JP2023528139A - ヒト核因子e2関連因子2の発現ベクター及び発現ベクターの適用 - Google Patents
ヒト核因子e2関連因子2の発現ベクター及び発現ベクターの適用 Download PDFInfo
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Abstract
Description
(a)配列番号1に記載のヌクレオチド配列、及び
(b)配列番号1に記載のヌクレオチド配列と95%以上、好ましくは98%以上、より好ましくは99%以上の同一性を有するヌクレオチド配列、からなる群から選択される、ヌクレオチド配列を提供する。
(a)細胞の抗炎症反応または抗酸化反応を活性化すること、
(b)神経節細胞の生存率を高めること、
(c)神経節細胞のアポトーシスを防止するまたは遅延させること、
(d)網膜神経節細胞におけるNrf2遺伝子の安定した高い発現を引き起こすこと、及び
(e)疾患細胞からフリーラジカルを除去すること
からなる群から選択される1つ以上の項目のためにも使用される。
本開示をより容易に理解することができるように、ある特定の用語が最初に定義される。本出願で使用される場合、本明細書で別途明確に記載されない限り、以下の用語は各々、以下に付与される意味を有する。他の定義は、本出願を通じて記載される。
アデノ随伴ウイルス(adeno-associated virus、AAV)、別名、アデノ随伴ウイルス(adeno associated virus)は、Parvoviridae科のディペンドウイルス属に属し、これまでに発見された最も単純な構造を有する一本鎖DNA欠損ウイルスのクラスであり、その複製にはヘルパーウイルス(通常、アデノウイルス)の関与が必要である。これは、2つの逆方向末端反復(ITR)におけるcap遺伝子及びrep遺伝子をコードする。ITRは、ウイルス複製及びパッケージングにおいて決定的な役割を果たす。cap遺伝子はウイルスカプシドタンパク質をコードし、rep遺伝子はウイルスの複製及び組み込みに関与する。AAVは、様々な細胞に感染することができる。
本発明によって解決されるべき技術的課題は、先行技術における核因子E2関連因子2(Nrf2)タンパク質のトランスフェクション効率の低さ及び治療効果の低さという技術的欠陥を克服することである。本発明は、核因子E2関連因子2(Nrf2)タンパク質ならびにその調製方法及び適用を提供する。本発明の最適化されたNrf2遺伝子配列(配列番号1)がNrf2タンパク質の発現をより効率的にし、それにより、より多くのNrf2タンパク質が患者の視神経節細胞において生理的役割を果たすことが研究により見出される。
本発明は、Nrf2ヌクレオチド配列の最適化(配列番号1に記載のもの、本発明では最適化Nrf2遺伝子/核酸と称される)を行い、この配列は特別に最適化され、それ故に、図1aに示されるように、転写効率及び翻訳効率が著しく改善され、ここで、最適化Nrf2配列と非最適化Nrf2配列の相同性は75%である。
本発明は、本発明の最適化Nrf2コード配列を含むNrf2タンパク質の発現ベクターも提供する。
本発明は、(a)請求項2に記載のベクターと、(b)薬学的に許容される担体または賦形剤とを含む調製物または組成物を提供する。
(a)細胞の抗炎症反応または抗酸化反応を活性化すること、
(b)神経節細胞の生存率を高めること、
(c)神経節細胞のアポトーシスを防止するまたは遅延させること、
(d)網膜神経節細胞におけるNrf2遺伝子の安定した高い発現を引き起こすこと、及び
(e)疾患細胞からフリーラジカルを除去すること
からなる群から選択される1つ以上の項目のために使用される。
1.本発明の組換えヒト核因子E2関連因子2(Nrf2)タンパク質のコード遺伝子配列は、特別に最適化されている。Nrf2の非最適化DNAコード配列と比較して、転写効率及び翻訳効率が有意に改善され、コドン使用がより効率的になり、最適化配列のNrf2タンパク質発現レベルが有意に改善され、生物学的活性が高い。
1.1 ベクターの構築
ヒトNrf2ヌクレオチド配列(配列番号2に記載のもの)を得た後、Nrf2ヌクレオチド配列最適化(配列番号1に記載のもの)を行った。核酸配列最適化には、コドン使用バイアス、発現を助長しない二次構造(ヘアピン構造など)の排除、GC含有量、CpGジヌクレオチド含有量、mRNA二次構造、潜在スプライス部位、初期ポリアデニル化部位、内部リボソーム進入及び結合部位、ネガティブCpGアイランド、RNA不安定性領域、反復(直接反復、逆方向反復など)、ならびにクローニングに影響を及ぼし得る制限部位の変化が含まれる。最適化配列(配列番号1)にCla I及びXba Iの2つの制限部位を付加するか、または新規遺伝子及びpAAVプラスミドベクターに設計したプライマーでのPCR増幅によって生成された生成物を、それぞれ、Cla I及びXba I二重酵素消化に供し、その後、消化産物を回収し、T4 DNAリガーゼで一晩ライゲーションし、その後、ライゲーション産物をコンピテント細胞に形質転換して、組換えpAAV-Nrf2を得た。細菌液体シーケンシングのために単一のクローンを選択し、シーケンシングによって得た配列を配列アラインメント分析に供して、エンハンサー/プロモーター、遺伝子配列、ポリA、及びウイルスパッケージングITR配列を含む、図7に示される、正しい配列を有する組換えアデノ随伴ウイルスプラスミドpAAV-Nrf2の取得に成功した。図7a、図7b、図7c、及び図7dは、それぞれ、pAAV-CMV-Nrf2_天然プラスミド、pAAV-CMV-Nrf2_最適化プラスミド、pAAV-CAG-Nrf2_最適化プラスミド、及びpAAV-SYN-Nrf2_最適化プラスミドのプロファイルである。
pAdHelperプラスミド、pAAV-r2c5プラスミド、pAAV-Nrf2プラスミドを293T細胞にトランスフェクトし(225cm2の細胞培養フラスコに播種)、細胞を48時間後に採取した。細胞をPBS中に再懸濁し、3回凍結融解した。
クロロホルム処理-PEG/NaCl析出-クロロホルム抽出の3つのステップを使用してrAAV2/2-Nrf2ウイルスを単離し、濃縮し、精製した。全回収率=最終生成物中のウイルス粒子の数/出発材料中のウイルス粒子の数。
実験材料:SYBR II(takara)、標的断片プライマー(20uM)、ウイルスをパッケージングするための標的プラスミド(既知の濃度)、試験するウイルス、8チューブPCRストリップ(Bio-red)。
2.1 マウスの眼の硝子体への組換えアデノ随伴ウイルスの注射
マウスを5%抱水クロラールの腹腔内注射により麻酔し、マウスを麻酔した後、マウスの眼の外側及び眼球を洗浄し、消毒した。インスリン針を使用してマウスの強角膜縁の下に穴を開け、組換えアデノ随伴ウイルスベクター及び薬学的に許容される担体または賦形剤からなる1~2μlの組換えアデノ随伴ウイルス調製物をマイクロシリンジで硝子体内注射した。マウスを、自由に食餌させた標準環境で維持し、マウスの眼の状態を毎日観察した。
投薬の3週間後、眼球を取り出した状態で脊椎を折ることによってマウスを屠殺し、網膜を氷上で速やかに剥離し、液体窒素下で保存した。RIPA緩衝液で網膜組織を溶解した後、Nrf2タンパク質の発現レベルをWB実験によって検出した。図1aは、rAAV2/2-Nrf2_天然ウイルス及びrAAV2/2-Nrf2_最適化ウイルスがマウスの網膜に感染した後のタンパク質レベルの比較であり、配列最適化後のベクターのタンパク質発現レベルは、最適化前の5倍以上高い。
3.1 マウス急性視神経損傷モデル
成体BL6/C57マウスを5%抱水クロラールの腹腔内注射によって麻酔した。マウスを動物手術台上に固定し、眼皮膚をヨードフォアで消毒した。眼球の結膜組織を強角膜縁から強膜に沿って切断し、眼球と結膜との間で鈍的後方分離(bluntly backward separation)を行い、脊椎筋肉を露出させた。この分離により視神経が完全に露出し、その後、視神経を、鉗子の終端を幅1mmに保った状態で逆自動ロック式エルボー鉗子を用いて、眼球の2mm後方で10秒間保持した。術後観察を行い、眼軟膏を使用して、眼底出血、水晶体損傷、及び眼内炎症を有するマウスを除外した。
5匹の非モデル化マウスを正常群として使用した。モデル化に成功したマウスを、硝子体内注射のために3つの群に分けた。対照群にrAAV2/2-mCherryウイルスを注射し(n=7)、実験群AにrAAV2/2-Nrf2_天然ウイルスを注射し(n=8)、実験群BにrAAV2/2-Nrf2_最適化ウイルスを注射した(n=8)。
脊椎を折ることによってマウスを屠殺し、眼球を取り出し、固定剤で20分間固定し、その後、網膜を剥離し、4枚の花弁状の花びらに切断し、4%パラホルムアルデヒドに4℃で一晩固定した。得られた材料を1%TritonX-100で2時間穿孔し、ブロッキング溶液で1時間ブロックした。その後、得られた材料を、一次抗体としてTUJ1抗体(神経節細胞特異的抗体)と4℃で一晩インキュベートし、二次抗体と室温で2時間インキュベートした。核をDAPIで15分間染色した後、載置した。
4.1 DBA/2Jマウスの硝子体内注射
7ヶ月齢のDBA/2Jマウス(眼内圧>18mmHg)を3つの群に分け、6匹のマウスにモデル群としてPBSを注射し、他の2つの群に、それぞれ、rAAV2/2-CAG-Nrf2_最適化ウイルス(n=7)及びrAAV2/2-SYN-Nrf2_最適化ウイルス(n=7)を単眼硝子体内注射した。同じ月齢の5匹のC57/BL6マウスを正常群として使用した。
眼底検査及び眼内圧検査を術後3~5日毎に行った。すべてのマウスの眼に明らかな異常は認められず、結膜充血、分泌、眼内炎、または眼内圧の明らかな変化は認められなかった。
投薬の5ヶ月後、脊椎を折ることによってマウスを屠殺し、網膜を神経節細胞の免疫蛍光染色及び計数のために採取した。図3に示される結果は、モデル群のマウスにおける生存神経節細胞の密度(971±146 RGC/mm2、図3b)が正常群(1506±152 RGC/mm2、図3a)よりも有意に低かった(P<0.05)ことを示す。rAAV2/2-CAG-Nrf2_最適化群(1426±179 RGC/mm2、図3c)及びrAAV2/2-SYN-Nrf2_最適化群(1384±88 RGC/mm2、図3d)における神経節細胞の密度は、モデル群(1506±152 RGC/mm2、図3b)と比較して有意に増加し(P<0.05)、薬物群のモデル化マウスにおける網膜神経節細胞に対する保護効果を示した。しかしながら、rAAV2/2-CAG-Nrf2_最適化群とrAAV2/2-SYN-Nrf2_最適化群との間に有意差はなかった。図3eは、各群におけるマウスの単位網膜面積当たりのRGC細胞数の統計的結果を示す。
眼球を、凍結切片及びNrf2抗体での免疫蛍光染色を行うために採取した。網膜におけるrAAV2/2-CAG-Nrf2_最適化及びrAAV2/2-SYN-Nrf2_最適化の発現レベルを検出した。図4に示される結果は、マウスの網膜をrAAV2/2-CAG-Nrf2_最適化に感染させた後、Nrf2タンパク質が網膜神経節細胞のみならず、双極細胞及びアマクリン細胞でも強く発現したことを示す(図4a)。しかしながら、Nrf2タンパク質は、rAAV2/2-SYN-Nrf2_最適化による網膜感染後では、網膜神経節細胞で主に発現し(図4b)、より高い特異性を有した。
5.1 ウサギ眼への硝子体内注射
27匹のニュージーランド白ウサギを、PBS群、実験群A、及び実験群Bを含む3つの群に分け、その後、50uLの1×1012vg/mlのrAAV2/2-CAG-Nrf2_最適化及びrAAV2/2-SYN-Nrf2_最適化を、それぞれ、角膜縁から3mmに位置する毛様体扁平部を穿刺した後に硝子体内注射して、硝子体腔に入れた。
3つの群のウサギを、術後1、3、7、及び30日目に細隙灯及び眼内圧に関して検査した。すべてのウサギに明らかな異常は認められず、結膜充血、分泌、眼内炎、または眼内圧の増加は認められなかった。術後1ヶ月のウサギの眼底写真の結果を図5に示す。対照群におけるウサギの眼底(図5a)と比較して、実験群A(図5b)及び実験群B(図5c)におけるウサギの網膜血管及び視神経に明らかな合併症または損傷はなく、通常の標準硝子体内注射が明らかな炎症反応または他の合併症を引き起こさないことを示した。
ウサギ眼球切片のH&E染色の結果は、実験群A(図6b)、実験群B(図6c)、及び対照群(図6a)における網膜神経節細胞数が基本的に同じであり、かつ階層構造が完全であることを示し、Nrf2_最適化組換え遺伝子が安全であり、網膜に損傷を与えないことを示した。
pAdHelperプラスミド、pAAV-r2c2プラスミド、pAAV-Nrf2プラスミドを293T細胞にトランスフェクトし(225cm2の細胞培養フラスコに播種)、細胞を48時間後に採取した。細胞をPBS中に再懸濁し、3回凍結融解した。
投薬の5ヶ月後、脊椎を折ることによってマウスを屠殺し、網膜を神経節細胞の免疫蛍光染色及び計数のために採取した。図3に示される結果は、モデル群のマウスにおける生存神経節細胞の密度(971±146 RGC/mm2、図3b)が正常群(1506±152 RGC/mm2、図3a)よりも有意に低かった(P<0.05)ことを示す。rAAV2/2-CAG-Nrf2_最適化群(1426±179 RGC/mm2、図3c)及びrAAV2/2-SYN-Nrf2_最適化群(1384±88 RGC/mm2、図3d)における神経節細胞の密度は、モデル群(971±146 RGC/mm2、図3b)と比較して有意に増加し(P<0.05)、薬物群のモデル化マウスにおける網膜神経節細胞に対する保護効果を示した。しかしながら、rAAV2/2-CAG-Nrf2_最適化群とrAAV2/2-SYN-Nrf2_最適化群との間に有意差はなかった。図3eは、各群におけるマウスの単位網膜面積当たりのRGC細胞数の統計的結果を示す。
Claims (10)
- ヒト核因子E2関連因子2(Nrf2)タンパク質をコードするヌクレオチド配列であって、
(a)配列番号1に記載のヌクレオチド配列、及び
(b)配列番号1に記載のヌクレオチド配列と95%以上、好ましくは98%以上、より好ましくは99%以上の同一性を有するヌクレオチド配列
からなる群から選択される、前記ヌクレオチド配列。 - 請求項1に記載のヌクレオチド配列を含む、ベクター。
- 宿主細胞であって、請求項2に記載のベクターを含むか、またはその染色体に組み込まれた請求項1に記載の外因性ヌクレオチド配列を有する、前記宿主細胞。
- 視神経関連疾患の治療のための調製物または組成物の調製における、請求項2に記載のベクターの使用。
- 前記調製物または組成物が、
(a)細胞の抗炎症反応または抗酸化反応を活性化すること、
(b)神経節細胞の生存率を高めること、
(c)神経節細胞のアポトーシスを防止するまたは遅延させること、
(d)網膜神経節細胞におけるNrf2遺伝子の安定した高い発現を引き起こすこと、及び
(e)疾患細胞からフリーラジカルを除去すること
からなる群から選択される1つ以上の項目のためにも使用される、請求項4に記載の使用。 - 前記視神経関連疾患が視神経損傷を含む、請求項4に記載の使用。
- 前記視神経関連疾患が、緑内障、常染色体優性視神経萎縮症(DOA)、レーバー遺伝性視神経症(LHON)、虚血性視神経症、またはそれらの組み合わせからなる群から選択される、請求項4に記載の使用。
- (a)請求項2に記載のベクターと、(b)薬学的に許容される担体または賦形剤と、を含む、医薬調製物。
- 前記医薬調製物中の前記ベクターの含有量が1×109~1×1016ウイルス/ml、好ましくは1×1010~1×1012ウイルス/mlである、請求項8に記載の医薬調製物。
- 組換えヒト核因子E2関連因子2(Nrf2)タンパク質を調製する方法であって、請求項3に記載の宿主細胞を培養して、前記組換えヒト核因子E2関連因子2(Nrf2)タンパク質を得るステップを含む、前記方法。
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