to the subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; The steps include intravenously administering a second dose of the chemically modified mRNA encapsulated within the LNP to the subject in order to reduce or inhibit the activation of undesirable immune cells in the subject, and A method that includes this. (Item 67) A method for reducing or inhibiting undesirable immune cell activation in a subject after repeated administration of messenger RNA (mRNA) encoding a target polypeptide, (i) The step of intravenously administering to the subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, The step of the chemically modified mRNA comprising one or more modified nucleobases; (ii) The step of detecting the level of activation of immune cells in a sample derived from the subject; (iii) Once the level of immune cell activation in the sample has decreased, the subject is intravenously administered a second dose of the chemically modified mRNA encapsulated in the LNP to reduce or inhibit the unwanted immune cell activation in the subject. A method that includes this. (Item 68) The method according to item 66 or 67, wherein the reduction or inhibition of the undesirable activation of immune cells is the reduction or inhibition of B cell activation. (Item 69) The method according to item 66 or 67, wherein the reduction or inhibition of the activation of the aforementioned undesirable immune cells causes a reduction or inhibition of cytokine production. (Item 70) A method for reducing or inhibiting the acceleration of blood clearance in a subject who has been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to the subject chemically modified mRNA encoding the target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, to reduce or inhibit the acceleration of blood clearance in the subject after repeated administration. (Item 71) A method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a target polypeptide encapsulated within lipid nanoparticles (LNPs), comprising the steps of: intravenously administering a first dose of the chemically modified mRNA encapsulated within the lipid nanoparticles (LNPs) to the subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; The step of intravenously administering a second dose of the chemically modified mRNA encapsulated within the LNP to the subject in order to reduce or inhibit the acceleration of blood clearance in the subject. A method that includes this. (Item 72) The method according to any one of items 70 to 71, wherein the mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), does not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs. (Item 73) The method according to any one of items 70 to 72, for determining a reduction or inhibition of accelerated blood clearance compared to a control dose of chemically modified mRNA lacking at least one microRNA binding site, encapsulated in lipid nanoparticles (LNPs). (Item 74) The method according to any one of items 70 to 73, wherein acceleration of blood clearance is reduced or inhibited without corresponding reduction or inhibition of the expression of the target polypeptide encoded by the chemically modified mRNA. (Item 75) The method according to any one of items 70 to 74, wherein the interval between two consecutive doses is less than two weeks. (Item 76) The method according to item 75, wherein the interval between two consecutive doses is less than one week. (Item 77) A method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in a subject that has been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to the subject chemically modified mRNA encoding the target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject after repeated administration. (Item 78) A method for reducing or inhibiting production of IgM molecules that recognize polyethylene glycol (PEG) in a subject administered a messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the step of: intravenously administering to the subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for a microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; intravenously administering to the subject a second dose of the chemically modified mRNA encapsulated in an LNP, so as to reduce or inhibit production of IgM molecules that recognize PEG in the subject; A method comprising. (Item 79) The method according to any one of Items 70 to 78, wherein the chemically modified mRNA comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR comprising the at least one microRNA binding site, and a 3' tailing region of linked nucleosides. (Item 80) The method according to Item 79, wherein the chemically modified mRNA comprises a 5'UTR and a 3'UTR that are heterologous to the open reading frame. (Item 81) The method according to any one of Items 70 to 80, wherein the mRNA is fully modified. (Item 82) The mRNA is selected from pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m5C), 1-methylpseudouridine (m 1 ψ), 1-methylpseudouridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 C). The method according to any one of items 70 to 81, which comprises C). (Item 83) The mRNA comprises pseudouridine (ψ), N1-methylpseudouridine (m 1 ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydropseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methylpseudouridine, 4-thiopseudouridine, 5-azauridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine, or a combination thereof. The method according to any one of items 70 to 82, which comprises any of the foregoing. (Item 84) The mRNA comprises 1-methylpseudouridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C), pseudouridine (ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof. The method according to any one of items 70 to 83, which comprises any of the foregoing. (Item 85) The method according to any one of items 70 to 84, wherein the microRNA binding site binds to a microRNA expressed in myeloid cells. (Item 86) The method according to any one of items 70 to 84, wherein the microRNA binding site is a miR-142 microRNA binding site. (Item 87) The method according to item 86, wherein the miR-142 microRNA binding site includes the sequence shown in SEQ ID NO: 3. (Item 88) The method according to any one of items 70 to 84, wherein the microRNA binding site binds to a microRNA expressed in a plasmacytoid dendritic cell. (Item 89) The method according to any one of items 70 to 84, wherein the microRNA binding site is a miR-126 microRNA binding site. (Item 90) The method according to item 89, wherein the miR-126 microRNA binding site includes the sequence shown in SEQ ID NO: 26. (Item 91) The method according to any one of items 70 to 84, wherein the microRNA binding site binds to microRNA expressed within a macrophage. (Item 92) The method according to any one of items 70 to 84, wherein the microRNA binding site is a miR-155 microRNA binding site. (Item 93) The method according to item 92, wherein the miR-155 microRNA binding site includes the sequence shown in SEQ ID NO: 35. (Item 94) The method according to any one of items 70 to 93, wherein the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein. (Item 95) The method according to any one of items 70 to 94, wherein the chemically modified mRNA includes at least two microRNA binding sites. (Item 96) The method according to item 95, wherein at least one of the microRNA binding sites is a miR-126 microRNA binding site. (Item 97) The method according to item 96, wherein the chemically modified mRNA includes a miR-126 binding site and a second microRNA binding site for miR selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 98) The method according to item 96, wherein the chemically modified mRNA includes a miR-126 binding site and a miR-142 binding site. (Item 99) The method according to any one of items 70 to 98, wherein the lipid nanoparticles are liposomes. (Item 100) The method according to any one of items 70 to 98, wherein the lipid nanoparticles include cationic lipids and / or ionized lipids. (Item 101) The method according to item 100, wherein the cationic lipid and / or ionized lipid is DLin-KC2-DMA or DLin-MC3-DMA. (Item 102) A modified messenger RNA (mmRNA) encoding a target polypeptide, wherein the mmRNA comprises at least two different microRNA (miR) binding sites, the microRNA is expressed in hematopoietic immune cells or in cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines, and the mmRNA comprises one or more modified nucleobases. (Item 103) The hematopoietic immune cells are lymphoid cells such as T cells, B cells, or NK cells, as specified in item 102, and the mMRNA is as described above. (Item 104) The mmRNA described in item 102, wherein the hematopoietic immune cells are myeloid cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, or platelets. (Item 105) The aforementioned hematopoietic immune cells are hematopoietic progenitor cells, and the mmRNA is as described in item 102. (Item 106) The mmRNA described in item 102, wherein the cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines are endothelial cells. (Item 107) The mmRNA described in any one of items 102 to 106, wherein the microRNA is abundant within the same target cell type or within different target cell types. (Item 108) The aforementioned microRNA is an mmRNA described in any one of items 102 to 106, which is abundant in multiple target cell types. (Item 109) The mmRNA described in item 102, wherein the mmRNA includes at least one first microRNA binding site of a large number of microRNAs in hematopoietic immune cells and at least one second microRNA binding site of a large number of microRNAs in endothelial cells. (Item 110) The mmRNA described in item 102, wherein the mmRNA comprises at least one first microRNA binding site of a microRNA abundant in B cells and at least one second microRNA binding site of a microRNA abundant in endothelial cells. (Item 111) The mmRNA described in item 102, wherein the mmRNA comprises at least one first microRNA binding site of a microRNA abundant in plasmacytoid dendritic cells and at least one second microRNA binding site of a microRNA abundant in endothelial cells. (Item 112) mmRNA as described in any one of items 102 to 111, comprising multiple copies of a first microRNA binding site and at least one copy of a second microRNA binding site. (Item 113) The mmRNA described in item 112, containing two copies of the first microRNA binding site. (Item 114) mmRNA as described in any one of items 102 to 111, including the first microRNA binding site and the second microRNA binding site of the same microRNA. (Item 115) The mmRNA described in item 114, wherein the microRNA binding sites are those of the 3p arm and 5p arm of the same microRNA. (Item 116) The mmRNA described in item 102, wherein the microRNA is selected from the group consisting of miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, and miR-26a. (Item 117) The mmRNA described in item 102, wherein the microRNA is selected from the group consisting of miR126-3p, miR-142-3p, miR-142-5p, and miR-155. (Item 118) mmRNA as described in item 102, wherein at least one microRNA binding site is a miR-126 binding site. (Item 119) mmRNA as described in item 102, wherein at least one microRNA binding site is a miR-142 binding site. (Item 120) mmRNA as described in item 102, wherein one microRNA binding site is a miR-126 binding site, and the second microRNA binding site is for a microRNA selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 121) mmRNA as described in item 102, comprising at least one miR-126-3p binding site and at least one miR-142-3p binding site. (Item 122) mmRNA as described in item 102, comprising at least one miR-142-3p binding site and at least one miR-142-5p binding site. (Item 123) mmRNA as described in item 102, comprising at least three different microRNA binding sites, wherein at least one of the microRNA binding sites is a miR-126 binding site. (Item 124) mmRNA as described in item 102, comprising at least three different microRNA binding sites, wherein at least one of the microRNA binding sites is a miR-142 binding site. (Item 125) mmRNA as described in item 102, comprising at least one miR-126-3p binding site, at least one miR-142-3p, and a third microRNA binding site selected from the group consisting of miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24, and miR-27. (Item 126) mmRNA as described in item 102, comprising at least one miR-126-3p binding site, at least one miR-142-3p binding site, and at least one miR-155 binding site. (Item 127) mmRNA as described in item 102, comprising at least one miR-126-3p binding site, at least one miR-142-3p binding site, at least one miR-142-5p binding site, and at least one miR-155 binding site. (Item 128) The mmRNA described in any of the preceding items, wherein the microRNA binding site is located within the 5'UTR, within the 3'UTR, or both within the 5'UTR and 3'UTR of the mmRNA. (Item 129) The mmRNA described in item 128, wherein the microRNA binding site is located within the 3'UTR of the mmRNA. (Item 130) The mmRNA described in item 128, wherein the microRNA binding site is located within the 5'UTR of the mmRNA. (Item 131) The mmRNA described in item 128, wherein the microRNA binding site is located both within the 5'UTR and 3'UTR of the mmRNA. (Item 132) The mmRNA according to item 128, wherein at least one microRNA binding site is located within the 3'UTR directly adjacent to the stop codon of the coding region of the mmRNA. (Item 133) The mmRNA described in item 128, wherein at least one microRNA binding site is located within the 3'UTR 70-80 bases downstream of the stop codon of the coding region of the mmRNA. (Item 134) The mmRNA according to item 128, wherein at least one microRNA binding site is located within the 5'UTR immediately preceding the start codon of the coding region of the mmRNA. (Item 135) The mmRNA according to item 128, wherein at least one microRNA binding site is located within the 5'UTR, 15 to 20 nucleotides preceding the start codon of the coding region of the mmRNA. (Item 136) The mmRNA according to item 128, wherein at least one microRNA binding site is located within the 5'UTR, 70-80 nucleotides preceding the start codon of the coding region of the mmRNA. (Item 137) mmRNA as described in item 128, containing multiple copies of the same microRNA binding site located directly adjacent to each other or with spacers of less than 5 nucleotides, 5-10, 10-15, or 15-20 nucleotides. (Item 138) The mmRNA described in item 128, comprising multiple copies of the same microRNA binding site located within the 3'UTR, wherein the first microRNA binding site is located directly adjacent to a stop codon, and the second and third microRNA binding sites are located 30 to 40 bases downstream of the first microRNA binding site. (Item 139) The mmRNA described in item 128, comprising two copies of a first microRNA binding site and one copy of a second microRNA binding site located within the 3'UTR, wherein the first copy of the first microRNA binding site is located directly adjacent to a stop codon, the second microRNA binding site is located 30-40 bases downstream of the first copy of the first microRNA binding site, and the second copy of the first microRNA binding site is located 30-40 bases downstream of the second microRNA binding site. (Item 140) The aforementioned mmRNA is a fully modified mmRNA as described in any of the preceding items. (Item 141) Pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m5C), 1-methylpseudridine (m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine(s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 mmRNA as described in any of the preceding items, including C). (Item 142) Pseudouridine (ψ), N1-methylpseuduridine (m 1 mmRNA as described in any of the preceding entries, including ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine, or a combination thereof. (Item 143) 1-Methylpseudridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) mmRNA as described in any of the preceding items, including pseudouridine(ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof. (Item 144) The target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein, as described in any of the preceding items, wherein the mMRNA is one of the preceding items. (Item 145) Lipid nanoparticles containing the mmRNA described in one of the preceding items. (Item 146) Lipid nanoparticles, including liposomes, as described in item 145. (Item 147) Lipid nanoparticles as described in item 145, comprising cationic lipids and / or ionized lipids. (Item 148) Lipid nanoparticles according to item 147, wherein the cationic lipid and / or ionized lipid is DLin-KC2-DMA or DLin-MC3-DMA. (Item 149) A pharmaceutical composition comprising the mmRNA according to any one of items 102 to 144, or the lipid nanoparticle according to any one of items 145 to 148, and a pharmaceutically acceptable carrier, diluent, or excipient. (Item 150) The mmRNA according to any one of items 102 to 144, the lipid nanoparticle according to any one of items 145 to 148, or the pharmaceutical composition according to item 149, for use in reducing or inhibiting an anti-drug antibody response or inhibiting drug-related toxicity in a subject in need of reducing or inhibiting an anti-drug antibody response or inhibiting drug-related toxicity. (Item 151) The mmRNA according to any one of items 102 to 144, the lipid nanoparticle according to any one of items 145 to 148, or the pharmaceutical composition according to item 149, for use in reducing or inhibiting undesirable immune cell activation or reducing or inhibiting undesirable cytokine production in a subject in need of reducing or inhibiting undesirable immune cell activation or reducing or inhibiting undesirable cytokine production. (Item 152) The mmRNA according to any one of items 102 to 144, the lipid nanoparticle according to any one of items 145 to 148, or the pharmaceutical composition according to item 149, for use in reducing or inhibiting accelerated blood clearance in a subject in need of reducing or inhibiting accelerated blood clearance. (Item 153) The mmRNA according to any one of items 102 to 144, the lipid nanoparticle according to any one of items 145 to 148, or the pharmaceutical composition according to item 149, for use in reducing or inhibiting production of IgM molecules that recognize polyethylene glycol (PEG) in a subject in need of reducing or inhibiting production of IgM molecules that recognize polyethylene glycol (PEG). BRIEF DESCRIPTION OF THE DRAWINGS
[0075] [Figure 1]Figure 1 is a bar graph showing the levels of human erythropoietin (hEPO) protein in cynomolgus monkeys treated with the indicated doses of mmRNA encoding hEPO (but lacking a miR binding site) or recombinant hEPO protein as a positive control, 6 hours after injection on the indicated day of treatment.
[0076] [Figure 2] Figure 2 is a schematic diagram of an mRNA construct without insertion of a miR site in the 3'UTR and an mRNA construct with insertion of a miR site in the 3'UTR.
[0077] [Figure 3] Figure 3 is a graph comparing anti-hEPO antibody levels in cynomolgus monkeys treated with 0.2 mg / kg of mmRNA encoding PBS or hEPO, which either lack or contain a miR-142-3p binding site in the 3'UTR of the construct. Animals positive for the anti-drug antibody (ADA) response are indicated.
[0078] [Figure 4] Figures 4A-C are graphs showing protein expression levels (Figure 4A), B cell frequency (Figure 4B), and activated B cell frequency (Figure 4C) in mice treated with 0.05 mg / kg of mmRNA encoding hEPO, which either lack or contain a miR-142-3p binding site, a miR-126 binding site, or both miR-142-3p and miR-126 binding sites in the 3'UTR of the construct.
[0079] [Figure 5]Figures 5A and 5B are graphs showing protein expression levels in mice treated with a single dose of 0.2 mg / kg (Figure 5A) or 1 mg / kg (Figure 5B) of a mRNA encoding hEPO that either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0080] [Figure 6] Figures 6A and 6B are graphs showing protein expression levels in mice treated with two doses of 0.2 mg / kg (Figure 6A) or 1 mg / kg (Figure 6B) of a mRNA encoding hEPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0081] [Figure 7] Figure 7 presents a graph showing the frequency of B cells in mice treated with a single dose of the indicated dose (0.2 mg / kg or 1 mg / kg) of an mmRNA encoding hEPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0082] [Figure 8] Figure 8 presents a graph showing the frequency of activated B cells in mice treated with a single dose of the indicated dose (0.2 mg / kg or 1 mg / kg) of an mMRNA encoding hEPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the construct's 3'UTR.
[0083] [Figure 9]Figure 9 presents a graph showing the frequency of B cells in mice treated with two doses of the indicated dose (0.2 mg / kg or 1 mg / kg) of an mMRNA encoding hEPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0084] [Figure 10] Figure 10 presents a graph showing the frequency of activated B cells in mice treated with two doses of the indicated dose (0.2 mg / kg or 1 mg / kg) of an mMRNA encoding hEPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the construct's 3'UTR.
[0085] [Figure 11] Figures 11A and 11B are graphs showing IL-6 levels in mice treated with a single dose of 0.2 mg / kg (Figure 11A) or 1 mg / kg (Figure 11B) of a mRNA encoding hEPO that either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0086] [Figure 12] Figures 12A and 12B are graphs showing IL-6 levels in mice treated with two doses of 0.2 mg / kg (Figure 12A) or 1 mg / kg (Figure 12B) of a mRNA encoding hEPO that either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0087] [Figure 13]Figures 13A to 13C are graphs showing IL-6 levels (Figure 13A), TNF-α levels (Figure 13B), and IFN-γ levels (Figure 13C) in mice treated with two administrations of 0.2 mg / kg mmRNA encoding hEPO, which lacks or contains a miR-142-3p binding site, a miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0088] [Figure 14] Figures 14A to 14C are graphs showing IL-6 levels (Figure 14A), TNF-α levels (Figure 14B), and IFN-γ levels (Figure 14C) in mice treated with two administrations of 1 mg / kg mmRNA encoding hEPO, which lacks or contains a miR-142-3p binding site, a miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0089] [Figure 15] Figures 15A to 15B are graphs showing the expression level of luciferase (Luc) measured by whole-body luminescence in mice treated with 0.2 mg / kg mmRNA encoding Luc, which lacks or contains a miR-142-3p binding site, a miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct, for 1 week (Figure 15A) or 2 weeks (Figure 15B).
[0090] [Figure 16] Figure 16 presents a graph showing the frequency of total B cells in mice treated with 0.2 mg / kg mmRNA encoding Luc, which lacks or contains a miR-142-3p binding site, a miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0091] [Figure 17] Figure 17 presents a graph showing the frequency of activated B cells in mice treated with 0.2 mg / kg of mmol RNA encoding Luc, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0092] [Figure 18A-B] Figures 18A-C are graphs showing the IL-6 levels (Figure 18A), TNF-α levels (Figure 18B), and IFN-γ levels (Figure 18C) secreted in mice treated with 0.2 mg / kg of mmol RNA encoding Luc, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the 3'UTR of the construct. [Figure 18C] Figures 18A-C are graphs showing the IL-6 levels (Figure 18A), TNF-α levels (Figure 18B), and IFN-γ levels (Figure 18C) secreted in mice treated with 0.2 mg / kg of mmol RNA encoding Luc, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the 3'UTR of the construct.
[0093] [Figure 19] Figures 19A and 19B are graphs showing the serum EPO expression levels of mice treated for one week (Figure 19A) or two weeks (Figure 19B) with 0.2 mg / kg of mMRNA encoding EPO, which either lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0094] [Figure 20]Figure 20 presents a graph showing the frequency of total B cells in mice treated for one week with 0.2 mg / kg of EPO-encoding mmol RNA, which lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0095] [Figure 21] Figure 21 presents a graph showing the frequency of activated B cells in mice treated for one week with 0.2 mg / kg of mRNA encoding EPO, which lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0096] [Figure 22] Figure 22 presents a graph showing the frequency of total B cells in mice treated for two weeks with 0.2 mg / kg of EPO-encoding mMRNA, which lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0097] [Figure 23] Figure 23 presents a graph showing the frequency of activated B cells in mice treated for two weeks with 0.2 mg / kg of mRNA encoding EPO, which either lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0098] [Figure 24A]Figures 24A-C are graphs showing the IL-6 levels (Figure 24A), TNF-α levels (Figure 24B), and IFN-γ levels (Figure 24C) secreted in mice treated for two weeks with 0.2 mg / kg of mRNA encoding EPO, which either lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof. [Figure 24B] Figures 24A-C are graphs showing the IL-6 levels (Figure 24A), TNF-α levels (Figure 24B), and IFN-γ levels (Figure 24C) secreted in mice treated for two weeks with 0.2 mg / kg of mRNA encoding EPO, which either lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof. [Figure 24C] Figures 24A-C are graphs showing the IL-6 levels (Figure 24A), TNF-α levels (Figure 24B), and IFN-γ levels (Figure 24C) secreted in mice treated for two weeks with 0.2 mg / kg of mRNA encoding EPO, which either lacks or contains multiple copies or combinations of the miR-142-3p binding site, the miR-142-5p binding site, the miR-155-5p binding site, or a combination thereof.
[0099] [Figure 25] Figures 25A and 25B are graphs showing the percentage of CD27+CD19+B cells in spleen CD19+B cells (Figure 25A) and the expression level of CD27 in CD27+CD19+B cells (Figure 25B) in mice treated with mRNA encoding EPO that lacks or contains the miR-142 binding site, the miR-126 binding site, or miR-142 and miR-126 binding sites.
[0100] [Figure 26]Figures 26A and 26B are graphs showing the frequency of all CD11c+ cells (Figure 26A) and the percentage of activated dendritic cells (CD11c+CD70+CD86+ cells) (Figure 26B) in spleen cells of mice treated with mRNA encoding EPO that lacks or contains the miR-142 binding site, the miR-126 binding site, or miR-142 and miR-126 binding sites.
[0101] [Figure 27] Figure 27 presents a graph showing the growth levels of naive B cells in the presence of plasmacytoid dendritic cells (pDCs) isolated from mice treated with mRNA encoding EPO that lacks or contains the miR-142 binding site, the miR-126 binding site, or the miR-142 and miR-126 binding sites.
[0102] [Figure 28A-B] Figures 28A-C are graphs showing serum anti-PEG IgM antibody levels in mice treated with EPO-encoding mMRNAs that either lack or contain the miR-142 binding site, the miR-126 binding site, or both miR-142 and miR-126 binding sites, after two doses (Figure 28A), three doses (Figure 28B), or four doses (Figure 28C). [Figure 28C] Figures 28A-C are graphs showing serum anti-PEG IgM antibody levels in mice treated with EPO-encoding mMRNAs that either lack or contain the miR-142 binding site, the miR-126 binding site, or both miR-142 and miR-126 binding sites, after two doses (Figure 28A), three doses (Figure 28B), or four doses (Figure 28C).
[0103] [Figure 29]Figure 29 presents a graph showing serum EPO expression levels in mice treated for 6 weeks with 0.2 mg / kg of mRNA encoding EPO, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both the miR-142-3p and miR-126 binding sites within the construct's 3'UTR.
[0104] [Figure 30] Figure 30 presents a graph showing the serum luciferase (Luc) expression levels of mice treated for 5 weeks with 0.2 mg / kg of mmol RNA encoding Luc, which either lacks or contains the miR-142-3p binding site, the miR-126 binding site, or both miR-142-3p and miR-126 binding sites within the construct's 3'UTR.
[0105] [Figure 31] Figures 31A-D are graphs showing the eGFP expression levels in primary hepatocytes transfected with equimolar mixtures of Luc mRNA constructs and eGFP mRNA constructs within LNPs at doses of 7.5 ng (Figure 31A), 15 ng (Figure 31B), 50 ng (Figure 31C), or 100 ng (Figure 31D), wherein the mRNA constructs either do not contain recognizable miR sites (control), contain 1X miR-122 binding sites or 3X miR-122 binding sites, or contain putative mRNA (control) with sequences similar to both the eGFP sequence and the Luc sequence.
[0106] [Figure 32]Figures 32A-D are graphs showing the confluence phase percentage (as a measure of caspase-mediated toxicity) of primary hepatocytes transfected with caspase mRNA constructs at doses of 7.5 ng (Figure 32A), 15 ng (Figure 32B), 50 ng (Figure 32C), or 100 ng (Figure 32D), containing either no recognizable miR sites (control), a 1X miR-122 binding site or a 3X miR-122 binding site, or a caspase-like sequence-containing putative mRNA without a start codon (control).
[0107] [Figure 33] Figure 33 shows a graph illustrating the hEPO expression levels in cynomolgus monkeys administered mRNA constructs that either did not contain a recognizable miR site (control), contained a 1X miR-142-3p binding site, or contained a 3X miR-142-3p binding site.
[0108] [Figure 34] Figure 34 presents a bar graph showing eGFP expression levels in RAW264.7 cells transfected with mRNA constructs that either do not contain a recognizable miR site (control), contain a miR-142-3p binding site in the 3'UTR (1X or 3X), a miR-142-3p binding site in the 5'UTR (inserted into P1, P2, or P3), or contain a miR-142-3p binding site in both the 3'UTR and 5'UTR.
[0109] [Figure 35] Figure 35 presents a bar graph showing the expression levels of hEPO in primary hepatocytes transfected with mRNA constructs that either do not contain a recognizable miR site (control), contain a miR-122 binding site in the 3'UTR (1X or 3X), a miR-122 binding site in the 5'UTR (inserted in P1, P2, or P3), or contain a miR-122 binding site in both the 3'UTR and 5'UTR. [Modes for carrying out the invention]
[0110] One challenge associated with the clinical use of protein-based therapeutics is the development of undesirable anti-drug antibody (ADA) responses, in which the patient's immune system generates antibodies against the therapeutic agent (for reviews, see, for example, Subramanyam, M. (2006), J. Immunotoxicol., Vol. 3: pp. 151-156; De Groot, AS and Scott, DW (2007), Trends Immunol., Vol. 28: pp. 482-490; Nechansky, A. and Kircheis, R. (2010), Expert Opin. Drug. Discov., Vol. 5: pp. 1067-1079). The development of ADA responses has been reported for both recombinant antibody-mediated and non-antibody-mediated biological agents (see, for example, Brickelmaier, M. et al. (1999), J. Immunol., Methods, Vol. 227: pp. 121-135; Ruf, P. et al. (2010), Br. J. Clin. Pharmacol., Vol. 69: pp. 617-625; Lundkvist, M. et al. (2013), Mult. Scler., Vol. 19: pp. 757-764). ADA responses can interfere with or neutralize the effects of therapeutic agents, thereby affecting pharmacokinetics and efficacy. Neutralizing antibodies (NABs) are generally more important than non-neutralizing conjugating antibodies (BABs), but both can have clinical consequences.
[0111] Furthermore, allergic reactions, complement activation, and other adverse events are also associated with the development of ADA, which often affects drug safety. Therefore, ADA is an important factor in the ability to use biological agents for long-term treatment.
[0112] The use of modified mRNA, such as mRNA (mmRNA), as a therapeutic agent offers an attractive alternative to protein-based therapeutics. mRNA therapeutics offer several advantages over protein-based therapeutic technologies, including fidelity to encoded protein characteristics (because proteins are produced by the body's own translation machinery), a highly sensitive and finely tuneable pharmacokinetic profile (protein expression can be transient, which can be advantageous for controlling pharmacokinetics and administration in some therapies), a superior safety profile (as demonstrated in various vaccine clinical trials), intracytoplasmic functionality that does not require nuclear translocation, resulting in protein translation almost immediately after mRNA administration, thus eliminating the risk of genomic integration, as well as ease of manufacture, such as the ease with which mRNA can be produced through diverse and common in vitro processes, such as in vitro transcription reactions that do not require organisms. Furthermore, mRNA can be designed to have self-adjuvanting properties, for example in vaccine applications, or to avoid immunogenic activation, for example in therapeutic applications. However, it has now been discovered that the administration of mRNA encoding a target protein can also lead to the development of an anti-drug antibody response against the mRNA-encoded protein, particularly when mRNA administration directly or indirectly results in the expression of the encoded protein within immune cells, such as the spleen. Surprisingly, however, it has also been confirmed that the integration of at least one microRNA (miRNA) binding site to a miR expressed within immune cells (e.g., within immune cells and / or spleen cells of peripheral lymphoid organs) into mRNA encoding a target protein can reduce the anti-drug antibody response against the target protein when the mRNA is administered to a subject.
[0113] Accordingly, this disclosure presents methods for reducing or inhibiting anti-drug antibody (ADA) responses to proteins of interest by post-transcriptional regulation within immune system tissues, such as the spleen. This disclosure also presents methods for reducing drug-related toxicity in subjects by incorporating at least one microRNA (miRNA) binding site to miRs expressed in immune cells into mRNA (e.g., mmRNA) encoding a protein of interest. Preferred microRNA binding sites used in the methods of this disclosure are those that bind to miRs that are abundantly or preferentially expressed in immune cells (e.g., in immune cells of peripheral lymphoid organs and / or spleen cells). A particularly preferred microRNA binding site is that for miR-142-3p. Another particularly preferred microRNA binding site is that for miR-126.
[0114] As described in Example 1, in vivo studies in which cynomolgus monkeys were administered an mRNA construct encoding human erythropoietin (hEPO) resulted in the observation that hEPO levels in the animals decreased over time. Furthermore, reticulocytopenia and reduced hematopoiesis in the bone marrow were also observed. These results suggest the possibility of anti-drug antibody development in the animals, which was confirmed by ELISA analysis of the serum. While not bound by theory in any way, prior findings with the mRNA delivery system used in the study (using LNPs, which are lipid nanoparticles) supported the idea that mRNA is primarily distributed to the liver, but also to the spleen, and therefore this distribution may result in enhanced immunity against the proteins produced by the mRNA. Here again, though not bound by theory or mechanism, the expression of encoded proteins within the spleen (delivered either directly via bloodstream based on LNP distribution to the spleen, or indirectly via specialized antigen-presenting cells) may lead to T cell-dependent antibody production via the presentation of appropriate epitopes (i.e., derived from the target protein) to T cells.
[0115] To address this, as described in Example 2, we designed further mRNA constructs containing at least one microRNA (miRNA) binding site for miRs expressed in immune cells (e.g., at least one binding site for miR-142-3p). In vivo administration of the miR-containing mRNA constructs resulted in a significant reduction in the development of the ADA response in recipient animals.
[0116] Another challenge associated with the clinical use of protein-based therapeutics in this art is the development of undesirable immune cell activation (e.g., B cell activation) in response to therapeutic proteins, resulting in immune-mediated side effects. However, it has now been discovered that administration of mRNA encoding a protein of interest, particularly when the mRNA administration directly or indirectly results in the expression of the encoded protein in immune cells, such as splenic cells, can also lead to the development of undesirable immune cell activation (e.g., B cell activation, including cytokine production). Surprisingly, however, it has also been shown that the incorporation of at least one binding site, particularly at least one miR-126 binding site and / or miR-142 binding site, into mRNA for microRNAs (miRNAs) expressed in peripheral lymphoid tissues and / or endothelial cells can reduce or inhibit undesirable immune cell activation when the mRNA is administered to a subject. Accordingly, this disclosure presents compositions and methods for reducing or inhibiting undesirable immune cell activation, particularly through post-transcriptional regulation within immune system tissues such as peripheral lymphoid organs or the spleen, when using mRNA-based therapeutic agents.
[0117] The experiments described in Example 3 confirmed that the incorporation of a miR-126 binding site, a miR-142 (e.g., miR-142-3p) binding site, or a combination of the two sites, into an mRNA construct encoding the target protein resulted in a reduction in the frequency of activated B cells and a reduction in the levels of cytokine (IL-6, TNF-α, IFN-γ) production in animals treated with the construct compared to animals treated with a construct lacking the miR binding site. The effect of the miR-126 binding site alone was more potent than that of the miR-142 binding site alone, with the strongest effect observed when the two sites were used in combination. The frequency of B cell activation and cytokine production are indicators of the induction of an early immune response in vivo, including an antibody response. Thus, these results suggest that the incorporation of the miR-126 binding site into mRNA constructs (either alone or in combination with the miR-142-3p binding site) can lead to a reduction in the development of the ADA response to the encoded protein in recipient animals.
[0118] While not bound by theory in any way, the incorporation of the miR-126 binding site into mRNA constructs may lead to a reduction or inhibition of immune cell activation via one or more possible mechanisms, based on the miR-126 expression pattern. MicroRNA-126 is known to be highly and selectively expressed in plasmacytoid dendritic cells (pDCs) and to regulate the maturation, survival, and effector function of these cells (Agudo, J. et al. (2014), Nat. Immunol., Vol. 15: pp. 54-62; Cella, M. and Trinchieri, G. (2014), Nat. Immunol., Vol. 15: pp. 8-9). Plasmacytoid dendritic cells (pDCs) make up less than 0.1% of peripheral blood mononuclear cells and 0.4-0.6% of all splenocytes. When activated, they differentiate into dendritic cells, produce interferon, and are used as a link between innate and acquired immunity, as well as potentially playing a role in antigen presentation (for reviews on pDCs, see, for example, Jegalian, AG et al. (2009), Adv. Anat. Pathol., Vol. 16: pp. 392-404; Reizis, B. et al. (2011), Annu. Rev. Immunol., Vol. 29: pp. 163-183; Tel, J. et al. (2012), Cancer Immunol. Immunotherap., Vol. 61: pp. 1279-1288). Furthermore, pDCs are also involved in promoting B cell activation and differentiation, as well as stimulating cytokine production (see, for example, Douag, I. et al. (2009), J. Immunol., Vol. 182: pp. 1991-2001; Ding, C. et al. (2009), J. Immunol., Vol. 183: pp. 7140-7149; Gujer, C. et al. (2011), J. Leukoc. Biol., Vol. 89: pp. 811-821).Therefore, the reduction in B cell activation frequency and cytokine production observed upon incorporation of the miR-126 binding site into the mRNA construct may result from, for example, the inhibition of pDC antigen presentation function and / or the inability of pDCs to produce an effective response to foreign nucleic acids and / or the inhibition of pDC maturation and survival, which leads to reduced promotion of B cell activation and reduced cytokine production, and subsequently, the overall consequence of these effects is a reduction in the ADA response to the protein encoded by the mRNA construct in vivo.
[0119] In addition, it is known that miR-126 is expressed in endothelial cells (see, for example, Fish, JE et al. (2008), Dev. Cell., Vol. 15: pp. 272-284; Wang, S. et al. (2008), Dev. Cell., Vol. 15: pp. 261-271). Therefore, the effect of incorporating the miR-126 binding site into the mRNA construct may be related to the abundance of miR-126 in endothelial cells. Thus, incorporating the miR-126 binding site into the mRNA construct can lead to a reduction in the expression of proteins encoded in endothelial cells in vivo, resulting in a reduction in antigen presentation by endothelial cells, which in turn leads to a synchronous reduction in the frequency of B cell activation and cytokine production, and ultimately to a reduction in the ADA response to the encoded proteins in vivo.
[0120] As demonstrated in Example 6, the incorporation of the miR-142 and / or miR-126 binding sites into the mRNA construct is CD11c + A reduction in the total frequency of dendritic cells, and CD11c + Activated dendritic cells (CD11c) within a population of spleen cells + CD70 + CD86 + This results in a reduction in the frequency of cells. In contrast, the incorporation of the miR-142 and / or miR-126 binding sites into the mRNA construct leads to spleen CD19 + CD27 in B cells + CD19+ It does not affect the frequency of B cells, CD27 + CD19 + It also did not affect the expression levels of CD27 within the B cell population. Furthermore, incubation with pDCs isolated from mice treated with the miR binding site-containing construct resulted in reduced growth of naive B cells compared to treatment with mRNA constructs lacking the miR binding site. Therefore, the data from this experiment support the proposed mechanism by which the inhibition of B cell activation and cytokine production in mice treated with modified mRNA constructs containing one or a combination of miR binding sites arises from a decrease in pDC frequency and / or activation, thereby resulting in reduced B cell stimulation, which also results from a decrease in CD70-CD27 interaction or reduced cytokine secretion by dendritic cells.
[0121] In the use of lipid-containing compounds and compositions, such as lipid nanoparticles (LNPs), with respect to therapeutic agents, e.g., modified mRNA, there is also a separate challenge (i.e., accelerated blood clearance (ABC)) where the drug is rapidly cleared from the blood during second and subsequent administrations. The mechanism involves the recognition of lipid-containing compounds or compositions (e.g., LNPs) by B cells, particularly B1a cells, including the recognition of lipid components such as phosphatidylcholine via CD36 and / or TLR recognition. Activated B1a cells secrete intrinsic IgM, particularly IgM, which can contribute to ABC (e.g., via acute-phase response mechanisms). The phospholipid component (e.g., DSPC) of lipid-containing compounds or compositions (e.g., LNPs) can also activate, for example, circulating platelets. Activated platelets aggregate and bind to macrophages, which can subsequently release inflammatory cytokines and migrate to the spleen. Blockage of lipid-containing compounds or compositions (e.g., LNPs) into the spleen occurs almost immediately after administration.
[0122] ABC has been found to be mediated, at least partially, by B cells, specifically B1a cells. These B cells are responsible for the secretion of multireactive innate IgM antibodies, meaning they are capable of binding to a variety of antigens, although their affinity for each is usually relatively low. Upon administration of the first dose of a drug, B1a cells bind to the drug and become activated, thereby secreting innate IgM that binds to the drug, such as phosphatidylcholine. Subsequently, a second or subsequent administration of a lipid-containing compound or composition is targeted by circulating IgM and rapidly cleared. Conventional B cells, referred herein as B2 cells or CD19(+) B cells, are also involved in ABC. Specifically, conventional B cells can also elicit an IgM response first, followed by an IgG response, which is synchronized with a memory response. Conventional B cells react to administered drugs and polyethylene glycol (PEG), contributing to the IgM (and ultimately, IgG) that mediates ABC. Previous solutions to this problem focused on suppressing the immune response in subjects administered LNP compositions. In particular, co-medication regimens (e.g., antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, corticosteroids, etc.) were used to suppress the immune system. However, it has now been discovered that incorporating at least one microRNA binding site to microRNAs expressed in immune cells (e.g., miR-126, miR-142, miR-155, and combinations thereof) into a modified mRNA construct can reduce or inhibit ABC when a lipid-containing compound or a composition containing modified mRNA is administered to a subject. Specifically, it has been found that incorporating at least one microRNA binding site into modified mRNA may reduce or inhibit the growth and / or activation of plasmacytoid dendritic cells, and / or reduce or inhibit the production of anti-PEG IgM. For example, as demonstrated in Example 7, the incorporation of at least one miR binding site into an mRNA construct results in a decrease in serum anti-PEG IgM antibody levels in mice administered with a lipid-containing compound or a composition containing an mRNA construct.
[0123] There are several possible mechanisms by which the incorporation of at least one microRNA binding site, delivered by a lipid-containing compound or composition, into a modified mRNA construct, as described herein, results in the reduction or inhibition of ABC. In one embodiment, the mechanism of action of the miRNA binding site is that the miRNA binding site within the construct acts as a microRNA "sponge," "absorbing" the microRNAs that bind to the binding site. This can result in the de-dynchronization of specific microRNAs against their native targets, as these microRNAs become less / not at all available to regulate them. This scenario mimics the knockdown / knockout effect of microRNAs. In examples where proper regulation of the native targets of microRNAs is necessary for a cell's ability to act as an effective immune cell, this microRNA sponge effect makes it impossible for the cell to produce an immune response. De-dynchronization of endogenous targets by microRNAs can also lead to stress responses (e.g., responses of unfolded proteins) by disrupting cellular homeostasis (e.g., calcium signaling). Alternatively, the integration of a microRNA binding site into mRNA may suppress the expression of this mRNA within specific microRNA-containing cell types. Furthermore, integration of the microRNA binding site may lead to mRNA degradation before sensors such as TLR7 can recognize it. These latter two mechanisms rely on the cleavage of mRNA containing one or more binding sites to microRNAs (miRs) expressed in immune cells, mediated by RNA-induced silencing complexes (RISCs). These mechanisms may also act cooperatively to produce the miR-mediated and observed effects described herein.
[0124] Regardless of the mechanism involved, the effect resulting from the incorporation of at least one microRNA binding site into the mRNA construct, as described herein, is that immune cells that recognize lipid-containing compounds or compositions (e.g., pDCs, B cells (e.g., circulating B cells), macrophages) are not activated and therefore do not migrate to the spleen to activate B cells (e.g., splenic B cells). In addition, cytokine production (e.g., IL-6) is also reduced or inhibited, thereby further preventing the activation of immune cells. The reduction or inhibition of B cell activation results in the reduction or inhibition of innate IgM (e.g., by B1a cells), IgM, and IgG. The production of these molecules is essential for ABC, and therefore the reduction or inhibition of their production reduces or inhibits ABC as a whole.
[0125] Accordingly, this disclosure presents methods for reducing or inhibiting ABC when using a lipid-containing compound or composition that includes modified mRNA encoding a polypeptide of interest.
[0126] The various aspects of this disclosure are further described in the following subsections.
[0127] mRNA This disclosure presents isolated RNA, in particular mRNA, e.g., chemically modified mRNA, that encodes the polypeptide of interest and contains at least one microRNA binding site (e.g., a miR-126 binding site and / or a miR-142 binding site). In other embodiments, this disclosure presents RNA, e.g., chemically modified RNA, that contains at least one microRNA binding site (e.g., a miR-126 binding site and / or a miR-142 binding site) but does not necessarily encode the polypeptide of interest. The latter RNA may also contain a miR-126 binding site and / or a miR-142 binding site, although it may also lack other typical mRNA features (such as those described below).
[0128] RNA may be naturally occurring RNA or non-naturally occurring RNA, for example, mRNA. mRNA comprises one or more modified nucleobases, modified nucleosides, or modified nucleotides, which may be referred to as "chemically modified mRNA," but also herein as "modified mRNA" or "mmRNA." A "nucleoside" as used herein is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof, combined with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also herein referred to as a "nucleobase"). A "nucleotide" as used herein is defined as a nucleoside containing a phosphate group.
[0129] mRNA may contain a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and / or a coding region (e.g., an open reading frame). mRNA may contain any suitable number of base pairs, including hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands (e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000) of base pairs. Any number (e.g., all, some, or zero) of nucleobases, nucleosides, or nucleotides may be analogues of canonical molecular species, may be substituted, may be modified, or may otherwise be unspontaneously occurring. In certain embodiments, all of a particular nucleotide type or nucleobase type may be modified.
[0130] In one embodiment, the mRNA includes a first flanking region located at the 5' end of the open reading frame (coding region) and a second flanking region located at the 3' end of the open reading frame (coding region), in which case the first flanking region includes a 5' untranslated region (5'UTR) and the second flanking region includes a 3' untranslated region (3'UTR). In one embodiment, the 5'UTR and 3'UTR of the mRNA are not derived from the same molecular species. In one embodiment, the 5'UTR and / or 3'UTR of the mRNA are not derived from beta-globin. In one embodiment, the 5' untranslated region is heterogeneous with respect to the coding region of the mRNA. In another embodiment, the 3' untranslated region is heterogeneous with respect to the coding region of the mRNA. In yet another embodiment, the 5' and 3' untranslated regions are heterogeneous with respect to the coding region of the mRNA. In yet another embodiment, the mRNA includes at least two stop codons.
[0131] A non-limiting example of a 5'UTR sequence suitable for use in mRNA constructs is shown in SEQ ID NO: 53. A non-limiting example of a 3'UTR sequence suitable for use in mRNA constructs is shown in SEQ ID NO: 30. Other suitable 5'UTRs and 3'UTRs suitable for use in mRNA constructs are also known in the art.
[0132] For example, suitable 5'UTRs include the β-globin gene (e.g., Kariko et al. (2008), Mol. Therap., Vol. 16: pp. 1833-1840; see US8,278,063, US9,012,219), the α-globin gene (e.g., see US9,012,219), the human cytochrome b-245α polypeptide gene (CYBA) (e.g., Ferizi et al. (2015), Lab. Chip., Vol. 23: pp. 1456-1464), and the hydroxysteroid (17-β) dehydrogenase gene (HSD17B4) (e.g., Thess et al. (2015), Mol. Therap., Vol. 23: pp. 1456-1464; see WO2015 / 024667), TOP gene (e.g., see WO2015 / 101414, WO2015 / 101415, WO2015 / 062738, WO2015 / 024667, WO2015 / 024667), ribosomal protein large 32 (L32) gene (e.g., see WO2015 / 101414, WO2015 / 101415, WO2015 / 062738), and ATP51 gene (e.g., see WO2015 / 024667) 5'UTR, as well as tobacco etch virus (TEV) (e.g., Katalin et al. (2012), Mol. Therap., Vol. 20: pp. 948-953; see US8, 278, 063, US9, 012, 219), Venezuelan encephalitis virus (VEEV) (see, e.g., Andries et al. (2015), J. Control Release, Vol. 217: pp. 337-344), and CMV i.e., early 1 (IE1) gene (see, e.g., US20140206753, WO2014 / 089486, WO2013 / 185069, WO2014 / 144196, WO2014 / 152659, WO2014 / 152940, WO2014 / 152774, WO2014 / 153052), including viral 5'UTRs. Synthetic 5'UTRs are described and suitable for use (see, for example, Mandal and Rossi (2013), Nat. Protocol, Vol. 5: pp. 68-82).
[0133] In addition, for example, a suitable 3'UTR is the β-globin gene (e.g., Kariko et al. (2008), Mol. See Therap., Vol. 16: pp. 1833-1840; US8,278,063; US9,012,219; WO2007 / 036366, US2011 / 0065103, WO2011 / 015347, WO2012 / 072096, WO2013 / 143555, WO2014 / 071963), α-globin gene (see, for example, US9,012,219; WO2015 / 101414, WO2015 / 101415, WO2015024667), human cytochrome b-245α polypeptide gene (CYBA) (see, for example, Ferizi et al. (2015), Lab. See Chip., Vol. 23: pp. 1456-1464), albumin gene (e.g., Thess et al. (2015), Mol. Therap., Vol. 23: pp. 1456-1464), human growth hormone (hGH) gene (e.g., see US20140206753, WO2013 / 185069, WO2014 / 089486, WO2014 / 144196, WO2014 / 152659, WO2014152940, WO2014 / 152774, WO2014 / 153052), ribosomal rps9 protein gene (e.g., WO2015 This includes viral 3'UTRs, including those derived from the FIG4 gene (see, for example, WO2015 / 101415), the human albumin 7 gene (see, for example, WO2015 / 101415, WO2015 / 101414, WO2015 / 06273, WO2015 / 024667, WO2105 / 062737), and 3'UTRs derived from Venezuelan encephalitis virus (VEEV) (see, for example, Andries et al. (2015), J. Control Release, Vol. 217: pp. 337-344).
[0134] In some embodiments, the mRNA described herein may include a 5' cap structure, a chain termination nucleotide, a Kozak sequence (also known as the Kozak consensus sequence), a stem-loop, a poly(A) sequence, and / or a polyadenylation signal. In other embodiments, the mRNA lacks a poly(A) sequence and / or a polyadenylation signal and contains alternative structures for stabilizing the mRNA.
[0135] A 5' cap structure or 5' cap molecular species is a compound containing two nucleoside moieties linked by a linker, and can be selected from spontaneously occurring caps, non-spontaneously occurring caps or cap analogs, or anti-reverse cap analogs (ARCA). A cap molecular species may contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap consists of a guanine nucleotide and a 7-methylated guanine (G) nucleotide linked by a triphosphate linkage at their 5' positions, for example, generally m 7 It is written as GpppG, m 7 This may include G(5')ppp(5')G. The cap molecular species may also be an anti-reverse cap analog. A non-restrictive list of possible cap molecular species is given by m 7 GpppG, m 7 Gpppm 7 G, m 7 3'dGpppG, m2 7,O3’ GpppG, m2 7,O3’ GppppG, m2 7,O2’ GppppG, m 7 Gpppm 7 G, m 7 3'dGpppG, m2 7,O3’ GpppG, m2 7,O3’ GppppG, and m2 7,O2’It contains GppppG. In various embodiments, the mRNA may contain a 5' terminal cap selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methylguanosine, 2'fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNAguanosine, and 2-azidoguanosine. In one embodiment, the 5' terminal cap is Cap1.
[0136] mRNA may, in addition to or instead, contain chain-terminating nucleosides. For example, chain-terminating nucleosides may include nucleosides deoxygenated at the 2' and / or 3' positions of their sugar groups. Such molecular species may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, as well as 2',3'-dideoxy nucleosides such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, for example, the incorporation of a chain termination nucleotide at the 3' end into mRNA may result in mRNA stabilization, as described, for example, in International Patent Publication WO2013 / 103659.
[0137] mRNA may, in addition to or instead, contain stem loops, such as histone stem loops. Stem loops may contain 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may contain 4, 5, 6, 7, or 8 nucleotide base pairs. Stem loops can be located within any region of mRNA. For example, a stem loop may be located within an untranslated region (5' or 3' untranslated region), within a coding region, within a polyA sequence or polyA tail, before or after these. In some embodiments, stem loops may affect one or more functions of mRNA, such as translation initiation, translation efficiency, and / or transcription termination.
[0138] mRNA may instead, or in addition, contain a poly(A) sequence and / or a polyadenylation signal. The poly(A) sequence may consist entirely or nearly entirely of adenine nucleotides or their analogues or derivatives. The poly(A) sequence may be a tail positioned adjacent to the 3' untranslated region of the mRNA. In some embodiments, the poly(A) sequence may affect the extrusion of mRNA from the nucleus, translation, and / or stability.
[0139] In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region, along with an intervening sequence containing an internal ribosome entry site (IRES) sequence that enables the initiation of translation between the first and second coding regions, or an intervening sequence encoding a self-cleaving peptide such as a 2A peptide. IRES sequences and 2A peptides are typically used to enhance the expression of multiple proteins from the same vector. Various IRES sequences are known and available in the art, and for example, IRES sequences containing encephalomyocarditis virus IRESs can be used.
[0140] In one embodiment, the polynucleotide of the present disclosure may include a sequence encoding a self-cleaving peptide. The self-cleaving peptide may be, but is not limited to, a 2A peptide. Various 2A peptides are known and available in the art, and 2A peptides that can be used include, for example, the foot-and-mouth disease virus (FMDV) 2A peptide, the equine rhinitis virus type A 2A peptide, the Thosea asigna virus 2A peptide, and the porcine rhinitis virus type I 2A peptide. 2A peptides are used by some viruses so that, by ribosome skipping, two discontinuous proteins are produced from one translation event as a result of the normal peptide bond being impaired in the 2A peptide sequence, generating two proteins from one transcript. As a non-limiting example, a 2A peptide may have the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 4), a fragment thereof, or a variant thereof. In one embodiment, the 2A peptide is cleaved between the last glycine and the last proline. As another non-limiting example, the polynucleotides of this disclosure may include polynucleotide sequences encoding a 2A peptide having the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 4), a fragment thereof, or a variant thereof. An example of a polynucleotide sequence encoding a 2A peptide is GGAAGGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 5). In one exemplary embodiment, the 2A peptide is encoded by the following sequence: 5'-TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC-3' (SEQ ID NO: 6). The polynucleotide sequence of the 2A peptide can be modified or codon-optimized by the methods described herein and / or methods known in the art.
[0141] In one embodiment, this sequence can be used to isolate two or more coding regions of the target polypeptide. As a non-limiting example, the sequence encoding the 2A peptide may be located between a first coding region A and a second coding region B (A-2Apep-B). The presence of the 2A peptide results in the cleavage of a single long protein into protein A, protein B, and the 2A peptide. Protein A and protein B may be the same peptide, different peptides, or the target polypeptide.
[0142] modified mRNA In some embodiments, the mRNA of this disclosure comprises one or more modified nucleobases, modified nucleosides, or modified nucleotides (referred to as "chemically modified mRNA," but also referred to herein as "modified mRNA" or "mmRNA"). In some embodiments, modified mRNA may have useful properties compared to standard unmodified mRNA, including enhanced stability, intracellular storage, enhanced translation, and / or substantial absence of induction of the innate immune response in the cell into which the mRNA is introduced. Thus, the use of modified mRNA may result in increased efficiency of protein production, increased intracellular storage of nucleic acids, and reduced immunogenicity.
[0143] In some embodiments, the mRNA contains one or more (e.g., one, two, three, or four) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA contains one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may reduce the degradation of the mRNA within the cell into which it is introduced compared to the corresponding unmodified mRNA.
[0144] In one embodiment, the mRNA includes at least one nucleoside (or nucleotide) modification. In another embodiment, the mRNA includes at least one modification compared to the chemical structure of a ribonucleoside that is A, G, U, or C. In yet another embodiment, the mRNA is (a) a first region of a linked nucleoside comprising a first region encoding the target polypeptide; (b) A first flanking region located 5'-side to the first region, comprising a 5' untranslated region (5'UTR) and at least one 5' terminal cap; (c) A second flanking region located 3' to the first region, comprising a 3' untranslated region (3'UTR) and a 3' tailing sequence of a linked nucleoside, The polynucleotide comprises a second flanking region containing at least one chemically modified nucleoside and It is an isolated polynucleotide containing [the specified element].
[0145] In some embodiments, the modified nucleobase is modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine(ψ), pyridine-4-onyribonucleoside, 5-azauridine, 6-azauridine, 2-thio-5-azauridine, and 2-thiouridine(s) 2 U), 4-thiouridine (s 4 U), 4-thiopseudolidine, 2-thiopseudolidine, 5-hydroxyuridine (ho 5 U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), Uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethylpseudridine, 5-carboxyhydroxymethyluridine (chm 5U), 5-carboxyhydroxymethyluridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpsuduridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thiopsuduridine, 5-methyluridine (m 5 U, i.e., having the nucleobase deoxythymine), 1-methylpseudridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudridine (m 1 s 4 ψ), 4-thio-1-methylpseuduridine, 3-methylpseuduridine (m 3 ψ), 2-thio-1-methylpseuduridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyldihydrouridine(m 5D) 2-Thiodihydrouridine, 2-Thiodihydropsuduridine, 2-Methoxyuridine, 2-Methoxy-4-Thiouridine, 4-Methoxypsuduridine, 4-Methoxy-2-Thiopsuduridine, N1-Methylpsuduridine, 3-(3-Amino-3-Carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudridine(ψm), 2-thio-2'-O-methyluridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 It contains Um), 1-thiouridine, deoxythymidine, 2'-F-arauridine, 2'-Furidine, 2'-OH-arauridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.
[0146] In some embodiments, the modified nucleobase is modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-azacytidine, 6-azacytidine, pseudoisocytidine, and 3-methylcytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formylcytidine (f 5 C), N4-methylcytidine (m4 C), 5-methylcytidine (m 5 C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5 C) 1-methylpseudoisocytidine, pyrrolocitidine, pyrroloceudoisocytidine, 2-thiocytidine (s 2 C), 2-thio-5-methylcytidine, 4-thiopseudoisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebralin, 5-azazebralin, 5-methylzebralin, 5-azazebralin, 2-aza-2-thiozebralin, 2-thiozebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methylpseudoisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methylcytidine (Cm), 5,2'-O-dimethylcytidine (m 5 Cm), N4-acetyl-2'-O-methylcytidine (ac 4 Cm), N4,2'-O-dimethylcytidine (m 4 Cm), 5-formyl-2'-O-methylcytidine (f 5 Cm), N4,N4,2'-O-trimethylcytidine (m 4 It contains 2Cm), 1-thiocytidine, 2'-F-alacytidine, 2'-F-cytidine, and 2'-OH-alacytidine.
[0147] In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides having modified adenine include α-thioadenosine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine (m 1A) 2-methyladenine (m 2 A) N6-methyladenosine (m 6 A) 2-methylthio-N6-methyladenosine (ms 2 m 6 A) N6-isopentenyladenosine (i 6 A) 2-methylthio-N6-isopentenyladenosine (ms 2 i 6 A) N6-(cis-hydroxyisopentenyl)adenosine (io 6 A) 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A) N6-Glycinylcarbamoyladenosine (g 6 A) N6-Threonylcarbamoyladenosine (t 6 A) N6-methyl N6-threonylcarbamoyladenosine (m 6 t 6 A) 2-methylthio-N6-threonylcarbamoyladenosine (ms 2 g 6 A) N6,N6-dimethyladenosine (m 6 2A), N6-hydroxynorvalylcarbamoyladenosine (hn 6 A) 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine (ms 2 hn 6 A) N6-acetyladenosine (ac 6 A) 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, α-thioadenosine, 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m 6 Am), N6,N6,2'-O-trimethyladenosine (m 6 2Am), 1,2'-O-dimethyladenosine (m 1 It contains Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-araadenosine, 2'-F-adenosine, 2'-OH-araadenosine, and N6-(19-amino-pentaoxanoneadecyl)-adenosine.
[0148] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include α-thioguanosine, inosine(I), and 1-methylinosine(m). 1 I) Wyosin (imG), Methylwyosin (mimG), 4-Demethylwyosin (imG-14), Isowyosin (imG2), Wybutosin (yW), Peroxywybutosin (o2yW), Hydroxywybutosin (OhyW), Unmodified Hydroxywybutosin (OhyW * ), 7-deazaguanosine, queosin (Q), epoxyqueosin (oQ), galactosyl eosin (galQ), mannosilceosin (manQ), 7-cyano-7-deazaguanosine (preQ0), 7-aminomethyl-7-deazaguanosine (preQ1), alkaeosin (G + ), 7-deaza-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-azaguanosine, 7-methylguanosine (m 7 G), 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine (m 1 G), N2-methylguanosine (m 2 G), N2,N2-dimethylguanosine (m 2 2G), N2,7-dimethylguanosine (m 2,7 G), N2,N2,7-dimethylguanosine (m 2,2,7 G), 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, α-thioguanosine, 2'-O-methylguanosine (Gm), N2-methyl-2'-O-methylguanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methylguanosine (m 2 2Gm), 1-methyl-2'-O-methylguanosine (m 1 Gm), N2,7-dimethyl-2'-O-methylguanosine (m 2,7Gm), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (m 1 It contains Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thioguanosine, O6-methylguanosine, 2'-F-araguanosine, and 2'-F-guanosine.
[0149] In some embodiments, the mMRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleobases (for example, two, three, or four combinations of the aforementioned modified nucleobases).
[0150] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudridine (m 1 ψ) is 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mmRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0151] In some embodiments, the modified nucleobase is modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine are N4-acetylcytidine (ac 4 C), 5-methylcytidine (m 5 C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5 C), 1-methylpseudoisocytidine, 2-thiocytidine (s 2C) comprising 2-thio-5-methylcytidine. In some embodiments, the mMRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0152] In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 7-deazaadenine and 1-methyladenosine (m 1 A) 2-methyladenine (m 2 A) N6-methyladenosine (m 6 A) is included. In some embodiments, the mMRNA of this disclosure comprises one or more combinations of the modified nucleobases described above (for example, two, three, or four combinations of the modified nucleobases described above).
[0153] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine(I), 1-methylinosine(m) 1 I) Wyosin (imG), Methylwyosin (mimG), 7-Deazaguanosine, 7-Cyano-7-Deazaguanosine (preQ0), 7-Aminomethyl-7-Deazaguanosine (preQ1), 7-Methylguanosine (m 7 G), 1-methylguanosine (m 1 G) comprises 8-oxoguanosine and 7-methyl-8-oxoguanosine. In some embodiments, the mMRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0154] In some embodiments, the modified nucleobase is 1-methylpseudolidine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5C) Pseudouridine(ψ), α-thioguanosine, or α-thioadenosine. In some embodiments, the mMRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleobases (e.g., two, three, or four combinations of the aforementioned modified nucleobases).
[0155] In some embodiments, the mmRNA contains pseudouridine (ψ). In some embodiments, the mmRNA contains pseudouridine (ψ) and 5-methylcytidine (m 5 C) is included. In some embodiments, the mmRNA is 1-methylpseudridine (m 1 It contains ψ). In some embodiments, the mmRNA is 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C) is included. In some embodiments, the mMRNA is 2-thiouridine (s 2 U) is included. In some embodiments, the mmRNA is 2-thiouridine and 5-methylcytidine (m 5 C) is included. In some embodiments, the mmRNA is 5-methoxyuridine (mo 5 It contains U). In some embodiments, RNA is, for example, 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C) is included. In some embodiments, the mMRNA contains 2'-O-methyluridine. In some embodiments, the mMRNA contains 2'-O-methyluridine and 5-methylcytidine (m 5 C) is included. In some embodiments, the mmRNA is N6-methyladenosine (m 6 A) is included. In some embodiments, the mmRNA is N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 Includes C).
[0156] In certain embodiments, the mMRNA of this disclosure is uniformly modified with respect to a specific modification (i.e., fully modified, modified throughout the entire sequence). For example, the mMRNA may be modified with 1-methylpseuduridine (m 1ψ) or 5-methylcytidine (m 5 C) can be uniformly modified, which means that all uridine or cytosine residues in the mMRNA sequence are each modified with 1-methylpseudridine (m 1 ψ) or 5-methylcytidine (m 5 This means replacing with C). Similarly, the mMRNAs disclosed herein can be uniformly modified by replacing any type of nucleoside residue present in the sequence with modifying residues such as those specified above.
[0157] In some embodiments, the mRNA of this disclosure can be modified within the coding region (e.g., the open reading frame encoding the polypeptide). In other embodiments, the mRNA can be modified in regions other than the coding region. For example, in some embodiments, one or both of them provide 5'-UTR and / or 3'-UTR, which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present within the coding region.
[0158] Examples of nucleoside modifications and combinations thereof that may be present in the mMRNA of this disclosure include, but are not limited to, those described in PCT Patent Application Publications WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.
[0159] The mMRNAs of this disclosure may include combinations of modifications to sugars, nucleobases, and / or internucleoside junctions. These combinations may include any one or more modifications described herein.
[0160] Examples of modified nucleosides and combinations of modified nucleosides are shown in Tables 1 and 2 below. These combinations of modified nucleotides can be used to form the mMRNAs of this disclosure. In certain embodiments, the native nucleotides of the mMRNAs of this disclosure may be partially or completely replaced by the modified nucleosides. As an unrestricted example, the native nucleotide uridine may be replaced with a modified nucleoside described herein. In another unrestricted example, the native nucleotide uridine may be partially replaced (for example, about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9% of the native uridine) with at least one of the modified nucleosides disclosed herein. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]
[0161] In accordance with this disclosure, the polynucleotides of this disclosure may be synthesized to include a combination of modifications or a single modification from Table 1 or Table 2.
[0162] When listing single modifications, the listed nucleoside or nucleotide represents 100 percent of those A, U, G, or C that are modified nucleotides or nucleosides. When listing percentages, these percentages represent the percentage of those specific A, U, G, or C triphosphates that are nucleobases out of the total amount of A, U, G, or C triphosphates present. For example, the combination: 25% 5-aminoallyl CTP + 75% CTP / 25% 5-methoxy UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine triphosphates are 5-aminoallyl CTP, 75% of the cytosine is CTP, while 25% of the uracil is 5-methoxy UTP, 75% of the uracil is UTP. If modified UTPs are not listed, naturally occurring ATP, UTP, GTP, and / or CTP are used for 100% of the nucleotide sites found within the polynucleotide. In this example, all GTP and ATP nucleotides are considered unmodified.
[0163] The mRNA or region thereof in this disclosure may be an optimized codon. Codon optimization methods are known in the art and may be useful for a variety of purposes: to match codon frequencies in a host organism to ensure proper folding; to bias GC content to increase mRNA stability or reduce secondary structure; to minimize tandem repeat codons or nucleotide sequences that may impair gene construction or expression; to customize transcriptional and translational regulatory regions; to insert or remove trafficking sequences in proteins; to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein; to add, remove, or shuffle protein domains; to insert or delete restriction sites; to modify ribosome binding sites and mRNA degradation sites; to adjust translation rates to enable proper folding of diverse protein domains; or to reduce or eliminate problematic secondary structures within polynucleotides. In the art, codon optimization tools, algorithms, and services are publicly known, and non-limiting examples include GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or services by patented methods. In one embodiment, for example, an optimization algorithm is used to optimize an mRNA sequence to optimize expression in a mammalian cell or to enhance mRNA stability.
[0164] In certain embodiments, the Disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any of the polynucleotide sequences described herein.
[0165] The mRNAs of this disclosure can be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthesis methods. Enzymatic (IVT), solid-phase, liquid-phase, combination synthesis, small-region synthesis, and ligation methods can be utilized. In one embodiment, mRNA is produced using an enzymatic synthesis method by IVT. Methods for producing polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062, which is incorporated herein by reference in its entirety. Accordingly, this disclosure also includes polynucleotides, such as DNA, constructs, and vectors, which can be used to transcribe the mRNAs described herein in vitro.
[0166] Non-natural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications may be on internucleoside junctions, purine or pyrimidine bases, or sugars. In certain embodiments, the modifications may be introduced at the ends of the polynucleotide chain, at other locations within the polynucleotide chain, by chemical synthesis, or by polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT application PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, Vol. 76, pp. 99–134 (1998).
[0167] Using enzymatic or chemical ligation methods, polynucleotides or their regions can be conjugated with different functional parts, such as targeting agents or delivery agents, fluorescent labels, liquids, or nanoparticles. Conjugation of polynucleotides and modified polynucleotides is reviewed in Goodchild, Bioconjugate Chemistry, Vol. 1 (No. 3), pp. 165-187 (1990).
[0168] MicroRNA binding sites for miRs expressed in immune cells A microRNA (or miRNA) is a non-coding RNA of 19–25 nucleotides in length (generally 19–23 nucleotides, most typically 22 nucleotides) that binds to the 3'UTR of a nucleic acid molecule and downregulates gene expression posttranslation by reducing the stability of the nucleic acid molecule or inhibiting translation. The mRNAs of this disclosure may include one or more microRNA target sequences or microRNA target sites, microRNA binding sequences or microRNA binding sites, sequences complementary to a microRNA sequence, or microRNA seed regions or sequences complementary to a microRNA seed sequence. Such sequences may correspond to any known microRNA, such as those taught in U.S. Publications US2005 / 0261218 and U.S. Publications US2005 / 0059005, whose contents are incorporated herein by reference in their entirety. A microRNA sequence contains a "seed" region or seed sequence, i.e., a sequence within the 2-8 region of a mature microRNA, which has perfect Watson-Crick complementarity with the miRNA target sequence. The bases of the microRNA seed region or seed sequence are perfectly complementary to the target sequence. MicroRNAs are enzymatically derived from the region of an RNA transcript and folded back to form a short hairpin structure often called pre-miRNA (precursor miRNA). Precursor miRNAs typically have a 2-nucleotide overhang at their 3' end, containing a 3' hydroxyl group and a 5' phosphate group. This precursor mRNA is processed in the nucleus and then transported to the cytoplasm, where it is further processed by DICER (RNase III enzyme) to form a mature microRNA of approximately 22 nucleotides. The mature microRNA is then incorporated into a ribonuclear particle to form RISC, an RNA-induced silencing complex that mediates gene silencing.The nomenclature for mature miRNAs recognized in the art typically indicates the pre-miRNA arm from which the mature miRNA is derived, where "5p" means the microRNA originates from the 5-prime arm of the pre-miRNA hairpin, and "3p" means the microRNA originates from the 3-prime end of the pre-miRNA hairpin. In this specification, a numbered miR may refer to one of two mature microRNAs (e.g., 3p microRNA or 5p microRNA) that originate from opposing arms of the same pre-miRNA. All miRs referred to herein are intended to include both the 3p and 5p arms, per sequence, unless specifically designated by the 3p or 5p designation.
[0169] In some embodiments, the mRNA of this disclosure may include one or more microRNA (miRNA) binding sites. As used herein, the term “microRNA (miRNA) binding site” refers to a sequence within a polynucleotide, e.g., within DNA or an RNA transcript, that is sufficiently complementary to all or a region of the miRNA to interact with, associate with, or bind to the miRNA. In exemplary embodiments, the miRNA binding site is incorporated within the mRNA, e.g., within the 5'UTR and / or 3'UTR of the mRNA. A sequence of a miR binding site that is sufficiently complementary to the miR means that the degree of complementarity is sufficient to facilitate miR-mediated regulation of mRNA, e.g., miR-mediated suppression or degradation of mRNA translation. In exemplary embodiments of this disclosure, a sequence of a miR binding site that is sufficiently complementary to the miR means that the degree of complementarity is sufficient to facilitate miR-mediated degradation of mRNA, e.g., miR-guided RISC-mediated cleavage of mRNA. The miR binding site may have complementarity to, for example, a 19-25 nucleotide miR sequence, a 19-23 nucleotide miR, or most typically, a 22 nucleotide miR sequence. The miR binding site may be complementary to only a portion of the miR, for example, a portion that is 1, 2, 3, or 4 nucleotides shorter than the naturally occurring miR. If the desired regulation is mRNA degradation, full or complete complementarity (e.g., fully or completely complementary over all or a significant portion of the naturally occurring miR) is preferred. In some embodiments, the miRNA binding site includes a sequence that is complementary (e.g., partially or completely complementary) to the miRNA seed sequence. In certain embodiments, the miRNA binding site includes a sequence that is completely complementary to the miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that is complementary (e.g., partially or completely complementary) to the miRNA sequence. In certain embodiments, the miRNA binding site includes a sequence that is completely complementary to the miRNA sequence. In some embodiments, the miRNA binding site is fully complementary to the miRNA sequence, except for 1, 2, or 3 nucleotide substitutions, terminal additions, and / or truncations.
[0170] One or more miR-binding sequences can be incorporated into the mRNA of this disclosure for one or more of a variety of different purposes. For example, the incorporation of one or more miRNA-binding sites into the mRNA of this disclosure may target the molecule for degradation or reduced translation, provided that the miRNA in question is targetable (e.g., expressed in a target cell or tissue). In some embodiments, the incorporation of one or more miRNA-binding sites into the mRNA of this disclosure may reduce the risk of off-target effects during delivery of nucleic acid molecules and / or enable tissue-specific regulation of the expression of polypeptides encoded by the mRNA. In yet other embodiments, the incorporation of one or more miRNA-binding sites into the mRNA of this disclosure may modulate immune responses during nucleic acid delivery in vivo. In further embodiments, the incorporation of one or more miRNA-binding sites into the mRNA of this disclosure may also modulate accelerated blood clearance (ABC) of lipid-containing compounds and compositions described herein.
[0171] Representative miRNAs were selected based on their expression and abundance in cells known to express TLR7 / TLR8 and / or secrete cytokines, such as hematopoietic immune cells including B cells, T cells, macrophages, and dendritic cells, as well as endothelial cells and platelets. Therefore, the set of miRNAs included miRs that may contribute partially to the immunogenicity of these cells, such that the integration of the corresponding miR site into mRNA can lead to mRNA destabilization and / or suppression of translation of these mRNAs within specific cell types. Non-limiting representative examples include miR-142, miR-144, miR-150, miR-155, and miR-223, which are specific to many hematopoietic cells; miR-142, miR-150, miR-16, and miR-223, which are expressed in B cells; miR-223, miR-451, miR-26a, and miR-16, which are expressed in hematopoietic progenitor cells; and miR-126, which is expressed in plasmacytoid dendritic cells, platelets, and endothelial cells. For further discussion of tissue-mediated miR expression, see, for example, Teruel-Montoya, R. et al. (2014), PLoS One, vol. 9: e102259; Landgraf, P. et al. (2007), Cell, vol. 129: pp. 1401-1414; and Bissels, U. et al. (2009), RNA, vol. 15: pp. 2375-2384. As evidenced, the integration of any single miR site into the 3'UTR and / or 5'UTR can mediate such effects in multiple cell types of interest (e.g., miR-142 is abundant in both B cells and dendritic cells).
[0172] Targeting the same cell type with multiple miRs and incorporating binding sites for both the 3p and 5p arms is beneficial when both are abundant (for example, both miR-142-3p and miR142-5p are abundant in hematopoietic stem cells). Thus, for example, in a particular embodiment, the mRNA construct contains two or more (e.g., two, three, four, or more) miR binding sites, which are derived from (i) the group consisting of miR-142, miR-144, miR-150, miR-155, and miR-223 (expressed in many hematopoietic cells); or (ii) the group consisting of miR-142, miR-150, miR-16, and miR-223 (expressed in B cells); or the group consisting of miR-223, miR-451, miR-26a, and miR-16 (expressed in hematopoietic progenitor cells).
[0173] Furthermore, it is also beneficial to combine diverse miRs to simultaneously target multiple target cell types (for example, combining miR-142 and miR-126 to target many hematopoietic cells and endothelial cells). Thus, for example, in a particular embodiment, the mRNA construct has two or more (e.g., two, three, four, or more) miR binding sites, where (i) at least one of the miRs targets hematopoietic cells (e.g., miR-142, miR-144, miR-150, miR-155, or miR-223), and at least one of the miRs targets plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126). (ii) at least one of the miRs targets B cells (e.g., miR-142, miR-150, miR-16, or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126); or (iii) at least one of the miRs targets hematopoietic progenitor cells (e.g., miR-223, miR-451, miR-26a, or miR-16), (iv) At least one of the miRs targets plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126); or (iv) At least one of the miRs targets hematopoietic cells (e.g., miR-142, miR-144, miR-150, miR-155, or miR-223) and at least one of the miRs targets B cells (e.g., miR-142, miR-150, miR-16, or miR-223), and the miR At least one of them is an miR binding site that targets plasmacytoid dendritic cells, platelets, or endothelial cells (e.g., miR-126); or it contains any other possible combination of the four aforementioned classes of miR binding sites (i.e., miR binding sites that target the hematopoietic system, miR binding sites that target B cells, miR binding sites that target hematopoietic progenitor cells, and / or miR binding sites that target plasmacytoid dendritic cells / platelets / endothelial cells).
[0174] Therefore, in one embodiment, the mRNA may contain one or more miR-binding sequences that bind to one or more miRs expressed in conventional immune cells or in any cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs and / or spleen cells and / or endothelial cells) to modulate the immune response. It has now been found that the incorporation of one or more miRs expressed in conventional immune cells or in any cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs and / or spleen cells and / or endothelial cells) into mRNA reduces or inhibits immune cell activation (e.g., B cell activation, as measured by the frequency of activated B cells) and / or cytokine production (e.g., production of IL-6, IFN-γ and / or TNFα). It has now been discovered that the incorporation of one or more miRs into mRNA, expressed within conventional immune cells or in any cells expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., within immune cells of peripheral lymphoid organs and / or spleen cells and / or endothelial cells), can reduce or inhibit the anti-drug antibody (ADA) response to the target protein encoded by the mRNA.
[0175] In another embodiment, the mRNA may contain one or more miR-binding sequences that bind to one or more miRs expressed in conventional immune cells or in any cell expressing TLR7 and / or TLR8 and secreting pro-inflammatory cytokines and / or chemokines (e.g., in immune cells of peripheral lymphoid organs and / or spleen cells and / or endothelial cells) to modulate the acceleration of blood clearance of mRNA delivered to a lipid-containing compound or composition. It has now been found that the incorporation of one or more miR-binding sites into mRNA reduces or inhibits the acceleration of blood clearance (ABC) of lipid-containing compounds or compositions for use in mRNA delivery. It has now also been discovered that the incorporation of one or more miR binding sites into mRNA reduces serum levels of anti-PEG anti-IgM (e.g., by reducing or inhibiting the rapid production of polyethylene glycol (PEG)-recognizing IgM by B cells), and / or reduces or inhibits the growth and / or activation of plasmacytoid dendritic cells after administration of a lipid-containing compound or composition containing mRNA.
[0176] Such miR sequences may correspond to any known microRNA expressed in immune cells, including but not limited to the microRNAs taught in U.S. Publications US2005 / 0261218 and US2005 / 0059005, which are incorporated herein by reference in their entirety. Non-exclusive examples of miRs expressed in immune cells include miRs expressed in splenic cells, myeloid cells, dendritic cells, plasmacytoid dendritic cells, B cells, T cells, and / or macrophages. For example, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, and miR-27 are expressed in myeloid cells, miR-155 is expressed in dendritic cells, B cells, and T cells, miR-146 is upregulated in macrophages upon TLR stimulation, and miR-126 is expressed in plasmacytoid dendritic cells. In certain embodiments, miRs are expressed abundantly or preferentially in immune cells. For example, miR-142 (miR-142-3p and / or miR-142-5p), miR-126 (miR-126-3p and / or miR-126-5p), miR-146 (miR-146-3p and / or miR-146-5p), and miR-155 (miR-155-3p and / or miR155-5p) are highly expressed in immune cells. Since these microRNA sequences are known in the art, those skilled in the art can easily design binding or target sequences to which these microRNAs bind based on Watson-Crick complementarity.
[0177] Therefore, in various embodiments, the mRNA includes at least one microRNA binding site for a miR selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27. In another embodiment, the mRNA includes at least two miR binding sites for a microRNA expressed in an immune cell. In various embodiments, the mRNA includes one to four, one, two, three, or four miR binding sites for a microRNA expressed in an immune cell. In another embodiment, the mRNA includes three miR binding sites. These miR binding sites may be for a microRNA selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27, and combinations thereof. In one embodiment, the mRNA contains two or more copies (e.g., two, three, or four) of the same miR binding site expressed in an immune cell, for example, two or more copies of a miR binding site selected from the group of miRs consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
[0178] For example, in one embodiment, the mRNA contains three copies of the same miR binding site. As described in Example 8, in certain embodiments, the use of three copies of the same miR binding site may exhibit beneficial properties compared to the use of a single miR binding site. Non-limiting examples of 3'UTR sequences containing three miR binding sites are shown in SEQ ID NO: 38 (three miR-142-3p binding sites), SEQ ID NO: 40 (three miR-142-5p binding sites), and SEQ ID NO: 54 (three miR-122 binding sites).
[0179] In another embodiment, the mRNA contains two or more (e.g., two, three, or four) copies of at least two different miR binding sites expressed in the immune cell. Non-limiting examples of 3'UTR sequences containing two or more different miR binding sites are shown in SEQ ID NO: 33 (one miR-142-3p binding site and one miR-126-3p binding site), SEQ ID NO: 47 (one miR-142-3p binding site and one miR-122-5p binding site), SEQ ID NO: 41 (two miR-142-5p binding sites and one miR-142-3p binding site), and SEQ ID NO: 44 (two miR-155-5p binding sites and one miR-142-3p binding site).
[0180] In another embodiment, the mRNA includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-142-3p. In various embodiments, the mRNA includes binding sites for miR-142-3p and miR-155 (miR-155-3p or miR-155-5p), miR-142-3p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-3p and miR-126 (miR-126-3p or miR-126-5p).
[0181] In another embodiment, the mRNA includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-126-3p. In various embodiments, the mRNA includes binding sites for miR-126-3p and miR-155 (miR-155-3p or miR-155-5p), miR-126-3p and miR-146 (miR-146-3p or miR-146-5p), or miR-126-3p and miR-142 (miR-142-3p or miR-142-5p).
[0182] In another embodiment, the mRNA includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-142-5p. In various embodiments, the mRNA includes binding sites for miR-142-5p and miR-155 (miR-155-3p or miR-155-5p), miR-142-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-5p and miR-126 (miR-126-3p or miR-126-5p).
[0183] In yet another embodiment, the mRNA includes at least two miR binding sites for microRNA expressed in immune cells, in which case one of the miR binding sites is for miR-155-5p. In various embodiments, the mRNA includes binding sites for miR-155-5p and miR-142 (miR-142-3p or miR-142-5p), miR-155-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-155-5p and miR-126 (miR-126-3p or miR-126-5p).
[0184] In exemplary embodiments, one or more miR binding sites are located in the 3'UTR, in the 5'UTR, or in either the 3'UTR or 5'UTR, such that the mRNA has the desired properties. The miR binding site may be located in the 3'UTR immediately after the stop codon of the coding region in the mRNA construct (or immediately after the last stop codon if there are multiple copies of the stop codon in the construct), or it may be located further downstream of the stop codon, in which case a 3'UTR base is present between the stop codon and the miR binding site. For example, three non-limiting examples of possible insertion sites for miR in the 3'UTR are shown in SEQ ID NOs. 48, 49, and 50, which show 3'UTR sequences in which the miR-142-3p site is inserted into one of the three different possible insertion sites in the 3'UTR, respectively. Furthermore, one or more miR binding sites may be located in one or more possible insertion sites within the 5'UTR. For example, three non-limiting examples of possible insertion sites for miRs within the 5'UTR are further described in Example 9, shown in SEQ ID NOs. 55, 56, and 57, which show 5'UTR sequences in which the miR-142-3p site is inserted into one of three different possible insertion sites within the 5'UTR, respectively. In addition, SEQ ID NOs. 58, 59, and 60 also show 5'UTR sequences in which the miR-122 site is inserted into one of three different possible insertion sites within the 5'UTR, respectively.
[0185] In one embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a stop codon, and at least one microRNA binding site is located within 1 to 100 nucleotides of the 3'UTR after the stop codon. In another embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a stop codon, and at least one microRNA binding site for miR expressed in immune cells is located within 30 to 50 nucleotides of the 3'UTR after the stop codon. In yet another embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a stop codon, and at least one microRNA binding site for miR expressed in immune cells is located within at least 50 nucleotides of the 3'UTR after the stop codon. In other embodiments, the codon-optimized open reading frame encoding the polypeptide of interest includes a stop codon, and at least one microRNA binding site for miRs expressed in immune cells is located within the 3'UTR immediately following the stop codon, or within 15-20 nucleotides of the 3'UTR after the stop codon, or within 70-80 nucleotides of the 3'UTR after the stop codon. In other embodiments, the 3'UTR includes more than one miR binding site (e.g., two to four miR binding sites), in which case spacer regions (e.g., 10-100, 20-70, or 30-50 nucleotides in length) may be present between each miR binding site. In yet another embodiment, the 3'UTR includes spacer regions between the ends of the miR binding sites and the nucleotides of the poly-A tail. For example, spacer regions of 10-100, 20-70, or 30-50 nucleotides in length may be located between the ends of the miR binding sites and the beginning of the poly-A tail.
[0186] In one embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a start codon, and at least one microRNA binding site is located within 1 to 100 nucleotides of the 5'UTR, upstream of the start codon. In another embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a start codon, and at least one microRNA binding site for the miR expressed in immune cells is located within 10 to 50 nucleotides of the 5'UTR, upstream of the start codon. In yet another embodiment, the codon-optimized open reading frame encoding the target polypeptide includes a start codon, and at least one microRNA binding site for the miR expressed in immune cells is located within at least 25 nucleotides of the 5'UTR, upstream of the start codon. In other embodiments, the codon-optimized open reading frame encoding the polypeptide of interest includes a start codon, and at least one microRNA binding site for a miR expressed in an immune cell is located within the 5'UTR immediately preceding the start codon, or within 15-20 nucleotides of the 5'UTR preceding the start codon, or within 70-80 nucleotides of the 5'UTR preceding the start codon. In other embodiments, the 5'UTR includes more than one miR binding site (e.g., two to four miR binding sites), in which case spacer regions (e.g., 10-100, 20-70, or 30-50 nucleotides in length) may be present between each miR binding site.
[0187] In one embodiment, the 3'UTR contains more than one stop codon, in which case at least one miR binding site is located downstream of the stop codon. For example, the 3'UTR may contain one, two, or three stop codons. Non-limiting examples of triple stop codons that can be used include UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA, and UAGUAGUAG. Within the 3'UTR, for example, there may be one, two, three, or four miR binding sites, such as the miR-142-3p binding site, which may be located directly adjacent to the stop codon or located any number of nucleotides downstream of the last stop codon. If the 3'UTR contains multiple miR binding sites, these binding sites may be located directly adjacent to each other (i.e., sequentially) within the construct, or alternatively, spacer nucleotides may be located between each binding site.
[0188] In one embodiment, the 3'UTR includes three stop codons, with a single miR-142-3p binding site located downstream of a third stop codon. Non-limiting examples of sequences of the 3'UTR having three stop codons and a single miR-142-3p binding site located at different positions downstream of the last stop codon are shown in SEQ ID NOs. 31 and 48-50.
[0189] In one embodiment, the mMRNA comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR containing at least one microRNA binding site for miRs expressed in immune cells, and a 3' tailing region of a nucleoside to be ligated. In various embodiments, the 3'UTR contains one to four, at least two, one, two, three, or four microRNA binding sites for miRs expressed in immune cells, preferably in large numbers or preferentially in immune cells.
[0190] In one embodiment, at least one miR expressed in an immune cell is a miR-142-3p microRNA binding site. In one embodiment, the miR-142-3p microRNA binding site includes the sequence shown in SEQ ID NO: 3. In one embodiment, the 3'UTR of the mRNA containing the miR-142-3p microRNA binding site includes the sequence shown in SEQ ID NO: 2.
[0191] In one embodiment, at least one miR expressed in an immune cell is a miR-126 microRNA binding site. In one embodiment, the miR-126 binding site is a miR-126-3p binding site. In one embodiment, the miR-126-3p microRNA binding site includes the sequence shown in SEQ ID NO: 26. In one embodiment, the 3'UTR of the mmRNA containing the miR-126-3p microRNA binding site includes the sequence shown in SEQ ID NO: 27.
[0192] Non-limiting exemplary sequences of miRs to which the microRNA binding sites of this disclosure may bind include: miR-142-3p (SEQ ID NO: 8), miR-142-5p (SEQ ID NO: 9), miR-146-3p (SEQ ID NO: 10), miR-146-5p (SEQ ID NO: 11), miR-155-3p (SEQ ID NO: 12), miR-155-5p (SEQ ID NO: 13), miR-126-3p (SEQ ID NO: 14), miR-126-5p (SEQ ID NO: 15) This includes miR-16-3p (SEQ ID NO: 16), miR-16-5p (SEQ ID NO: 17), miR-21-3p (SEQ ID NO: 18), miR-21-5p (SEQ ID NO: 19), miR-223-3p (SEQ ID NO: 20), miR-223-5p (SEQ ID NO: 21), miR-24-3p (SEQ ID NO: 22), miR-24-5p (SEQ ID NO: 23), miR-27-3p (SEQ ID NO: 24), and miR-27-5p (SEQ ID NO: 25). In the art, other suitable miR sequences that are expressed in immune cells (e.g., excessively or preferentially expressed in immune cells) are known and available, for example, in the University of Manchester's microRNA database, miRBase. The binding sites for any of the aforementioned miRs are designed based on Watson-Crick complementarity to the miR, typically 100% complementarity to the miR, and can be inserted into the mRNA constructs of this disclosure as described herein.
[0193] In yet another embodiment, the therapeutic window and / or differential expression (e.g., tissue-specific expression) of the polypeptide of this disclosure can be altered by incorporating the miRNA binding site into the mRNA encoding the polypeptide. Tissues in which microRNAs are known to regulate mRNA and thereby regulate protein expression include, but are not limited to, the liver (e.g., miR-122), muscle (e.g., miR-133, miR-206, and miR-208), endothelial cells (e.g., miR-17-92 and miR-126), myeloid cells (e.g., miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, and miR-27), adipose tissue (e.g., let-7 and miR-30c), heart (e.g., miR-1d and miR-149), kidney (e.g., miR-192, miR-194, and miR-204), and lung epithelial cells (e.g., let-7, miR-133, and miR-126). Therefore, in various embodiments, the mRNA may contain one or more binding sites to any of the aforementioned miRs, either alone or in combination, thereby modulating the expression of the target protein it encodes in a tissue.
[0194] For example, mRNA may contain one or more miRNA-binding sites to which miRNAs are highly expressed within one tissue type compared to those within another tissue type. In another example, mRNA may contain one or more miRNA-binding sites to which miRNAs are less expressed in cancer cells compared to non-cancerous cells from the same tissue of origin. When present in cancer cells expressing such low levels of miRNAs, the polypeptide encoded by the mRNA would typically show increased expression. If the polypeptide is capable of inducing apoptosis, this could result in preferential cytotoxicity of cancer cells compared to normal cells.
[0195] For example, liver cancer cells (e.g., hepatocellular carcinoma cells) typically express lower levels of miR-122 compared to normal liver cells. Therefore, mRNA encoding a polypeptide and containing at least one miR-122 binding site (e.g., within the 3'-UTR of the mRNA) would typically express relatively low levels of polypeptide in normal liver cells and relatively high levels in liver cancer cells. If the polypeptide is capable of inducing apoptosis, this could lead to preferential cell killing of liver cancer cells (e.g., hepatocellular carcinoma cells) compared to normal liver cells.
[0196] Therefore, as a non-limiting example of the integration of a miR-binding site into mRNA that modulates the expression of the protein of interest encoded by a tissue, the mRNA of this disclosure may include at least one miR-122-binding site. For example, the mRNA of this disclosure may include a miR-122-binding site comprising a miR-122 seed sequence and a sequence that is partially or completely complementary to it. In some embodiments, the miR-122 seed sequence may correspond to nucleotides 2-7 of miR-122. In some embodiments, the miR-122 seed sequence may be 5'-GGAGUG-3'. In some embodiments, the miR-122 seed sequence may be nucleotides 2-8 of miR-122. In some embodiments, the miR-122 seed sequence may be 5'-GGAGUGU-3'. In some embodiments, the miR-122 binding site includes the nucleotide sequence 5'-UAUUUAGUGUGAUAAUGGCGUU-3' (SEQ ID NO: 45) or 5'-CAAACACCAUUGUCACACUCCA-3' (SEQ ID NO: 46), or their complements. In some embodiments, incorporation of at least one miR-122 binding site into mRNA may reduce the expression of the mRNA-encoded polypeptide in normal hepatocytes compared to other cell types that express low levels of miR-122. In other embodiments, incorporation of at least one miR-122 binding site into mRNA may enable increased expression of the mRNA-encoded polypeptide in liver cancer cells (e.g., hepatocellular carcinoma cells) compared to normal hepatocytes.
[0197] In yet another embodiment, the mRNA (e.g., its 3'UTR) may contain at least one miR-binding site for miRs expressed in immune cells to reduce or inhibit immune activation (e.g., B cell activation, cytokine production, ADA response) during nucleic acid delivery in vivo, and may contain at least one miR-binding site for modulating the expression of the target protein encoded by the tissue. For example, in one embodiment, the mRNA may contain a miR-122 binding site to enable increased expression of the mRNA-encoded polypeptide in liver cancer cells (e.g., hepatocellular carcinoma cells) compared to normal liver cells, and may also contain one or more miR-binding sites for miRs expressed in immune cells, selected from the group consisting of, for example, miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
[0198] In another embodiment, the mRNA (e.g., its 3'UTR) may contain at least one miR binding site to reduce or inhibit the acceleration of blood clearance by, for example, B cells reducing or inhibiting IgM production to, for example, PEG, and / or reducing or inhibiting pDC growth and / or activation, and may contain at least one miR binding site to modulate the expression of the target protein encoded by the tissue. For example, in one embodiment, the mRNA may contain a miR-122 binding site to enable increased expression of the mRNA-encoded polypeptide in liver cancer cells (e.g., hepatocellular carcinoma cells) compared to normal liver cells, and may also contain one or more miR binding sites selected from the group consisting of, for example, miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, and miR-27.
[0199] In one embodiment, the mRNA includes a miR-122 binding site and a miR-142-3p binding site. In another embodiment, the mRNA includes a miR-122 binding site and a miR-142-5p binding site. In another embodiment, the mRNA includes a miR-122 binding site and a miR-126-3p binding site. In another embodiment, the mRNA includes a miR-122 binding site and a miR-155-5p binding site. In another embodiment, the mRNA includes a miR-122 binding site and a miR-126-3p binding site. In another embodiment, the mRNA includes a miR-122 binding site, a miR-142 (miR-142-3p or 142-5p) binding site, and a miR-126 (miR-126-3p or miR-126-5p) binding site. In another embodiment, the mRNA includes a miR-122 binding site, a miR-142 (miR-142-3p or 142-5p) binding site, and a miR-155 (miR-155-3p or miR-155-5p) binding site. In yet another embodiment, the mRNA includes a miR-122 binding site, a miR-126 (miR-126-3p or 126-5p) binding site, and a miR-155 (miR-155-3p or miR-155-5p) binding site. In yet another embodiment, the mRNA includes a miR-122 binding site, a miR-142 (miR-142-3p or miR-142-5p) binding site, a miR-126 (miR-126-3p or 126-5p) binding site, and a miR-155 (miR-155-3p or miR-155-5p) binding site. In any of these embodiments, the miR-122 binding site may be a miR-122-5p binding site.
[0200] A non-restrictive example of a 3'UTR sequence containing both miR-142-3p and miR-122-5p binding sites is shown in SEQ ID NO: 47. The structure of the 3'UTR of SEQ ID NO: 47 includes three stop codons at its 5' end, a single miR-142-3p binding site immediately following this, a spacer nucleotide following downstream, and then a single miR-122-5p binding site. The distance between the miR binding sites (e.g., miR-142-3p and miR-122-5p) can vary considerably, and numerous different constructs have been investigated for different arrangements of the two miR binding sites, but all constructs were functional. In certain embodiments, the nucleotide spacer is located between the two miR binding sites of RISC, long enough to allow binding to each binding site. In one embodiment, the two miR binding sites are located approximately 40 bases apart from each other, and the total length of the 3'UTR is approximately 100-110 bases.
[0201] Target protein The mRNAs of this disclosure may encode a protein of interest, typically a protein having therapeutic properties for use in a subject. The protein of interest may be essentially any protein that can be encoded by the mRNA. In particular, the protein of interest may be a protein that stimulates the activation of immune cells (e.g., B cell activation), such as inducing an anti-drug antibody (ADA) response in a subject, and therefore is desirable to reduce or inhibit the activation of immune cells in a subject (e.g., reduce the ADA response). In various embodiments, the protein of interest may be, for example, a therapeutic protein, a cytokine, a growth factor, an antibody, or a fusion protein. Non-limiting examples of therapeutic proteins include, for example, blood factors (such as factors VIII and VII), complement factors, low-density lipoprotein receptors (LDLRs), and MUT1. Non-limiting examples of cytokines include, for example, interleukins, interferons, chemokines, lymphokines, and the like. Non-exclusive examples of growth factors include erythropoietin, EGF, PDGF, FGF, TGF, IGF, TNF, CSF, MCSF, and GMCSF. Non-exclusive examples of antibodies include, for example, adalimumab, infliximab, rituximab, ipilimumab, tocilizumab, canakinumab, itorizumab, and tralokinumab. Non-exclusive examples of fusion proteins include, for example, etanercept, abatacept, and baratacept.
[0202] In one embodiment, the target protein is human erythropoietin. In one embodiment, the mRNA includes a microRNA binding site that binds to miR-142-3p, such as mRNA encoding human erythropoietin and having the sequence shown in SEQ ID NO: 1. In another embodiment, the mRNA includes a microRNA binding site that binds to miR-126, such as mRNA encoding human erythropoietin and having the sequence shown in SEQ ID NO: 28. In yet another embodiment, the mRNA includes a microRNA binding site that binds to miR-142-3p and a microRNA binding site that binds to miR-126, such as mRNA encoding human erythropoietin and having the sequence shown in SEQ ID NO: 29. In yet another embodiment, the target protein is LDLR (for use in cholesterol inhibition). In yet another embodiment, the target protein is MUT1 (for use in the treatment of methylmalonic acidemia (MMA)). In yet another embodiment, the target protein encoded by the mRNA is a therapeutic antibody, including but not limited to the antibodies listed above.
[0203] nanoparticles The mRNAs of this disclosure can be formulated, for example, within nanoparticles or other delivery vehicles to protect them from degradation when delivered to a subject. Exemplary nanoparticles are described in Panyam, J. and Labhasetwar, V. (2003), Adv. Drug Deliv. Rev., Vol. 55, pp. 329-347; and Peer, D. et al. (2007), Nature Nanotech., Vol. 2, pp. 751-760. In certain embodiments, the RNAs of this disclosure, e.g., mRNAs, are encapsulated within nanoparticles. In certain embodiments, nanoparticles are particles having at least one size (e.g., diameter) of less than or equal to 1000 nM, less than or equal to 500 nM, or less than or equal to 100 nM. In certain embodiments, the nanoparticles include lipids. Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any of a number of lipids may be present, including cationic lipids and / or ionized lipids, anionic lipids, neutral lipids, amphiphilic lipids, PEGylated lipids, and / or structural lipids. Such lipids may be used individually or in combination. In certain embodiments, the lipid nanoparticles include one or more RNAs as described herein, e.g., mRNA, e.g., mmRNA, which encodes the polypeptide of interest and has at least one microRNA binding site for miR expressed in immune cells.
[0204] In some embodiments, the mRNA lipid nanoparticle formulations described herein may comprise one or more (e.g., one, two, three, four, five, six, seven, or eight) cationic lipids and / or ionized lipids. Such cationic lipids include 3-(didodecylamino)-N1,N1,4-tridedecyl-1-piperazinediethaneamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridedecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraazaoctatricontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2 -Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3 -[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy ]Octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)); N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,NN-triethylammonium chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.Cl"); 3-β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate This includes, but is not limited to, trifluoracetate ("DOSPA"), dioctadecylamideglycylcarboxyspermine ("DOGS"), 1,2-dioleoyl-3-dimethylammoniumpropane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). In addition, a number of commercially available preparations of cationic and / or ionized lipids can also be used, such as LIPOFECTIN® (commercially available from GIBCO / BRL, including DOTMA and DOPE) and LIPOFECTAMINE® (commercially available from GIBCO / BRL, including DOSPA and DOPE). KL10, KL22, and KL25 are described, for example, in U.S. Patent No. 8,691,750, which is incorporated herein by reference in its entirety. In certain embodiments, the lipid is DLin-MC3-DMA or DLin-KC2-DMA.
[0205] Anionic lipids suitable for use in the lipid nanoparticles of this disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine (ethanoloamine), N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups linked to neutral lipids.
[0206] Natural lipids suitable for use in the lipid nanoparticles of this disclosure include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside. Lipids having various acyl chain groups with varying chain length and saturation are available or can be isolated or synthesized by known techniques. In addition, lipids having mixtures of saturated and unsaturated fatty acid chains can also be used. In some embodiments, the neutral lipids used in this disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipids may consist of phospholipids with other head groups, such as sphingomyelin, dihydrosphingomyelin, or serine and inositol.
[0207] In some embodiments, amphiphilic lipids are incorporated into the nanoparticles of the Disclosure. Exemplary amphiphilic lipids suitable for use in the nanoparticles of the Disclosure include, but are not limited to, sphingolipids, phospholipids, and aminolipids. In some embodiments, the phospholipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 Selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.Other phosphorus-free compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxy acids, can also be used. In addition, such amphiphilic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
[0208] In some embodiments, the lipid component of the nanoparticles of this disclosure may include one or more PEGylated lipids. PEGylated lipids (also known as PEG lipids or PEG-modified lipids) are lipids modified with polyethylene glycol. The lipid component may include one or more PEGylated lipids. The PEGylated lipids can be selected from an indefinite group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEGylated lipids may be PEG-c-DOMG lipids, PEG-DMG lipids, PEG-DLPE lipids, PEG-DMPE lipids, PEG-DPPC lipids, or PEG-DSPE lipids.
[0209] The lipid nanoparticles of this disclosure may contain one or more structural lipids. Exemplary and non-limiting structural lipids that may be present in the lipid nanoparticles of this disclosure include cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol.
[0210] In some embodiments, one or more mRNAs of the Disclosure can be formulated in lipid nanoparticles having diameters of approximately 1 nm to approximately 900 nm, for example, approximately 1 nm to approximately 100 nm, approximately 1 nm to approximately 200 nm, approximately 1 nm to approximately 300 nm, approximately 1 nm to approximately 400 nm, approximately 1 nm to approximately 500 nm, approximately 1 nm to approximately 600 nm, approximately 1 nm to approximately 700 nm, approximately 1 nm to approximately 800 nm, or approximately 1 nm to approximately 900 nm. In some embodiments, the nanoparticles may have diameters of approximately 10 nm to approximately 300 nm, approximately 20 nm to approximately 200 nm, approximately 30 nm to approximately 100 nm, or approximately 40 nm to approximately 80 nm. In some embodiments, the nanoparticles may have diameters of approximately 30 nm to approximately 300 nm, approximately 40 nm to approximately 200 nm, approximately 50 nm to approximately 150 nm, approximately 70 nm to approximately 110 nm, or approximately 80 nm to approximately 120 nm. In one embodiment, mRNA is approximately 10-20 nm, 10-30 nm, 10-40 nm, 10-50 nm, 10-60 nm, 10-70 nm, 10-80 nm, 10-90 nm, 20-30 nm, 20-40 nm, 20-50 nm, 20-60 nm, 20-70 nm, 20-80 nm, 20-90 nm, 20-100 nm, 30-40 nm, 30-50 nm, 30-60 nm, 30-70 nm, 30-80 nm, 30-90 nm, 30-100 nm, 40-50 nm, and 40-60 nm. It can be formulated in lipid nanoparticles having a diameter of about 10 to about 100 nm, including a range between about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm, etc., but not limited to these ranges. In one embodiment, mRNA can be formulated into lipid nanoparticles having diameters of approximately 30 nm to 300 nm, 40 nm to 200 nm, 50 nm to 150 nm, 70 nm to 110 nm, or 80 nm to 120 nm, including the ranges in between.
[0211] In some embodiments, lipid nanoparticles may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, or greater than 950 nm.
[0212] In some embodiments, the particle size of lipid nanoparticles can be increased and / or decreased. Varying particle size can help counteract biological responses, including but not limited to inflammation, and can also enhance the biological effect of mRNA delivered to the patient or subject.
[0213] In certain embodiments, it is desirable to target the nanoparticles of this disclosure, such as lipid nanoparticles, using cell type and / or tissue type-specific targeting moieties. In some embodiments, the targeting moieties can be used to target the nanoparticles to specific cells, tissues, and / or organs. In certain embodiments, the nanoparticles comprise one or more mRNAs as described herein and the targeting moiety. Exemplary and non-limiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin), and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single-chain Fv(scFv) fragments, Fab' fragments, or F(ab')2 fragments), single-domain antibodies, camelid antibodies and their fragments, human antibodies and their fragments, monoclonal antibodies, and polyspecific antibodies (e.g., bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. The targeting moiety may include a whole polypeptide (e.g., a peptide or protein) or a fragment thereof. Typically, the targeting portion is located on the outer surface of the nanoparticle so that it can be used for interaction with a target, such as a cell surface receptor. In the art, various different targeting portions and targeting methods are known and available, including, for example, those described in Sapra et al., Prog. Lipid Res., Vol. 42 (No. 5): pp. 439-462, 2003; and Abra et al., J. Liposome Res., Vol. 12: pp. 1-3, 2002.
[0214] In some embodiments, lipid nanoparticles (e.g., liposomes) may include a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains (e.g., Allen et al., Biochimica et Biophysica Acta, Vol. 1237: pp. 99-108, 1995; DeFrees et al., Journal of the American Chemistry Society, Vol. 118: pp. 6101-6104, 1996; Blume et al., Biochimica et Biophysica Acta, Vol. 1149: pp. 180-184, 1993; Klibanov et al., Journal of Liposome Research, Vol. 2: pp. 321-334, 1992; U.S. Patent No. 5,013,556; Zalipsky, Bioconjugate Chemistry, Vol. 4: pp. 296-299, 1993; Zalipsky, FEBS). See Letters, Vol. 353: pp. 71-74, 1994; Zalipsky, Stealth Liposomes, Chapter 9 (edited by Lasic and Martin), CRC Press, Boca Raton Fla., 1995. One method involves linking a targeting moiety for targeting lipid nanoparticles to the polar head group of the lipid forming the nanoparticles. Another method involves attaching the targeting moiety to the distal end of a PEG chain that forms a hydrophilic polymer coating (see, for example, Klibanov et al., Journal of Liposome Research, Vol. 2: pp. 321-334, 1992; Kirpotin et al., FEBS Letters, Vol. 388: pp. 115-118, 1996).
[0215] Standard methods can be used to couple one or more targeting moieties. For example, phosphatidylethanolamine can be used, which can be activated to conjugate a targeting moiety or a derivatized lipophilic compound, such as lipid-derivatized bleomycin. For example, antibody-targeting liposomes can be constructed using liposomes incorporating protein A (see, e.g., Renneisen et al., J. Bio. Chem., vol. 265: pp. 16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA), vol. 87: pp. 2448-2451, 1990). Other examples of antibody conjugations are disclosed in U.S. Patent No. 6,027,726. Examples of targeting moieties may also include other polypeptides that are specific to intracellular components, including antigens associated with neoplasms or tumors. Polypeptides used as targeting molecules can be attached to liposomes via covalent bonds (see, for example, Heath, Covalent Attachment of Proteins to Liposomes, Vol. 149, Methods in Enzymology, pp. 111-119 (Academic Press, Inc., 1987)). Other targeting methods include the biotin-avidin system.
[0216] In some embodiments, the lipid nanoparticles of this disclosure include a targeting portion that targets the lipid nanoparticles to cells including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, endothelial cells, lung cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial surface cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary and metastatic tumor cells). In certain embodiments, the targeting portion targets the lipid nanoparticles to hepatocytes. In other embodiments, the targeting portion targets the lipid nanoparticles to colon cells. In some embodiments, the targeting portion targets the lipid nanoparticles to liver cancer cells (e.g., hepatocellular carcinoma cells) or colorectal cancer cells (e.g., primary and metastatic tumors).
[0217] Pharmaceutical composition This disclosure includes pharmaceutical compositions comprising mRNA or nanoparticles (e.g., lipid nanoparticles) as described herein, in combination with one or more pharmaceutically acceptable excipients, carriers, or diluents. In certain embodiments, the mRNA is present within nanoparticles, for example, lipid nanoparticles. In certain embodiments, the mRNA or nanoparticles are present in the pharmaceutical composition. In various embodiments, the mRNA present in the pharmaceutical composition is encapsulated within nanoparticles, for example, lipid nanoparticles.
[0218] The pharmaceutical composition may optionally contain one or more further active substances, such as therapeutic and / or prophylactic substances. The pharmaceutical compositions of this disclosure may be sterile and / or pyrogenically free. General considerations in the formulation and / or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein in its entirety by reference). In certain embodiments, the pharmaceutical composition may contain mRNA and lipid nanoparticles, or a complex thereof.
[0219] Formulations comprising the pharmaceutical compositions described herein may be prepared by any method known or to be developed in the art of pharmacology. Generally, such preparations include the steps of associating the active ingredient with excipients and / or one or more other accessory components, and then, if necessary and / or desirable, dividing, shaping, and / or packaging the product into desired single-dose or multi-dose dose units.
[0220] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any further components in a pharmaceutical composition according to this disclosure will vary depending on the identification, size, and / or condition of the subject being treated, and will also vary further depending on the route of administration of the composition. For example, a composition may contain between 0.1% and 100%, e.g., between 0.5% and 70%, between 1% and 30%, between 5% and 80%, or at least 80% (w / w) of the active ingredient. In some embodiments, the active agent is the protein of interest and mRNA encoding at least one microRNA binding site to a miR expressed in an immune cell, such as a miR-142-3p binding site.
[0221] The mRNAs of this disclosure can be formulated with one or more excipients to (1) increase stability; (2) increase transfection into cells; (3) enable sustained or delayed release (e.g., of mRNA from a depot formulation); (4) alter in vivo distribution (e.g., target mRNA to a specific tissue or cell type); (5) increase translation of mRNA-encoded polypeptides in vivo; and / or (6) alter the release profile of mRNA-encoded polypeptides in vivo. In addition to conventional excipients such as any solvent and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, and preservatives, the excipients of the present disclosure may include, without limitation, lipidoids, liposomes, lipid nanoparticles (e.g., liposomes and micelles), polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, carbohydrates, mRNA-transfected cells (e.g., for transplantation into subjects), hyaluronidases, nanoparticle mimetic agents, and combinations thereof. Thus, each formulation of the present disclosure may contain one or more excipients in amounts that, together, increase mRNA stability, increase mRNA transfection into cells, increase the expression of mRNA-encoded polypeptides, and / or alter the release profile of mRNA-encoded polypeptides. Furthermore, the mRNA of the present disclosure can be formulated using self-assembling nucleic acid nanoparticles.
[0222] In the art, a variety of excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known (see Remington: The Science and Practice of Pharmacy, 21st edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006, which is incorporated herein by reference in its entirety). The use of conventional excipient media may be considered within the scope of this disclosure unless any conventional excipient media may be incompatible with the substance or its derivatives, for example, by causing any undesirable biological effect or by interacting with any other component of the pharmaceutical composition in any other harmful way. Excipients may include, for example, anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, pigments (colorants), softeners, emulsifiers, fillers (diluents), thin-film forming agents or coatings, flow enhancers (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, crystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, alpha starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
[0223] In some embodiments, the formulations described herein may contain at least one pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts that may be incorporated into the formulations of this disclosure include, but are not limited to, acid-added salts, alkali salts or alkaline earth metal salts, mineral salts or organic salts of basic residues such as amines; and alkali salts or organic salts of acidic residues such as carboxylic acids. Typical acid-added salts include acetate, acetic acid, adipine, alginate, ascorbate, aspartate, and benzenesulfonate. (acid), benzoate, bicarbonate, borate, butyrate, camphor, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptoneate, glycerophosphate, hemisulfate, heptaneate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfide This includes salts, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, valers, etc. Typical alkali salts or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium, as well as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine, and also include non-toxic ammonium, quaternary ammonium, and amine cations.
[0224] In some embodiments, the formulations described herein may contain at least one type of polynucleotide. In non-limiting examples, the formulations may contain one, two, three, four, five, or more than five of the mRNAs described herein.
[0225] For example, liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may include diluents such as water or other solvents, solubilizers, and emulsifiers, as well as inert diluents commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral composition may include adjuvants such as humectants, emulsifiers, and / or suspending agents. In certain embodiments, for parenteral administration, the composition is mixed with a solubilizer such as CREMAPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or a combination thereof.
[0226] Preparations for injection, such as sterile aqueous or sterile oily suspensions for injection, can be formulated according to known techniques using suitable dispersants, humectants, and / or suspending agents. Sterile preparations for injection may be sterile injection solutions, sterile injection suspensions, and / or sterile injection emulsions in non-toxic, parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be assisted include water, Ringer's solution according to USP, and isotonic sodium chloride solutions. Conventionally, sterile fixative oils have also been assisted as solvents or suspension media. For this purpose, any non-irritating fixative oil, including synthetic monoglycerides or synthetic diglycerides, may be assisted. Fatty acids, such as oleic acid, may also be used in the preparation of injections. Injectable formulations can also be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.
[0227] In some embodiments, a pharmaceutical composition containing at least one mRNA described herein is administered to a mammal (e.g., a human). While this specification primarily describes pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans are well understood to make them suitable for administration to a variety of animals, and those skilled in the art in the field of veterinary pharmacology can design and / or carry out such modifications, if any, by prescribed experiments only. Subjects to which the pharmaceutical composition is intended to be administered include, but are not limited to, humans and / or other primates; mammals, including commercially active mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially active birds such as poultry, chickens, ducks, geese, and / or turkeys. In certain embodiments, a subject is subjected to two or more mRNAs described herein, for example, a first mRNA encoding a first target polypeptide and containing at least one microRNA binding site to a miR expressed in an immune cell, and a second mRNA encoding a second target polypeptide and containing at least one microRNA binding site to a miR expressed in an immune cell. In certain embodiments, the first mRNA and the second mRNA are subjected to the subject simultaneously or at different time points, for example, sequentially. In certain embodiments, the first mRNA and the second mRNA are subjected to the subject in the same pharmaceutical composition or formulation, for example, to facilitate uptake of both mRNAs by the same cell.
[0228] Inhibition of immune cell activation and cytokine production The method of this disclosure, by incorporating at least one miR-126 (e.g., miR-126-3p) binding site and / or miR-142 (e.g., miR-142-3p) binding site into an mRNA construct, enables reduction or inhibition of unwanted immune cell activation and / or cytokine production in subjects treated with an mRNA-based therapeutic agent, such as unwanted immune cell activation and / or cytokine production stimulated by the polypeptide of interest (e.g., the therapeutic agent) encoded by the mRNA-based therapeutic agent. In one embodiment, immune cell activation is lymphocyte activation. In one embodiment, immune cell activation is B cell activation. In another embodiment, immune cell activation is T cell activation. In yet another embodiment, immune cell activation is macrophage activation, dendritic cell activation, NK cell activation, basophil activation, or eosinophil activation.
[0229] In one embodiment, the reduction or inhibition of undesirable immune cell activation is determined compared to a control dose of mmRNA lacking at least one miR-126 microRNA binding site or miR-142 microRNA binding site. In various embodiments, the activation of immune cells is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least about 60%, at least about 70%, at least about 80%, about 5% to 50%, about 10% to 50%, about 15% to 50%, about 20% to 50%, about 25% to 50%, about 10% to 80%, about 10% to 70%, about 10% to 60%, about 20% to 80%, about 20% to 70%, about 20% to 60%, about 20% to 40%, or about 25% to 75%.
[0230] The level of immune cell activation can be assessed by essentially any method established in the art for evaluating immune cell activation, such as the frequency of activated immune cell populations, typically evaluated by detecting cells expressing cell surface activation markers, or the production level of one or more cytokines indicating immune cell activation. In one embodiment, immune cell activation is B cell activation, in which case the level of B cell activation is determined by measuring the frequency of activated B cells, such as the frequency of activated B cells across a spleen B cell population. B cell surface markers indicating B cell activation are well known in the art (see, for example, Maddalay, R. et al. (2010), FEBS Letters, vol. 584: pp. 4883-4894). In one embodiment, B cell activation is defined as CD19 + CD86 + CD69 + This is determined by the frequency of B cells. In another embodiment, immune cell activation is B cell activation, in which case the level of B cell activation is determined by the secretion of cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) in the serum of the treated subject. In one embodiment, B cell activation is determined, for example, by the secretion of IL-6 in the serum of the treated subject. In other embodiments, the production of undesirable cytokines that are reduced or inhibited is, for example, the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) in the serum of the treated subject. In another embodiment, the production of undesirable cytokines that are reduced or inhibited is the production of interleukin-6 (IL-6).
[0231] In one embodiment, immune cell activation is the activation of plasmacytoid dendritic cells (pDCs), in which case the level of pDC activation is determined by measuring the frequency of activated pDCs, such as the frequency of activated pDCs across a spleen pDC population. In the art, pDC surface markers indicating activation are well known (see, for example, Dzionek, A. et al. (2002), Hum Immunol., vol. 63 (no. 12): pp. 1133-1148). In one embodiment, pDC activation is defined as CD11c + CD70 + CD86 + It is determined by the cell frequency.
[0232] In one embodiment, the activation of undesirable immune cells and / or the production of undesirable cytokines is reduced without a corresponding decrease in the expression of the polypeptide of interest encoded by mRNA. Thus, the method of the present disclosure makes it possible to inhibit or reduce the activation of immune cells (e.g., B cell activation, cytokine production) in a subject treated with the mRNA encoding the polypeptide of interest, which is a therapeutic agent, without significantly affecting the expression level of the therapeutic agent in the subject.
[0233] A standard metric that can be used in the methods of this disclosure is a measure of the ratio of the level or amount of encoded polypeptide (protein) produced in a cell, tissue, or organism to the level or amount of one or more (or panel) of cytokines whose expression is induced in a cell, tissue, or organism as a result of administration of or contact with chemically modified mRNA. Herein, such a ratio is referred to as the protein:cytokine ratio or "PC" ratio. A higher PC ratio indicates a more effective chemically modified mRNA (polynucleotide encoding the protein being measured). Preferred PC ratios in this disclosure, based on cytokines, may be greater than 1, greater than 10, greater than 100, greater than 1000, greater than 10,000, or more. Modified mRNA with a higher PC ratio than modified mRNA with a different construct or with an unmodified construct is preferred.
[0234] The PC ratio can be further qualified by the percentage of modifications present in the mRNA. For example, protein production standardized against 100% modified mRNA can be used to determine protein production (or risk) as a function of cytokines or the cytokine profile.
[0235] In one embodiment, the disclosure presents a method for determining the relative efficacy of any particular modified mRNA containing at least one miR-126 binding site and / or miR-142 binding site, across chemical reactions, cytokines, or modification percentages, by comparing the PC ratio of the modified mRNA containing a miR-126 binding site and / or miR-142 binding site with the PC ratio of the same construct without a miR-126 binding site and / or miR-142 binding site.
[0236] In one embodiment, the expression level of the target polypeptide encoded by mMRNA in the serum of a mammal (e.g., human) may be at least 50 pg / ml at least 2 hours after administration. In another embodiment, the expression level of the target polypeptide encoded by mMRNA in the serum of a mammal (e.g., human) may remain above 50 pg / ml for at least 72 hours after administration. In yet another embodiment, the expression level of the target polypeptide encoded by mMRNA in the serum of a mammal (e.g., human) may remain above 60 pg / ml for at least 72 hours after administration.
[0237] Inhibition of B1a cells / Inhibition of accelerated blood clearance The spleen has long been involved in ABC (Abstract Cell Activation), but the precise mechanisms contributing to ABC remain unclear. The spleen consists of the red pulp (rich in red blood cells), the white pulp (rich in lymphocytes), and the marginal zone (located between the red and white pulp and outside the marginal sinus). Antigens that enter the spleen are retained in the marginal zone, where blood flow is reduced to allow interaction between the antigen and immune effector cells (e.g., B cells) (Harm et al., Infect. Immuno., Vol. 64: pp. 4220-4225, 1996). The spleen's role in accelerating blood clearance is considered important. In vivo distribution data support that lipid-containing compounds or compositions (e.g., LNPs) are taken up by the spleen (data not shown). Histological assessment shows rapid uptake of LNPs into the marginal zone after administration (e.g., IV administration). Within the spleen, LNPs interact with splenic B cells and can contribute to various aspects of the immune response to LNPs. For example, certain components of LNPs (e.g., PEG components) interact with CD19+ B cells in the spleen, resulting in the production of LNP component-specific IgG and / or IgM molecules, which in turn lead to binding, internalization, membrane fusion, and / or activation of such cells. This can, for example, lead to accelerated blood clearance of LNPs upon second or subsequent administration.
[0238] Surprisingly, this specification also confirms that certain cells of the immune system, namely pDCs, also contribute to the ABC phenomenon. This specification confirms that the incorporation of miR binding sites (e.g., miR-126 binding sites) can result in a reduction of undesirable immune responses (e.g., ADA) to mRNA encapsulated in LNPs, e.g., proteins encoded by modified mRNA. Now, surprisingly, it is also confirmed that the incorporation of miR binding sites, particularly miR-126 binding sites, can result in a further reduction of undesirable immune responses to mRNA-encapsulating LNPs. miR-126 (e.g., miR-126-3p) is highly expressed in pDCs and is, in fact, upregulated upon pDC activation. In response to nucleic acids, pDCs synergistically increase B cell activation and increase other forms of activation through cytokine secretion and plasma cell activation. Furthermore, pDCs with "low miR-126-3p" (e.g., pDCs in which miR-126 is knocked down or knocked out) exhibit impaired activation (e.g., they are unable to produce an effective immune response to nucleic acids, resulting in IFN-α / β secretion, IL-6 secretion, etc., and are unable to migrate to the spleen upon activation). As supported herein, incorporation of the miR-126 binding site into mRNA results in low B cell activation and low serum IL-6 with repeated administration of LNP-encapsulated mRNA over several weeks. Protein expression is maintained over similar administration schedules. Surprisingly, the anti-PEG IgM response is dramatically reduced with repeated administration over several weeks. Thus, the unexpected benefit of incorporation of the miR-126 binding site into mRNA, particularly LNP-encapsulated mRNA, is the reduction of ABC.
[0239] Within the spleen, for example, in the marginal zone of the spleen, some of these key immune cells may interact directly or indirectly, for example, as a result of cytokine production (e.g., IL-6).
[0240] Without being constrained by theory, this disclosure presents evidence that miRs expressed in immune cells within the marginal zone participate in accelerating blood clearance. When the mRNA of this disclosure contains one or more miR-binding sites that bind to one or more miRs expressed in immune cells, the miR of interest is downregulated (e.g., antagonistized and / or degraded). Incorporation of at least one miR-binding site that binds to at least one miR expressed in immune cells results in a decrease in the production of IgM molecules capable of binding to lipid components (e.g., PEG lipids) compared to mRNA without at least one miR-binding site. Given the known role of IgM molecules in accelerating blood clearance, the ability of miR-binding sites that bind to miRs expressed in immune cells to inhibit or reduce IgM molecule production specifically points to a crucial role in accelerating blood clearance of miRs expressed in immune cells within the marginal zone of the spleen.
[0241] The method of this disclosure enables the reduction or inhibition of accelerated blood clearance in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), the method comprising the step of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit accelerated blood clearance in the subject after repeated administration.
[0242] In other embodiments, the first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) is administered intravenously to a subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and the second dose of chemically modified mRNA encapsulated in LNPs is administered intravenously to a subject to reduce or inhibit the acceleration of blood clearance in the subject, thereby reducing or inhibiting the acceleration of blood clearance in the subject.
[0243] In some embodiments, a subject is administered chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, thereby reducing or inhibiting the acceleration of blood clearance in the subject after multiple administrations of messenger RNA (mRNA) encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs).
[0244] In another embodiment, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, so as to reduce or inhibit the acceleration of blood clearance in the subject when subsequent administration of mRNA is performed.
[0245] In a further embodiment, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA contains one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, thereby reducing or inhibiting the acceleration of blood clearance in the subject after repeated administration.
[0246] In further embodiments, the following are performed: a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) is administered intravenously to a subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and a second dose of chemically modified mRNA encapsulated in LNPs is administered intravenously to a subject, wherein the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to LNPs, thereby reducing or inhibiting the acceleration of blood clearance in subjects administered with messenger RNA (mRNA) encoding the target polypeptide encapsulated in lipid nanoparticles (LNPs).
[0247] In another embodiment, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA contains one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, thereby reducing or inhibiting the acceleration of blood clearance in the subject after multiple administrations of the messenger RNA (mRNA) encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs).
[0248] In some embodiments, the administration of a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), reduces or inhibits the acceleration of blood clearance in the subject upon subsequent administration of mRNA, wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA contains one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs.
[0249] In some embodiments, the Disclosure provides a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of PEG-recognizing IgM molecules in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs).
[0250] In some embodiments, the first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) is administered intravenously to a subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and the second dose of chemically modified mRNA encapsulated in LNPs is administered intravenously to a subject to reduce or inhibit the production of polyethylene glycol (PEG)-recognizing IgM molecules in the subject, thereby reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in the subject administered with messenger RNA (mRNA) encoding the target polypeptide encapsulated in lipid nanoparticles (LNPs).
[0251] In some embodiments, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, so as to reduce or inhibit the production of polyethylene glycol (PEG)-recognizing IgM molecules in the subject after one or more subsequent administrations of the messenger RNA (mRNA) encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs).
[0252] In another embodiment, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, so as to reduce or inhibit the production of polyethylene glycol (PEG)-recognizing IgM molecules in the subject upon administration of subsequent mRNA administration.
[0253] In further embodiments, the present disclosure presents a method for reducing or inhibiting B1a cell activation in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit B1a cell activation in subjects after repeated administration.
[0254] In some embodiments, the first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) is administered intravenously to a subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and the second dose of chemically modified mRNA encapsulated in LNPs is administered intravenously to a subject to reduce or inhibit B1a cell activation in the subject, thereby reducing or inhibiting B1a cell activation in the subject.
[0255] In another embodiment, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, thereby reducing or inhibiting B1a cell activation in the subject after multiple administrations of messenger RNA (mRNA) encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs).
[0256] In some embodiments, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, thereby reducing or inhibiting the activation of B1a cells in the subject upon administration of subsequent mRNA.
[0257] In further embodiments, the present disclosure presents a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs).
[0258] In some embodiments, the first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) is administered intravenously to a subject, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and the second dose of chemically modified mRNA encapsulated in LNPs is administered intravenously to a subject to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject, thereby reducing or inhibiting the activation of plasmacytoid dendritic cells in subjects administered with messenger RNA (mRNA) encoding the target polypeptide encapsulated in lipid nanoparticles (LNPs).
[0259] In some embodiments, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, thereby reducing or inhibiting the activation of plasmacytoid dendritic cells in the subject after multiple administrations of messenger RNA (mRNA) encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs).
[0260] In some embodiments, a subject is administered a chemically modified mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA contains one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject when subsequent mRNA administration is performed.
[0261] In further embodiments, mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), does not activate B cells and / or induce the production of IgM molecules capable of binding to LNPs. In some embodiments, mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), does not activate B cells. In other embodiments, mRNA encoding the target polypeptide, encapsulated within lipid nanoparticles (LNPs), does not induce the production of IgM molecules capable of binding to LNPs.
[0262] In some embodiments, the reduction or inhibition of accelerated blood clearance is determined compared to a control dose of chemically modified mRNA lacking at least one microRNA binding site, encapsulated in lipid nanoparticles (LNPs). In other embodiments, the reduction or inhibition of accelerated blood clearance is achieved without corresponding reduction or inhibition of the expression of the polypeptide of interest encoded by the chemically modified mRNA.
[0263] In further embodiments, the interval between two consecutive doses is less than two weeks. In some embodiments, the interval between two consecutive doses is less than one week.
[0264] In some embodiments, the IgM molecule recognizes polyethylene glycol (PEG).
[0265] Method of Disclosure In one embodiment, the present disclosure relates to a method for reducing or inhibiting an anti-drug antibody response in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one microRNA binding site (e.g., miR-142-3p and / or miR-126-3p) to a miR expressed in an immune cell, and the mmRNA comprises one or more modified nucleobases, to reduce or inhibit the anti-drug antibody response to the polypeptide of interest in the subject. As described above, in various embodiments, the mmRNA may comprise, for example, two or more, one to four, one, two, three, or four binding sites to one or more miRs expressed in an immune cell. In certain embodiments, the mmRNA comprises at least two binding sites to at least two different miRs expressed in an immune cell. For example, the mmRNA may include a first binding site for miR-142-3p and a second binding site for a different miR expressed in immune cells, such as miR-155, miR-146 (miR-146-3p and / or miR-146-5p), or miR-126. Alternatively, the mmRNA may include a first binding site for miR-126 (e.g., miR-126-3p) and a second binding site for a different miR expressed in immune cells, such as miR-142 (miR-142-3p and / or miR-142-5p), miR-155, or miR-146 (miR-146-3p and / or miR-146-5p). In one embodiment, the mmRNA includes a first binding site for miR-142-3p and a second binding site for miR-126.
[0266] In relevant embodiments, a subject is subjected to or administered with nanoparticles containing mmol RNA (e.g., lipid nanoparticles). In further relevant embodiments, a subject is subjected to or administered with a pharmaceutical composition of the present disclosure. In certain embodiments, the pharmaceutical composition contains mmol RNA encoding the polypeptide of interest and having at least one miR binding site as described herein, or contains nanoparticles containing mmol RNA. In certain embodiments, the mmol RNA is present within the nanoparticles, for example, within the lipid nanoparticles. In certain embodiments, the mmol RNA or nanoparticles are present in the pharmaceutical composition.
[0267] In one embodiment, the mmRNA is encapsulated in lipid nanoparticles and administered intravenously. In another embodiment, the mmRNA is administered by weekly injection (e.g., intravenous injection via a pump). In yet another embodiment, the mmRNA is administered by weekly injection for at least four weeks.
[0268] In another embodiment, the present disclosure presents a method for reducing or inhibiting an anti-drug antibody response to a polypeptide of interest after repeated administration to a subject, comprising the steps of: intravenously administering to the subject a first dose of modified mRNA (mmRNA) encoding the polypeptide of interest encapsulated in an LNP, wherein the mmRNA comprises at least one binding site to a miR expressed in an immune cell (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site), and the mmRNA comprises one or more modified nucleobases; and intravenously administering to the subject a second dose of the LNP-encapsulated mmRNA to reduce or inhibit the anti-drug antibody response to the polypeptide of interest in the subject.
[0269] In another aspect, the present disclosure relates to a method for reducing or inhibiting an anti-drug antibody response after repeated administration of the target polypeptide to a subject, (i) The step of intravenously administering to a subject a first dose of a modified mRNA (mmRNA) encoding the target polypeptide, which is encapsulated within an LNP, wherein the mmRNA comprises at least one microRNA binding site (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site) for miR expressed in immune cells, mmRNA includes one or more modified nucleobases in the step; (ii) The step of detecting the level of anti-drug antibodies in a sample derived from the subject; (iii) Once the level of anti-drug antibodies in the sample has decreased, the subject is intravenously administered a second dose of the mmol RNA encapsulated in the LNP to reduce or inhibit the subject's anti-drug antibody response to the target polypeptide. I will present a method that includes this.
[0270] Given the ability of the methods of this disclosure to reduce or inhibit the expression of a target protein encoded by the mmRNA in the spleen of a subject to which the mmRNA is administered, the disclosure further presents methods for reducing the toxicity of mmRNA-based therapeutic agents. Accordingly, in another embodiment, the disclosure presents a method for reducing or inhibiting drug-related toxicity in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one binding site to a miR expressed in an immune cell (e.g., a miR-142-3p microRNA binding site and / or a miR-126 microRNA binding site), and the mmRNA comprises one or more modified nucleobases, so as to reduce or inhibit drug-related toxicity to the polypeptide of interest in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is a decrease in blood cell count (cytopenia) in the subject. In one embodiment, the drug-related toxicity to the polypeptide of interest is autoimmunity in the subject. In one embodiment, the drug-related toxicity to the target polypeptide is complement-mediated activity in the subject. In another embodiment, the drug-related toxicity to the target polypeptide is a decrease in hematopoiesis in the subject. In yet another embodiment, the drug-related toxicity may be, for example, nephrotoxicity or hepatotoxicity.
[0271] In another aspect, the present disclosure relates to a method for reducing or inhibiting unwanted immune cell activation in a subject administered RNA, e.g., messenger RNA (mRNA), comprising the step of administering RNA, e.g., mRNA (e.g., chemically modified mRNA or mmRNA) to a subject, wherein the mRNA, e.g., chemically modified RNA or mmRNA comprises at least one miR-126 microRNA binding site and / or a miR-142 microRNA binding site, and the mRNA, e.g., chemically modified mRNA or mmRNA comprises one or more modified nucleobases, to reduce or inhibit unwanted immune cell activation in the subject. In another aspect, the present disclosure relates to a method for reducing or inhibiting the production of undesirable cytokines in a subject administered RNA, e.g., messenger RNA (mRNA), comprising the steps of administering RNA, e.g., mRNA (e.g., chemically modified mRNA or mmRNA) to a subject, wherein the mRNA, e.g., chemically modified mRNA or mmRNA comprises at least one miR-126 microRNA binding site and / or a miR-142 microRNA binding site, and the mRNA, e.g., chemically modified mmRNA comprises one or more modified nucleobases, to reduce or inhibit the production of undesirable cytokines in the subject.
[0272] As described above, in various embodiments, chemically modified mRNA (referred to as mmRNA) may contain, for example, two or more, one to four, one, two, three, or four binding sites for one or more miRs expressed in an immune cell. In certain embodiments, the mmRNA may contain at least two binding sites for at least two different miRs expressed in an immune cell. For example, the mmRNA may contain a first binding site for miR-126 and a second binding site for a different miR expressed in an immune cell, such as miR-142 (miR-142-3p and / or miR-142-5p), miR-155, or miR-146 (miR-146-3p and / or miR-146-5p). Alternatively, the mMRNA may include a first binding site for miR-142 (miR-142-3p and / or miR-142-5p) and a second binding site for a different miR expressed in immune cells, such as miR-126, miR-155, or miR-146 (miR-146-3p and / or miR-146-5p). In one embodiment, the mMRNA includes a first binding site for miR-142-3p and a second binding site for miR-126.
[0273] In certain embodiments, the mRNA encodes a target polypeptide (e.g., a therapeutic agent), in which case the activation of undesirable immune cells occurs in response to the target polypeptide.
[0274] In relevant embodiments, a subject is subjected to or administered with nanoparticles containing mRNA, for example, mmRNA (e.g., lipid nanoparticles). In further relevant embodiments, a subject is subjected to or administered with the pharmaceutical composition of the present disclosure. In certain embodiments, the pharmaceutical composition contains mmRNA encoding the polypeptide of interest and having at least one miR binding site as described herein, or contains nanoparticles containing mmRNA. In certain embodiments, the mmRNA is present within the nanoparticles, for example, within the lipid nanoparticles. In certain embodiments, the mmRNA or nanoparticles are present in the pharmaceutical composition.
[0275] In one embodiment, mRNA, for example, mmRNA, is encapsulated in lipid nanoparticles and administered intravenously. In another embodiment, mRNA, for example, mmRNA, is administered by weekly injection (for example, intravenous injection via a pump). In yet another embodiment, mRNA, for example, mmRNA, is administered by weekly injection for at least four weeks.
[0276] In another embodiment, the present disclosure presents a method for reducing or inhibiting unwanted immune cell activation (e.g., lymphocyte activation, B cell activation) or unwanted cytokine production in a subject administered messenger RNA (mRNA), comprising the steps of: intravenously administering to the subject a first dose of mRNA encapsulated in an LNP, e.g., chemically modified mRNA (mmRNA), wherein the mRNA, e.g., mmRNA comprises at least one miR-126 microRNA binding site and / or a miR-142 microRNA binding site, and the mRNA, e.g., mmRNA comprises one or more modified nucleobases; and intravenously administering to the subject a second dose of mRNA encapsulated in an LNP, e.g., mmRNA, to reduce or inhibit unwanted immune cell activation or unwanted cytokine production in the subject.
[0277] In certain embodiments, the mRNA encodes a polypeptide of interest (e.g., a therapeutic agent), in which case undesirable activation of immune cells and / or production of undesirable cytokines occur in response to the polypeptide of interest.
[0278] In another aspect, the present disclosure provides a method for reducing or inhibiting unwanted immune cell activation (e.g., lymphocyte activation, B cell activation) or unwanted cytokine production in a subject after repeated administration of messenger RNA (mRNA) to the subject, (i) The step of intravenously administering to a subject a first dose of mRNA encapsulated within an LNP, for example, chemically modified mRNA (mmRNA), wherein the mRNA, for example, mmRNA, comprises at least one miR-126 microRNA binding site and / or a miR-142 microRNA binding site. The mRNA, for example, mMRNA, is a step in which one or more modified nucleobases are present; (ii) A step of detecting the level of activation of immune cells in a sample derived from the subject; (iii) Once the level of immune cell activation in the sample has decreased, the subject is intravenously administered a second dose of mRNA encapsulated in the LNP, for example, mMRNA, to reduce or inhibit unwanted immune cell activation or the production of unwanted cytokines in the subject. I will present a method that includes this.
[0279] In certain embodiments, mRNA, for example, mmRNA, encodes a polypeptide of interest (e.g., a therapeutic agent), in which case undesirable activation of immune cells or production of undesirable cytokines occurs in response to the polypeptide of interest.
[0280] ADA assay ADA assays (bioassays) can be used to assay both neutralizing antibodies (NABs) and non-neutralizing conjugated antibodies (BABs). NAB assays may include both cell-based assays, such as cell growth assays, biomarker assays, gene expression assays, gene reporter assays, antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and complement-dependent cytotoxicity (CDC) assays, as well as non-cell-based assays, such as competitive ligand binding (CLBA) assays, surface plasmon resonance (SPR), enzyme immunoassays (ELISA), electrochemiluminescence (ECL), such as electrochemiluminescence immunoassay (ECLIA), DELFIA® (dissociation-enhanced lanthanide fluorescent immunoassay), Gyros® anti-drug antibody (ADA) immunoassay, fluorescent enzyme immunoassay (FEIA), and ristocetin-induced platelet aggregation (RIPA).
[0281] In exemplary embodiments, a treatment regimen may include performing one or more ADA assays before or during the treatment regimen. In exemplary embodiments, the ADA assay is a NAB assay. In such cases, the bioassay should be related to the mechanism of action of the product, and other assays would not be useful for the clinical pharmacological effects of the NAB. In preferred embodiments, a cell-based NAB is featured in the treatment regimen of the present disclosure. If a cell-based neutralization assay is not feasible / available, a competitive ligand binding assay or alternative method may be suitable. However, if these are used, it is preferable that the assay appropriately supports the neutralizing ability / potency.
[0282] In addition to the direct measurement of the ADA response, the level of immune cell activation can also be assessed as a measure of the development of the antibody response. The level of immune cell activation can be assessed by essentially any method established in the art for evaluating immune cell activation, such as the frequency of activated immune cell populations, which is typically evaluated by detecting cells expressing cell surface activation markers, or the production level of one or more cytokines that indicate immune cell activation. In one embodiment, immune cell activation is B cell activation, in which case the level of B cell activation is determined by measuring the frequency of activated B cells, such as the frequency of activated B cells across a spleen B cell population. In the art, B cell surface markers that indicate B cell activation are well known (see, e.g., Maddalay, R. et al. (2010), FEBS Letters, vol. 584: pp. 4883-4894). In one embodiment, B cell activation is measured by CD19 + CD86 + CD69 + This is determined by the frequency of B cells. In another embodiment, immune cell activation is B cell activation, in which case the level of B cell activation is determined by the secretion of cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) in the serum of the treated subject. In one embodiment, B cell activation is determined, for example, by the secretion of IL-6 in the serum of the treated subject. In other embodiments, the production of undesirable cytokines that are reduced or inhibited is, for example, the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) in the serum of the treated subject. In another embodiment, the production of undesirable cytokines that are reduced or inhibited is the production of interleukin-6 (IL-6).
[0283] Administration of pharmaceutical compositions The pharmaceutical compositions comprising one or more RNAs, such as mRNA, of this disclosure can be administered to a subject by any suitable route. In some embodiments, the compositions of the present disclosure are administered by one or more of various routes, including parenteral routes (e.g., subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular injection, intraarticular injection, intra-arterial injection, intrasynovial injection, intrasternal injection, intrathecal injection, intralesional injection, or intracranial injection, and any suitable injection method), oral routes, transdermal or intradermal routes, interdermal routes, intrarectal routes, intravaginal routes, topical routes (e.g., powders, ointments, creams, gels, lotions, and / or drops), transmucosal routes, transnasal routes, intraoral routes, intraintestinal routes, intravitreal routes, intratumoral routes, sublingual routes, and intranasal routes; by intratracheal infusion, intrabronchial infusion, and / or inhalation; as oral sprays and / or oral powders, nasal sprays, and / or nasal aerosols, and / or via portal vein catheters. In some embodiments, the composition may be administered intravenously, intramuscularly, intradermally, intra-arterially, intratumorally, subcutaneously, or by inhalation. However, this disclosure encompasses the delivery of the composition of this disclosure by any appropriate route, taking into account possible advances in the science of drug delivery. Generally, the most appropriate route of administration will depend on a variety of factors, including the nature of the pharmaceutical composition containing one or more mRNAs (e.g., its stability in various internal environments, such as blood flow and the gastrointestinal tract) and the patient's condition (e.g., whether the patient can tolerate a particular route of administration). In one embodiment, the composition is administered parenterally. In another embodiment, the composition is administered intravenously. In yet another embodiment, the composition is administered intratumorally.
[0284] In a particular embodiment, the composition of the present disclosure contains approximately 0.0001 mg / kg to approximately 10 mg / kg, approximately 0.001 mg / kg to approximately 10 mg / kg, approximately 0.005 mg / kg to approximately 10 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 2 mg / kg to approximately 10 mg / kg, approximately 5 mg / kg to approximately 10 mg / kg, approximately 0.0001 mg / kg to approximately 5 mg / kg, approximately 0.001 mg / kg to approximately 5 mg / kg, approximately 0.005 mg / kg to approximately 5 mg / kg, The mRNA or nanoparticles of this disclosure can be administered at dose levels sufficient to deliver approximately 0.01 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 2 mg / kg to approximately 5 mg / kg, approximately 0.0001 mg / kg to approximately 1 mg / kg, approximately 0.001 mg / kg to approximately 1 mg / kg, approximately 0.005 mg / kg to approximately 1 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, or approximately 0.1 mg / kg to approximately 1 mg / kg (a dose of 1 mg / kg yields 1 mg of mRNA or nanoparticles per kg of body weight of the subject). In certain embodiments, the mRNA or nanoparticles of this disclosure can be administered at doses of approximately 0.005 mg / kg to approximately 5 mg / kg. In certain embodiments, the mRNA or nanoparticles of this disclosure can be administered at doses of approximately 0.002 mg / kg to approximately 2 mg / kg. In certain embodiments, the mRNA or nanoparticles of the Disclosure may be administered in doses of approximately 0.02 mg / kg to approximately 0.2 mg / kg.
[0285] The administration may be carried out once or multiple times daily in the same or different amounts to obtain the desired mRNA level, expression, and / or effect (e.g., therapeutic effect). The desired dose may be delivered, for example, three times daily, twice daily, once daily, every other day, every three days, weekly, every other week, every three weeks, or every four weeks. In certain embodiments, the desired dose may be delivered using multiple doses (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more doses). In some embodiments, a single dose may be administered, for example, before or after surgery, or in the case of an acute disease, disorder, or condition.
[0286] A therapeutically effective dose level, a prophylactically effective dose level, or any other appropriate dose level specific to any particular patient will depend on a variety of factors, including, if any, the severity and identification of the disorder being treated; one or more mRNAs being invoked; the specific composition being invoked; the patient's age, weight, overall health, sex, and diet; the number of doses, route of administration, and elimination rate of the specific pharmaceutical composition being invoked; the duration of treatment; drugs used in combination with or concurrently with the specific pharmaceutical composition being invoked; and similar factors well known in the medical technology field.
[0287] In some embodiments, the pharmaceutical compositions of the Disclosure may be administered in combination with other agents, such as other therapeutic agents, prophylactic agents, and / or diagnostic agents. “In combination with” is not intended to imply that the agents must be administered simultaneously and / or formulated together for delivery, but these delivery methods are also within the scope of the Disclosure. For example, one or more compositions containing one or more different mRNAs may be administered in combination. The compositions may also be administered synchronously with, before, or after one or more other desired therapeutic or medical procedures. Generally, each agent is administered in a dose and / or time schedule determined for that agent. In some embodiments, the Disclosure encompasses the delivery of the compositions of the Disclosure, or imaging compositions, diagnostic compositions, or prophylactic compositions, in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their elimination, and / or alter their distribution in the body.
[0288] Exemplary therapeutic agents that may be administered in combination with the compositions of this disclosure include, but are not limited to, cytotoxic agents, chemotherapeutic agents, and other therapeutic agents. Cytotoxic agents may include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, meitansinoids, rashelmycin, and analogs thereof. Radioactive ions may also be used as therapeutic agents and may include, for example, radioactive iodine, strontium, phosphorus, palladium, cesium, iridium, cobalt, yttrium, samarium, and praseodymium. Other therapeutic agents may include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil, and decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, rashelmycin, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), and cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mitramycin, and anthramycin), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and meitansinoids).
[0289] When employing a combination regimen, the suitability of a particular combination of treatments (therapeutic drugs or procedures) should be taken into consideration for the desired therapeutic drug and / or procedure, as well as the desired therapeutic effect to be achieved. It should also be noted that the employed treatments may achieve the desired effect on the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve a different effect (for example, control of any adverse effects).
[0290] Other Embodiments This disclosure relates to the following embodiments.
[0291] In some embodiments, the Disclosure relates to a method for reducing or inhibiting an anti-drug antibody response in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one miR-142-3p microRNA binding site, and the mmRNA comprises one or more modified nucleobases.
[0292] In other embodiments, the present disclosure relates to a method for reducing or inhibiting an anti-drug antibody response to a polypeptide of interest after repeated administration to a subject, comprising the steps of: intravenously administering to a subject a first dose of modified mRNA (mmRNA) encoding the polypeptide of interest, encapsulated within an LNP, wherein the mmRNA comprises at least one miR-142-3p microRNA binding site and the mmRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of the LNP-encapsulated mmRNA to reduce or inhibit an anti-drug antibody response to the polypeptide of interest in the subject.
[0293] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the anti-drug antibody response after repeated administration of the target polypeptide to a subject, (i) The step of intravenously administering to the subject a first dose of a modified mRNA (mmRNA) encoding the target polypeptide, which is encapsulated within an LNP, wherein the mmRNA contains at least one miR-142-3p microRNA binding site, mmRNA includes one or more modified nucleobases in the step; (ii) The step of detecting the level of anti-drug antibodies in a sample derived from the subject; (iii) Once the level of anti-drug antibodies in the sample has decreased, the subject is intravenously administered a second dose of the mmol RNA encapsulated in the LNP to reduce or inhibit the subject's anti-drug antibody response to the target polypeptide. Regarding methods including
[0294] In certain embodiments, the Disclosure relates to a method for reducing or inhibiting drug-related toxicity in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one miR-142-3p microRNA binding site and the mmRNA comprises one or more modified nucleobases, so as to reduce or inhibit drug-related toxicity to the polypeptide of interest in the subject.
[0295] In other embodiments, the Disclosure relates to a method for reducing or inhibiting drug-related toxicity in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one miR-126 microRNA binding site and the mmRNA comprises one or more modified nucleobases, so as to reduce or inhibit drug-related toxicity to the polypeptide of interest in the subject.
[0296] In some embodiments of the aforementioned embodiments, the drug-related toxicity to the target polypeptide is a decrease in blood cell count (cytopenia) in the subject. In other embodiments of the aforementioned embodiments, the drug-related toxicity to the target polypeptide is autoimmunity in the subject. In further embodiments of the aforementioned embodiments, the drug-related toxicity to the target polypeptide is complement-mediated action in the subject. In some embodiments of the aforementioned embodiments, the drug-related toxicity to the target polypeptide is a decrease in hematopoiesis in the subject. In other embodiments of the aforementioned embodiments, the drug-related toxicity is nephrotoxicity or hepatotoxicity.
[0297] In some embodiments, the Disclosure relates to a method for reducing or inhibiting an anti-drug antibody response in a subject, comprising the step of administering to the subject a modified messenger RNA (mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprises at least one miR-126 microRNA binding site, and the mmRNA comprises one or more modified nucleobases.
[0298] In other embodiments, the present disclosure relates to a method for reducing or inhibiting an anti-drug antibody response to a polypeptide of interest after repeated administration to a subject, comprising the steps of: intravenously administering to a subject a first dose of modified mRNA (mmRNA) encoding the polypeptide of interest, encapsulated within an LNP, wherein the mmRNA comprises at least one miR-126 microRNA binding site and comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of the LNP-encapsulated mmRNA to reduce or inhibit an anti-drug antibody response to the polypeptide of interest in the subject.
[0299] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the anti-drug antibody response after repeated administration of the target polypeptide to a subject, (i) The step of intravenously administering to the subject a first dose of a modified mRNA (mmRNA) encoding the target polypeptide, which is encapsulated within an LNP, wherein the mmRNA contains at least one miR-126 microRNA binding site, mmRNA includes one or more modified nucleobases in the step; (ii) The step of detecting the level of anti-drug antibodies in a sample derived from the subject; (iii) Once the level of anti-drug antibodies in the sample has decreased, the subject is intravenously administered a second dose of the mmol RNA encapsulated in the LNP to reduce or inhibit the subject's anti-drug antibody response to the target polypeptide. Regarding methods including
[0300] In some embodiments, the Disclosure relates to a method for reducing or inhibiting unwanted immune cell activation in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest, comprising the step of administering to the subject the chemically modified mRNA encoding the polypeptide of interest, wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit unwanted immune cell activation in the subject.
[0301] In some embodiments of the aforementioned set of embodiments, the reduction or inhibition of undesirable immune cell activation is determined by comparison with a control dose of chemically modified mRNA lacking at least one microRNA binding site. In other embodiments of the aforementioned set of embodiments, the reduction or inhibition of undesirable immune cell activation is the reduction or inhibition of lymphocyte activation.
[0302] In some embodiments of the aforementioned set of embodiments, the reduction or inhibition of lymphocyte activation is the reduction or inhibition of B cell activation. In other embodiments of the aforementioned set of embodiments, the reduction or inhibition of B cell activation is the reduction or inhibition of CD19 + CD86 + CD69 + This is determined by the frequency of B cells.
[0303] In some embodiments of the aforementioned embodiments, the reduction or inhibition of undesirable immune cell activation leads to a reduction or inhibition of cytokine production. In some embodiments of the aforementioned embodiments, immune cell activation is reduced by at least 10%. In further embodiments of the aforementioned embodiments, immune cell activation is reduced by at least 25%. In some embodiments of the aforementioned embodiments, immune cell activation is reduced by at least 50%. In other embodiments of the aforementioned embodiments, immune cell activation is reduced without a corresponding reduction in the expression of the target polypeptide encoded by chemically modified mRNA.
[0304] In some embodiments, the Disclosure relates to a method for reducing or inhibiting unwanted immune cell activation in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest, comprising the steps of: intravenously administering to the subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to the subject a second dose of chemically modified mRNA encapsulated in LNPs to reduce or inhibit unwanted immune cell activation in the subject.
[0305] In other embodiments, the present disclosure relates to a method for reducing or inhibiting undesirable immune cell activation in a subject after repeated administration of messenger RNA (mRNA) encoding a target polypeptide, (i) The step of intravenously administering a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs) to a subject, wherein the chemically modified mRNA contains at least one microRNA binding site for microRNA expressed in immune cells, The chemically modified mRNA includes one or more modified nucleobases in the step; (ii) A step of detecting the level of activation of immune cells in a sample derived from the subject; (iii) Once the level of immune cell activation in the sample has decreased, the subject is intravenously administered a second dose of chemically modified mRNA encapsulated in the LNP to reduce or inhibit the unwanted immune cell activation in the subject. Regarding methods including
[0306] In some embodiments of the aforementioned aspects, the reduction or inhibition of undesirable immune cell activation reduces or inhibits B cell activation. In some embodiments of the aforementioned aspects, the reduction or inhibition of undesirable immune cell activation leads to a reduction or inhibition of cytokine production.
[0307] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the production of undesirable cytokines in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest, comprising the step of administering to the subject a chemically modified mRNA encoding the polypeptide of interest, wherein the chemically modified mRNA comprises at least one microRNA binding site for a microRNA expressed in an immune cell, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of undesirable cytokines in the subject.
[0308] In some embodiments of the aforementioned set of embodiments, the reduction or inhibition of undesirable cytokine production is determined by comparing the administration of a control to a microRNA expressed in immune cells with a chemically modified mRNA lacking at least one microRNA binding site. In other embodiments of the aforementioned set of embodiments, the reduction or inhibition of cytokine production is the reduction or inhibition of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ). In some embodiments of the aforementioned set of embodiments, the reduction or inhibition of cytokine production is the reduction or inhibition of interleukin-6 (IL-6) production.
[0309] In some embodiments of the aforementioned embodiments, cytokine production is reduced by at least 10%. In some embodiments of the aforementioned embodiments, cytokine production is reduced by at least 25%. In some embodiments of the aforementioned embodiments, cytokine production is reduced by at least 50%. In some embodiments of the aforementioned embodiments, cytokine production is reduced without a corresponding reduction in the expression of the target polypeptide encoded by chemically modified mRNA.
[0310] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject who has been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), the method comprising the step of administering to a subject chemically modified mRNA encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the acceleration of blood clearance in the subject after repeated administration.
[0311] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of: intravenously administering to a subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of chemically modified mRNA encapsulated in LNPs to reduce or inhibit the acceleration of blood clearance in the subject.
[0312] In other embodiments, the Disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject who has received multiple doses of messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject the chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, to reduce or inhibit the acceleration of blood clearance in the subject during one or more subsequent doses.
[0313] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the acceleration of blood clearance in the subject when subsequent administration of mRNA is performed.
[0314] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject who has been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA comprises one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, so as to reduce or inhibit the acceleration of blood clearance in the subject after repeated administration.
[0315] In other embodiments, the present disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of: intravenously administering to a subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of chemically modified mRNA encapsulated in LNPs, wherein the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, so as to reduce or inhibit the acceleration of blood clearance in the subject.
[0316] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject who has received multiple doses of messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA comprises one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs, so as to reduce or inhibit the acceleration of blood clearance in the subject during one or more subsequent doses.
[0317] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the acceleration of blood clearance in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated within lipid nanoparticles (LNPs), comprising the steps of administering to a subject the chemically modified mRNA encoding a polypeptide of interest encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, the chemically modified mRNA comprises one or more modified nucleobases, and the LNPs do not activate B cells and / or induce the production of IgM molecules capable of binding to the LNPs.
[0318] In other embodiments, the Disclosure relates to a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA-binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject after repeated administration.
[0319] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of: intravenously administering to a subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA-binding site for microRNA expressed in an immune cell, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of chemically modified mRNA encapsulated in LNPs to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject.
[0320] In other embodiments, the Disclosure relates to a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in subjects who have received multiple doses of messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA-binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject after one or more subsequent doses.
[0321] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the production of polyethylene glycol (PEG)-recognizing IgM molecules in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject the chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the production of PEG-recognizing IgM molecules in the subject upon subsequent mRNA administration.
[0322] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting B1a cell activation in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a target polypeptide encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit B1a cell activation in the subject after repeated administration.
[0323] In some embodiments, the Disclosure relates to a method for reducing or inhibiting B1a cell activation in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of: intravenously administering to a subject a first dose of chemically modified mRNA encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to a subject a second dose of chemically modified mRNA encapsulated in LNPs to reduce or inhibit B1a cell activation in the subject.
[0324] In other embodiments, the Disclosure relates to a method for reducing or inhibiting B1a cell activation in subjects who have received multiple doses of messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, to reduce or inhibit B1a cell activation in the subject during one or more subsequent doses.
[0325] In some embodiments, the Disclosure relates to a method for reducing or inhibiting B1a cell activation in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject the chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit B1a cell activation in the subject upon subsequent mRNA administration.
[0326] In other embodiments, the Disclosure relates to a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in subjects who have been repeatedly administered messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject after repeated administration.
[0327] In a further embodiment, the present disclosure relates to a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in a subject administered with messenger RNA (mRNA) encoding a target polypeptide encapsulated within lipid nanoparticles (LNPs), the method comprising: intravenously administering to the subject a first dose of chemically modified mRNA encapsulated within lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases; and intravenously administering to the subject a second dose of chemically modified mRNA encapsulated within LNPs to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject.
[0328] In some embodiments, the Disclosure relates to a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in subjects who have received multiple doses of messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the step of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject after one or more subsequent doses.
[0329] In other embodiments, the Disclosure relates to a method for reducing or inhibiting the activation of plasmacytoid dendritic cells in a subject administered with messenger RNA (mRNA) encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), comprising the steps of administering to a subject chemically modified mRNA encoding a polypeptide of interest encapsulated in lipid nanoparticles (LNPs), wherein the chemically modified mRNA comprises at least one microRNA binding site for microRNA expressed in immune cells, and the chemically modified mRNA comprises one or more modified nucleobases, so as to reduce or inhibit the activation of plasmacytoid dendritic cells in the subject upon subsequent mRNA administration.
[0330] In some embodiments of the aforementioned model, mRNA encoding the target polypeptide encapsulated within lipid nanoparticles (LNPs) does not activate B cells and / or does not induce the production of IgM molecules capable of binding to LNPs. In some embodiments of the aforementioned model, mRNA encoding the target polypeptide encapsulated within lipid nanoparticles (LNPs) does not activate B cells. In some embodiments of the aforementioned model, mRNA encoding the target polypeptide encapsulated within lipid nanoparticles (LNPs) does not induce the production of IgM molecules capable of binding to LNPs.
[0331] In some embodiments of the aforementioned aspects, the reduction or inhibition of accelerated blood clearance is determined by comparing it to a control dose of chemically modified mRNA lacking at least one microRNA binding site, encapsulated in lipid nanoparticles (LNPs). In some embodiments of the aforementioned aspects, the reduction or inhibition of accelerated blood clearance is achieved without corresponding reduction or inhibition of the expression of the target polypeptide encoded by the chemically modified mRNA. In some embodiments of the aforementioned aspects, the interval between two consecutive doses is less than two weeks. In some embodiments of the aforementioned aspects, the interval between two consecutive doses is less than one week.
[0332] In some embodiments of the aforementioned model, the IgM molecule recognizes polyethylene glycol (PEG).
[0333] In any of the embodiments described herein, the mmRNA described herein is encapsulated in lipid nanoparticles and administered intravenously. In any of the embodiments described herein, the mmRNA described herein is administered by weekly injection.
[0334] In any of the embodiments described herein, the mmRNA described herein comprises a 5'UTR, a codon-optimized open reading frame encoding the polypeptide of interest, a 3'UTR containing at least one miR-142-3p microRNA binding site, and a 3' tailing region of the ligated nucleoside. In some embodiments, the mmRNA described herein comprises a 5'UTR and a 3'UTR that are heterogeneous with respect to the coding region. In some embodiments, the mmRNA described herein is fully modified. In some embodiments, the mmRNA described herein is fully modified for a specific chemical modification.
[0335] In any of the embodiments described herein, the mMRNAs described herein are pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m5C), and 1-methylpseudridine (m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine(s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 Includes C).
[0336] In any of the embodiments described herein, the mmRNA described herein is pseudouridine (ψ), N1-methylpseudridine (m 1 ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseuduridine, 2-thio-5-azauridine, 2-thiodihydropseuduridine, 2-thiodihydrouridine, 2-thiopseuduridine, 4-methoxy-2-thiopseuduridine, 4-methoxypseuduridine, 4-thio-1-methylpseuduridine, 4-thiopseuduridine, 5-azauridine, dihydropseuduridine, 5-methoxyuridine, or 2'-O-methyluridine, or combinations thereof.
[0337] In some embodiments, the mmRNA described herein is 1-methylpseudridine (m 1 ψ), 5-methoxyuridine (mo 5 U), 5-methylcytidine (m 5 C) Contains pseudouridine(ψ), α-thioguanosine, or α-thioadenosine, or a combination thereof.
[0338] In any of the embodiments described above, the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein.
[0339] In any of the embodiments described above, the lipid nanoparticles are liposomes. In some embodiments, the lipid nanoparticles include cationic lipids and / or ionized lipids. In some embodiments, the cationic lipids and / or ioni...
Claims
1. A messenger RNA (mRNA) encoding a target polypeptide, wherein the mRNA comprises a 3' untranslated region (UTR) having at least one miR-142 binding site and at least one miR-126 binding site, and the mRNA comprises one or more modified nucleobases.
2. The mRNA according to claim 1, wherein the 3'UTR comprises two, three, or four miR-142 binding sites.
3. The mRNA according to claim 1 or 2, wherein the 3'UTR comprises two, three, or four miR-126 binding sites.
4. The mRNA according to any one of claims 1 to 3, wherein the at least one miR-142 binding site is a miR-142-3p binding site.
5. The mRNA according to any one of claims 1 to 3, wherein the at least one miR-142 binding site is a miR-142-5p binding site.
6. The mRNA according to any one of claims 1 to 5, wherein the at least one miR-126 binding site is a miR-126-3p binding site.
7. The mRNA according to any one of claims 1 to 5, wherein the at least one miR-126 binding site is a miR-126-5p binding site.
8. mRNA encoding a target polypeptide, wherein the mRNA comprises a 3'UTR having at least one miR-142-3p binding site and at least one miR-126-3p binding site, and the mRNA comprises one or more modified nucleobases.
9. The mRNA according to any one of claims 4, 6 to 8, wherein the 3'UTR comprises two, three, or four miR-142-3p binding sites.
10. The mRNA according to any one of claims 6, 8, or 9, wherein the 3'UTR comprises two, three, or four miR-126-3p binding sites.
11. The mRNA according to any one of claims 4, 6 to 10, wherein the miR-142-3p binding site includes the sequence of Sequence ID No.
3.
12. The mRNA according to any one of claims 6, 8 to 11, wherein the miR-126-3p binding site includes the sequence of SEQ ID NO:
26.
13. The one or more modified nucleobases are pseudouridine (ψ), pseudouridine (ψ) and 5-methylcytidine (m 5 C), 1-methylpseudridine (m 1 ψ), 1-methylpseudridine (m 1 ψ) and 5-methylcytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine and 5-methylcytidine (m 5 C), 5-methoxyuridine (mo 5 U), 5-methoxyuridine (mo 5 U) and 5-methylcytidine (m 5 C), 2'-O-methyluridine, 2'-O-methyluridine and 5-methylcytidine (m 5 C), N6-methyladenosine (m 6 A), or N6-methyladenosine (m 6 A) and 5-methylcytidine (m 5 The mRNA according to any one of claims 1 to 12, selected from C).
14. The mRNA according to any one of claims 1 to 13, wherein all uracil nucleo bases in the mRNA are modified uracil nucleo bases.
15. The mRNA according to claim 14, wherein the modified uracil nucleobase is selected from pseudouridine (ψ), 1-methylpseudridine (m1ψ), and 5-methoxyuridine (mo5U).
16. The mRNA according to claim 14, wherein the modified uracil nucleobase is 1-methylpseudridine (m 1 ψ).
17. The mRNA according to any one of claims 1 to 16, wherein all cytosine nucleobases in the mRNA are modified cytosine nucleobases.
18. The mRNA according to claim 17, wherein the modified cytosine nucleobase is 5-methylcytidine.
19. The mRNA according to any one of claims 1 to 18, wherein the target polypeptide is a therapeutic protein, cytokine, growth factor, antibody, or fusion protein.
20. Lipid nanoparticles comprising mRNA according to any one of claims 1 to 19, wherein the lipid nanoparticles comprise ionized lipids, structural lipids, phospholipids, and PEG lipids.
21. A pharmaceutical composition comprising lipid nanoparticles according to claim 20 and a pharmaceutically acceptable carrier, diluent, or excipient.
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