JPWO2020247452A5 - - Google Patents
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これは、アミロイドーシスを非常に急速に発症する最初の報告されたインビボモデルであり、2ヶ月齢のベータスリップマウスでアミロイド沈着が観察された。次に、抗TTR抗体で共染色して複屈折沈着物を共標識し、それらがTTR沈着によって引き起こされることを確認するための実験が行われる。他の器官や組織に沈着物があるかどうかを分析するために、追加の実験が行われる。
本発明は、例えば、以下の項目を提供する。
(項目1)
ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む非ヒト動物であって、Ttrコード配列と非コード配列の両方を含む前記内因性Ttr遺伝子座の領域が削除され、TTRコード配列と非コード配列の両方を含む対応するヒトTTR配列で置き換えられており、前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含む、非ヒト動物。
(項目2)
前記コードされたトランスサイレチンタンパク質がヒトトランスサイレチンタンパク質と最適に整列している場合、前記変異が、前記ヒトトランスサイレチンタンパク質の残基L58に対応する残基を、前記ヒトトランスサイレチンタンパク質の残基L55に対応する残基が通常占める位置に配置する、ベータ鎖Dの3残基シフトを引き起こす、項目1に記載の非ヒト動物。
(項目3)
前記コードされたトランスサイレチンタンパク質が前記ヒトトランスサイレチンタンパク質と最適に整列している場合、前記変異が、前記ヒトトランスサイレチンタンパク質のG53S/E54D/L55Sに対応する三重変異である、項目2に記載の非ヒト動物。
(項目4)
前記三重変異が、前記対応するヒトTTR配列にある、項目3に記載の非ヒト動物。
(項目5)
前記遺伝子改変された内因性Ttr遺伝子座が、前記内因性Ttrプロモーターを含み、前記ヒトTTR配列が、前記内因性Ttrプロモーターに作動可能に連結されている、先行項目のいずれか一項に記載の非ヒト動物。
(項目6)
前記内因性Ttr遺伝子座の少なくとも1つのイントロンおよび少なくとも1つのエキソンが削除され、前記対応するヒトTTR配列で置き換えられている、先行項目のいずれか一項に記載の非ヒト動物。
(項目7)
前記内因性Ttr遺伝子座の全Ttrコード配列が削除され、前記対応するヒトTTR配列で置き換えられている、先行項目のいずれか一項に記載の非ヒト動物。
(項目8)
Ttr開始コドンからTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、項目7に記載の非ヒト動物。
(項目9)
前記遺伝子改変された内因性Ttr遺伝子座が、ヒトTTR3’非翻訳領域を含む、先行項目のいずれか一項に記載の非ヒト動物。
(項目10)
内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられていない、先行項目のいずれか一項に記載の非ヒト動物。
(項目11)
前記Ttr開始コドンから前記Ttr終止コドンまでの前記内因性Ttr遺伝子座の領域が削除され、前記対応するヒトTTR配列およびヒトTTR3’非翻訳領域を含むヒトTTR配列で置き換えられており、かつ
前記内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、かつ
前記内因性Ttrプロモーターが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、先行項目のいずれか一項に記載の非ヒト動物。
(項目12)
(i)前記遺伝子改変された内因性Ttr遺伝子座の前記ヒトTTR配列が、配列番号14に記載の配列と、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含むか、または
(ii)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号9に記載の配列と、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含むタンパク質をコードし、
(iii)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号10に記載の配列と少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含むコード配列を含むか、または
(iv)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号12もしくは13に記載の配列と少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含む、項目11に記載の非ヒト動物。
(項目13)
前記遺伝子改変された内因性Ttr遺伝子座が、シグナルペプチドを含むトランスサイレチン前駆体タンパク質をコードし、前記シグナルペプチドをコードする前記内因性Ttr遺伝子座の領域が削除されておらず、前記対応するヒトTTR配列で置き換えられていない、項目1~6のいずれか一項に記載の非ヒト動物。
(項目14)
前記内因性Ttr遺伝子座の第1のエキソンが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、項目13に記載の非ヒト動物。
(項目15)
前記内因性Ttr遺伝子座の第1のエキソンおよび第1のイントロンが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、項目14に記載の非ヒト動物。
(項目16)
第2のTtrエキソンの開始からTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、項目13~15のいずれか一項に記載の非ヒト動物。
(項目17)
前記遺伝子改変された内因性Ttr遺伝子座が、ヒトTTR3’非翻訳領域を含む、項目13~16のいずれか一項に記載の非ヒト動物。
(項目18)
前記第2のTtrエキソンから前記Ttr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列およびヒトTTR3’非翻訳領域で置き換えられており、かつ
前記内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、かつ
前記内因性Ttrプロモーターが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、項目13~17のいずれか一項に記載の非ヒト動物。
(項目19)
前記遺伝子改変された内因性Ttr遺伝子座が、選択カセットまたはレポーター遺伝子を含まない、先行項目のいずれか一項に記載の非ヒト動物。
(項目20)
前記非ヒト動物が、前記遺伝子改変された内因性Ttr遺伝子座についてホモ接合性である、先行項目のいずれか一項に記載の非ヒト動物。
(項目21)
前記非ヒト動物が哺乳動物である、先行項目のいずれか一項に記載の非ヒト動物。
(項目22)
前記非ヒト動物がラットまたはマウスである、項目21に記載の非ヒト動物。
(項目23)
前記非ヒト動物がマウスである、項目22に記載の非ヒト動物。
(項目24)
前記非ヒト動物が、その生殖系列において前記遺伝子改変された内因性Ttr遺伝子座を含む、先行項目のいずれか一項に記載の非ヒト動物。
(項目25)
前記非ヒト動物が、対照の野生型非ヒト動物または変異を伴わない遺伝子改変された内因性Ttr遺伝子座を含む非ヒト動物と比較して活動亢進的である、先行項目のいずれか一項に記載の非ヒト動物。
(項目26)
前記活動亢進が、オープンフィールド試験における総距離、総活動量、もしくは総立ち上がり(total rearing)のうちの1つ以上またはすべてによって測定される、項目25に記載の非ヒト動物。
(項目27)
前記非ヒト動物が、後肢ジストニアを示す、先行項目のいずれか一項に記載の非ヒト動物。
(項目28)
前記非ヒト動物が、アミロイド沈着を含む、先行項目のいずれか一項に記載の非ヒト動物。
(項目29)
前記非ヒト動物が、坐骨神経にアミロイド沈着を含む、項目28に記載の非ヒト動物。
(項目30)
前記非ヒト動物が、約2ヶ月齢までにアミロイドーシスを発症する、項目28または29に記載の非ヒト動物。
(項目31)
ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む非ヒト動物細胞であって、Ttrコード配列と非コード配列の両方を含む前記内因性Ttr遺伝子座の領域が削除され、TTRコード配列と非コード配列の両方を含む対応するヒトTTR配列で置き換えられており、前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含む、非ヒト動物細胞。
(項目32)
遺伝子改変された内因性Ttr遺伝子座を含む非ヒト動物ゲノムであって、Ttrコード配列と非コード配列の両方を含む前記内因性Ttr遺伝子座の領域が削除され、TTRコード配列と非コード配列の両方を含む対応するヒトTTR配列で置き換えられており、前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含む、非ヒト動物ゲノム。
(項目33)
遺伝子改変された内因性Ttr遺伝子座を生成するための標的化ベクターであって、Ttrコード配列と非コード配列の両方を含む前記内因性Ttr遺伝子座の領域が削除され、TTRコード配列と非コード配列の両方を含む対応するヒトTTR配列で置き換えられており、前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含み、前記標的化ベクターが、前記内因性Ttr遺伝子座で5’標的配列を標的とする5’相同性アーム、および前記内因性Ttr遺伝子座で3’標的配列を標的とする3’相同性アームに隣接する対応するヒトTTR配列を含む挿入核酸を含む、標的化ベクター。
(項目34)
遺伝子改変された非ヒト動物Ttr遺伝子であって、Ttrコード配列と非コード配列の両方を含む内因性Ttr遺伝子の領域が削除され、TTRコード配列と非コード配列の両方を含む対応するヒトTTR配列で置き換えられており、前記遺伝子改変された非ヒト動物のTtr遺伝子が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含む、遺伝子改変された非ヒト動物Ttr遺伝子。
(項目35)
インビボでのヒトTTR標的化試薬の活性を評価する方法であって、
(a)項目1~30のいずれか一項に記載の非ヒト動物に前記ヒトTTR標的化試薬を投与することと、
(b)前記非ヒト動物における前記ヒトTTR標的化試薬の活性を評価することと、を含む、方法。
(項目36)
導入が、アデノ随伴ウイルス(AAV)媒介送達、脂質ナノ粒子(LNP)媒介送達、または流体力学的送達(HDD)を含む、項目35に記載の方法。
(項目37)
前記導入が、LNP媒介送達を含む、項目36に記載の方法。
(項目38)
前記導入が、AAV8媒介送達を含む、項目36に記載の方法。
(項目39)
ステップ(b)が、前記非ヒト動物から肝臓を単離することと、前記肝臓における前記ヒトTTR標的化試薬の活性を評価することとを含む、項目35~38のいずれか一項に記載の方法。
(項目40)
ステップ(b)が、前記肝臓以外の器官または組織における前記ヒトTTR標的化試薬の活性を評価することをさらに含む、項目39に記載の方法。
(項目41)
前記評価が、前記遺伝子改変されたTtr遺伝子座の改変を評価することを含む、項目35~40のいずれか一項に記載の方法。
(項目42)
前記評価が、前記遺伝子改変されたTtr遺伝子座によってコードされるTtrメッセンジャーRNAの発現を評価することを含む、項目35~41のいずれか一項に記載の方法。
(項目43)
前記評価が、前記遺伝子改変されたTtr遺伝子座によってコードされるTTRタンパク質の発現を評価することを含む、項目35~42のいずれか一項に記載の方法。
(項目44)
前記TTRタンパク質の発現を評価することが、前記非ヒト動物における前記TTRタンパク質の血清レベルを測定することを含む、項目43に記載の方法。
(項目45)
前記活性が、前記非ヒト動物の前記肝臓において評価される、項目41~43のいずれか一項に記載の方法。
(項目46)
前記評価が、活動亢進を評価することを含む、項目35~45のいずれか一項に記載の方法。
(項目47)
前記評価が、後肢ジストニアを評価することを含む、項目35~46のいずれか一項に記載の方法。
(項目48)
前記評価が、アミロイド沈着を評価することを含む、項目35~47のいずれか一項に記載の方法。
(項目49)
前記評価が、坐骨神経におけるアミロイド沈着を評価することを含む、項目48に記載の方法。
(項目50)
前記評価が、未処置の対照非ヒト動物との比較で行われる、項目35~49のいずれか一項に記載の方法。
(項目51)
前記ヒトTTR標的化試薬が、ヒトTTR遺伝子の領域を標的化するように設計されたヌクレアーゼ剤を含む、項目35~50のいずれか一項に記載の方法。
(項目52)
前記ヌクレアーゼ剤が、Casタンパク質および前記ヒトTTR遺伝子のガイドRNA標的配列を標的化するように設計されたガイドRNAを含む、項目51に記載の方法。
(項目53)
前記Casタンパク質が、Cas9タンパク質である、項目52に記載の方法。
(項目54)
前記ヒトTTR標的化試薬が、外因性ドナー核酸を含み、前記外因性ドナー核酸が、前記ヒトTTR遺伝子と組換えるように設計されている、項目35~53のいずれか一項に記載の方法。
(項目55)
前記外因性ドナー核酸が、一本鎖オリゴデオキシヌクレオチド(ssODN)である、項目54に記載の方法。
(項目56)
前記ヒトTTR標的化試薬が、抗原結合タンパク質を含む、項目35~55のいずれか一項に記載の方法。
(項目57)
前記ヒトTTR標的化試薬が、RNAi剤またはアンチセンスオリゴヌクレオチドを含む、項目35~56のいずれか一項に記載の方法。
(項目58)
インビボでのヒトTTR標的化試薬の活性を最適化する方法であって、
(I)項目35~57のいずれか一項に記載の方法を、ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む第1の非ヒト動物において、1回目に実行することと、
(II)可変要素(variable)を変更し、ステップ(I)の前記方法を、ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む第2の非ヒト動物において、変更された可変要素を用いて2回目に実行することと、
(III)ステップ(I)の前記ヒトTTR標的化試薬の前記活性を、ステップ(II)の前記ヒトTTR標的化試薬の前記活性と比較し、より高い有効性、より高い精度、より高い一貫性、またはより高い特異性をもたらす方法を選択することと、を含む、方法。
(項目59)
ステップ(II)における前記変更された可変要素が、前記ヒトTTR標的化試薬を前記非ヒト動物に導入する送達方法である、項目58に記載の方法。
(項目60)
ステップ(II)における前記変更された可変要素が、前記ヒトTTR標的化試薬を前記非ヒト動物に導入する投与経路である、項目58に記載の方法。
(項目61)
ステップ(II)における前記変更された可変要素が、前記非ヒト動物に導入された前記ヒトTTR標的化試薬の濃度または量である、項目58に記載の方法。
(項目62)
ステップ(II)における前記変更された可変要素が、前記非ヒト動物に導入された前記ヒトTTR標的化試薬の形態である、項目58に記載の方法。
(項目63)
ステップ(II)における前記変更された可変要素が、前記非ヒト動物に導入された前記ヒトTTR標的化試薬である、項目58に記載の方法。
(項目64)
項目1~30のいずれか一項に記載の非ヒト動物を作製する方法であって、
(I)(a)遺伝子改変された内因性Ttr遺伝子座を含むように、多能性非ヒト動物細胞のゲノムを改変することと、
(b)前記遺伝子改変された内因性Ttr遺伝子座を含む前記遺伝子改変された多能性非ヒト動物細胞を同定または選択することと、
(c)前記遺伝子改変された多能性非ヒト動物細胞を非ヒト動物宿主胚に導入することと、
(d)代理母に前記非ヒト動物宿主胚を懐胎させることと、を含むか、または
(II)(a)遺伝子改変された内因性Ttr遺伝子座を含むように、非ヒト動物の1細胞期胚のゲノムを改変することと、
(b)前記遺伝子改変された内因性Ttr遺伝子座を含む前記遺伝子改変された非ヒト動物の1細胞期胚を選択することと、
(c)代理母に前記遺伝子改変された非ヒト動物の1細胞期胚を懐胎させることと、を含む、方法。
This is the first reported in vivo model of very rapid onset of amyloidosis, with amyloid deposits observed in 2 month old beta slip mice. Experiments are then performed to co-label birefringent deposits by co-staining with an anti-TTR antibody and confirm that they are caused by TTR deposits. Additional experiments will be performed to analyze whether there are deposits in other organs and tissues.
The present invention provides, for example, the following items.
(Item 1)
A non-human animal comprising an endogenous Ttr locus genetically modified in its genome, wherein regions of said endogenous Ttr locus comprising both Ttr coding and non-coding sequences have been deleted, and TTR coding and non-coding sequences and wherein said genetically modified endogenous Ttr locus comprises a mutation causing a beta-strand D shift of the encoded transthyretin protein.
(Item 2)
If the encoded transthyretin protein is optimally aligned with the human transthyretin protein, then the mutation replaces the residue corresponding to residue L58 of the human transthyretin protein with the human transthyretin protein. 2. The non-human animal of item 1, which causes a 3-residue shift in the beta chain D that places the residue corresponding to residue L55 of L5 in the position normally occupied.
(Item 3)
Item 2, wherein when said encoded transthyretin protein is optimally aligned with said human transthyretin protein, said mutation is a triple mutation corresponding to G53S/E54D/L55S of said human transthyretin protein. Non-human animals as described in .
(Item 4)
4. The non-human animal of item 3, wherein said triple mutation is in said corresponding human TTR sequence.
(Item 5)
3. Any one of the preceding items, wherein said genetically modified endogenous Ttr locus comprises said endogenous Ttr promoter, and wherein said human TTR sequence is operably linked to said endogenous Ttr promoter. non-human animal.
(Item 6)
3. The non-human animal of any one of the preceding items, wherein at least one intron and at least one exon of said endogenous Ttr locus have been deleted and replaced with said corresponding human TTR sequence.
(Item 7)
3. The non-human animal of any one of the preceding items, wherein the entire Ttr coding sequence of said endogenous Ttr locus has been deleted and replaced with said corresponding human TTR sequence.
(Item 8)
8. The non-human animal of item 7, wherein said region of said endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted and replaced with the corresponding human TTR sequence.
(Item 9)
The non-human animal of any one of the preceding items, wherein said genetically modified endogenous Ttr locus comprises a human TTR 3' untranslated region.
(Item 10)
3. The non-human animal of any one of the preceding items, wherein the endogenous Ttr 5' untranslated region has not been deleted and replaced with said corresponding human TTR sequence.
(Item 11)
the region of the endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted and replaced with a human TTR sequence including the corresponding human TTR sequence and a human TTR 3' untranslated region; and
the endogenous Ttr 5' untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and
3. The non-human animal of any one of the preceding items, wherein said endogenous Ttr promoter has not been deleted and replaced with said corresponding human TTR sequence.
(Item 12)
(i) said human TTR sequence of said genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least contain 99% or 100% identical sequences, or
(ii) the genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the sequence set forth in SEQ ID NO:9 encode proteins containing % identical sequences,
(iii) the genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the sequence set forth in SEQ ID NO: 10 contains a coding sequence containing the same sequence, or
(iv) the genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% with the sequence set forth in SEQ ID NO: 12 or 13; 12. Non-human animal according to item 11, comprising 100% identical sequences.
(Item 13)
wherein said genetically modified endogenous Ttr locus encodes a transthyretin precursor protein comprising a signal peptide, the region of said endogenous Ttr locus encoding said signal peptide has not been deleted, and said corresponding Non-human animal according to any one of items 1-6, which is not replaced with a human TTR sequence.
(Item 14)
14. The non-human animal of item 13, wherein the first exon of said endogenous Ttr locus has not been deleted and replaced with said corresponding human TTR sequence.
(Item 15)
15. The non-human animal of item 14, wherein the first exon and first intron of said endogenous Ttr locus have not been deleted and replaced with said corresponding human TTR sequence.
(Item 16)
16. Any one of items 13-15, wherein said region of said endogenous Ttr locus from the start of the second Ttr exon to the Ttr stop codon is deleted and replaced with said corresponding human TTR sequence. non-human animal.
(Item 17)
17. The non-human animal of any one of items 13-16, wherein said genetically modified endogenous Ttr locus comprises a human TTR 3' untranslated region.
(Item 18)
said region of said endogenous Ttr locus from said second Ttr exon to said Ttr stop codon has been deleted and replaced with said corresponding human TTR sequence and human TTR 3' untranslated region; and
the endogenous Ttr 5' untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and
18. The non-human animal of any one of items 13-17, wherein said endogenous Ttr promoter has not been deleted and replaced with said corresponding human TTR sequence.
(Item 19)
3. The non-human animal of any one of the preceding items, wherein said genetically modified endogenous Ttr locus does not comprise a selection cassette or reporter gene.
(Item 20)
3. The non-human animal of any one of the preceding items, wherein said non-human animal is homozygous for said genetically modified endogenous Ttr locus.
(Item 21)
A non-human animal according to any one of the preceding items, wherein said non-human animal is a mammal.
(Item 22)
22. The non-human animal of item 21, wherein said non-human animal is a rat or mouse.
(Item 23)
23. The non-human animal of item 22, wherein said non-human animal is a mouse.
(Item 24)
3. The non-human animal of any one of the preceding items, wherein said non-human animal comprises said genetically modified endogenous Ttr locus in its germline.
(Item 25)
3. Any one of the preceding items, wherein said non-human animal is hyperactive compared to a control wild-type non-human animal or a non-human animal comprising a genetically modified endogenous Ttr locus without mutation. Non-human animals as described.
(Item 26)
26. The non-human animal of item 25, wherein said hyperactivity is measured by one or more or all of total distance, total activity, or total rearing in an open field test.
(Item 27)
A non-human animal according to any one of the preceding items, wherein said non-human animal exhibits hindlimb dystonia.
(Item 28)
A non-human animal according to any one of the preceding items, wherein said non-human animal comprises amyloid deposits.
(Item 29)
29. The non-human animal of item 28, wherein said non-human animal comprises amyloid deposits in the sciatic nerve.
(Item 30)
30. The non-human animal of item 28 or 29, wherein said non-human animal develops amyloidosis by about 2 months of age.
(Item 31)
A non-human animal cell comprising an endogenous Ttr locus genetically modified in its genome, wherein a region of said endogenous Ttr locus containing both Ttr coding and non-coding sequences is deleted, resulting in a TTR coding sequence and a non-coding sequence. wherein the genetically modified endogenous Ttr locus comprises a mutation that causes a shift of the beta-strand D of the encoded transthyretin protein. cell.
(Item 32)
A non-human animal genome comprising an endogenous Ttr locus that has been genetically modified, wherein a region of said endogenous Ttr locus comprising both Ttr coding and non-coding sequences has been deleted, resulting in a TTR coding sequence and a non-coding sequence. A non-human animal genome that has been replaced with a corresponding human TTR sequence containing both, wherein said genetically modified endogenous Ttr locus comprises a mutation that causes a beta-strand D shift of the encoded transthyretin protein.
(Item 33)
A targeting vector for generating a genetically modified endogenous Ttr locus, wherein a region of said endogenous Ttr locus comprising both Ttr coding and non-coding sequences is deleted and TTR coding and non-coding sequences are deleted. wherein the genetically modified endogenous Ttr locus contains a mutation that causes a shift of the beta-strand D of the encoded transthyretin protein; a vector flanked by a 5' homology arm that targets a 5' target sequence at said endogenous Ttr locus and a 3' homology arm that targets a 3' target sequence at said endogenous Ttr locus; A targeting vector comprising an insert nucleic acid comprising a human TTR sequence.
(Item 34)
A genetically modified non-human animal Ttr gene wherein a region of the endogenous Ttr gene containing both Ttr coding and non-coding sequences has been deleted and a corresponding human TTR sequence containing both TTR coding and non-coding sequences and wherein said genetically modified non-human animal Ttr gene comprises a mutation that causes a shift of the beta chain D of the encoded transthyretin protein.
(Item 35)
A method for evaluating the activity of a human TTR targeting reagent in vivo, comprising:
(a) administering the human TTR targeting reagent to the non-human animal of any one of items 1-30;
(b) evaluating the activity of said human TTR targeting reagent in said non-human animal.
(Item 36)
36. The method of item 35, wherein introducing comprises adeno-associated virus (AAV)-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, or hydrodynamic delivery (HDD).
(Item 37)
37. The method of item 36, wherein said introducing comprises LNP-mediated delivery.
(Item 38)
37. The method of item 36, wherein said introducing comprises AAV8-mediated delivery.
(Item 39)
39. The method of any one of items 35-38, wherein step (b) comprises isolating liver from said non-human animal and assessing activity of said human TTR targeting reagent in said liver. Method.
(Item 40)
40. The method of item 39, wherein step (b) further comprises assessing activity of said human TTR targeting reagent in said non-hepatic organ or tissue.
(Item 41)
41. The method of any one of items 35-40, wherein said assessing comprises assessing alteration of said genetically altered Ttr locus.
(Item 42)
42. The method of any one of items 35-41, wherein said evaluating comprises evaluating expression of Ttr messenger RNA encoded by said genetically modified Ttr locus.
(Item 43)
43. The method of any one of items 35-42, wherein said evaluating comprises evaluating expression of a TTR protein encoded by said genetically modified Ttr locus.
(Item 44)
44. The method of item 43, wherein assessing expression of said TTR protein comprises measuring serum levels of said TTR protein in said non-human animal.
(Item 45)
44. The method of any one of items 41-43, wherein said activity is assessed in said liver of said non-human animal.
(Item 46)
46. The method of any one of items 35-45, wherein said assessing comprises assessing hyperactivity.
(Item 47)
47. The method of any one of items 35-46, wherein said evaluating comprises evaluating hindlimb dystonia.
(Item 48)
48. The method of any one of items 35-47, wherein said assessing comprises assessing amyloid deposition.
(Item 49)
49. The method of item 48, wherein said assessing comprises assessing amyloid deposits in the sciatic nerve.
(Item 50)
50. The method of any one of items 35-49, wherein said evaluation is performed in comparison to an untreated control non-human animal.
(Item 51)
51. The method of any one of items 35-50, wherein said human TTR targeting reagent comprises a nuclease agent designed to target a region of the human TTR gene.
(Item 52)
52. The method of item 51, wherein said nuclease agent comprises a Cas protein and a guide RNA designed to target a guide RNA target sequence of said human TTR gene.
(Item 53)
53. The method of item 52, wherein the Cas protein is a Cas9 protein.
(Item 54)
54. The method of any one of items 35-53, wherein said human TTR targeting reagent comprises an exogenous donor nucleic acid, said exogenous donor nucleic acid being designed to recombine with said human TTR gene.
(Item 55)
55. The method of item 54, wherein said exogenous donor nucleic acid is a single stranded oligodeoxynucleotide (ssODN).
(Item 56)
56. The method of any one of items 35-55, wherein said human TTR targeting reagent comprises an antigen binding protein.
(Item 57)
57. The method of any one of items 35-56, wherein said human TTR targeting reagent comprises an RNAi agent or an antisense oligonucleotide.
(Item 58)
A method of optimizing the activity of a human TTR targeting reagent in vivo, comprising:
(I) performing the method of any one of items 35-57 for a first time in a first non-human animal whose genome contains a genetically modified endogenous Ttr locus;
(II) altering the variable and repeating the method of step (I) in a second non-human animal containing a genetically modified endogenous Ttr locus in its genome using the altered variable; executing a second time, and
(III) comparing said activity of said human TTR targeting reagent of step (I) with said activity of said human TTR targeting reagent of step (II) for greater efficacy, greater precision, greater consistency; , or selecting a method that provides higher specificity.
(Item 59)
59. The method of item 58, wherein said altered variable in step (II) is a delivery method that introduces said human TTR targeting reagent to said non-human animal.
(Item 60)
59. The method of item 58, wherein said altered variable in step (II) is a route of administration for introducing said human TTR targeting reagent to said non-human animal.
(Item 61)
59. The method of item 58, wherein the altered variable in step (II) is the concentration or amount of the human TTR targeting reagent introduced into the non-human animal.
(Item 62)
59. The method of item 58, wherein said altered variable in step (II) is in the form of said human TTR targeting reagent introduced into said non-human animal.
(Item 63)
59. The method of item 58, wherein said altered variable in step (II) is said human TTR targeting reagent introduced into said non-human animal.
(Item 64)
A method for producing a non-human animal according to any one of items 1 to 30,
(I)(a) modifying the genome of a pluripotent non-human animal cell to contain a genetically modified endogenous Ttr locus;
(b) identifying or selecting said genetically modified pluripotent non-human animal cell comprising said genetically modified endogenous Ttr locus;
(c) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo;
(d) gestating said non-human animal host embryo in a surrogate mother, or
(II) (a) modifying the genome of a one-cell stage embryo of a non-human animal to contain a genetically modified endogenous Ttr locus;
(b) selecting a one-cell stage embryo of said genetically modified non-human animal comprising said genetically modified endogenous Ttr locus;
(c) gestating a one-cell stage embryo of said genetically modified non-human animal in a surrogate mother.
Claims (26)
前記遺伝子改変された内因性Ttr遺伝子座が、前記内因性Ttrプロモーターを含み、前記ヒトTTR配列が、前記内因性Ttrプロモーターに作動可能に連結されており、
前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含み、
前記コードされたトランスサイレチンタンパク質が前記ヒトトランスサイレチンタンパク質と最適に整列している場合、前記変異が、前記ヒトトランスサイレチンタンパク質のG53S/E54D/L55Sに対応する三重変異であり、ここで:
(I)前記内因性Ttr遺伝子座の全Ttrコード配列が削除され、前記対応するヒトTTR配列で置き換えられており、Ttr開始コドンからTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、または
(II)前記遺伝子改変された内因性Ttr遺伝子座が、シグナルペプチドを含むトランスサイレチン前駆体タンパク質をコードし、前記シグナルペプチドをコードする前記内因性Ttr遺伝子座の領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、そして第2のTtrエキソンの開始からTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、
げっ歯動物。 A rodent comprising an endogenous Ttr locus genetically modified in its genome, wherein a region of said endogenous Ttr locus comprising both Ttr coding and non-coding sequences is deleted and TTR coding and non-coding sequences are deleted. has been replaced with the corresponding human TTR sequence containing both
wherein said genetically modified endogenous Ttr locus comprises said endogenous Ttr promoter, said human TTR sequence is operably linked to said endogenous Ttr promoter;
said genetically modified endogenous Ttr locus comprises a mutation that causes a shift of the beta chain D of the encoded transthyretin protein;
when said encoded transthyretin protein is optimally aligned with said human transthyretin protein, said mutation is a triple mutation corresponding to G53S/E54D/L55S of said human transthyretin protein, wherein :
(I) the entire Ttr coding sequence of said endogenous Ttr locus has been deleted and replaced with said corresponding human TTR sequence, and said region of said endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted; and replaced with said corresponding human TTR sequence, or
(II) the genetically modified endogenous Ttr locus encodes a transthyretin precursor protein containing a signal peptide, and the region of the endogenous Ttr locus encoding the signal peptide has not been deleted; not replaced with the corresponding human TTR sequence, and wherein the region of the endogenous Ttr locus from the start of the second Ttr exon to the Ttr stop codon is deleted and replaced with the corresponding human TTR sequence there is
rodent .
内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられていない、請求項2に記載のげっ歯動物。 said genetically modified endogenous Ttr locus comprises a human TTR 3' untranslated region, and/or
3. The rodent of claim 2, wherein the endogenous Ttr 5' untranslated region has not been deleted and replaced with the corresponding human TTR sequence.
前記内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、かつ
前記内因性Ttrプロモーターが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、先行請求項のいずれか一項に記載のげっ歯動物。 the region of the endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted and replaced with a human TTR sequence comprising the corresponding human TTR sequence and a human TTR 3' untranslated region; and the endogenous Ttr 5' untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and the endogenous Ttr promoter has not been deleted and replaced with the corresponding human TTR sequence , a rodent according to any one of the preceding claims.
(ii)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号9に記載の配列と、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含むタンパク質をコードし、
(iii)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号10に記載の配列と少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含むコード配列を含むか、または
(iv)前記遺伝子改変された内因性Ttr遺伝子座が、配列番号12もしくは13に記載の配列と少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、少なくとも99%、もしくは100%同一の配列を含む、請求項4に記載のげっ歯動物。 (i) said human TTR sequence of said genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least comprises a sequence that is 99% or 100% identical, or (ii) said genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96% with the sequence set forth in SEQ ID NO:9, encoding a protein comprising a sequence that is at least 97%, at least 98%, at least 99%, or 100% identical;
(iii) the genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% the sequence set forth in SEQ ID NO: 10 or (iv) said genetically modified endogenous Ttr locus is at least 90%, at least 95%, at least 96%, at least 5. The rodent of claim 4 , comprising 97%, at least 98%, at least 99%, or 100% identical sequences.
前記内因性Ttr5’非翻訳領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、かつ
前記内因性Ttrプロモーターが削除されておらず、前記対応するヒトTTR配列で置き換えられていない、請求項6~8のいずれか一項に記載のげっ歯動物。 said region of said endogenous Ttr locus from said second Ttr exon to said Ttr stop codon has been deleted and replaced with said corresponding human TTR sequence and human TTR 3' untranslated region; and said endogenous Ttr5 'the untranslated region has not been deleted and replaced with the corresponding human TTR sequence, and the endogenous Ttr promoter has not been deleted and replaced with the corresponding human TTR sequence, Item 9. The rodent according to any one of items 6 to 8 .
(II)前記げっ歯動物が、後肢ジストニアを示す;および/または
(III)前記げっ歯動物が、アミロイド沈着を含み、任意選択で、前記げっ歯動物が、坐骨神経にアミロイド沈着を含み、任意選択で、前記げっ歯動物が、約2ヶ月齢までにアミロイドーシスを発症する、先行請求項のいずれか一項に記載のげっ歯動物。 (I) said rodent is hyperactive compared to a control wild-type rodent or a rodent comprising a genetically modified endogenous Ttr locus without mutation ; said hyperactivity is measured by one or more or all of total distance, total activity, or total rearing in an open field test; and/or
(II) the rodent exhibits hindlimb dystonia; and/or
(III) said rodent comprises amyloid deposits, optionally said rodent comprises amyloid deposits in the sciatic nerve, optionally said rodent has amyloidosis by about 2 months of age; A rodent according to any one of the preceding claims , which develops disease .
前記遺伝子改変された内因性Ttr遺伝子座が、前記内因性Ttrプロモーターを含み、前記ヒトTTR配列が、前記内因性Ttrプロモーターに作動可能に連結されており、
前記遺伝子改変された内因性Ttr遺伝子座が、コードされたトランスサイレチンタンパク質のベータ鎖Dのシフトを引き起こす変異を含み、
前記コードされたトランスサイレチンタンパク質が前記ヒトトランスサイレチンタンパク質と最適に整列している場合、前記変異が、前記ヒトトランスサイレチンタンパク質のG53S/E54D/L55Sに対応する三重変異であり、ここで:
(I)前記内因性Ttr遺伝子座の全Ttrコード配列が削除され、前記対応するヒトTTR配列で置き換えられており、Ttr開始コドンからTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、または
(II)前記遺伝子改変された内因性Ttr遺伝子座が、シグナルペプチドを含むトランスサイレチン前駆体タンパク質をコードし、前記シグナルペプチドをコードする前記内因性Ttr遺伝子座の領域が削除されておらず、前記対応するヒトTTR配列で置き換えられておらず、そして第2のTtrエキソンの開始からTtr終止コドンまでの前記内因性Ttr遺伝子座の前記領域が削除され、前記対応するヒトTTR配列で置き換えられている、げっ歯動物細胞。 1. A rodent cell comprising an endogenous Ttr locus genetically modified in its genome, wherein a region of said endogenous Ttr locus comprising both Ttr coding and non-coding sequences has been deleted, resulting in TTR coding and non-coding sequences. has been replaced with the corresponding human TTR sequence containing both
wherein said genetically modified endogenous Ttr locus comprises said endogenous Ttr promoter, said human TTR sequence is operably linked to said endogenous Ttr promoter;
said genetically modified endogenous Ttr locus comprises a mutation that causes a shift of the beta chain D of the encoded transthyretin protein;
when said encoded transthyretin protein is optimally aligned with said human transthyretin protein, said mutation is a triple mutation corresponding to G53S/E54D/L55S of said human transthyretin protein, wherein :
(I) the entire Ttr coding sequence of said endogenous Ttr locus has been deleted and replaced with said corresponding human TTR sequence, and said region of said endogenous Ttr locus from the Ttr start codon to the Ttr stop codon has been deleted; and replaced with said corresponding human TTR sequence, or
(II) the genetically modified endogenous Ttr locus encodes a transthyretin precursor protein containing a signal peptide, and the region of the endogenous Ttr locus encoding the signal peptide has not been deleted; not replaced with the corresponding human TTR sequence, and wherein the region of the endogenous Ttr locus from the start of the second Ttr exon to the Ttr stop codon is deleted and replaced with the corresponding human TTR sequence There are rodent cells.
(a)請求項1~15のいずれか一項に記載のげっ歯動物に前記ヒトTTR標的化試薬を投与することと、
(b)前記げっ歯動物における前記ヒトTTR標的化試薬の活性を評価することと、を含む、方法。 A method for evaluating the activity of a human TTR targeting reagent in vivo, comprising:
(a) administering the human TTR targeting reagent to the rodent of any one of claims 1-15 ;
(b) evaluating the activity of said human TTR targeting reagent in said rodent .
任意選択で、前記投与が、AAV8媒介送達を含む、請求項17に記載の方法。 said administration comprises adeno-associated virus (AAV)-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, or hydrodynamic delivery (HDD);
18. The method of claim 17 , optionally wherein said administering comprises AAV8-mediated delivery .
(I)前記遺伝子改変されたTtr遺伝子座の改変を評価すること;ならびに/あるいは
(II)前記遺伝子改変されたTtr遺伝子座によってコードされるTtrメッセンジャーRNAの発現を評価すること、または前記遺伝子改変されたTtr遺伝子座によってコードされるTTRタンパク質の発現を評価すること
を含み、
任意選択で、前記TTRタンパク質の発現を評価することが、前記げっ歯動物における前記TTRタンパク質の血清レベルを測定することを含む、請求項17~19のいずれか一項に記載の方法。 the evaluation is
(I) assessing alteration of said genetically altered Ttr locus ; and/or
(II) assessing expression of Ttr messenger RNA encoded by said genetically modified Ttr locus or assessing expression of TTR protein encoded by said genetically modified Ttr locus;
including
Optionally, the method of any one of claims 17-19 , wherein assessing expression of said TTR protein comprises measuring serum levels of said TTR protein in said rodent.
任意選択で、前記評価が、坐骨神経におけるアミロイド沈着を評価することを含む、請求項17~20のいずれか一項に記載の方法。 said evaluating comprises evaluating hyperactivity , hindlimb dystonia or amyloid deposition ;
Optionally, the method of any one of claims 17-20 , wherein said evaluating comprises evaluating amyloid deposits in the sciatic nerve .
(I)ヒトTTR遺伝子の領域を標的化するように設計されたヌクレアーゼ剤であって、任意選択で、前記ヌクレアーゼ剤が、Casタンパク質および前記ヒトTTR遺伝子のガイドRNA標的配列を標的化するように設計されたガイドRNAを含み、任意選択で、前記Casタンパク質が、Cas9タンパク質である、ヌクレアーゼ剤;
(II)外因性ドナー核酸であって、前記外因性ドナー核酸が、前記ヒトTTR遺伝子と組換えるように設計されており、任意選択で、前記外因性ドナー核酸が、一本鎖オリゴデオキシヌクレオチド(ssODN)である、外因性ドナー核酸;
(III)抗原結合タンパク質;あるいは
(IV)RNAi剤またはアンチセンスオリゴヌクレオチド
を含む、請求項17~22のいずれか一項に記載の方法。 said human TTR targeting reagent comprising:
(I) a nuclease agent designed to target a region of the human TTR gene, optionally such that said nuclease agent targets a Cas protein and a guide RNA target sequence of said human TTR gene; a nuclease agent comprising a designed guide RNA, optionally wherein said Cas protein is a Cas9 protein;
(II) an exogenous donor nucleic acid, wherein said exogenous donor nucleic acid is designed to recombine with said human TTR gene; optionally said exogenous donor nucleic acid comprises a single-stranded oligodeoxynucleotide ( ssODN), an exogenous donor nucleic acid;
(III) an antigen binding protein; or
(IV) RNAi agents or antisense oligonucleotides
A method according to any one of claims 17 to 22 , comprising
(I)請求項17~23のいずれか一項に記載の方法を、ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む第1のげっ歯動物において、1回目に実行することと、
(II)可変要素(variable)を変更し、ステップ(I)の前記方法を、ゲノムに遺伝子改変された内因性Ttr遺伝子座を含む第2のげっ歯動物において、変更された可変要素を用いて2回目に実行することと、
(III)ステップ(I)の前記ヒトTTR標的化試薬の前記活性を、ステップ(II)の前記ヒトTTR標的化試薬の前記活性と比較し、より高い有効性、より高い精度、より高い一貫性、またはより高い特異性をもたらす方法を選択することと、を含む、方法。 A method of optimizing the activity of a human TTR targeting reagent in vivo, comprising:
(I) performing the method of any one of claims 17-23 for a first time in a first rodent whose genome contains a genetically modified endogenous Ttr locus;
(II) altering the variable and repeating the method of step (I) in a second rodent containing a genetically modified endogenous Ttr locus in its genome with the altered variable; executing a second time, and
(III) comparing said activity of said human TTR targeting reagent of step (I) with said activity of said human TTR targeting reagent of step (II) for greater efficacy, greater precision, greater consistency; , or selecting a method that provides higher specificity.
(I)(a)遺伝子改変された内因性Ttr遺伝子座を含むように、げっ歯動物胚性幹(ES)細胞のゲノムを改変することと、
(b)前記遺伝子改変された内因性Ttr遺伝子座を含む前記遺伝子改変されたげっ歯動物ES細胞を同定または選択することと、
(c)前記遺伝子改変されたげっ歯動物ES細胞をげっ歯動物宿主胚に導入することと、
(d)代理げっ歯動物母に前記げっ歯動物宿主胚を懐胎させることと、を含むか、または
(II)(a)遺伝子改変された内因性Ttr遺伝子座を含むように、げっ歯動物の1細胞期胚のゲノムを改変することと、
(b)前記遺伝子改変された内因性Ttr遺伝子座を含む前記遺伝子改変されたげっ歯動物の1細胞期胚を選択することと、
(c)代理げっ歯動物母に前記遺伝子改変されたげっ歯動物の1細胞期胚を懐胎させることと、を含む、方法。 A method of producing a rodent according to any one of claims 1 to 15 , comprising
(I)(a) modifying the genome of rodent embryonic stem (ES) cells to contain a genetically modified endogenous Ttr locus;
(b) identifying or selecting said genetically modified rodent ES cells comprising said genetically modified endogenous Ttr locus;
(c) introducing the genetically modified rodent ES cells into a rodent host embryo;
(d) gestating said rodent host embryo in a surrogate rodent mother; or modifying the genome of a one-cell stage embryo;
(b) selecting said genetically modified rodent one-cell stage embryos containing said genetically modified endogenous Ttr locus;
(c) gestating the genetically modified rodent one-cell stage embryo in a surrogate rodent mother.
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BR112021022722A2 (en) | 2019-06-07 | 2022-01-04 | Regeneron Pharma | Non-human animal, non-human animal cell, non-human animal genome, humanized non-human animal albumin gene, targeting vector, method of evaluating the activity of a reagent, and, method of optimizing the activity of a reagent |
WO2021108363A1 (en) | 2019-11-25 | 2021-06-03 | Regeneron Pharmaceuticals, Inc. | Crispr/cas-mediated upregulation of humanized ttr allele |
EP4125348A1 (en) | 2020-03-23 | 2023-02-08 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use |
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2020
- 2020-06-03 JP JP2021568280A patent/JP2022534867A/en active Pending
- 2020-06-03 US US16/891,571 patent/US11891618B2/en active Active
- 2020-06-03 CN CN202080038549.3A patent/CN113874510A/en active Pending
- 2020-06-03 KR KR1020217040893A patent/KR20220016869A/en unknown
- 2020-06-03 EP EP20747218.4A patent/EP3801011A1/en active Pending
- 2020-06-03 AU AU2020286382A patent/AU2020286382A1/en active Pending
- 2020-06-03 CA CA3137761A patent/CA3137761A1/en active Pending
- 2020-06-03 WO PCT/US2020/035859 patent/WO2020247452A1/en unknown
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