JP2024001178A - 血流の途絶に起因する神経損傷の治療のための機能性ニューロンの再生 - Google Patents
血流の途絶に起因する神経損傷の治療のための機能性ニューロンの再生 Download PDFInfo
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Abstract
Description
本出願は、2017年2月28日に出願した米国仮特許出願第62/464,469号;及び2017年6月13日に出願した米国仮特許出願第62/518,914号に基づく優先権を主張する。各出願の全内容は、参照により本明細書に組み込まれる。
本発明は、国立衛生研究所により交付された助成金番号AG045656の下で政府支援を得てなされた。政府は、本発明に特定の権利を有する。
本発明は、個々の対象のCNSにおける正常な血流の途絶の影響を治療するための組成物及び方法に関する。
脳機能は、ニューロンとそれらの周囲のグリア細胞との間の微妙なバランスに依存している。重度の虚血性損傷の後、ニューロンは、直ちに又は二次的な損傷により徐々に死滅するが、それらの隣接するグリア細胞は活性化され、増殖が開始され得るため、神経再生を最終的に阻害するグリア性瘢痕組織が生じる。本明細書に記載のNeuroD1介在性in vivo細胞変換技術は、反応性グリア細胞を機能性ニューロンに直接的に変換することによって、CNSにおける正常な血流の途絶から生じた損傷領域における神経機能を回復させる。本明細書に記載された本発明のグリアからニューロンへのin vivo変換技術は、脳卒中を治療するための生着及び試行に関する「古典的な」外因性幹細胞の投与と比較して、多くの利点を有する。
材料及び方法
脳卒中のマウスモデル及びウイルス注射
野生型(WT)FVB/NJ及びGFAP-GFPトランスジェニックマウスを、本研究で記載する実験の大部分に対して用いた。GFAP-GFPトランスジェニックマウスは、Zhuo, L.ら、1997、Dev Biol 187、36~42ページに記載されているJackson Laboratory[FVB/N-Tg(GFAPGFP)14Mes/J]から購入し、FVB/NJマウス(Jackson Laboratory)と交配した。エンドセリン-1(1-31)を、Horie, N.ら、2008、J. Neurosci. Methods 173、286~290ページ;及びRoome, R.B.ら、2014、J. Neurosci. Methods、233、34~44ページに記載されているように、成体WT FVB/NJ又はGFAPA-GFPトランスジェニックマウス(28~40g、5~10ヶ月齢)の運動皮質に注射し、局所的な虚血性損傷を生じさせた。
hGFAPプロモーターはpDRIVE-hGFAPプラスミド(InvivoGen, Inc.)から入手し、pAAV-MCS(Cell Biolab)のMluIとSacIIとの間に挿入し、CMVプロモーターを置き換えた。Cre遺伝子は、PCRによってhGFAP-Cre(Addgene plasmid #40591)から取得し、pAAV MCSのEcoRI部位とSal1部位との間に挿入し、pAAV-hGFAP::Creベクターを作製した。
組換えAAV9は、293AAV細胞(Cell Biolabs)で産生させた。簡単に説明すると、ポリエチレンイミン(PEI、直鎖、分子量25,000)をトリプルプラスミド:pAAV発現ベクター、pAAV9-RC(Cell Biolab)、及びpHelper(Cell Biolab)のトランスフェクションに使用した。
pCAG-NeuroD1-IRES-GFP及びpCAG-GFPは、Guo, Z.ら、2014、Cell Stem Cell 14、188~202ページに記載されている。レトロウイルス粒子をパッケージングするため、gpgヘルパー不含ヒト胎児腎臓(HEK)細胞を、水疱性口内炎ウイルス糖タンパク質(VSV-G)ベクターと一緒に標的プラスミドでトランスフェクトし、NeuroD1又はGFPを発現するレトロウイルスを産生した。レトロウイルス粒子の力価は、HEK細胞の形質導入後に決定して、約107粒子/mlであった。
マウス脳の浮遊凍結切片の免疫組織化学は、Guo, Z.ら、2014、Cell Stem Cell 14、188~202ページに記載されているように実施した。2.5%アベルチンを用いて動物を麻酔し、人工脳脊髄液(ACSF)を経心的に灌流して脳組織中の血液を洗い流した。次いで、脳を解剖し、トリミングし、後固定のために、4℃で一晩、4%パラホルムアルデヒド(PFA)に入れた。固定後、脳組織をビブラトーム(Leica)により40μmの切片に切断した。
深く麻酔したマウスに、上記のように人工脳脊髄液を用いて灌流し、続いてPBS中の0.5mg/mlのスルホ-NHS-LC-ビオチン15mlを灌流した。免疫組織化学検査のために、脳切片を、PBS中1:800に希釈したテキサスレッドストレプトアビジン(Vector SA-5006)+0.3%トリトン+2.5%正常ヤギ又はロバ血清と共に室温で1時間インキュベートし、続いて通常のマウント手順を実施した。
脳切片の記録は、Guo, Z.ら、2014、Cell Stem Cell 14、188~202ページ;及びWu, Z.ら、2014、Nat. Commun. 5, 4159ページに記載されている方法と同様に実施した。AAV注射の2~3か月後、マウスを2.5%アベルチンで麻酔し、次いで、NMDG系の切断溶液(mMで):93 NMDG、93 HC1、2.5 KC1、1.25 NaH2PO4、30 NaHCO3、20 HEPES、15 グルコース、12 N-アセチル-L-システイン、5 アスコルビン酸ナトリウム、2 チオ尿素、3 ピルビン酸ナトリウム、7 MgSO4、0.5 CaCl2、pH7.3~7.4、300 mOsmo、95%O2/5%CO2で通気、灌流した。
餌ペレット回収試験
マウスにおけるすべての行動実験では、重度の運動障害を誘発するために、ET-1(1-31)を前肢運動皮質の2点に注射した。2点の座標は次のとおりである:(i)+0.2mm AP、+/-1.35mm ML、(ii)+0.38mm AP、+/-2.45mm ML(+ML又は-MLは、脳卒中前のペレット回収トレーニングにおいて優勢な前肢の対側に基づいた)。
グリッド歩行試験は、Baskin, Y.K.ら、2003、J. Neurosci. Methods 129、87~93ページ;及びClarkson, A.N.ら、2010、Nature 468、305~309ページに記載されているのと同様であった。
シリンダー試験は、Roome, R.B.ら、2014、J. Neurosci. Methods、233、34~44ページに記載されている方法に基づいた。この試験には、マウスの立ち上がり、前肢による透明シリンダー(直径10cm、高さ15cm)の側壁へのタッチのビデオ録画を含めた。各動物を透明な板上のシリンダーに入れ、カメラを用いて下からビデオ記録した。マウスが立ち上がり、側壁に30回よりも多くタッチすることを試みた後に実験は停止したが、これは通常約3~4分間続いた。
損傷コア(約2mm×2mmの正方形)の周囲の皮質組織を灌流後に取得し、液体窒素中で瞬間凍結した。RNA抽出は、Macherey-Nagel NucleoSpin RNAキットを使用して実行し、RNA濃度はNanoDropにより測定した。cDNAは、Quanta Biosciences qScript cDNA supermixを使用して合成した。
切片ゴルジキット(Bieoenno Tech、LLC)をゴルジ染色に使用した。簡単に説明すると、マウスをaCSFで灌流し、続いてキットの固定液を灌流した。脳を取り出し、5日間含浸させ、200μmのビブラトームで切片化し、染色し、キットのプロトコールに基づいてマウントし、DPXマウント剤で覆った。4×及び100×の明視野画像を、Keyence BZ-9000螢光顕微鏡を使用して得た。
皮質領域サイズ分析
皮質領域は、Keyence BZ-9000蛍光顕微鏡において4×レンズで得られた画像を使用して定量化した。最も明白な脳卒中損傷を有する注射点(+0.2 AP)の周囲の3つの切片を、画像取得のために選択した。DAPI及びNeuNシグナルを使用して、皮質上方境界と下方界面を帯状皮質と共に特定した。正中線から3mm外側までの皮質領域をImageJで測定した。
GFAP又はNeuNと同時染色したmCherry/GFPの40×共焦点画像を、4、7、及び17dpiでの定量化に使用した。各動物について、4つの画像を脳梗塞付近のウイルス感染領域で動物ごとに得た。GFAP比については、mCherry/GFP及びGFAPに二重陽性であった細胞を、Zeiss共焦点ソフトウェアZenを使用してカウントした。すべてのmCherry又はGFP陽性細胞(すべてのウイルス感染細胞)のうち二重陽性細胞のパーセンテージを計算した。同様の方法をNeuN比に適用した。
皮質ニューロンマーカー(Tbr1、Emx1、Satb2、PV、GABA)と同時染色したGFP(NeuroD1-GFP)の40×共焦点画像を、60dpiでの定量化に使用した。各動物について、3つの画像を、GFPシグナルを示すウイルス感染領域で得た。すべてのGFP陽性細胞(NeuroD1-GFP感染細胞)のうちのGFP、及び個々のマーカーについての二重陽性細胞のパーセンテージをそれぞれ計算した。
グリアマーカー(LCN2、CSPG、AQP4及びIba1)及びニューロン突起マーカー(Map2、SMI312、VGluT1及びMBP)の単層共焦点画像を、17dpiでの定量化に使用した。63×レンズを使用したSMI312を除き、40×レンズをほとんどのマーカーに使用した。3つの画像を、脳卒中梗塞に近いウイルス感染領域(脳卒中コアボーダーから500μm以内)及び皮質厚全体の中間で得た。1つの画像は、正規化のために比較的健全な領域で得た。健全な領域のバックグラウンドに基づいて閾値を設定した後、ImageJを使用して、画像領域全体の強度とカバー領域を定量化した。3つの画像の結果を使用して、各動物についての平均データを取得した。
NeuN及びPVの細胞数は、正中線の外側500μm~2500μmの皮質領域内で、Zeiss LSM800共焦点顕微鏡のタイル機能によって得られたNeuN又はPV免疫染色の画像でカウントした。共焦点ソフトウェアZenを細胞カウントに使用した。最も明白な脳卒中損傷を有する注射点の周囲の切片(+0.2 AP)を得られた画像に使用した。
NeuN免疫染色の40×共焦点画像を17dpiでの定量化に使用した。図4Aに示したように、3つの画像を、脳卒中梗塞に近いウイルス感染領域(脳卒中コアボーダーから500μm以内)で得た。1μm間隔で10個の切片でZスタック機能を使用した。共焦点ソフトウェアZenを細胞カウントに使用した。NeuroD1群においては、GFP(NeuroD1-GFP)及びNeuN(変換されたニューロン)の両方についての陽性細胞又はNeuN陽性GFP陰性(既存のニューロン)をカウントした。GFP対照群については、NeuN陽性細胞の総数をカウントした。
sEPSC及びsIPSCは、Mini Analysis Program(Synaptosoft、New Jersey、USA)を使用して分析した。大きな事象の複数の検出を回避するため、分析結果を自動検出後、更に視覚的にチェックした。異なる群の間のsEPSCとsIPSCの周波数を比較するため、平均値を取得する前に、各細胞について200を超える事象又は少なくとも3分の記録期間をサンプリングした。
Ly6C数の定量化については、単層40×画像をZeiss共焦点により得た。Ly6Cの強度をImageJで測定し、バックグラウンドよりも3倍高い強度を有する血管を陽性シグナルとして同定した。梗塞周辺領域の3つの画像におけるLy6C+血管の数を定量化し、各マウスについて平均化した。
統計分析は、Prism 6(GraphPad)ソフトウェアを使用して実施した。示したすべての実験は、少なくとも3匹の動物において繰り返し、代表的なデータを示している。群間の有意性を決定するため、比較は、示したように、対応のある両側スチューデントt検定、又は反復測定ANOVA試験を使用して行った。ウイルス注射前に重度の脳卒中について第一次スクリーニングをした後、動物を、欠損レベルが一致する群に無作為に割り当てた。定量化用のすべての画像は、1人の研究者によって取得し、動物の状態及びアイデンティティーを知らない別の研究者によって定量化された。上述のように、マウス行動試験は盲検様式で実施した。
NeuroD1は脳卒中誘発反応性星状細胞をニューロンに変換する
この実施例において、in vivoでの細胞変換が虚血性脳卒中後の機能的な脳修復を達成できるかどうかについて調査した。血管収縮ペプチドのエンドセリン-1(ET-1)によって誘発した局所脳卒中モデルを使用し、げっ歯動物において一貫した局所虚血性損傷を生じさせた(Fuxeら、1997; Hughesら、2003; Roomeら、2014; Windleら、2006)。
細胞変換後の神経炎症の低減
脳卒中領域におけるNeuroD1感染後の星状細胞からニューロンへの変換に伴って、脳卒中領域におけるGFAPシグナルの有意な低減が観察された(図5Aを参照)。NeuroD1感染反応性星状細胞の>70%がニューロンに変換され、GFAPシグナルがそれに応じて減少するので、このことは予測される。しかし、またこれによって、変換された領域で星状細胞が枯渇されるのではないかという疑問が生じる。これに答えるため、星状細胞からニューロンへの変換がほとんど終了した際、ウイルス注射後17日目(dpi; 27dps)に、NeuroD1 AAVに高度に感染された梗塞に近い脳卒中領域(*)を試験した。
神経再生は神経保護に結びつく
神経炎症の低減と一致して、NeuN陽性ニューロンの数は、NeuroD1感染領域において有意に増加するようであった(図7Aを参照)。驚くべきことに、損傷コアに近い脳卒中領域においては、NeuroD1-GFP標識ニューロンが検出されただけでなく、GFP陽性(図7A、矢印を参照)でもNeuroD1-陽性でもない(図7B、矢印を参照)、多数の非変換ニューロンも検出された。
脳卒中後の機能的神経回路の再構成
in vivoでの細胞変換に関する極めて重要な問題の1つは、グリア変換ニューロンが機能的神経回路を形成することができる否かである。この質問に答えるため、NeuroD1介在性の細胞変換後の脳卒中領域における形態学的構造を試験した。脳卒中及びウイルス注射の後、対照及びNeuroD1治療マウスの両方において、7、17、40、及び60dpiでの虚血損傷運動皮質を試験した(図9Aを参照)。
細胞変換後の血管及び血液脳関門のレスキュー
NeuroD1治療が脳卒中領域において脳組織の喪失をレスキューし神経回路を形成することができる場合、細胞変換後に血管及び血液脳関門を回復させることができるであろうか?
この質問に答えるため、脳卒中領域における血管及び血管に接触して血液脳関門を形成する星状細胞エンドフィートを試験した(図11A~図11Hを参照)。脳卒中のない正常脳においては、血管(Ly6C、内皮細胞及び単球マーカーによって標識)の周囲を包む星状細胞エンドフィートのマーカーアクアポリン4(AQP4、水チャネル)は、無傷の血液脳関門(BBB)を示した(図11A、上段を参照)。反対に、ET-1(1-31)誘発性局所脳卒中の1週間後、血管と星状細胞エンドフィートの両方で有意な破壊が観察された(図11A、下段を参照)。
運動障害及び記憶障害の機能的なレスキュー
NeuroD1治療後の脳卒中領域における有意水準の神経再生を考慮して、この治療が脳卒中によって引き起こされた機能障害をレスキューすることができるかどうかを判定する実験を実施した。様々な動物行動試験に関する予備試験の後に、マウスの前肢機能に対する詳細な分析を、以下の3つの試験を使用して実施した:餌ペレット回収試験、グリッド歩行試験、及びシリンダー試験(図13A、行動試験パラダイムを参照)。
NeuroD1介在性のin vivoでのグリアからニューロンへの変換後の脳卒中領域においては、グリアの形態と機能の両方がNeuroD1感染領域で顕著に改善された。ニューロン変換後のかなりの数の星状細胞は、反応性星状細胞の増殖能力と一致している。NeuroD1発現領域における肥大性から通常の分岐したアストログリア細胞形態への改善は、反応性グリア細胞がより少ない損傷シグナルに暴露されること、新しく生成された星状細胞の反応性が低いこと、又はその両方を示唆している。
in vivoでのグリアからニューロンへの変換はまた、げっ歯類モデルにおける虚血性脳卒中によって引き起こされた運動障害を機能的にレスキューすることもできる。この機能レスキューは、NeuroD1治療後の細胞変換及び回路再構築の両方を含む構造変化に基づく。NeuroD1-GFP標識ニューロンと非標識ニューロンとの混合物がNeuroD1治療後の脳卒中領域において観察された。これは、修復された脳回路が、新しく生成されたニューロンと古い成熟ニューロンの両方の統合であることを示唆している。
項目1。それを必要とする個々の対象のCNSにおける正常な血流の途絶の影響を治療する方法であって、正常な血流が途絶された領域に治療有効用量の外因性NeuroD1を投与することを含む、方法。
配列番号1-ヒトNeuroD1タンパク質をコードするヒトNeuroD1核酸配列-終止コドンを含む1071ヌクレオチド
SEQUENCE LISTING
<110> The Penn State Research Foundation
<120> REGENERATING FUNCTIONAL NEURONS FOR TREATMENT OF NEURAL INJURY
CAUSED BY DISRUPTION OF BLOOD FLOW
<130> PA23-496
<150> US 62/464,469
<151> 2017-02-28
<150> US 62/518,914
<151> 2017-06-13
<160> 9
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gacgacctcg aagccatgaa cgcagaggag gactcactga ggaacggggg agaggaggag 180
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Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala
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Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
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atgaccaaat catacagcga gagcgggctg atgggcgagc ctcagcccca aggtccccca 60
agctggacag atgagtgtct cagttctcag gacgaggaac acgaggcaga caagaaagag 120
gacgagcttg aagccatgaa tgcagaggag gactctctga gaaacggggg agaggaggag 180
gaggaagatg aggatctaga ggaagaggag gaagaagaag aggaggagga ggatcaaaag 240
cccaagagac ggggtcccaa aaagaaaaag atgaccaagg cgcgcctaga acgttttaaa 300
ttaaggcgca tgaaggccaa cgcccgcgag cggaaccgca tgcacgggct gaacgcggcg 360
ctggacaacc tgcgcaaggt ggtaccttgc tactccaaga cccagaaact gtctaaaata 420
gagacactgc gcttggccaa gaactacatc tgggctctgt cagagatcct gcgctcaggc 480
aaaagccctg atctggtctc cttcgtacag acgctctgca aaggtttgtc ccagcccact 540
accaatttgg tcgccggctg cctgcagctc aaccctcgga ctttcttgcc tgagcagaac 600
ccggacatgc ccccgcatct gccaaccgcc agcgcttcct tcccggtgca tccctactcc 660
taccagtccc ctggactgcc cagcccgccc tacggcacca tggacagctc ccacgtcttc 720
cacgtcaagc cgccgccaca cgcctacagc gcagctctgg agcccttctt tgaaagcccc 780
ctaactgact gcaccagccc ttcctttgac ggacccctca gcccgccgct cagcatcaat 840
ggcaacttct ctttcaaaca cgaaccatcc gccgagtttg aaaaaaatta tgcctttacc 900
atgcactacc ctgcagcgac gctggcaggg ccccaaagcc acggatcaat cttctcttcc 960
ggtgccgctg cccctcgctg cgagatcccc atagacaaca ttatgtcttt cgatagccat 1020
tcgcatcatg agcgagtcat gagtgcccag cttaatgcca tctttcacga ttag 1074
<210> 4
<211> 357
<212> PRT
<213> Mus musculus
<400> 4
Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro
1 5 10 15
Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 30
Glu His Glu Ala Asp Lys Lys Glu Asp Glu Leu Glu Ala Met Asn Ala
35 40 45
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Glu Glu Asp Glu
50 55 60
Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Gln Lys
65 70 75 80
Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu
85 90 95
Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn
100 105 110
Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly
145 150 155 160
Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu
165 170 175
Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro
180 185 190
Arg Thr Phe Leu Pro Glu Gln Asn Pro Asp Met Pro Pro His Leu Pro
195 200 205
Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro
210 215 220
Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe
225 230 235 240
His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe
245 250 255
Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro
260 265 270
Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu
275 280 285
Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro
290 295 300
Ala Ala Thr Leu Ala Gly Pro Gln Ser His Gly Ser Ile Phe Ser Ser
305 310 315 320
Gly Ala Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser
325 330 335
Phe Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn
340 345 350
Ala Ile Phe His Asp
355
<210> 5
<211> 1053
<212> DNA
<213> Mus musculus
<400> 5
gcagtgtgga gacacaccca ctttccccaa gggctcctgc tcccccaagt gatcccctta 60
tcctccgtgc taagatgaca ccgaggttgc agtccttacc tttgaaagca gccacaaggg 120
cgtgggggtg cacaccttta atcccagcac tcgggaggca gaggcaggca gatttctgag 180
ttcgagacca gcctggtcta caaagtgaat tccaggacag ccagggctat acagagaaac 240
cctgtcttga aaaaaaaaga gaaagaaaaa agaaaaaaaa aaatgaaagc agccacatct 300
aaggactacg tggcacagga gagggtgagt ccctgagagt tcagctgctg ccctgtctgt 360
tcctgtaaat ggcagtgggg tcatgggaaa gtgaaggggc tcaaggtatt ggacacttcc 420
aggataatct tttggacgcc tcaccctgtg ccaggaccaa ggctgagctt ggcaggctca 480
gaacagggtg tcctgttctt ccctgtctaa aacattcact ctcagcttgc tcacccttcc 540
ccagacaagg aagctgcaca gggtctggtg ttcagatggc tttggcttac agcaggtgtg 600
ggtgtggggt aggaggcagg gggtaggggt gggggaagcc tgtactatac tcactatcct 660
gtttctgacc ctctaggact cctacagggt tatgggagtg gacaggcagt ccagatctga 720
gctgctgacc cacaagcagt gccctgtgcc tgccagaatc caaagccctg ggaatgtccc 780
tctggtcccc ctctgtcccc tgcagccctt cctgttgctc aaccttgcac agttccgacc 840
tgggggagag agggacagaa atcttgccaa gtatttcaac agaatgtact ggcaattact 900
tcatggcttc ctggacttgg taaaggatgg actaccccgc ccaacagggg ggctggcagc 960
caggtaggcc cataaaaagc ccgctgggga gtcctcctca ctctctgctc ttcctcctcc 1020
agcacacatc agacctagta gctgtggaaa cca 1053
<210> 6
<211> 1672
<212> DNA
<213> Homo sapiens
<400> 6
gtctgcaagc agacctggca gcattgggct ggccgccccc cagggcctcc tcttcatgcc 60
cagtgaatga ctcaccttgg cacagacaca atgttcgggg tgggcacagt gcctgcttcc 120
cgccgcaccc cagcccccct caaatgcctt ccgagaagcc cattgagtag ggggcttgca 180
ttgcacccca gcctgacagc ctggcatctt gggataaaag cagcacagcc ccctaggggc 240
tgcccttgct gtgtggcgcc accggcggtg gagaacaagg ctctattcag cctgtgccca 300
ggaaagggga tcaggggatg cccaggcatg gacagtgggt ggcagggggg gagaggaggg 360
ctgtctgctt cccagaagtc caaggacaca aatgggtgag gggactgggc agggttctga 420
ccctgtggga ccagagtgga gggcgtagat ggacctgaag tctccaggga caacagggcc 480
caggtctcag gctcctagtt gggcccagtg gctccagcgt ttccaaaccc atccatcccc 540
agaggttctt cccatctctc caggctgatg tgtgggaact cgaggaaata aatctccagt 600
gggagacgga ggggtggcca gggaaacggg gcgctgcagg aataaagacg agccagcaca 660
gccagctcat gcgtaacggc tttgtggagc tgtcaaggcc tggtctctgg gagagaggca 720
cagggaggcc agacaaggaa ggggtgacct ggagggacag atccaggggc taaagtcctg 780
ataaggcaag agagtgccgg ccccctcttg ccctatcagg acctccactg ccacatagag 840
gccatgattg acccttagac aaagggctgg tgtccaatcc cagcccccag ccccagaact 900
ccagggaatg aatgggcaga gagcaggaat gtgggacatc tgtgttcaag ggaaggactc 960
caggagtctg ctgggaatga ggcctagtag gaaatgaggt ggcccttgag ggtacagaac 1020
aggttcattc ttcgccaaat tcccagcacc ttgcaggcac ttacagctga gtgagataat 1080
gcctgggtta tgaaatcaaa aagttggaaa gcaggtcaga ggtcatctgg tacagccctt 1140
ccttcccttt tttttttttt ttttttgtga gacaaggtct ctctctgttg cccaggctgg 1200
agtggcgcaa acacagctca ctgcagcctc aacctactgg gctcaagcaa tcctccagcc 1260
tcagcctccc aaagtgctgg gattacaagc atgagccacc ccactcagcc ctttccttcc 1320
tttttaattg atgcataata attgtaagta ttcatcatgg tccaaccaac cctttcttga 1380
cccaccttcc tagagagagg gtcctcttga ttcagcggtc agggccccag acccatggtc 1440
tggctccagg taccacctgc ctcatgcagg agttggcgtg cccaggaagc tctgcctctg 1500
ggcacagtga cctcagtggg gtgaggggag ctctccccat agctgggctg cggcccaacc 1560
ccaccccctc aggctatgcc agggggtgtt gccaggggca cccgggcatc gccagtctag 1620
cccactcctt cataaagccc tcgcatccca ggagcgagca gagccagagc at 1672
<210> 7
<211> 1610
<212> DNA
<213> Mus musculus
<400> 7
aactgagagt ggaggggcac agaagagccc aagaggctcc ttaggttgtg tggagggtac 60
aatatgtttg ggctgagcaa cccagagcca gactttgtct ggctggtaag agacagaggt 120
gcctgctatc acaatccaag ggtctgcttg aggcagagcc agtgcaaagg atgtggttag 180
agccagcctg gtgtactgaa gaggggcgaa gagcttgagt aaggagtctc agcggtggtt 240
tgagaggcag ggtggttaat ggagtagctg caggggagaa tccttgggag ggagcctgca 300
ggacagagct ttggtcagga agtgatgggc atgtcactgg accctgtatt gtctctgact 360
tttctcaagt aggacaatga ctctgcccag ggagggggtc tgtgacaagg tggaagggcc 420
agaggagaac ttctgagaag aaaaccagag gccgtgaaga ggtgggaagg gcatgggatt 480
cagaacctca ggcccaccag gacacaaccc caggtccaca gcagatgggt gaccttgcat 540
gtctcagtca ccagcattgt gctccttgct tatcacgctt gggtgaagga aatgacccaa 600
atagcataaa gcctgaaggc cgggactagg ccagctaggg cttgcccttc ccttcccagc 660
tgcactttcc ataggtccca ccttcagcag attagacccg cctcctgctt cctgcctcct 720
tgcctcctca ctcatgggtc tatgcccacc tccagtctcg ggactgaggc tcactgaagt 780
cccatcgagg tctggtctgg tgaatcagcg gctggctctg ggccctgggc gaccagttag 840
gttccgggca tgctaggcaa tgaactctac ccggaattgg gggtgcgggg aggcggggag 900
gtctccaacc cagccttttg aggacgtgcc tgtcgctgca cggtgctttt tatagacgat 960
ggtggcccat tttgcagaag ggaaagccgg agccctctgg ggagcaaggt ccccgcaaat 1020
ggacggatga cctgagcttg gttctgccag tccacttccc aaatccctca ccccattcta 1080
gggactaggg aaagatctcc tgattggtca tatctggggg cctggccgga gggcctccta 1140
tgattggaga gatctaggct gggcgggccc tagagcccgc ctcttctctg cctggaggag 1200
gagcactgac cctaaccctc tctgcacaag acccgagctt gtgcgccctt ctgggagctt 1260
gctgcccctg tgctgactgc tgacagctga ctgacgctcg cagctagcag gtacttctgg 1320
gttgctagcc cagagccctg ggccggtgac cctgttttcc ctacttcccg tctttgacct 1380
tgggtaagtt tctttttctt ttgtttttga gagaggcacc cagatcctct ccactacagg 1440
cagccgctga accttggatc ctcagctcct gccctgggaa ctacagttcc tgcccttttt 1500
ttcccacctt gagggaggtt ttccctgagt agcttcgact atcctggaac aagctttgta 1560
gaccagcctg ggtctccgga gagttgggat taaaggcgtg caccaccacc 1610
<210> 8
<211> 1387
<212> DNA
<213> Homo sapiens
<400> 8
ctctggtttc aagaccaata ctcataaccc ccacatggac caggcaccat cacacctgag 60
cactgcactt agggtcaaag acctggcccc acatctcagc agctatgtag actagctcca 120
gtcccttaat ctctctcagc ctcagtttct tcatctgcaa aacaggtctc agtttcgttg 180
caaagtatga agtgctgggc tgttactggt caaagggaag agctgggaag agggtgcaag 240
gtggggttgg gctggagatg ggctggagca gatagatgga gggacctgaa tggaggaagt 300
aaaccaaggc ccggtaacat tgggactgga cagagaacac gcagatcctc taggcaccgg 360
aagctaagta acattgccct ttctcctcct gtttgggact aggctgatgt tgctgcctgg 420
aagggagcca gcagaagggc cccagcctga agctgttagg tagaagccaa atccagggcc 480
agatttccag gaggcagcct cgggaagttg aaacacccgg attcaggggt caggaggcct 540
gggcttctgg caccaaacgg ccagggacct actttccacc tggagtcttg taagagccac 600
tttcagcttg agctgcactt tcgtcctcca tgaaatgggg gaggggatgc tcctcaccca 660
ccttgcaagg ttattttgag gcaaatgtca tggcgggact gagaattctt ctgccctgcg 720
aggaaatcca gacatctctc ccttacagac agggagactg aggtgaggcc cttccaggca 780
gagaaggtca ctgttgcagc catgggcagt gccccacagg acctcgggtg gtgcctctgg 840
agtctggaga agttcctagg ggacctccga ggcaaagcag cccaaaagcc gcctgtgagg 900
gtggctggtg tctgtccttc ctcctaaggc tggagtgtgc ctgtggaggg gtctcctgaa 960
ctcccgcaaa ggcagaaagg agggaagtag gggctgggac agttcatgcc tcctccctga 1020
gggggtctcc cgggctcggc tcttggggcc agagttcagg gtgtctgggc ctctctatga 1080
ctttgttcta agtctttagg gtggggctgg ggtctggccc agctgcaagg gccccctcac 1140
ccctgcccca gagaggaaca gccccgcacg ggccctttaa gaaggttgag ggtgggggca 1200
ggtgggggag tccaagcctg aaacccgagc gggcgcgcgg gtctgcgcct gccccgcccc 1260
cggagttaag tgcgcggaca cccggagccg gcccgcgccc aggagcagag ccgcgctcgc 1320
tccactcagc tcccagctcc caggactccg ctggctcctc gcaagtcctg ccgcccagcc 1380
cgccggg 1387
<210> 9
<211> 9232
<212> DNA
<213> Artificial Sequence
<220>
<223> Construct including CAG promoter and encoding NeuroD1 and green
fluoresecent protein separated by an IRES.
<400> 9
gatccggcca ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg 60
gccattgcat acgttgtatc catatcataa tatgtacatt tatattggct catgtccaac 120
attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc 180
attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc 240
tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt 300
aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 360
cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 420
taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca 480
gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa 540
tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa 600
tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc 660
cccattgacg caaatgggcg gtaggcatgt acggtgggag gtctatataa gcagagctca 720
ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg 780
ggtacccgta ttcccaataa agcctcttgc tgtttgcatc cgaatcgtgg tctcgctgtt 840
ccttgggagg gtctcctctg agtgattgac tacccacgac gggggtcttt catttggggg 900
ctcgtccggg atttggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc 960
tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgtttga tgttatgcgc 1020
ctgcgtctgt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac 1080
gagttctgaa cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt 1140
tgtggcccga cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt 1200
ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt 1260
tggaaccgaa gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc 1320
tgtctgactg tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta 1380
agtttgacct taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat 1440
gtcaagaaga gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga 1500
tggccgcgag acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt 1560
tcacctggcc cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg 1620
gcttttgacc cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt 1680
cctccatccg ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc 1740
ctttatccag ccctcactcc ttctctaggc gccggaattc gatgtcgaca ttgattattg 1800
actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 1860
cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 1920
ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 1980
caatgggtgg actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 2040
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 2100
tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 2160
accatgggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 2220
acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 2280
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 2340
gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 2400
ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgttgc 2460
cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc gccccggctc tgactgaccg 2520
cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 2580
tggtttaatg acggctcgtt tcttttctgt ggctgcgtga aagccttaaa gggctccggg 2640
agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg 2700
agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 2760
tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg 2820
gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg 2880
ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc gagttgctga 2940
gcacggcccg gcttcgggtg cggggctccg tgcggggcgt ggcgcggggc tcgccgtgcc 3000
gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga 3060
gggctcgggg gaggggcgcg gcggccccgg agcgccggcg gctgtcgagg cgcggcgagc 3120
cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa 3180
atctggcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcgggcgaa 3240
gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg 3300
ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg gggacggctg ccttcggggg 3360
ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct 3420
aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttgttgtg 3480
ctgtctcatc attttggcaa agaattcgct agcggatccg gccgcctcgg ccaccggtcg 3540
ccaccatcgc caccatgacc aaatcataca gcgagagcgg gctgatgggc gagcctcagc 3600
cccaaggtcc cccaagctgg acagatgagt gtctcagttc tcaggacgag gaacacgagg 3660
cagacaagaa agaggacgag cttgaagcca tgaatgcaga ggaggactct ctgagaaacg 3720
ggggagagga ggaggaggaa gatgaggatc tagaggaaga ggaggaagaa gaagaggagg 3780
aggaggatca aaagcccaag agacggggtc ccaaaaagaa aaagatgacc aaggcgcgcc 3840
tagaacgttt taaattaagg cgcatgaagg ccaacgcccg cgagcggaac cgcatgcacg 3900
ggctgaacgc ggcgctggac aacctgcgca aggtggtacc ttgctactcc aagacccaga 3960
aactgtctaa aatagagaca ctgcgcttgg ccaagaacta catctgggct ctgtcagaga 4020
tcctgcgctc aggcaaaagc cctgatctgg tctccttcgt acagacgctc tgcaaaggtt 4080
tgtcccagcc cactaccaat ttggtcgccg gctgcctgca gctcaaccct cggactttct 4140
tgcctgagca gaacccggac atgcccccgc atctgccaac cgccagcgct tccttcccgg 4200
tgcatcccta ctcctaccag tcccctggac tgcccagccc gccctacggc accatggaca 4260
gctcccacgt cttccacgtc aagccgccgc cacacgccta cagcgcagct ctggagccct 4320
tctttgaaag ccccctaact gactgcacca gcccttcctt tgacggaccc ctcagcccgc 4380
cgctcagcat caatggcaac ttctctttca aacacgaacc atccgccgag tttgaaaaaa 4440
attatgcctt taccatgcac taccctgcag cgacgctggc agggccccaa agccacggat 4500
caatcttctc ttccggtgcc gctgcccctc gctgcgagat ccccatagac aacattatgt 4560
ctttcgatag ccattcgcat catgagcgag tcatgagtgc ccagcttaat gccatctttc 4620
acgattaggt ttaaacgcgg ccgcgcccct ctccctcccc cccccctaac gttactggcc 4680
gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc 4740
cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta 4800
ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag 4860
ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga 4920
accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg 4980
caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 5040
ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta 5100
tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa 5160
aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat 5220
atggccacaa ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 5280
gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 5340
gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 5400
ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 5460
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 5520
cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 5580
ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 5640
atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 5700
aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 5760
gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 5820
cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 5880
gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 5940
ctgtacaagt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt 6000
attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat 6060
catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg 6120
tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt 6180
gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact 6240
ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc 6300
tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg 6360
tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc 6420
tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg 6480
cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc 6540
tccccgcctg gaattcgagc tcgagcttgt taacatcgat aaaataaaag attttattta 6600
gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 6660
taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa 6720
ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 6780
cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat 6840
ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 6900
ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 6960
aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 7020
cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc 7080
cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 7140
acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 7200
ggggtctttc atttccgact tgtggtctcg ctgccttggg agggtctcct ctgagtgatt 7260
gactacccgt cagcgggggt cttcacatgc agcatgtatc aaaattaatt tggttttttt 7320
tcttaagtat ttacattaaa tggccatagt tgcattaatg aatcggccaa cgcgcgggga 7380
gaggcggttt gcgtattggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 7440
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 7500
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 7560
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 7620
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 7680
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 7740
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 7800
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 7860
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 7920
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 7980
tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 8040
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 8100
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 8160
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 8220
aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 8280
tttaaattaa aaatgaagtt tgcggccggc cgcaaatcaa tctaaagtat atatgagtaa 8340
acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 8400
tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 8460
ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 8520
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 8580
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 8640
aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 8700
ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 8760
ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 8820
gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 8880
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 8940
cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga 9000
actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 9060
ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 9120
tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 9180
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca at 9232
Claims (28)
- それを必要とする個々の対象のCNSにおける正常な血流の途絶の影響を治療する方法であって、正常な血流が途絶された領域に治療有効用量の外因性NeuroD1を投与することを含む、方法。
- 外因性NeuroD1を投与することが、NeuroD1をコードする核酸を含む発現ベクターをその領域に送達することを含む、請求項1に記載の方法。
- 外因性NeuroD1を投与することが、NeuroD1をコードする核酸を含む組換えウイルス発現ベクターをその領域に送達することを含む、請求項1又は2に記載の方法。
- 外因性NeuroD1を発現することが、NeuroD1をコードする核酸を含む組換えアデノ随伴ウイルス発現ベクターをその領域に送達することを含む、請求項1から3のいずれか一項に記載の方法。
- 外因性NeuroD1を発現することが、1)NeuroD1をコードする核酸を含む組換えアデノ随伴ウイルス発現ベクター、及び2)部位特異的リコンビナーゼをコードする核酸を含む組換えアデノ随伴ウイルス発現ベクターの効果的な「フリップ切除」組換え発現ベクターの組合せをその領域に送達することを含む、請求項1から4のいずれか一項に記載の方法。
- NeuroD1が、その領域に送達される唯一の外因的に発現される転写因子である、請求項1から5のいずれか一項に記載の方法。
- 外因性NeuroD1が、反応性星状細胞が存在する場合、CNSにおける正常な血流が途絶された後に一度に投与される、請求項1から6のいずれか一項に記載の方法。
- 外因性NeuroD1が、グリア細胞が反応性である場合、CNSにおける正常な血流が途絶された後に一度に投与される、請求項1から7のいずれか一項に記載の方法。
- CNSにおける正常な血流の途絶が、虚血、血栓症、塞栓症、出血、脳震盪、ブラスト、脳侵入、腫瘍、炎症、感染症、血管の慢性疾患媒介による制限、及び任意のそれらの2つ以上の組合せからなる群から選択される障害によるものである、請求項1から8のいずれか一項に記載の方法。
- CNSにおける正常な血流の途絶が、虚血性脳卒中;出血性脳卒中;脳動脈瘤;外傷性脳損傷;脳震盪;ブラスト;脳侵入;腫瘍;炎症;感染症;外傷性脊髄損傷;虚血性若しくは出血性の脊髄症(脊髄梗塞);心拍停止若しくは重度の低血圧(ショック)によって引き起こされる全脳虚血;低酸素、低血糖症若しくは貧血によって引き起こされる低酸素性虚血性脳症;感染性心内膜炎若しくは心房粘液腫によって引き起こされるCNS塞栓症;線維軟骨性塞栓性脊髄症;小児白血病によって引き起こされるCNS血栓症;ネフローゼ症候群(腎臓病)、慢性炎症性疾患、妊娠、エストロゲン系避妊薬の使用、脳膜炎、脱水によって引き起こされる脳静脈洞血栓症;又は任意のそれらの2つ以上の組合せからなる群から選択される障害によるものである、請求項1から9のいずれか一項に記載の方法。
- 治療有効用量のNeuroD1を投与することが、NeuroD1タンパク質をコードする核酸配列を含む組換え発現ベクターを投与することを含み、NeuroD1タンパク質をコードする核酸配列が、配列番号2又はその機能的断片をコードする核酸配列、配列番号4又はその機能的断片をコードする核酸配列、配列番号1又はその機能的断片、配列番号3又はその機能的断片、及び配列番号2又は配列番号4と70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%又はそれ以上の同一性を有するタンパク質又はその機能的断片をコードする核酸配列からなる群から選択される核酸配列を含む、請求項1から10のいずれか一項に記載の方法。
- 治療有効用量のNeuroD1を投与することが、損傷領域内又はその付近における定位注入を含む、請求項1から11のいずれか一項に記載の方法。
- NeuroD1をコードする核酸配列がユビキタスプロモーターに作動可能に連結されている、請求項1から12のいずれか一項に記載の方法。
- 部位特異的リコンビナーゼをコードする核酸配列がグリア細胞特異的プロモーターに作動可能に連結されている、請求項5から12のいずれか一項に記載の方法。
- グリア細胞特異的プロモーターがGFAPプロモーターである、請求項14に記載の方法。
- GFAPプロモーターがヒトGFAPプロモーターである、請求項15に記載の方法。
- 対象における治療の有効性を評価することを更に含む、請求項1から16のいずれか一項に記載の方法。
- 対象における治療の有効性を評価することが、電気生理学的アッセイ、血流アッセイ、組織構造アッセイ、機能アッセイ、及びそれらの任意の2つ以上の組合せから選択されるアッセイを含む、請求項17に記載の方法。
- 対象における治療の有効性を評価することが脳波を含む、請求項17に記載の方法。
- 対象における治療の有効性を評価することが、近赤外分光法及びfMRIからなる群から選択される血流のアッセイを含む、請求項17に記載の方法。
- 対象における治療の有効性を評価することが、MRI、PETスキャン、CATスキャン及び超音波からなる群から選択される組織構造のアッセイを含む、請求項17に記載の方法。
- 対象における治療の有効性を評価することが、行動アッセイを含む、請求項17に記載の方法。
- 対象における治療の有効性を評価することが、治療有効用量の外因性NeuroD1を投与する前に実施されるアッセイを含む、請求項17に記載の方法。
- NeuroD1をコードする核酸を含むアデノ随伴ウイルス粒子を1010~1014個のアデノ随伴ウイルス粒子/担体1mlの濃度で含有する薬学的に許容される担体1~500μlを、正常な血流が途絶した領域において、0.1~5μl/分の制御された流速で対象に注射する、請求項1から23のいずれか一項に記載の方法。
- 血栓の除去、血流の促進、抗炎症剤の投与、酸化防止剤の投与、興奮毒性の低減、及びそれらの2つ以上からなる群から選択される治療を更に含む、請求項1から24のいずれか一項に記載の方法。
- 1)部位特異的リコンビナーゼをコードする核酸に作動可能に連結されているグリア細胞特異的プロモーターを含む組換えアデノ随伴アデノウイルス発現ベクター、及び2)NeuroD1をコードする核酸に作動可能に連結されているユビキタスプロモーターを含む組換えアデノ随伴アデノウイルス発現ベクター、を含む組成物であって、NeuroD1をコードする核酸が反転され、2組の部位特異的リコンビナーゼ認識部位に挟まれており、それによって、リコンビナーゼの作用がNeuroD1をコードする核酸を不可逆的に反転してNeuroD1が哺乳動物細胞で発現される、組成物。
- 実質的に本明細書に記載されているような、その必要のある個々の対象のCNSにおける正常な血流の途絶の影響を治療する方法。
- 実質的に本明細書に記載されているような、フリップ切除発現ベクターの組合せ。
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