We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

すべてのマウスは、コロラド州立大学の施設内動物管理使用委員会によって承認されたプロトコルに従って、ラボ動物ケア・インターナショナルの評価と認定協会の認定を受け、研究室動物資源で飼育し、維持しました。 LSPCsの調製<ol><li> 1 DOTAP（1,2-ジオレオイル-3-トリメチルアンモニウム - プロパン）：LSPCsのためのコレステロール比1を使用してください。 1クロロホルム：フラスコ内のメタノール溶液4ナノモルのリポソーム製剤については、DOTAPの2ナノモルと1の10ミリリットルへのコレステロールの2ナノモルを混ぜます。 </li><li>ドラフト内でN 2ガ ​​スを用いたメタノール溶液：クロロホルムの9ミリリットルを蒸発させます。ガスなしで一晩換気フード内の溶媒の最後の1ミリリットルを蒸発させます。フラスコの底に薄い脂質フィルムを観察します。 </li><li>熱、55℃に10％ショ糖溶液10mlを。 </li><li> GEながら、 すなわち、1ml /分、徐々に薄い脂質フィルム上に加熱された10％スクロース溶液を注ぎntlyフラスコを旋回。脂質分子はゲル - 液相に残るように55℃での脂質と10％のショ糖、フラスコを維持します。多重膜小胞（MLV）の形成での再水和をもたらします。 </li><li>脂質がリポソームをサイジングする前に、少なくとも1時間、55℃で再水和することができます。 </li><li> 1.0μmのフィルターを製造者の指示に従って、押出機を組み立てたり、サイジングのために1.0μmのシリンジフィルターを使用しています。 </li><li>押出機での注射器のいずれかに加熱されたリポソーム懸濁液の1ミリリットルを追加し、2つのシリンジ11時間の間のサスペンションを渡します。懸濁液を11回通過した後に開始し、注射器よりも反対注射器であることを確認します。 </li><li>サイズその後、0.45μmの0.2μmのフィルターを通して再びリポソームは、大型単層小胞（LUVを）を生成します。簡単にサイズ調整のために加熱し、リポソーム懸濁液を保管してください。 </li><li> 20μM（4200μlで4ナノモルリポソーム懸濁液（200μM）の20μLをインキュベート室温で10分間ナノモル合計）株式のsiRNA溶液。 </li><li> RTで10分間のsiRNA /リポソーム溶液と500μM（40ナノモル合計）株式RVG-9R液80μLをインキュベートします。 LSPCsはできるだけ早く使用されるべきであるが、調製後数時間まで使用することができます。調製後、数時間のために4℃で保存しLSPCs。 </li></ol> PALETSの調製<ol><li> 40：：DSP​​E（1,2-ジステアロイル-sn-グリセロ-3-ホスホエタノールアミン）のいずれかの5のモル比：コレステロール：DSPE-PEGまたはDOTAP：コレステロール：DSPE-PEG PALETSのための55を使用してください。 1クロロホルム：フラスコ内のメタノール溶液4ナノモルのリポソーム製剤については、2の10ミリリットルにDSPEまたはDOTAPのいずれかの2.2ナノモル、コレステロールの1.6ナノモル、及びDSPE-PEGの0.2ナノモルを混ぜます。 </li><li>ステップ1.1以上1.2で説明したように正確にリポソーム懸濁液を準備します。 1×PBS 10ml中DSPEリポソームを再水和を1ml /分を追加して、75℃に加熱しました。脂質は時のために75℃で再水和することを許可しますサイジングの前に少なくとも1時間。 </li><li>サイズステップ1.6から1.8に記載とおりにリポソーム懸濁液。 </li><li> 4ナノモルのそれぞれのピペットを20μl（200μM）リポソーム懸濁液の単一使用のアリコートへのDOTAPおよびDSPEリポソームはサイズされた後、およびベンチトップ凍結乾燥機（ 図2）を使用して、30分間凍結乾燥。 </li><li> DSPE PALETSリポソーム中に封入されるsiRNAをRTで10分間、硫酸プロタミンの26.6μM溶液を1.4μlの20μM（4ナノモル合計）株式のsiRNA溶液200μlをインキュベートします。 </li><li> siRNAの4ナノモルストック溶液の200μlでDOTAP PALETSリポソームを再水和またはsiRNA /プロタミン溶液を201.4μlのDSPE PALETSリポソームを再水和。 RTで10分間リポソーム/ siRNA溶液をインキュベートします。 </li><li> 60mMの1-エチル-3-（3-ジメチルアミノプロピル）カルボジイミド塩酸塩（EDC）の10μLをDOTAPとDSPE PALETS両方用リポソーム/ siRNA溶液の溶液を加えます。 </li><li>両方のDOTAPおよびDSPE PALETS用リポソーム/ siRNA溶液に150mMのN-ヒドロキシ（スルホNHS）溶液10μlを加えます。 </li><li> RTで2時間のsiRNA /リポソーム懸濁液とEDCおよびスルホNHSをインキュベートします。 </li><li> RTで10分間この溶液を架橋するsiRNA /リポソームと500μM（40ナノモル合計）株式RVG-9R液80μLをインキュベートします。 注：EDC /スルホNHSはPEG脂質に共有結合にRVG-9Rを可能にします。調製後、数時間以内にPALETSを使用してください。調製後、数時間のために4℃で保存しPALETS。 </li><li> PALETSのsiRNA封入効率を決定するには： <ol><li> 260nmで設定し、分光光度計を使用してプロタミン及びリポソームの添加前および後のsiRNAの濃度を測定します。 </li><li> 50kDaの遠心フィルターを使用してリポソーム/カプセル化されたsiRNAの懸濁液から封入されていないのsiRNAをフィルタリングします。 20分1,000×gで遠心分離します。 </li><li>の濃度を測定260nmで分光光度計を使用して、ろ液および保持液中のカプセル化されたsiRNAの濃度でカプセル化されていないのsiRNA。 </li></ol></li></ol> 3. LSPCsまたはPALETSをマウスに注射<ol><li>血管系を拡張するために熱ランプに5〜10分間、マウスを公開します。 </li><li> 5〜10分間注入前、および注入中に2から3パーセントイソフルオラン/酸素流量でマウスを麻酔。動物がもはや通院で、呼吸がつま先のピンチを実行して、減速しているときに麻酔を確認していません。尾静脈にアクセスするには、その背面または側面にマウスを置きます。麻酔下ながら乾燥を防ぐために、マウスの目に獣医軟膏を使用してください。 </li><li>ワイプ/ 70％エタノールで尾をスプレーし、26 Gインスリン注射器を用いて、マウスの尾静脈にLSPCsまたはPALETSの300μLを注入します。尾静脈に遠位に注入開始し、静脈が崩壊または破裂場合近位に移動します。 </li><li>それは胸骨横臥位を維持するまで、クリーンなケージにマウスを置きます。 MOを置かないでくださいそれは完全に回復するまで、他のケージの仲間と一緒に使用。マウスは、5〜15分で回復する必要があります。彼らは、回復に時間がかかる場合には、体温を維持するために加温パッド上又は加熱ランプの下に配置することができます。マウスは、過熱から保護するために注意深く監視する必要があります。 </li><li>麻酔が切れる確実にするために数時間注入後に動物を監視し、治療の全く悪影響はありません。 LSPCsまたはPALETSは、少なくとも24時間循環させます。 </li></ol>フローサイトメトリーを介して、タンパク質発現の4解析<ol><li> 15分間、20％CO 2の流量でマウスを安楽死させます。 </li><li>ハサミを用いてマウスの皮膚と頭蓋骨を切り取ることにより、脳の半分の半球を解剖。ピール離れて半分離れトングやハサミの先端を使って頭蓋骨から脳の半球。 </li><li>プレス半分のFACS緩衝液2.5ミリリットルを使用して、40μmのメッシュセルストレーナーを介して、各マウスからの脳の半球（1倍、シリンジのプランジャーを有するペトリ皿にPBS、1％ウシ胎児血清、10mMのEDTA）。 </li><li> FACS緩衝液のさらに2.5 mlの細胞ストレーナーとペトリ皿をすすぎます。氷上で15ミリリットルコニカルチューブに単一細胞懸濁液を入れてください。 </li><li>ピペットで350×gで5分間1.5 mlのマイクロ遠心チューブと遠心分離機に5ミリリットル単一細胞懸濁液100μl。上清を捨て、FACS緩衝液1ml中に細胞ペレットを再懸濁します。 5分間、350×gでスピン。 FACS緩衝液の別の1ミリリットルで繰り返します。氷上で細胞懸濁液を保管してください。 </li><li> 1100μlの細胞をインキュベート：FACS緩衝液中の0.5mg / mlのラット抗マウスFcブロックの100倍希釈を氷上で30〜60分間。ペレット化し、ステップ4.5のように細胞を洗浄します。氷上で細胞懸濁液を保管してください。 </li><li> 30〜60分間氷上でFACS緩衝液中で7％マウス血清中のマウスのPrP Cに対する20 / mlの蛍光抗体を100μlで細胞をインキュベートします。ペレット化し、ステップ4.5のように細胞を洗浄します。細胞懸濁液を保ちます冷やして。 </li><li>ペレットおよび1分RBC溶解緩衝液（1×PBS、155 mMのNH 4 Cl を 、12 mMの炭酸水素ナトリウム、0.1mMのEDTA）1ml中に再懸濁細胞。ステップ4.5のようにペレットとは、FACS緩衝液1mlに細胞を懸濁します。氷上で細胞懸濁液を保管してください。 </li><li>以前10記載されているように、フローサイトメーターを使用してのPrP Cの発現を解析。ゲートは、全細胞集団から細胞を生きています。ゲートのPrP C + MFI（蛍光強度中央値）を決定するために、生細胞集団由来の細胞。 </li></ol>

<ol>
	<li>Bolton, D. C., Mckinley, M. P., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+purifies+with+the+scrapie+prion.">Identification of a protein that purifies with the scrapie prion.</a> Science. 218 (4579), 1309-1311 (1982).</li><li>McKinley, M. P., Bolton, D. C., Pruisner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protease-resistant+protein+is+a+structural+component+of+the+scrapie+prion.">A protease-resistant protein is a structural component of the scrapie prion.</a> Cell. 35 (1), 57-62 (1983).</li><li>Pruisner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216 (4542), 136-144 (1982).</li><li>Jeffrey, M., McGovern, G., Sis&#242;, S., Gonz&#225;lez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+sub-cellular+pathology+of+prion+diseases:+relationship+between+morphological+changes,+accumulation+of+abnormal+prion+protein+and+clinical+disease.">Cellular and sub-cellular pathology of prion diseases: relationship between morphological changes, accumulation of abnormal prion protein and clinical disease.</a> Acta Neuropathologica. 121 (1), 113-134 (2011).</li><li>B&#252;eler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+devoid+of+PrP+are+resistant+to+scrapie.">Mice devoid of PrP are resistant to scrapie.</a> Cell. 73 (7), 1339-1347 (1993).</li><li>Pfeifer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivector-mediated+RNAi+efficiently+suppresses+prion+protein+and+prolongs+survival+of+scrapie-infected+mice.">Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice.</a> J Clin Invest. 116 (12), 3204-3210 (2006).</li><li>White, M., Farmer, M., Mirabile, I., Brandner, S., Collinge, J., Mallucci, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+treatment+with+RNAi+against+prion+protein+rescues+early+neuronal+dysfunction+and+prolongs+survival+in+mice.">Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice.</a> Proc Natl Acad Sci. 105 (29), 10238-10243 (2008).</li><li>Gallozzi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prnp+knockdown+in+transgenic+mice+using+RNA+interference.">Prnp knockdown in transgenic mice using RNA interference.</a> Transgenic Research. 17 (5), 783-791 (2008).</li><li>Mallucci, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cellular+prion+protein+reverses+early+cognitive+deficits+and+neurophysiological+dysfunction+in+prion-infected+mice.">Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice.</a> Neuron. 53 (3), 325-335 (2007).</li><li>Pulford, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-siRNA-peptide+complexes+cross+the+blood-brain+barrier+and+significantly+decrease+PrPC+on+neuronal+cells+PrPRes+in+infected+cultures.">Liposome-siRNA-peptide complexes cross the blood-brain barrier and significantly decrease PrPC on neuronal cells PrPRes in infected cultures.</a> PLoS One. 5 (6), (2010).</li><li>Malone, R. W., Felgner, P. L., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposome-mediated+RNA+transfection.">Cationic liposome-mediated RNA transfection.</a> Proc Natl Acad Sci. 86, 6077-6081 (1989).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+efficiency+of+cationic+liposome-mediated+intravenous+gene+delivery.">Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery.</a> Nat Biotechnol. 15, 167-173 (1997).</li><li>Freimark, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipids+enhance+cytokine+and+cell+influx+levels+in+the+lung+following+administration+of+plasmid:+cationic+lipid+complexes.">Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes.</a> J Immunol. 160 (9), 4580-4586 (1998).</li><li>Balzas, D. A., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+for+use+in+gene+delivery.">Liposomes for use in gene delivery.</a> J Drug Deliv. , (2011).</li><li>Srinivasan, C., Burgess, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+characterization+of+anionic+lipoplexes+for+gene+delivery.">Optimization and characterization of anionic lipoplexes for gene delivery.</a> J Control Release. 136 (1), 62-70 (2009).</li><li>Lakkaruju, A., Dubinsky, J. M., Low, W. C., Rahman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+are+protected+from+excitotoxic+death+by+p53+antisense+oligonucleotides+delivered+in+anionic+liposomes.">Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.</a> J Biol Chem. 276 (34), 32000-32007 (2001).</li><li>Tao, Y., Han, J., Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-targeting+gene+delivery+using+a+rabies+virus+glycoprotein+peptide+modulated+hollow+liposomes:+bio-behavioral+study.">Brain-targeting gene delivery using a rabies virus glycoprotein peptide modulated hollow liposomes: bio-behavioral study.</a> J Mater Chem. 22, 11808-11815 (2012).</li><li>Li, S., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+transfer+via+intravenous+administration+of+cationic+lipid-protamine-DNA+(LPD)+complexes.">In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.</a> Gene Ther. 4 (9), 891-900 (1997).</li><li>Li, S., Rizzo, M. A., Bhattacharya, S., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cationic+lipid-protamine-DNA+(LPD)+complexes+for+intravenous+gene+delivery.">Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery.</a> Gene Ther. 5 (7), 930-937 (1998).</li><li>Kumar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448, 39-43 (2007).</li><li>Voinea, M., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+of+'intelligent'+liposomes+for+efficient+delivery+of+drugs.">Designing of 'intelligent' liposomes for efficient delivery of drugs.</a> J Cell Mol Med. 6 (4), 465-474 (2002).</li><li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+are+protected+from+excitotoxic+death+by+p53+antisense+oligonucleotides+delivered+in+anionic+liposomes.">Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.</a> J. Biol. Chem. 276 (34), 32000-32007 (2001).</li><li>Li, S., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+transfer+via+intravenous+administration+of+cationic+lipid-protamine-DNA+(LPD)+complexes.">In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes.</a> Gene Ther. 4 (9), 891-900 (1997).</li><li>B&#252;eler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+development+and+behaviour+of+mice+lacking+the+neuronal+cell-surface+PrP+protein.">Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.</a> Nature. 356 (6370), 577-582 (1992).</li><li>Dykxhoorn, D., Novina, C., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+the+messenger:+short+RNAs+that+silence+gene+expression.">Killing the messenger: short RNAs that silence gene expression.</a> Nat Rev Mol Cell Bio. 4 (6), 457-467 (2003).</li><li>Graham, A., Ray, M., Perry, E., Jaros, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+nicotinic+acetylcholine+receptor+subunit+expression+in+the+human+hippocampus.">Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus.</a> J Chem Neuroanat. 25 (2), 97-113 (2003).</li><li>Kretzschmar, H. A., Prusiner, S. B., Stowring, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie+prion+proteins+are+synthesized+in+neurons.">Scrapie prion proteins are synthesized in neurons.</a> Am J Pathol. 122 (1), 1-5 (1986).</li><li>Moser, M., Colello, R., Pott, U., Oesch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+expression+of+the+prion+protein+gene+in+glial+cells.">Developmental expression of the prion protein gene in glial cells.</a> Neuron. 14 (3), 509-517 (1995).</li><li>Cronier, S., Laude, H., Peyrin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+can+infect+primary+cultured+neurons+and+astrocytes+and+promote+neuronal+cell+death.">Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.</a> Proc Natl Acad Sci. 101 (33), 12271-12276 (2004).</li><li>Diedrich, J., Bendheim, P., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrapie-associated+prion+protein+accumulates+in+astrocytes+during+scrapie+infection.">Scrapie-associated prion protein accumulates in astrocytes during scrapie infection.</a> Proc Natl Acad Sci. 88 (2), 375-379 (1991).</li></ol>

The authors declare that they have no competing financial interests.

このレポートでは、効率的にCNSに対するsiRNAをトランスポート2の標的送達システムを作成するためのプロトコルについて説明します。 CNSに対するsiRNAを送達する従来の方法は、脳、標的としたsiRNAの静脈内注射、または非標的リポソームのsiRNA複合体の静脈内注射に直接のsiRNA / shRNAのベクトルを注入含まれています。 CNSへのsiRNA / shRNAのベクターの注入は、標的タンパク質の発現レベルの減少...

プリオンは、CNSに影響を与える深刻な神経変性疾患です。プリオン病は、PrP RESと呼ばれる感染性異性体によって、正常な細胞プリオンタンパク質、のPrP Cのミスフォールディングに起因します。これらの疾患は、人間1-3でシカで羊、慢性消耗病におけるスクレイピー、牛における牛海綿状脳症を含む種の多種多様な影響を及ぼし、およびクロイツフェルト・ヤコブ病。プリオンは、シナプスの損失で始まる神経変性を引き起こし、空胞化、グリオーシス、ニューロン損失、およびプラーク沈着に進行します。結局、4個々の動物/の死に至ります。数十年にわたって、研究者は、プリオン病の進行を遅らせるまたは停止することを意味する化合物を調査しました。しかし、研究者は、成功した治療や効果的な全身送達ビヒクルのいずれかを見つけていません。 内因性のPrP Cの発現は、プリオン病の発症のために必要とされる5 </s&gt;アップ。したがって、減少または排除するのPrP Cの発現が遅延または疾患の改善をもたらすことができます。いくつかのグループは、プリオン病でのPrP C発現レベルの役割を調べるために、マウス脳組織に直接shRNAを発現のPrP Cの減少したレベルまたは注入レンチベクターを用いてトランスジェニックマウスを作成しました。これらの研究者は、プリオン病の進行性の神経病理の停止をもたらし、動物6-9の寿命を延長し、ニューロンのPrP Cの量を低減することがわかりました。我々は、マウス神経芽腫細胞10内のPrP RESのクリアランスのことのPrP C siRNA処理の結果を報告しています。これらの研究は療法の使用は、低分子干渉RNA（siRNA）、切断するmRNAのように、のPrP Cの発現レベルを減少させるために十分なプリオン病の進行を遅らせることができることを示唆しています。しかし、プリオン病について調査し、最も治療法は実用的ではない方法で配信されていました臨床設定インチこのために、siRNA療法は、CNSに静脈内に送達し、標的とする全身性送達システムを必要とします。 研究者らは、遺伝子治療製品の送達ビヒクルとしてのリポソームの使用を研究しました。カチオン性およびアニオン性脂質は、両方の、リポソームの形成に使用されます。カチオン性脂質とDNA / RNA間の電荷の差が効率的なパッケージングを可能にするためのカチオン性脂質は、より広くアニオン性脂質よりも使用されています。カチオン性脂質の他の利点は、他の脂質11-14より容易に細胞膜を通過することです。しかし、カチオン性脂質はアニオン性脂質13,14よりも免疫原性です。そのため、研究者らは、リポソーム中のアニオン性脂質にカチオンを使用してからシフトし始めています。遺伝子治療製品を効率的にDNA / RNA分子15-19を凝縮正に荷電したペプチドの硫酸プロタミンを用いてアニオン性リポソーム中にパッケージングすることができます。アニオン性LIPIので、DSは、彼らが循環時間を増加していることがあり、より多くの動物モデル13,14に許容され得るカチオン性脂質よりも免疫原性が低いです。リポソームは、リポソームに結合しているペプチドを標的に使用して特定の組織を標的とします。ニコチン性アセチルコリン受容体に結合するRVG-9R神経ペプチドは、CNS 17-20にsiRNAおよびリポソームを標的化するために使用されてきました。 このレポートは、3 siRNA送達車を生産するための、および神経細胞（ 図1）に対するsiRNAをパッケージ化して提供するためのプロトコルの概要を説明します。リポソームのsiRNAペプチド複合体（LSPCs）は、siRNAおよび静電リポソームの外表面に付着したRVG-9Rターゲティングペプチドを有するリポソームから構成されています。ペプチドは、RVG-9Rが共有脂質、PEG基に結合して、siRNAおよびリポソーム内に封入されたプロタミンで構成されている治療のsiRNA（PALETS）をカプセル化されたリポソームを取り上げました。 LSPCsとPALETを生成するには、以下の方法を使用してSは、のPrP C siRNAは、治癒または、実質的にプリオン病の病態の発症を遅延させるための途方もない約束を保持している神経細胞で90％、最大のPrP Cの発現を低下させます。

<table><tbody><tr><td>DOTAP lipid</td><td>Avanti Lipids</td><td>890890</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Lipids</td><td>700000</td><td></td></tr><tr><td>DSPE</td><td>Avanti Lipids</td><td>850715</td><td></td></tr><tr><td>DSPE-PEG</td><td>Avanti Lipids</td><td>880125</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>AC268320010</td><td></td></tr><tr><td>Methanol</td><td>EMD Millipore</td><td>113351</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;sub&gt; Gas</td><td>AirGas</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>Extruder</td><td>Avanti Lipids</td><td>610023</td><td></td></tr><tr><td>1.0, 0.4, and 0.2 &amp;#956;m filters</td><td>Avanti Lipids</td><td>610010, 610007, 610006</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Protamine sulfate</td><td>Fisher Scientific</td><td>ICN10275205</td><td></td></tr><tr><td>EDC</td><td>Thermo Scientific</td><td>22980</td><td>Aliquoted for single use</td></tr><tr><td>Sulfo-NHS</td><td>Thermo Scientific</td><td>24510</td><td>Aliquoted for single use</td></tr><tr><td>40 &amp;#956;m Cell Strainer</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Rat anti-mouse CD16/CD32 Fc block</td><td>BD Pharmigen</td><td>553141</td><td></td></tr><tr><td>Anti-PrP antibody (PRC5)</td><td>Proprietary - PRC</td><td></td><td></td></tr></tbody></table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

アニオンPALETS内のsiRNAのカプセル化の効率を高めるために、siRNAは、プロタミンと混合しました。 1（ 図3A）：1〜2：siRNAのための最高のプロタミン濃度を決定するために、siRNAは、1から、プロタミンの異なる濃度で混合しました。プロタミンを使用せずに、アニオン性リポソームで60〜65%のsiRNA封入効率がありました。プロタミンとサンプル：siRNAのモル比?...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

カチオン性およびアニオン性リポソームを用いてCNSへの治療用のsiRNAの送達

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.4K ビュー.  Colorado State University. The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases. 

ビデオ: Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

がん細胞への siRNA のエクソソームの準備

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

がん研究

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

siRNAの治療を送達するための多孔質シリコンマイクロ粒子

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

生物工学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

神経科学

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

生化学

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNAの脳特異的ノックダウンのための定位注射への単純な代替

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

体外の効率的な配信のカチオン性 Nanoliposomes の生成は、メッセンジャー RNA を転写

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

細胞質 siRNA 送達のための、中立的充電された pH 応答性高分子ナノ粒子の調製

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

中枢神経系への鼻腔内siRNA送達中におけるマウスの配置のための位置決め装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

免疫・感染学

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

カチオン性リポソームを介した特異的siRNAの再配達によるマウスの肺におけるIntersectin-1Sの長期サイレン。電子顕微鏡によるノックダウン効果の評価

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

生物学

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

ラット中枢神経系におけるアンチセンスオリゴヌクレオチドの子宮内送達

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...